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First Aid for the USMLE Step 1 2022,
32e Tao Le
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Contents
Contributing Authors vii General Acknowledgments xiii

Associate Authors viii How to Contribute xv
Faculty Advisors ix How to Use This Book xvii
Preface xi Selected USMLE Laboratory Values xviii
Special Acknowledgments xii First Aid Checklist for the USMLE Step 1 xx
` SECTION I G U I D E TO E F F I C I E N T E X A M P R E PA R AT I O N 1
Introduction 2 Test-Taking Strategies 19

USMLE Step 1—The Basics 2 Clinical Vignette Strategies 21
Learning Strategies 11 If You Think You Failed 22
Timeline for Study 14 Testing Agencies 22
Study Materials 18 References 23
` SECTION I SUPPLEMENT S P E C I A L S I T UAT I O N S 25
` SECTION II HIGH-YIELD GENERAL PRINCIPLES 27
How to Use the Database 28 Pathology 203

Biochemistry 31 Pharmacology 229
Immunology 93 Public Health Sciences 257
Microbiology 121
FAS1_2022_00_Frontmatter.indd 5 11/10/21 10:50 AM

` SECTION III H I G H - Y I E L D O R G A N S YS T E M S 281
Approaching the Organ Systems 282 Neurology and Special Senses 503
Cardiovascular 285 Psychiatry 575
Endocrine 331 Renal 601
Gastrointestinal 365 Reproductive 635
Hematology and Oncology 411 Respiratory 683
Musculoskeletal, Skin, and Connective Tissue 453 Rapid Review 713
` SECTION IV TO P - R AT E D R E V I E W R E S O U R C E S 7 37
How to Use the Database 738 Biochemistry 742

Question Banks 740 Cell Biology and Histology 742
Web and Mobile Apps 740 Microbiology and Immunology 742
Comprehensive 741 Pathology 743
Anatomy, Embryology, and Neuroscience 741 Pharmacology 743
Behavioral Science 742 Physiology 744
`
Abbreviations and Symbols 745 Index 771
Image Acknowledgments 753 About the Editors 828
vi
FAS1_2022_00_Frontmatter.indd 6 11/10/21 10:50 AM

HIGH-YIELD PRINCIPLES IN
Biochemistry
“The nitrogen in our DNA, the calcium in our teeth, the iron in our blood, ` Molecular 32
the carbon in our apple pies were made in the interiors of collapsing stars.
We are made of starstuff.” ` Cellular 44
—Carl Sagan
` Laboratory Techniques 50
“Biochemistry is the study of carbon compounds that crawl.”
—Mike Adams ` Genetics 54
“We think we have found the basic mechanism by which life comes from ` Nutrition 63
life.”
—Francis H. C. Crick ` Metabolism 71
DNA was the first three-dimensional Xerox machine.

—Kenneth Ewart Boulding
This high-yield material includes molecular biology, genetics, cell

biology, and principles of metabolism (especially vitamins, cofactors,
minerals, and single-enzyme-deficiency diseases). When studying
metabolic pathways, emphasize important regulatory steps and enzyme
deficiencies that result in disease, as well as reactions targeted by
pharmacologic interventions. For example, understanding the defect
in Lesch-Nyhan syndrome and its clinical consequences is higher yield
than memorizing every intermediate in the purine salvage pathway.
Do not spend time learning details of organic chemistry, mechanisms, or

physical chemistry. Detailed chemical structures are infrequently tested;
however, many structures have been included here to help students
learn reactions and the important enzymes involved. Familiarity with
the biochemical techniques that have medical relevance—such as
ELISA, immunoelectrophoresis, Southern blotting, and PCR—is
useful. Review the related biochemistry when studying pharmacology or
genetic diseases as a way to reinforce and integrate the material.
31
FAS1_2022_01-Biochem.indd 31 11/4/21 11:59 AM

32 SEC TION II Biochemistry  BIOCHEMISTRY—Molecular
` BIOCHEMISTRY—MOLECULAR
Chromatin structure DNA exists in the condensed, chromatin form to

fit into the nucleus. DNA loops twice around a
histone octamer to form a nucleosome (“beads
DNA double-helix on a string”). H1 binds to the nucleosome
and to “linker DNA,” thereby stabilizing the
chromatin fiber.
H1 histone DNA has ⊝ charge from phosphate groups.
(linker)
DNA Histones are large and have ⊕ charge from
lysine and arginine.
In mitosis, DNA condenses to form
Nucleosome Euchromatin Supercoiled chromosomes. DNA and histone synthesis
(H2A, H2B, structure occurs during S phase.
H3, H4) 2 Heterochromatin Mitochondria have their own DNA, which is
circular and does not utilize histones.
Metaphase
chromosome
Heterochromatin Condensed, appears darker on EM (labeled H Heterochromatin = highly condensed.

A
in A ; Nu, nucleolus). Sterically inaccessible, Barr bodies (inactive X chromosomes) may be
E thus transcriptionally inactive. methylation, visible on the periphery of nucleus.
H acetylation.
Nu
Euchromatin Less condensed, appears lighter on EM (labeled Eu = true, “truly transcribed.”

E in A ). Transcriptionally active, sterically Euchromatin is expressed.
accessible.
DNA methylation Changes the expression of a DNA segment DNA is methylated in imprinting.
without changing the sequence. Involved with Methylation within gene promoter (CpG islands)
aging, carcinogenesis, genomic imprinting, typically represses (silences) gene transcription.
transposable element repression, and X CpG methylation makes DNA mute.
chromosome inactivation (lyonization). Dysregulated DNA methylation is implicated in
Fragile X syndrome.
Histone methylation Usually causes reversible transcriptional Histone methylation mostly makes DNA mute.
suppression, but can also cause activation Lysine and arginine residues of histones can be
depending on location of methyl groups. methylated.
Histone acetylation Removal of histone’s ⊕ charge relaxed DNA Thyroid hormone receptors alter thyroid
coiling transcription. hormone synthesis by acetylation.
Histone acetylation makes DNA active.
Histone deacetylation Removal of acetyl groups tightened DNA Histone deacetylation may be responsible for the
coiling transcription. altered gene expression in Huntington disease.

Biochemistry  BIOCHEMISTRY—Molecular SEC TION II 33
Nucleotides Nucleoside = base + (deoxy)ribose (sugar).

Nucleotide = base + (deoxy)ribose + phosphate; 5′ end of incoming nucleotide bears the
linked by 3′-5′ phosphodiester bond. triphosphate (energy source for the bond).
α-Phosphate is target of 3′ hydroxyl attack.
Purines (A,G)—2 rings. Pure As Gold.

Pyrimidines (C,U,T)—1 ring. CUT the pyramid.
Thymine has a methyl.
Deamination reactions: C-G bond (3 H bonds) stronger than A-T bond
Cytosine uracil (2 H bonds). C-G content melting
Adenine hypoxanthine temperature of DNA. “C-G bonds are like
Guanine xanthine Crazy Glue.”
5-methylcytosine thymine
Amino acids necessary for purine synthesis (cats
Uracil found in RNA; thymine in DNA. purr until they GAG):
Methylation of uracil makes thymine. Glycine
Aspartate
Glutamine
Purine (A, G) Pyrimidine (C, U, T)
Nucleoside
CO2 Carbamoyl Aspartate
Aspartate Glycine phosphate
C N C
Phosphate
N C N C
C N10–Formyl- O-
O P O-
C C tetrahydrofolate C C
N N N N O
N10–Formyl- Nitrogenous base CH₂

Glutamine
tetrahydrofolate
Deoxyribose sugar
Nucleotide

De novo pyrimidine Various immunosuppressive, antineoplastic, and antibiotic drugs function by interfering with
and purine synthesis nucleotide synthesis:
Pyrimidine base production Purine base production or Pyrimidine synthesis:
(requires aspartate) Ribose 5-P reuse from salvage pathway Leflunomide: inhibits dihydroorotate
(de novo requires aspartate, dehydrogenase
Glutamine + CO2 glycine, glutamine, and THF)
2 ATP
5-fluorouracil (5-FU) and its prodrug
CPS2 (carbamoyl
phosphate capecitabine: form 5-F-dUMP, which inhibits
2 ADP + Pi + synthetase II) PRPP (phosphoribosyl
Glutamate pyrophosphate) synthetase thymidylate synthase ( dTMP)
Purine synthesis:
Carbamoyl 6-mercaptopurine (6-MP) and its prodrug
phosphate
Aspartate azathioprine: inhibit de novo purine
Leflunomide
6-MP, synthesis; azathioprine is metabolized via
PRPP
Orotic azathioprine purine degradation pathway and can lead to
acid
immunosuppression when administered with
UMP synthase UMP Mycophenolate, xanthine oxidase inhibitor
(impaired in IMP ribavirin
orotic aciduria) UDP Mycophenolate and ribavirin: inhibit inosine
ctas ide
reduucleot
Hydroxyurea AMP GMP monophosphate dehydrogenase

e
n
Purine and pyrimidine synthesis:

Ribo
dUDP CTP
Hydroxyurea: inhibits ribonucleotide
reductase
N5N10- dUMP
Methotrexate (MTX), trimethoprim (TMP),
Thymidylate
methylene THF
synthase
5-FU, and pyrimethamine: inhibit dihydrofolate

THF capecitabine
Dihydrofolate
DHF reductase ( deoxythymidine monophosphate
reductase dTMP [dTMP]) in humans (methotrexate),
bacteria (trimethoprim), and protozoa
MTX, TMP,
pyrimethamine (pyrimethamine)
CPS1 = m1tochondria, urea cycle, found in liver

and kidney cells
CPS2 = cytwosol, pyrimidine synthesis, found in
most cells

Purine salvage deficiencies
Nucleic acids Ribose 5-phosphate Nucleic acids

PRPP synthetase De novo synthesis
Nucleotides GMP IMP AMP
Cladribine, pentostatin
Lesch-Nyhan
syndrome
ADA
HGPRT APRT
Nucleosides Guanosine Inosine Adenosine
SCID
PRPP
Free bases Guanine PRPP Hypoxanthine Adenine
XO
Allopurinol
Xanthine
Febuxostat Degradation and salvage
XO
Uric acid
Urate oxidase (rasburicase)
Allantoin Excretion
ADA, adenosine deaminase; APRT, adenine phosphoribosyltransferase; HGPRT, hypoxanthine guanine phosphoribosyltransferase, XO, xanthine
oxidase; SCID, severe combined immune deficiency (autosomal recessive inheritance)
Adenosine deaminase ADA is required for degradation of adenosine One of the major causes of autosomal recessive
deficiency and deoxyadenosine. ADA dATP SCID.
ribonucleotide reductase activity
DNA precursors in cells lymphocytes.
Lesch-Nyhan Defective purine salvage due to absent HGPRT, HGPRT:
syndrome which converts hypoxanthine to IMP and Hyperuricemia
guanine to GMP. Compensatory in purine Gout
synthesis ( PRPP amidotransferase activity) Pissed off (aggression, self-mutilation)
excess uric acid production. X-linked Red/orange crystals in urine
recessive. Tense muscles (dystonia)
Findings: intellectual disability, self-mutilation, Treatment: allopurinol, febuxostat.
aggression, hyperuricemia (red/orange “sand”
[sodium urate crystals] in diaper), gout,
dystonia, macrocytosis.
Genetic code features

Unambiguous Each codon specifies only 1 amino acid.
Degenerate/ Most amino acids are coded by multiple codons. Exceptions: methionine (AUG) and tryptophan
redundant Wobble—codons that differ in 3rd (“wobble”) (UGG) encoded by only 1 codon.
position may code for the same tRNA/amino
acid. Specific base pairing is usually required
only in the first 2 nucleotide positions of
mRNA codon.
Commaless, Read from a fixed starting point as a continuous Exceptions: some viruses.
nonoverlapping sequence of bases.
Universal Genetic code is conserved throughout Exception in humans: mitochondria.
evolution.

DNA replication Occurs in 5′ 3′ direction (“5ynth3sis”) in continuous and discontinuous (Okazaki fragment) fashion.
Semiconservative. More complex in eukaryotes than in prokaryotes, but shares analogous enzymes.
Origin of Particular consensus sequence in genome AT-rich sequences (such as TATA box regions)
replication A where DNA replication begins. May be single are found in promoters and origins of
(prokaryotes) or multiple (eukaryotes). replication.
Replication fork B Y-shaped region along DNA template where
leading and lagging strands are synthesized.
Helicase C Unwinds DNA template at replication fork. Helicase halves DNA.
Deficient in Bloom syndrome (BLM gene
mutation).
Single-stranded Prevent strands from reannealing or degradation
binding proteins D by nucleases.
DNA Creates a single- (topoisomerase I) or double- In eukaryotes: irinotecan/topotecan inhibit
topoisomerases E (topoisomerase II) stranded break in the helix topoisomerase (TOP) I, etoposide/teniposide
to add or remove supercoils (as needed due to inhibit TOP II.
underwinding or overwinding of DNA). In prokaryotes: fluoroquinolones inhibit TOP II
(DNA gyrase) and TOP IV.
Primase F Makes an RNA primer on which DNA
polymerase III can initiate replication.
DNA polymerase III G Prokaryotes only. Elongates leading strand DNA polymerase III has 5′ 3′ synthesis and
by adding deoxynucleotides to the 3′ end. proofreads with 3′ 5′ exonuclease.
Elongates lagging strand until it reaches Drugs blocking DNA replication often have a
primer of preceding fragment. modified 3′ OH, thereby preventing addition of
the next nucleotide (“chain termination”).
DNA polymerase I H Prokaryotes only. Degrades RNA primer; Same functions as DNA polymerase III, also
replaces it with DNA. excises RNA primer with 5′ 3′ exonuclease.
DNA ligase I Catalyzes the formation of a phosphodiester Joins Okazaki fragments.
bond within a strand of double-stranded DNA. Ligase links DNA.
Telomerase Eukaryotes only. A reverse transcriptase (RNA- Upregulated in progenitor cells and also often in
dependent DNA polymerase) that adds DNA cancer; downregulated in aging and progeria.
(TTAGGG) to 3′ ends of chromosomes to avoid Telomerase TAGs for Greatness and Glory.
loss of genetic material with every duplication.
G 3'
E
DNA polymerase III 5'
Topoisomerase
C A
Helicase Origin of replication
Leading strand
B
Replication fork Lagging strand 3'
Okazaki fragment 5'
D
A
Area of interest Single-stranded RNA primer
Origin of replication binding protein
Leading strand Lagging strand I
F DNA ligase
Fork Fork Primase
movement movement
G
DNA polymerase III H
Lagging strand Leading strand
DNA polymerase I

DNA repair
Double strand
Nonhomologous end Brings together 2 ends of DNA fragments to 5´
Double strand break
3´ 5´
Double strand break
joining repair double-stranded breaks. 3´ 5´ 3´

5´
3´
Homology not required. Part of the DNA may be
lost or translocated. Nonhomologous end joining
Homologous Requires 2 homologous DNA duplexes. DoubleAstrand break Double strand break
5´ 3´ 5´ 3´
recombination strand from damaged dsDNA 3´ is repaired 5´ 3´
5´
5´
3´
using a complementary strand from intact 3´ 5´
homologous dsDNA as a template. Homologous recombination

Nonhomologous end joining
Defective in breast/ovarian cancers with BRCA1
or BRCA2 mutations and in Fanconi anemia.
Restores duplexes accurately without loss of
nucleotides. Homologous recombination
Single strand
Nucleotide excision Specific endonucleases remove the Occurs in G1 phase of cell cycle.
repair oligonucleotides containing damaged bases; Defective in xeroderma pigmentosum
DNA polymerase and ligase fill and reseal the (inability to repair DNA pyrimidine dimers
gap, respectively. Repairs bulky helix-distorting caused by UV exposure). Presents with dry
lesions (eg, pyrimidine dimers). skin, photosensitivity, skin cancer.
Base excision repair Base-specific Glycosylase removes altered base Occurs throughout cell cycle.
and creates AP site (apurinic/apyrimidinic). Important in repair of spontaneous/toxic
One or more nucleotides are removed by deamination.
AP-Endonuclease, which cleaves 5′ end. AP- “GEL Please.”
Lyase cleaves 3′ end. DNA Polymerase-β fills
the gap and DNA ligase seals it.
Mismatch repair Mismatched nucleotides in newly synthesized Occurs predominantly in S phase of cell cycle.
strand are removed and gap is filled and Defective in Lynch syndrome (hereditary
resealed. nonpolyposis colorectal cancer [HNPCC]).
UV exposure Pyrimidine dimer Deaminated C

T T
U G
A A G A
AP U
site
TT Endonucleases remove Glycosylase removes base Mismatched segment
G
damaged segment G removed
(AP site)
A A A
Endonuclease and lyase
G remove backbone segment
Newly replaced segment
T T C T
A A G A
Nucleotide excision repair Base excision repair Mismatch repair

Mutations in DNA Degree of change: silent << missense < nonsense < frameshift. Single nucleotide substitutions are
repaired by DNA polymerase and DNA ligase. Types of single nucleotide (point) mutations:
Transition—purine to purine (eg, A to G) or pyrimidine to pyrimidine (eg, C to T).
Transversion—purine to pyrimidine (eg, A to T) or pyrimidine to purine (eg, C to G).
Single nucleotide substitutions
Silent mutation Codes for same (synonymous) amino acid; often involves 3rd position of codon (tRNA wobble).
Missense mutation Results in changed amino acid (called conservative if new amino acid has similar chemical
structure). Examples: sickle cell disease (substitution of glutamic acid with valine).
Nonsense mutation Results in early stop codon (UGA, UAA, UAG). Usually generates nonfunctional protein. Stop the
nonsense!
Other mutations
Frameshift mutation Deletion or insertion of any number of nucleotides not divisible by 3 misreading of all
nucleotides downstream. Protein may be shorter or longer, and its function may be disrupted or
altered. Examples: Duchenne muscular dystrophy, Tay-Sachs disease.
Splice site mutation Retained intron in mRNA protein with impaired or altered function. Examples: rare causes of
cancers, dementia, epilepsy, some types of β-thalassemia, Gaucher disease, Marfan syndrome.
Original Silent Missense Nonsense Frameshift Frameshift
sequence mutation mutation mutation insertion deletion
T G
Coding DNA
5´
GAG GAA GTG TAG GA G GA C 3´
mRNA codon
5´
GAG GAA GUG UAG GAU GAC 3´
Amino acid Glu Glu Val Stop Asp Asp
Altered amino acids
Lac operon Classic example of a genetic response to an environmental change. Glucose is the preferred
metabolic substrate in E coli, but when glucose is absent and lactose is available, the lac operon is
activated to switch to lactose metabolism. Mechanism of shift:
Low glucose adenylate cyclase activity generation of cAMP from ATP activation of
catabolite activator protein (CAP) transcription.
High lactose unbinds repressor protein from repressor/operator site transcription.
Genes
CAP
Adenylate Lacl site P O LacZ LacY LacA
CAP cAMP cyclase Glucose DNA 5′ 3′
AUG AUG AUG

Messenger RNA
Binds CAP site, ATP
induces transcription
RNA
Lac operon STATE CAP polymerase
Low glucose
Lactose available
Lac genes strongly expressed
Lacl CAP site Promoter Operator LacZ LacY LacA Repressor protein
High glucose
o r, Lactose unavailable
e ra t o n Lac genes not expressed
Binds opnscripti
blocks tra
Low glucose
Repressor Lactose unavailable Lac genes not expressed
protein CAP
High glucose
site P O
Lactose available Very low (basal) expression
Allolactose Inactivated
(inducer) repressor

Functional Enhancer/
silencer Promoter 5´ UTR Open reading frame 3´ UTR Silencer
organization of a
eukaryotic gene Exon Intron Exon Intron Exon
DNA 5´ CAAT TATAAA GT AG GT AG AATAAA 3´

(coding strand)
CAAT Box TATA Box
Polyadenylation signal
Transcription start
Transcription
Exon Intron Exon Intron Exon
Pre-mRNA GU AG GU AG AAUAAA
Splicing
5´ cap
Protein coding region
Mature AAUAAA AAAAAA
mRNA
AUG start codon Stop
Poly-A tail
Translation
Protein
Regulation of gene expression

Promoter Site where RNA polymerase II and multiple Promoter mutation commonly results in
other transcription factors bind to DNA dramatic in level of gene transcription.
upstream from gene locus (AT-rich upstream
sequence with TATA and CAAT boxes, which
differ between eukaryotes and prokaryotes).
Enhancer DNA locus where regulatory proteins Enhancers and silencers may be located close to,
(“activators”) bind, increasing expression of a far from, or even within (in an intron) the gene
gene on the same chromosome. whose expression they regulate.
Silencer DNA locus where regulatory proteins
(“repressors”) bind, decreasing expression of a
gene on the same chromosome.
Epigenetics Changes made to gene expression (heritable Primary mechanisms of epigenetic change
mitotically/meiotically) without a change in include DNA methylation, histone
underlying DNA sequence. modification, and noncoding RNA.
RNA processing Initial transcript is called heterogeneous nuclear mRNA is transported out of nucleus to be
(eukaryotes) RNA (hnRNA). hnRNA is then modified and translated in cytosol.
becomes mRNA. mRNA quality control occurs at cytoplasmic
The following processes occur in the nucleus: processing bodies (P-bodies), which contain
Cap Coding
5'
Capping of 5′ end (addition of exonucleases, decapping enzymes, and
Gppp 7-methylguanosine cap; cotranscriptional) microRNAs; mRNAs may be degraded or
3' Polyadenylation of 3′ end (∼200 A’s poly-A stored in P-bodies for future translation.
HO-AAAAA
Tail
tail; posttranscriptional) Poly-A polymerase does not require a template.
Splicing out of introns (posttranscriptional) AAUAAA = polyadenylation signal. Mutation
Capped, tailed, and spliced transcript is called in polyadenylation signal early degradation
mRNA. prior to translation.

RNA polymerases
Eukaryotes RNA polymerase I makes rRNA, the most I, II, and III are numbered in the same order
common (rampant) type; present only in that their products are used in protein
nucleolus. synthesis: rRNA, mRNA, then tRNA.
RNA polymerase II makes mRNA (massive), α-amanitin, found in Amanita phalloides (death
microRNA (miRNA), and small nuclear RNA cap mushrooms), inhibits RNA polymerase II.
(snRNA). Causes dysentery and severe hepatotoxicity if
RNA polymerase III makes 5S rRNA, tRNA ingested.
(tiny). Dactinomycin inhibits RNA polymerase in both
No proofreading function, but can initiate prokaryotes and eukaryotes.
chains. RNA polymerase II opens DNA at
promoter site.
Prokaryotes 1 RNA polymerase (multisubunit complex) Rifamycins (rifampin, rifabutin) inhibit DNA-
makes all 3 kinds of RNA. dependent RNA polymerase in prokaryotes.
Splicing of pre-mRNA Part of process by which precursor mRNA (pre-mRNA) is transformed into mature mRNA. Introns
typically begin with GU and end with AG. Alterations in snRNP assembly can cause clinical
disease; eg, in spinal muscular atrophy, snRNP assembly is affected due to SMN protein
congenital degeneration of anterior horns of spinal cord symmetric weakness (hypotonia, or
“floppy baby syndrome”).
snRNPs are snRNA bound to proteins (eg, Smith [Sm]) to form a spliceosome that cleaves pre-
mRNA. Anti-U1 snRNP antibodies are associated with SLE, mixed connective tissue disease,
other rheumatic diseases.
Primary transcript combines with 5′ splice site U1 snRNP Branch point 3′ splice site
small nuclear ribonucleoproteins
U2 snRNP
(snRNPs) and other proteins to
form spliceosome. 5′ O P GU A AG P O 3′
Exon 1 Intron Exon 2
Spliceosome
Cleavage at 5′ splice site; lariat-

shaped (loop) intermediate is
generated. UG
P A
OH 3′ AG P O
Exon 1 Exon 2
Mature mRNA
Cleavage at 3′ splice site; lariat
is released to precisely remove
intron and join 2 exons.
P +
Exon 1 Exon 2 UG
P A
AG OH 3′

Introns vs exons Exons contain the actual genetic information Introns are intervening sequences and stay
coding for protein or functional RNA. in the nucleus, whereas exons exit and are
Introns do not code for protein, but are expressed.
important in regulation of gene expression. Alternative splicing—can produce a variety
Different exons are frequently combined by of protein products from a single hnRNA
alternative splicing to produce a larger number (heterogenous nuclear RNA) sequence (eg,
of unique proteins. transmembrane vs secreted Ig, tropomyosin
variants in muscle, dopamine receptors in the
brain, host defense evasion by tumor cells).
Exon 1 Exon 2 Exon 3 Exon 4 Exon 5 Exon 6

5′ 3′
DNA 3′ 5′
Transcription
hnRNA 5′ 3′
1 2 3 4 5 6
Splicing Alternative splicing
mRNA 5′ 3′ 5′ 3′ 5′ 3′
1 2 4 5 6 1 3 5 6 1 3 4 5 6
Translation
1 5 1 5 1 5
Proteins 6 6 6
4 4
2 3 3

tRNA
Structure 75–90 nucleotides, 2º structure, cloverleaf form, anticodon end is opposite 3′ aminoacyl end. All
tRNAs, both eukaryotic and prokaryotic, have CCA at 3′ end along with a high percentage of
chemically modified bases. The amino acid is covalently bound to the 3′ end of the tRNA. CCA
Can Carry Amino acids.
T-arm: contains the TΨC (ribothymidine, pseudouridine, cytidine) sequence necessary for tRNA-
ribosome binding. T-arm Tethers tRNA molecule to ribosome.
D-arm: contains Dihydrouridine residues necessary for tRNA recognition by the correct aminoacyl-
tRNA synthetase. D-arm allows Detection of the tRNA by aminoacyl-tRNA synthetase.
Attachment site: 3′-ACC-5′ is the amino acid ACCeptor site.
Charging Aminoacyl-tRNA synthetase (uses ATP; 1 unique enzyme per respective amino acid) and
binding of charged tRNA to the codon are responsible for the accuracy of amino acid selection.
Aminoacyl-tRNA synthetase matches an amino acid to the tRNA by scrutinizing the amino acid
before and after it binds to tRNA. If an incorrect amino acid is attached, the bond is hydrolyzed.
A mischarged tRNA reads the usual codon but inserts the wrong amino acid.
Structure Charging Pairing
(aminoacylation) (codon-anticodon)
Amino acid Amino acid
O O
Attachment site OH 3´ 3´ 3´
A A A
C C C
C C C
5´ 5´ 5´
IF2
T-arm
D-arm ATP AMP + PPi (initiation factor)
D D D
C Ψ T
Aminoacyl-tRNA C Ψ T C Ψ T
D D D
synthetase
Variable arm
Anticodon
loop U A C U A C Anticodon (5´-CAU-3´) U A C
Wobble
position C C C A U G A U A C
mRNA
Codon
(5´-AUG-3´)
Start and stop codons

mRNA start codon AUG. AUG inAUGurates protein synthesis.
Eukaryotes Codes for methionine, which may be removed
before translation is completed.
Prokaryotes Codes for N-formylmethionine (fMet). fMet stimulates neutrophil chemotaxis.
mRNA stop codons UGA, UAA, UAG. UGA = U Go Away.
Recognized by release factors. UA A = U Are Away.
UAG = U Are Gone.

Protein synthesis
Initiation 1. Eukaryotic initiation factors (eIFs) identify Eukaryotes: 40S + 60S 80S (even).
the 5′ cap. Prokaryotes: 30S + 50S 70S (prime).
2. eIFs help assemble the 40S ribosomal Synthesis occurs from N-terminus to
subunit with the initiator tRNA. C-terminus.
3. eIFs released when the mRNA and the
ATP—tRNA Activation (charging).
ribosomal 60S subunit assemble with the
GTP—tRNA Gripping and Going places
complex. Requires GTP.
(translocation).
Elongation Aminoacyl-tRNA binds to A site (except for
initiator methionine, which binds the P site), Think of “going APE”:
requires an elongation factor and GTP. A site = incoming Aminoacyl-tRNA.
rRNA (“ribozyme”) catalyzes peptide bond P site = accommodates growing Peptide.
formation, transfers growing polypeptide to E site = holds Empty tRNA as it Exits.
amino acid in A site. Elongation factors are targets of bacterial toxins
Ribosome advances 3 nucleotides toward 3′ (eg, Diphtheria, Pseudomonas).
end of mRNA, moving peptidyl tRNA to P
site (translocation). Shine-Dalgarno sequence—ribosomal binding
site in prokaryotic mRNA. Recognized by 16S
Termination Eukaryotic release factors (eRFs) recognize the
RNA in ribosomal subunit. Enables protein
stop codon and halt translation completed
synthesis initiation by aligning ribosome with
polypeptide is released from ribosome.
start codon so that code is read correctly.
Requires GTP.
60/50S
40/30S
M
R
M
Initiation M M H
Initiator tRNA
U A C
U A C 5´ A U G C A U G A U 3´ U A C G U A
mRNA
E P A
5´ A U G C A U G A U 3´
E P A
S Ribosome moves left to
right along mRNA
M Elongation M
H
U G A
G U A U A C
U A C Q G U A
Termination
A U G C A U G A U A U G C A U G A U
5´ 3´ 5´ 3´
E P A E P A
Posttranslational modifications
Trimming Removal of N- or C-terminal propeptides from zymogen to generate mature protein (eg,
trypsinogen to trypsin).
Covalent alterations Phosphorylation, glycosylation, hydroxylation, methylation, acetylation, and ubiquitination.
Chaperone protein Intracellular protein involved in facilitating and maintaining protein folding. In yeast, heat
shock proteins (eg, HSP60) are constitutively expressed, but expression may increase with high
temperatures, acidic pH, and hypoxia to prevent protein denaturing/misfolding.

44 SEC TION II Biochemistry  BIOCHEMISTRY—Cellular
` BIOCHEMISTRY—CELLULAR
Cell cycle phases Checkpoints control transitions between phases of cell cycle. This process is regulated by cyclins,
cyclin-dependent kinases (CDKs), and tumor suppressors. M phase (shortest phase of cell cycle)
includes mitosis (prophase, prometaphase, metaphase, anaphase, telophase) and cytokinesis
(cytoplasm splits in two). G1 is of variable duration.
REGULATION OF CELL CYCLE
Cyclin-dependent Constitutively expressed but inactive when not bound to cyclin.
kinases
Cyclin-CDK complexes Cyclins are phase-specific regulatory proteins that activate CDKs when stimulated by growth
factors. The cyclin-CDK complex can then phosphorylate other proteins (eg, Rb) to coordinate
cell cycle progression. This complex must be activated/inactivated at appropriate times for cell
cycle to progress.
Tumor suppressors p53 p21 induction CDK inhibition Rb hypophosphorylation (activation) G1-S
progression inhibition. Mutations in tumor suppressor genes can result in unrestrained cell
division (eg, Li-Fraumeni syndrome).
Growth factors (eg, insulin, PDGF, EPO, EGF) bind tyrosine kinase receptors to transition the cell
from G1 to S phase.
CELL TYPES
Permanent Remain in G0, regenerate from stem cells. Neurons, skeletal and cardiac muscle, RBCs.
Stable (quiescent) Enter G1 from G0 when stimulated. Hepatocytes, lymphocytes, PCT, periosteal cells.
Labile Never go to G0, divide rapidly with a short G1. Bone marrow, gut epithelium, skin, hair
Most affected by chemotherapy. follicles, germ cells.
Cell cycle arrest

p21 Cyclin
GO
Cyclin
CDK CDK
DNA damage Rb, p53 modulate G1
p21
G1 restriction point
th
ow
Gr
is
M
es
Li-Fraumeni syndrome
kin
to
(loss of function) Cy
Mito
p53
sis
HPV E6
IN TERPH
BCL-2 BCL-XL
Rb P
DNA
P
P
BAX/BAK
AS
Sy n
Rb E2F S
E
t he
is
s
Caspase activation E2F G2

Gene transcription
Apoptosis
(intrinsic pathway)

Biochemistry  BIOCHEMISTRY—Cellular SEC TION II 45
Rough endoplasmic Site of synthesis of secretory (exported) proteins N-linked glycosylation occurs in the
reticulum and of N-linked oligosaccharide addition to eNdoplasmic reticulum.
lysosomal and other proteins. Mucus-secreting goblet cells of small intestine
Nissl bodies (RER in neurons)—synthesize and antibody-secreting plasma cells are rich in
peptide neurotransmitters for secretion. RER.
Free ribosomes—unattached to any membrane; Proteins within organelles (eg, ER, Golgi bodies,
site of synthesis of cytosolic, peroxisomal, and lysosomes) are formed in RER.
mitochondrial proteins.
Smooth endoplasmic Site of steroid synthesis and detoxification of Hepatocytes and steroid hormone–producing
reticulum drugs and poisons. Lacks surface ribosomes. cells of the adrenal cortex and gonads are rich
Location of glucose-6-phosphatase (last step in in SER.
both glycogenolysis and gluconeogenesis).
Cell trafficking Golgi is distribution center for proteins and lipids from ER to vesicles and plasma membrane.
Posttranslational events in GOlgi include modifying N-oligosaccharides on asparagine, adding
O-oligosaccharides on serine and threonine, and adding mannose-6-phosphate to proteins for
lysosomal and other proteins.
Endosomes are sorting centers for material from outside the cell or from the Golgi, sending it to
lysosomes for destruction or back to the membrane/Golgi for further use.
I-cell disease (inclusion cell disease/mucolipidosis type II)—inherited lysosomal storage disorder
(autosomal recessive); defect in N-acetylglucosaminyl-1-phosphotransferase failure of the
Golgi to phosphorylate mannose residues ( mannose-6-phosphate) on glycoproteins enzymes
secreted extracellularly rather than delivered to lysosomes lysosomes deficient in digestive
enzymes buildup of cellular debris in lysosomes (inclusion bodies). Results in coarse facial
features, gingival hyperplasia, corneal clouding, restricted joint movements, claw hand deformities,
kyphoscoliosis, and plasma levels of lysosomal enzymes. Symptoms similar to but more severe
than Hurler syndrome. Often fatal in childhood.
Key: Signal recognition particle (SRP)—abundant,
brane
cytosolic ribonucleoprotein that traffics
mem polypeptide-ribosome complex from the
Clathrin sma
Pla
Secretory cytosol to the RER. Absent or dysfunctional
vesicle
COPI Late Early SRP accumulation of protein in cytosol.
endosome endosome
COPII Vesicular trafficking proteins

Lysosome COPI: Golgi Golgi (retrograde); cis-Golgi
Retrograde trans ER.
Anterograde COPII: ER cis-Golgi (anterograde). “Two
Golgi (COPII) steps forward (anterograde); one
apparatus (COPI) step back (retrograde).”
Clathrin: trans-Golgi lysosomes; plasma
membrane endosomes (receptor-mediated
cis
endocytosis [eg, LDL receptor activity]).
Rough
endoplasmic
reticulum
Nuclear envelope

46 SEC TION II Biochemistry  BIOCHEMISTRY—Cellular
Peroxisome Membrane-enclosed organelle involved in:

β-oxidation of very-long-chain fatty acids (VLCFA) (strictly peroxisomal process)
α-oxidation of branched-chain fatty acids (strictly peroxisomal process)
Catabolism of amino acids and ethanol
Synthesis of bile acids and plasmalogens (important membrane phospholipid, especially in
white matter of brain)
Zellweger syndrome—autosomal recessive disorder of peroxisome biogenesis due to mutated PEX
genes. Hypotonia, seizures, jaundice, craniofacial dysmorphia, hepatomegaly, early death.
Refsum disease—autosomal recessive disorder of α-oxidation buildup of phytanic acid due
to inability to degrade it. Scaly skin, ataxia, cataracts/night blindness, shortening of 4th toe,
epiphyseal dysplasia. Treatment: diet, plasmapheresis.
Adrenoleukodystrophy—X-linked recessive disorder of β-oxidation due to mutation in ABCD1
gene VLCFA buildup in adrenal glands, white (leuko) matter of brain, testes. Progressive
disease that can lead to adrenal gland crisis, progressive loss of neurologic function, death.
Proteasome Barrel-shaped protein complex that degrades ubiquitin-tagged proteins. Defects in the ubiquitin-
proteasome system have been implicated in some cases of Parkinson disease.
Cytoskeletal elements A network of protein fibers within the cytoplasm that supports cell structure, cell and organelle
movement, and cell division.
TYPE OF FILAMENT PREDOMINANT FUNCTION EXAMPLES
Microfilaments Muscle contraction, cytokinesis Actin, microvilli.
Intermediate Maintain cell structure Vimentin, desmin, cytokeratin, lamins, glial
filaments fibrillary acidic protein (GFAP), neurofilaments.
Microtubules Movement, cell division Cilia, flagella, mitotic spindle, axonal trafficking,
centrioles.
Microtubule Cylindrical outer structure composed of a Drugs that act on microtubules (microtubules
Positive
helical array of polymerized heterodimers get constructed very terribly):
end (+) of α- and β-tubulin. Each dimer has 2 GTP Mebendazole (antihelminthic)
Heterodimer bound. Incorporated into flagella, cilia, mitotic Griseofulvin (antifungal)
spindles. Also involved in slow axoplasmic Colchicine (antigout)
transport in neurons. Vinca alkaloids (anticancer)
Molecular motor proteins—transport cellular Taxanes (anticancer)
cargo toward opposite ends of microtubule. Negative end near nucleus.
Protofilament Retrograde to microtubule (+ −)—dynein. Positive end points to periphery.
Anterograde to microtubule (− +)—kinesin.
Negative Clostridium tetani toxin, herpes simplex virus, Ready? Attack!
end (–) poliovirus, and rabies virus use dynein for
retrograde transport to the neuronal cell body.

Cilia structure Motile cilia consist of 9 doublet + 2 singlet arrangement of microtubules (axoneme) A .
Basal body (base of cilium below cell membrane) consists of 9 microtubule triplets B with no
central microtubules.
Nonmotile (primary) cilia work as chemical signal sensors and have a role in signal transduction
and cell growth control. Dysgenesis may lead to polycystic kidney disease, mitral valve prolapse,
or retinal degeneration.
Axonemal dynein—ATPase that links peripheral 9 doublets and causes bending of cilium by
differential sliding of doublets.
Gap junctions enable coordinated ciliary movement.
A B
Dynein
arm
Microtubule
A
Microtubule
B
Nexin
Doublets
Triplets
Primary ciliary Also called Kartagener syndrome. Autosomal recessive. Dynein arm defect immotile cilia
dyskinesia dysfunctional ciliated epithelia.
Developmental abnormalities due to impaired migration and orientation (eg, situs inversus A , hearing
A
loss due to dysfunctional eustachian tube cilia); recurrent infections (eg, sinusitis, ear infections,
R L bronchiectasis due to impaired ciliary clearance of debris/pathogens); infertility ( risk of ectopic
pregnancy due to dysfunctional fallopian tube cilia, immotile spermatozoa).
Lab findings: nasal nitric oxide (used as screening test).
Sodium-potassium Na+/K+-ATPase is located in the plasma 2 strikes? K, you’re still in. 3 strikes? Nah, you’re
pump membrane with ATP site on cytosolic side. For out!
each ATP consumed, 2 K+ go in to the cell Cardiac glycosides (digoxin and digitoxin)
(pump dephosphorylated) and 3 Na+ go out of directly inhibit Na+/K+-ATPase indirect
the cell (pump phosphorylated). inhibition of Na+/Ca2+ exchange [Ca2+]i
cardiac contractility.
Extracellular
3Na+ 2K+
space
Plasma
membrane
P
Cytosol 2K+
3Na+ ATP ADP P

48 SECTION II Biochemistry  BIOCHEMISTRY—Cellular
Collagen Most abundant protein in the human body. Type I - Skeleton

Extensively modified by posttranslational Type II - Cartilage
modification. Type III - Arteries
Organizes and strengthens extracellular matrix. Type IV - Basement membrane
Types I to IV are the most common types in SCAB
humans.
Type I Most common (90%)—Bone (made by Type I: bone, tendone.
osteoblasts), Skin, Tendon, dentin, fascia, production in osteogenesis imperfecta type I.
cornea, late wound repair.
Type II Cartilage (including hyaline), vitreous body, Type II: cartwolage.
nucleus pulposus.
Type III Reticulin—skin, blood vessels, uterus, fetal Type III: deficient in vascular type of Ehlers-
tissue, early wound repair. Danlos syndrome (threE D).
Type IV Basement membrane/basal lamina (glomerulus, Type IV: under the floor (basement membrane).
cochlea), lens. Defective in Alport syndrome; targeted by
autoantibodies in Goodpasture syndrome.
Myofibroblasts are responsible for secretion
(proliferative stage) and wound contraction.
Collagen synthesis and structure
Fibroblast Preprocollagen S ynthesis—translation of collagen α

Pro α-chain backbone (Gly-X-Y) chains (preprocollagen)—usually Gly-X-Y
Nucleus Hydroxylation of proline and
(X is often proline or lysine and Y is often
OH
OH lysine (requires vitamin C) hydroxyproline or hydroxylysine). Collagen is
Collagen mRNA Sugar
Glycosylation
1/3 glycine; glycine content of collagen is less
Cytoplasm OH
OH variable than that of lysine and proline.
RER
Hydroxylation—hydroxylation
Triple helix formation
Procollagen (“hydroxCylation”) of specific proline
Golgi and lysine residues. Requires vitamin C;
deficiency scurvy.
Exocytosis
Glycosylation—glycosylation of pro-α-chain
Extracellular
space hydroxylysine residues and formation of
Cleavage of procollagen
C- and N-terminals procollagen via hydrogen and disulfide bonds
Tropocollagen (triple helix of 3 collagen α chains). Problems
forming triple helix osteogenesis imperfecta.
Self assembly into
collagen fibrils Exocytosis—exocytosis of procollagen into
extracellular space.
Proteolytic processing—cleavage of
disulfide-rich terminal regions of procollagen
Formation of cross-links
(stabilized by lysyl oxidase) insoluble tropocollagen.
Assembly and alignment—collagen assembles
Collagen fiber
in fibrils and aligns for cross-linking.
Cross-linking—reinforcement of staggered
tropocollagen molecules by covalent lysine-
hydroxylysine cross-linkage (by copper-
containing lysyl oxidase) to make collagen
fibrils. Cross-linking of collagen with age.
Problems with cross-linking Menkes disease.
FAS1_2022_01-Biochem.indd 48 11/8/21 1:04 PM

Osteogenesis Genetic bone disorder (brittle bone May be confused with child abuse.
imperfecta disease) caused by a variety of gene defects Treat with bisphosphonates to fracture risk.
A
(most commonly COL1A1 and COL1A2). Patients can’t BITE:
Most common form is autosomal dominant Bones = multiple fractures
with production of otherwise normal type I I (eye) = blue sclerae
collagen (altered triple helix formation). Teeth = dental imperfections
Upper Manifestations include: Ear = hearing loss
extremity Multiple fractures and bone deformities
B
(arrows in A ) after minimal trauma (eg,
during birth)
Blue sclerae B due to the translucent
connective tissue over choroidal veins
Some forms have tooth abnormalities,
including opalescent teeth that wear easily
due to lack of dentin (dentinogenesis
imperfecta)
Conductive hearing loss (abnormal ossicles)
Ehlers-Danlos Faulty collagen synthesis causing A B

syndrome hyperextensible skin A , hypermobile joints B ,
and tendency to bleed (easy bruising).
Multiple types. Inheritance and severity vary.
Can be autosomal dominant or recessive. May
be associated with joint dislocation, berry and
aortic aneurysms, organ rupture.
Hypermobility type (joint instability): most
common type.
Classical type (joint and skin symptoms):
caused by a mutation in type V collagen (eg,
COL5A1, COL5A2).
Vascular type (fragile tissues including vessels
[eg, aorta], muscles, and organs that are prone
to rupture [eg, gravid uterus]): mutations in
type III procollagen (eg, COL3A1).
Menkes disease X-linked recessive connective tissue disease caused by impaired copper absorption and transport
due to defective Menkes protein ATP7A (Absent copper), vs ATP7B in Wilson disease (copper
Buildup). Leads to activity of lysyl oxidase (copper is a necessary cofactor) defective collagen
cross-linking. Results in brittle, “kinky” hair, growth and developmental delay, hypotonia, risk of
cerebral aneurysms.

50 SEC TION II Biochemistry  Biochemistry—Laboratory Techniques
Elastin Stretchy protein within skin, lungs, large arteries, elastic ligaments, vocal cords, epiglottis,
ligamenta flava (connect vertebrae relaxed and stretched conformations).
Rich in nonhydroxylated proline, glycine, and lysine residues, vs the hydroxylated residues of
collagen.
Single Tropoelastin with fibrillin scaffolding.
elastin Stretch
Relax Cross-link
Cross-linking occurs extracellularly via lysyl oxidase and gives elastin its elastic properties.
molecule
Broken down by elastase, which is normally inhibited by α1-antitrypsin.
α1-Antitrypsin deficiency results in unopposed elastase activity, which can cause COPD.
Marfan syndrome—autosomal dominant (with variable expression) connective tissue disorder
A
affecting skeleton, heart, and eyes. FBN1 gene mutation on chromosome 15 (fifteen) results in
defective fibrillin-1, a glycoprotein that forms a sheath around elastin and sequesters TGF-β.
Findings: tall with long extremities; chest wall deformity (pectus carinatum [pigeon chest] or
pectus excavatum A ); hypermobile joints; long, tapering fingers and toes (arachnodactyly); cystic
medial necrosis of aorta; aortic root aneurysm rupture or dissection (most common cause of
death); mitral valve prolapse; risk of spontaneous pneumothorax.
Homocystinuria—most commonly due to cystathionine synthase deficiency leading to
homocysteine buildup. Presentation similar to Marfan syndrome with pectus deformity, tall
stature, arm:height ratio, upper:lower body segment ratio, arachnodactyly, joint hyperlaxity,
skin hyperelasticity, scoliosis.
Marfan syndrome Homocystinuria
INHERITANCE Autosomal dominant Autosomal recessive
INTELLECT Normal Decreased
VASCULAR COMPLICATIONS Aortic root dilatation Thrombosis
LENS DISLOCATION Upward/temporal (Marfan fans out) Downward/nasal
` BIOCHEMISTRY—LABORATORY TECHNIQUES
Polymerase chain Molecular biology lab procedure used to amplify a desired fragment of DNA. Useful as a diagnostic
reaction tool (eg, neonatal HIV, herpes encephalitis).
5' 3' 5' 3' 5' 3'
5' 3' 3' 5'

DNA primer
dNTP
Repeat
3' 5' 5' 3'
Double-stranded DNA
3' 5' 3' 5' 3' 5'
enaturation—DNA template, DNA primers, a heat-stable DNA polymerase, and

D
deoxynucleotide triphosphates (dNTPs) are heated to ~ 95ºC to separate the DNA strands.
Annealing—sample is cooled to ~ 55ºC. DNA primers anneal to the specific sequence to be
amplified on the DNA template.
Elongation—temperature is increased to ~ 72ºC. DNA polymerase adds dNTPs to the strand to
replicate the sequence after each primer.
Heating and cooling cycles continue until the amount of DNA is sufficient.

Biochemistry  Biochemistry—Laboratory Techniques SEC TION II 51
CRISPR/Cas9 A genome editing tool derived from bacteria. Consists of a guide RNA (gRNA) , which is
complementary to a target DNA sequence, and an endonuclease (Cas9), which makes a single- or
double-strand break at the target site . Imperfectly cut segments are repaired by nonhomologous
end joining (NHEJ) accidental frameshift mutations (“knock-out”) , or a donor DNA
sequence can be added to fill in the gap using homology-directed repair (HDR) .
Potential applications include removing virulence factors from pathogens, replacing disease-causing
alleles of genes with healthy variants (in clinical trials for sickle cell disease), and specifically targeting
tumor cells.
Cas9 gRNA
Donor DNA
NHEJ 3A 3B HDR
+
Frameshift/inactivation Edited sequence

(”knock-out”) (”knock-in”)
Blotting procedures
Southern blot 1. DNA sample is enzymatically cleaved into I: Parents
smaller pieces, which are separated on a gel
PEDIGREE
by electrophoresis, and then transferred to a

membrane.
II: Children
2. Membrane is exposed to labeled DNA probe
that anneals to its complementary strand. Aa Aa aa Aa AA Genotype
3. Resulting double-stranded, labeled piece
SOUTHERN BLOT
of DNA is visualized when membrane is Mutant
exposed to film or digital imager. Normal
Northern blot Similar to Southern blot, except that an RNA

sample is electrophoresed. Useful for studying SNoW DRoP:
mRNA levels, which are reflective of gene Southern = DNA
expression. Northern = RNA
Western = Protein
Western blot Sample protein is separated via gel electrophoresis
Northern blots detect splicing errors.
and transferred to a membrane. Labeled
antibody is used to bind to relevant protein.
Southwestern blot Identifies DNA-binding proteins (eg, c-Jun, Southern (DNA) + Western (protein) =
c-Fos [leucine zipper motif]) using labeled Southwestern (DNA-binding protein).
double-stranded DNA probes.

52 SEC TION II Biochemistry  Biochemistry—Laboratory Techniques
Flow cytometry Laboratory technique to assess size, granularity, Commonly used in workup of hematologic
and protein expression (immunophenotype) of abnormalities (eg, leukemia, paroxysmal
individual cells in a sample. nocturnal hemoglobinuria, fetal
RBCs in pregnant person’s blood) and
immunodeficiencies (eg, CD4+ cell count in
HIV).
Cells are tagged with antibodies specific to Fluorescent

label
surface or intracellular proteins. Antibodies
Antibody
are then tagged with a unique fluorescent
dye. Sample is analyzed one cell at a time by
Anti-CD3 Ab Cell
focusing a laser on the cell and measuring
light scatter and intensity of fluorescence.
Anti-CD8 Ab
Laser
Fluorescence
is detected; Laser makes
tor
labeled cells tec label fluoresce
De
are counted
Data are plotted either as histogram (one
measure) or scatter plot (any two measures, as
shown). In illustration: 104
Cells in left lower quadrant ⊝ for both CD8
and CD3. 103
Cells in right lower quadrant ⊕ for CD8
and ⊝ for CD3. In this example, right
CD3
102
lower quadrant is empty because all
CD8-expressing cells also express CD3. 101
Cells in left upper quadrant ⊕ for CD3 and
⊝ for CD8. 100
100 101 102 103 104
Cells in right upper quadrant ⊕ for both
CD8
CD8 and CD3.
Microarrays Array consisting of thousands of DNA oligonucleotides arranged in a grid on a glass or silicon chip.
The DNA or RNA samples being compared are attached to different fluorophores and hybridized
to the array. The ratio of fluorescence signal at a particular oligonucleotide reflects the relative
amount of the hybridizing nucleic acid in the two samples.
Used to compare the relative transcription of genes in two RNA samples. Can detect single
nucleotide polymorphisms (SNPs) and copy number variants (CNVs) for genotyping, clinical
genetic testing, forensic analysis, and cancer mutation and genetic linkage analysis when DNA is
used.
Enzyme-linked Immunologic test used to detect the presence of either a specific antigen or antibody in a patient’s
immunosorbent assay blood sample. Detection involves the use of an antibody linked to an enzyme. Added substrate
reacts with the enzyme, producing a detectable signal. Can have high sensitivity and specificity,
but is less specific than Western blot. Often used to screen for HIV infection.

Biochemistry  Biochemistry—Laboratory Techniques SEC TION II 53
Karyotyping Colchicine is added to cultured cells to halt A

chromosomes in metaphase. Chromosomes
are stained, ordered, and numbered according
to morphology, size, arm-length ratio, and
banding pattern (arrows in A point to extensive
abnormalities in a cancer cell).
Can be performed on a sample of blood, bone
marrow, amniotic fluid, or placental tissue.
Used to diagnose chromosomal imbalances
(eg, autosomal trisomies, sex chromosome
disorders).
Fluorescence in situ Fluorescent DNA or RNA probe binds A

hybridization to specific gene or other site of interest
on chromosomes (arrows in A point to
abnormalities in a cancer cell; each fluorescent
color represents a chromosome-specific probe).
Used for specific localization of genes and direct
visualization of chromosomal anomalies.
Microdeletion—no fluorescence on a
chromosome compared to fluorescence at
the same locus on the second copy of that
chromosome.
Translocation—fluorescence signal that
corresponds to one chromosome is found in
a different chromosome (two white arrows in
A show fragments of chromosome 17 that
have translocated to chromosome 19).
Duplication—a second copy of a
chromosome, resulting in a trisomy or
tetrasomy (two blue arrows in A duplicated
chromosomes 8, resulting in a tetrasomy).
Molecular cloning Production of a recombinant DNA molecule in a bacterial host. Useful for production of human
proteins in bacteria (eg, human growth hormone, insulin).
Steps:
1. Isolate eukaryotic mRNA (post-RNA processing) of interest.
2. Add reverse transcriptase (an RNA-dependent DNA polymerase) to produce complementary
DNA (cDNA, lacks introns).
3. Insert cDNA fragments into bacterial plasmids containing antibiotic resistance genes.
4. Transform (insert) recombinant plasmid into bacteria.
5. Surviving bacteria on antibiotic medium produce cloned DNA (copies of cDNA).

54 SEC TION II Biochemistry  BIOCHEMISTRY—Genetics
Gene expression Transgenic strategies in mice involve: Knock-out = removing a gene, taking it out.
modifications Random insertion of gene into mouse Knock-in = inserting a gene.
genome
Targeted insertion or deletion of gene Random insertion—constitutive expression.
through homologous recombination with Targeted insertion—conditional expression.
mouse gene
RNA interference Process whereby small non-coding RNA molecules target mRNAs to inhibit gene expression.
MicroRNA Naturally produced by cell as hairpin structures. Abnormal expression of miRNAs contributes
Loose nucleotide pairing allows broad to certain malignancies (eg, by silencing an
targeting of related mRNAs. When miRNA mRNA from a tumor suppressor gene).
binds to mRNA, it blocks translation of mRNA
and sometimes facilitates its degradation.
Small interfering Usually derived from exogenous dsRNA source Can be produced by transcription or
RNA (eg, virus). Once inside a cell, siRNA requires chemically synthesized for gene “knockdown”
complete nucleotide pairing, leading to highly experiments.
specific mRNA targeting. Results in mRNA
cleavage prior to translation.
` BIOCHEMISTRY—GENETICS
Genetic terms
TERM DEFINITION EXAMPLE
Codominance Both alleles contribute to the phenotype of the Blood groups A, B, AB; α1-antitrypsin
heterozygote. deficiency; HLA groups.
Variable expressivity Patients with the same genotype have varying Two patients with neurofibromatosis type 1 (NF1)
phenotypes. may have varying disease severity.
Incomplete Not all individuals with a disease show the BRCA1 gene mutations do not always result in
penetrance disease. breast or ovarian cancer.
% penetrance × probability of inheriting
genotype = risk of expressing phenotype.
Pleiotropy One gene contributes to multiple phenotypic Untreated phenylketonuria (PKU) manifests with
effects. light skin, intellectual disability, musty body odor.
Anticipation Increased severity or earlier onset of disease in Trinucleotide repeat diseases (eg, Huntington
succeeding generations. disease).
Loss of heterozygosity If a patient inherits or develops a mutation in Retinoblastoma and the “two-hit hypothesis,”
a tumor suppressor gene, the wild type allele Lynch syndrome (HNPCC), Li-Fraumeni
must be deleted/mutated/eliminated before syndrome.
cancer develops. This is not true of oncogenes.
Epistasis The allele of one gene affects the phenotypic Albinism, alopecia.
expression of alleles in another gene.
Aneuploidy An abnormal number of chromosomes; due to Down syndrome, Turner syndrome,
chromosomal nondisjunction during mitosis oncogenesis.
or meiosis.

Biochemistry  BIOCHEMISTRY—Genetics SEC TION II 55
Genetic terms (continued)

TERM DEFINITION EXAMPLE
Dominant negative Exerts a dominant effect. A heterozygote A single mutated p53 tumor suppressor gene
mutation produces a nonfunctional altered protein that results in a protein that is able to bind DNA
also prevents the normal gene product from and block the wild type p53 from binding to the
functioning. promoter.
Linkage Tendency for certain alleles to occur in close
disequilibrium proximity on the same chromosome more or
less often than expected by chance. Measured
in a population, not in a family, and often
varies in different populations.
Mosaicism Presence of genetically distinct cell lines in the McCune-Albright syndrome—due to Gs-protein
A
same individual. activating mutation. Presents with unilateral
Somatic mosaicism—mutation arises from café-au-lait spots A with ragged edges,
mitotic errors after fertilization and propagates polyostotic fibrous dysplasia (bone is replaced
through multiple tissues or organs. by collagen and fibroblasts), and at least one
Germline (gonadal) mosaicism—mutation only endocrinopathy (eg, precocious puberty).
in egg or sperm cells. If parents and relatives Lethal if mutation occurs before fertilization
do not have the disease, suspect gonadal (or (affecting all cells), but survivable in patients
germline) mosaicism. with mosaicism.
Locus heterogeneity Mutations at different loci result in the same Albinism, retinitis pigmentosa, familial
disease. hypercholesteremia.
Allelic heterogeneity Different mutations in the same locus result in β-thalassemia.
the same disease.
Heteroplasmy Presence of both normal and mutated mtDNA passed from mother to all children.
mtDNA, resulting in variable expression in
mitochondrially inherited disease.
Uniparental disomy Offspring receives 2 copies of a chromosome from Uniparental is euploid (correct number of
1 parent and no copies from the other parent. chromosomes). Most occurrences of uniparental
HeterodIsomy (heterozygous) indicates a meiosis disomy (UPD) normal phenotype. Consider
I error. IsodIsomy (homozygous) indicates a isodisomy in an individual manifesting a
meiosis II error or postzygotic chromosomal recessive disorder when only one parent is a
duplication of one of a pair of chromosomes, carrier. Examples: Prader-Willi and Angelman
and loss of the other of the original pair. syndromes.
Hardy-Weinberg If p and q represent the frequencies of alleles Hardy-Weinberg law assumptions include:
population genetics A and a, respectively, in a population, then No mutation occurring at the locus
p + q = 1: Natural selection is not occurring
A (p) a (q)
p2 = frequency of homozygosity for allele A Completely random mating
AA Aa
A (p)
(p2) (pq) q2 = frequency of homozygosity for allele a No net migration
Aa aa
2pq = frequency of heterozygosity (carrier Large population
a (q) frequency, if an autosomal recessive disease) If a population is in Hardy-Weinberg
(pq) (q2)
Therefore, the sum of the frequencies of these equilibrium, then the values of p and q remain
genotypes is p2 + 2pq + q2 = 1. constant from generation to generation.
The frequency of an X-linked recessive disease
in males = q and in females = q2.

Disorders of imprinting Imprinting—one gene copy is silenced by methylation, and only the other copy is expressed
parent-of-origin effects. The expressed copy may be mutated, may not be expressed, or may be
deleted altogether.
Prader-Willi syndrome Angelman syndrome
WHICH GENE IS SILENT? Maternally derived genes are silenced Paternally derived UBE3A is silenced
Disease occurs when the paternal allele is deleted Disease occurs when the maternal allele is
or mutated deleted or mutated
SIGNS AND SYMPTOMS Hyperphagia, obesity, intellectual disability, Seizures, Ataxia, severe Intellectual disability,
hypogonadism, hypotonia inappropriate Laughter
Set SAIL for Angel Island
CHROMOSOMES INVOLVED Chromosome 15 of paternal origin UBE3A on maternal copy of chromosome 15
NOTES 25% of cases are due to maternal uniparental 5% of cases are due to paternal uniparental
disomy disomy
POP: Prader-Willi, Obesity/overeating, Paternal MAMAS: Maternal allele deleted, Angelman
allele deleted syndrome, Mood, Ataxia, Seizures
P = Paternal
M = Maternal
Normal Mutation
Active gene
P M P M
Silenced gene (imprinting)
Gene deletion/mutation
Prader-Willi syndrome
Angelman syndrome

Modes of inheritance
Autosomal dominant Often due to defects in structural genes. Many Often pleiotropic (multiple apparently unrelated
generations, both males and females are affected. effects) and variably expressive (different
A a between individuals). Family history crucial
to diagnosis. With one affected (heterozygous)
a Aa aa
parent, each child has a 50% chance of being
a Aa aa
affected.
Autosomal recessive With 2 carrier (heterozygous) parents, on average: Often due to enzyme deficiencies. Usually seen
each child has a 25% chance of being affected, in only 1 generation. Commonly more severe
50% chance of being a carrier, and 25% chance than dominant disorders; patients often present
of not being affected nor a carrier. in childhood.
A a risk in consanguineous families.
Unaffected individual with affected sibling has
A AA Aa
2/3 probability of being a carrier.
a Aa aa
X-linked recessive Sons of heterozygous mothers have a 50% Commonly more severe in males. Females
chance of being affected. No male-to-male usually must be homozygous to be affected.
carrier transmission. Skips generations.
X X X X
X XX XX X XX XX
Y XY XY Y XY XY
X-linked dominant Transmitted through both parents. Children of Examples: fragile X syndrome, Alport syndrome,
affected mothers each have a 50% chance of hypophosphatemic rickets (also called X-linked
being affected. 100% of daughters and 0% of hypophosphatemia)—phosphate wasting at
sons of affected fathers will be affected. proximal tubule ricketslike presentation.
X X X X
X XX XX X XX XX
Y XY XY Y XY XY
Mitochondrial Transmitted only through the mother. All Caused by mutations in mtDNA.
inheritance offspring of affected females may show signs of Examples: mitochondrial myopathies, Leber
disease. hereditary optic neuropathy.
Variable expression in a population or even
within a family due to heteroplasmy.
= unaffected male; = affected male; = unaffected female; = affected female.

Autosomal dominant Achondroplasia, autosomal dominant polycystic kidney disease, familial adenomatous polyposis,
diseases familial hypercholesterolemia, hereditary hemorrhagic telangiectasia (Osler-Weber-Rendu
syndrome), hereditary spherocytosis, Huntington disease, Li-Fraumeni syndrome, Marfan syndrome,
multiple endocrine neoplasias, myotonic muscular dystrophy, neurofibromatosis type 1 (von
Recklinghausen disease), neurofibromatosis type 2, tuberous sclerosis, von Hippel-Lindau disease.
Autosomal recessive Mostly consist of enzyme defects. Oculocutaneous albinism, phenylketonuria, cystic fibrosis, sickle
diseases cell disease, Wilson disease, sphingolipidoses (except Fabry disease), hemochromatosis, glycogen
storage diseases, thalassemia, mucopolysaccharidoses (except Hunter syndrome), Friedreich
ataxia, Kartagener syndrome, ARPKD. Oh, please! Can students who score high grades tell me
features of the kidney disorder Autosomal Recessive Polycystic Kidney Disease?
Cystic fibrosis
GENETICS Autosomal recessive; defect in CFTR gene on chromosome 7; commonly a deletion of gene on
chromosome 7 (ΔF508). Most common lethal genetic disease in patients with European ancestry.
PATHOPHYSIOLOGY CFTR encodes an ATP-gated Cl− channel that secretes Cl− in lungs and GI tract, and reabsorbs
Cl− in sweat glands. Phe508 deletion misfolded protein improper protein trafficking and
protein retention in RER protein absent from cell membrane Cl− (and H2O) secretion;
intracellular Cl− results in compensatory Na+ reabsorption via epithelial Na+ channels
(ENaC) H2O reabsorption abnormally thick mucus secreted into lungs and GI tract. Na+
reabsorption also causes more negative transepithelial potential difference.
DIAGNOSIS Cl− concentration in pilocarpine-induced sweat test is diagnostic. Can present with contraction
alkalosis and hypokalemia (ECF effects analogous to a patient taking a loop diuretic) because
of ECF H2O/Na+ losses via sweating and concomitant renal K+/H+ wasting. immunoreactive
trypsinogen (newborn screening) due to clogging of pancreatic duct.
COMPLICATIONS Recurrent pulmonary infections (eg, S aureus [infancy and early childhood], P aeruginosa
[adulthood], allergic bronchopulmonary aspergillosis [ABPA]), chronic bronchitis and bronchiectasis
reticulonodular pattern on CXR, opacification of sinuses. Nasal polyps, nail clubbing.
Pancreatic insufficiency, malabsorption with steatorrhea, and fat-soluble vitamin deficiencies (A, D,
E, K) progressing to endocrine dysfunction (CF-related diabetes), biliary cirrhosis, liver disease.
Meconium ileus in newborns.
Infertility in males (absence of vas deferens, spermatogenesis may be unaffected) and subfertility in
females (amenorrhea, abnormally thick cervical mucus).
TREATMENT Multifactorial: chest physiotherapy, albuterol, aerosolized dornase alfa (DNase), and inhaled
hypertonic saline facilitate mucus clearance. Azithromycin used as anti-inflammatory agent.
Ibuprofen slows disease progression. Pancreatic enzyme replacement therapy (pancrelipase) for
pancreatic insufficiency.
Combination of lumacaftor or tezacaftor (each corrects misfolded proteins and improves their
transport to cell surface) with ivacaftor. (opens Cl– channels improved chloride transport).
Normal mucus Dehydrated mucus

CI– Na+ CI– Na+
CFTR ENaC Lumen
CI–
CI– H₂O Na+ H₂O Na+
Interstitium
Normal Sweat duct CF Normal Airway CF

X-linked recessive Bruton agammaglobulinemia, Duchenne and Becker muscular dystrophies, Fabry disease, G6PD
diseases deficiency, hemophilia A and B, Hunter syndrome, Lesch-Nyhan syndrome, ocular albinism,
ornithine transcarbamylase deficiency, Wiskott-Aldrich syndrome.
Females with Turner syndrome (45,XO) are more likely to have an X-linked recessive disorder.
X-inactivation (lyonization)—during development, one of the X chromosomes in each XX cell
is randomly deactivated and condensed into a Barr body (methylated heterochromatin). If
skewed inactivation occurs, XX individuals may express X-linked recessive diseases (eg, G6PD);
penetrance and severity of X-linked dominant diseases in XX individuals may also be impacted.
Muscular dystrophies
Duchenne X-linked recessive disorder typically due to Duchenne = deleted dystrophin.
A
frameshift deletions or nonsense mutations Dystrophin gene (DMD) is the largest
Muscle fibers truncated or absent dystrophin protein protein-coding human gene chance of
progressive myofiber damage. Weakness spontaneous mutation. Dystrophin helps
begins in pelvic girdle muscles and progresses to anchor muscle fibers to the extracellular
superiorly. Pseudohypertrophy of calf muscles matrix, primarily in skeletal and cardiac
due to fibrofatty replacement of muscle A . muscles. Loss of dystrophin myonecrosis.
Waddling gait. CK and aldolase; genetic testing confirms
Onset before 5 years of age. Dilated diagnosis.
cardiomyopathy is common cause of death. Calf
pseudohypertrophy
Gowers sign—patient uses upper extremities to
help stand up. Classically seen in Duchenne
muscular dystrophy, but also seen in other
muscular dystrophies and inflammatory
Lordosis
myopathies (eg, polymyositis).
Thigh
atrophy
Pushing on leg
to stand
Becker X-linked recessive disorder typically due to Deletions can cause both Duchenne and
non-frameshift deletions in dystrophin gene Becker muscular dystrophies. 2⁄3 of cases have
(partially functional instead of truncated). large deletions spanning one or more exons.
Less severe than Duchenne (Becker is better).
Onset in adolescence or early adulthood.
Myotonic dystrophy Autosomal dominant. Onset 20–30 years. CTG Cataracts, Toupee (early balding in males),
trinucleotide repeat expansion in the DMPK Gonadal atrophy.
gene abnormal expression of myotonin
protein kinase myotonia (eg, difficulty
releasing hand from handshake), muscle
wasting, cataracts, testicular atrophy, frontal
balding, arrhythmia.

60 SECTION II Biochemistry  BIOCHEMISTRY—Genetics
Mitochondrial diseases Rare disorders arising 2° to failure in oxidative phosphorylation. Tissues with energy requirements
are preferentially affected (eg, CNS, skeletal muscle).
Mitochondrial myopathies—include MELAS (mitochondrial encephalomyopathy with lactic
acidosis and strokelike episodes) and MERRF (myoclonic epilepsy with ragged red fibers). Light
microscopy with stain: ragged red fibers due to compensatory proliferation of mitochondria.
Electron microscopy: mitochondrial crystalline inclusions.
Leber hereditary optic neuropathy—mutations in complex I of ETC neuronal death in retina
and optic nerve subacute bilateral vision loss in teens/young adults (males > females). Usually
permanent. May be accompanied by neurologic dysfunction (eg, tremors, multiple sclerosis–like
illness).
Rett syndrome Sporadic disorder seen almost exclusively in females (affected males die in utero or shortly after
birth). Most cases are caused by de novo mutation of MECP2 on X chromosome. Symptoms of
Rett syndrome usually appear between ages 1–4 and are characterized by regression (“retturn”) in
motor, verbal, and cognitive abilities; ataxia; seizures; growth deceleration; and stereotyped hand-
wringing.
Fragile X syndrome X-linked dominant inheritance. Trinucleotide Trinucleotide repeat expansion [(CGG)n] occurs
repeats in FMR1 hypermethylation of during oogenesis.
cytosine residues expression. Premutation (50-200 repeats) tremor, ataxia,
Most common inherited cause of intellectual 1° ovarian insufficiency.
disability (Down syndrome is most common Full mutation (>200 repeats) postpubertal
genetic cause, but most cases occur macroorchidism (enlarged testes), long face
sporadically). with large jaw, large everted ears, autism,
mitral valve prolapse, hypermobile joints.
Self-mutilation is common and can be confused
with Lesch-Nyhan syndrome.
Trinucleotide repeat May show genetic anticipation (disease severity and age of onset in successive generations).
expansion diseases
DISEASE TRINUCLEOTIDE REPEAT MODE OF INHERITANCE MNEMONIC
Huntington disease (CAG)n AD Caudate has ACh and GABA
Myotonic dystrophy (CTG)n AD Cataracts, Toupee (early balding in males),
Gonadal atrophy in males, reduced fertility in
females
Fragile X syndrome (CGG)n XD Chin (protruding), Giant Gonads
Friedreich ataxia (GAA)n AR Ataxic GAAit

Autosomal trisomies Autosomal monosomies are incompatible with life due to a high chance of expression of recessive
traits for that chromosome. Incidence of trisomies: Down > Edwards > Patau.
Down syndrome Findings: intellectual disability, flat facies, Drinking age (21).
(trisomy 21) prominent epicanthal folds, single palmar Most common viable chromosomal disorder
crease, incurved 5th finger, gap between 1st 2 and most common cause of genetic
toes, duodenal atresia, Hirschsprung disease, intellectual disability.
congenital heart disease (eg, ASD), Brushfield First-trimester ultrasound commonly shows
spots (whitish spots at the periphery of the iris). nuchal translucency and hypoplastic nasal
Associated with early-onset Alzheimer disease bone. Markers for Down syndrome are hi up:
(chromosome 21 codes for amyloid precursor hCG, inhibin.
protein), risk of AML/ALL. risk of umbilical hernia (incomplete closure of
95% of cases due to meiotic nondisjunction, umbilical ring).
most commonly during meiosis I ( with The 5 A’s of Down syndrome:
Single palmar crease advanced maternal age: from 1:1500 in females Advanced maternal age
< 20 to 1:25 in females > 45). Atresia (duodenal)
4% of cases due to unbalanced Robertsonian Atrioventricular septal defect
translocation, most typically between Alzheimer disease (early onset)
chromosomes 14 and 21. Only 1% of cases are AML (<5 years of age)/ALL (>5 years of age)
due to postfertilization mitotic error.
Edwards syndrome Findings: PRINCE Edward—Prominent Election age (18).
(trisomy 18) occiput, Rocker-bottom feet, Intellectual 2nd most common autosomal trisomy resulting
disability, Nondisjunction, Clenched fists with in live birth (most common is Down syndrome).
overlapping fingers, low-set Ears, micrognathia In Edwards syndrome, every prenatal screening
(small jaw), congenital heart disease (eg, marker decreases.
VSD), omphalocele, myelomeningocele.
Death usually occurs by age 1.
Patau syndrome Findings: severe intellectual disability, rocker- Puberty at age 13.
(trisomy 13) bottom feet, microphthalmia, microcephaly, Defect in fusion of prechordal mesoderm
cleft lip/palate, holoprosencephaly, midline defects.
polydactyly, cutis aplasia, congenital heart
(pump) disease, polycystic kidney disease,
omphalocele. Death usually occurs by age 1.
Cutis aplasia
Nondisjunction in meiosis I Nondisjunction in meiosis II 1st trimester screening

Trisomy β-hCG PAPP-A
Meiosis I 21
18
Nondisjunction 13
Meiosis II 2nd trimester (quadruple) screening

Trisomy β-hCG Inhibin A Estriol AFP
Nondisjunction 21
18 — or
Gametes 13 — — — —
n+1 n+1 n–1 n–1 n n n–1 n+1
Trisomy Monosomy Normal Monosomy Trisomy
Noninvasive prenatal testing is recommended
over first- and second-trimester screening.

Genetic disorders by CHROMOSOME SELECTED EXAMPLES

chromosome 3 von Hippel-Lindau disease, renal cell carcinoma
4 ADPKD (PKD2), achondroplasia, Huntington disease
5 Cri-du-chat syndrome, familial adenomatous polyposis
6 Hemochromatosis (HFE)
7 Williams syndrome, cystic fibrosis
9 Friedreich ataxia, tuberous sclerosis (TSC1)
11 Wilms tumor, β-globin gene defects (eg, sickle cell disease, β-thalassemia), MEN1
13 Patau syndrome, Wilson disease, retinoblastoma (RB1), BRCA2
15 Prader-Willi syndrome, Angelman syndrome, Marfan syndrome
16 ADPKD (PKD1), α-globin gene defects (eg, α-thalassemia), tuberous sclerosis (TSC2)
17 Neurofibromatosis type 1, BRCA1, TP53 (Li-Fraumeni syndrome)
18 Edwards syndrome
21 Down syndrome
22 Neurofibromatosis type 2, DiGeorge syndrome (22q11)
X Fragile X syndrome, X-linked agammaglobulinemia, Klinefelter syndrome (XXY)
Robertsonian Chromosomal translocation that commonly

translocation involves chromosome pairs 21, 22, 13, 14,
and 15. One of the most common types of
translocation. Occurs when the long arms of
2 acrocentric chromosomes (chromosomes
with centromeres near their ends) fuse at the
centromere and the 2 short arms are lost.
Balanced translocations (no gain or loss of
Normal Robertsonian Unbalanced
significant genetic material) normally do gamete precursor translocation gamete precursor
not cause abnormal phenotype. Unbalanced
translocations (missing or extra genes)
can result in miscarriage, stillbirth, and
chromosomal imbalance (eg, Down syndrome,
Patau syndrome).
Cri-du-chat syndrome Cri du chat = cry of the cat. Congenital deletion on short arm of chromosome 5 (46,XX or XY,
5p−).
Findings: microcephaly, moderate to severe intellectual disability, high-pitched crying, epicanthal
folds, cardiac abnormalities (VSD). I cry when I am Very SaD.
Williams syndrome Congenital microdeletion of long arm of chromosome 7 (deleted region includes elastin gene).
Findings: distinctive “elfin” facies, intellectual disability, hypercalcemia, well-developed verbal
skills, extreme friendliness with strangers, cardiovascular problems (eg, supravalvular aortic
stenosis, renal artery stenosis).

Biochemistry  BIOCHEMISTRY—Nutrition SEC TION II 63
` BIOCHEMISTRY—NUTRITION
Essential fatty acids Polyunsaturated fatty acids that cannot be In contrast, consumption of trans-unsaturated
synthesized in the body and must be provided fatty acids (found in fast food) promotes
in the diet (eg, nuts/seeds, plant oils, seafood). cardiovascular disease by LDL and HDL.
Linoleic acid (omega-6) is metabolized to
arachidonic acid, which serves as the precursor
to leukotrienes and prostaglandins.
Linolenic acid (omega-3) and its metabolites
have cardioprotective and antihyperlipidemic
effects.
Vitamins: fat soluble A, D, E, K. Absorption dependent on bile Malabsorption syndromes with steatorrhea (eg,
emulsification, pancreatic secretions, and cystic fibrosis and celiac disease) or mineral
intact ileum. Toxicity more common than oil intake can cause fat-soluble vitamin
for water-soluble vitamins because fat-soluble deficiencies.
vitamins accumulate in fat.
Vitamins: water B1 (thiamine: TPP) Wash out easily from body except B12 and B9.
soluble B2 (riboflavin: FAD, FMN) B12 stored in liver for ~ 3–4 years. B9 stored in
B3 (niacin: NAD+) liver for ~ 3–4 months.
B5 (pantothenic acid: CoA) B-complex deficiencies often result in
B6 (pyridoxine: PLP) dermatitis, glossitis, and diarrhea.
B7 (biotin) Can be coenzymes (eg, ascorbic acid) or
B9 (folate) precursors to coenzymes (eg, FAD, NAD+).
B12 (cobalamin)
C (ascorbic acid)

64 SEC TION II Biochemistry  BIOCHEMISTRY—Nutrition
Vitamin A Includes retinal, retinol, retinoic acid.

FUNCTION Antioxidant; constituent of visual pigments Retinol is vitamin A, so think retin-A (used
(retinal); essential for normal differentiation topically for wrinkles and Acne).
of epithelial cells into specialized tissue Found in liver and leafy vegetables.
(pancreatic cells, mucus-secreting cells); Supplementation in vitamin A-deficient measles
prevents squamous metaplasia. patients may improve outcomes.
Use oral isotretinoin to treat severe cystic acne.
Use all-trans retinoic acid to treat acute
promyelocytic leukemia.
DEFICIENCY Night blindness (nyctalopia); dry, scaly skin

A
(xerosis cutis); dry eyes (xerophthalmia);
conjunctival squamous metaplasia Bitot
spots (keratin debris; foamy appearance
on conjunctiva A ); corneal degeneration
(keratomalacia); immunosuppression.
EXCESS Acute toxicity—nausea, vomiting, ICP (eg, Teratogenic (cleft palate, cardiac abnormalities),
vertigo, blurred vision). therefore a ⊝ pregnancy test and two forms of
Chronic toxicity—alopecia, dry skin (eg, contraception are required before isotretinoin
scaliness), hepatic toxicity and enlargement, (vitamin A derivative) is prescribed.
arthralgias, and idiopathic intracranial Isotretinoin is teratogenic.
hypertension.
Vitamin B1 Also called thiamine.

FUNCTION In thiamine pyrophosphate (TPP), a cofactor for several dehydrogenase enzyme reactions (Be APT):
Branched-chain ketoacid dehydrogenase
α-Ketoglutarate dehydrogenase (TCA cycle)
Pyruvate dehydrogenase (links glycolysis to TCA cycle)
Transketolase (HMP shunt)
DEFICIENCY Impaired glucose breakdown ATP depletion worsened by glucose infusion; highly aerobic tissues
(eg, brain, heart) are affected first. In patients with chronic alcohol overuse or malnutrition, give
thiamine before dextrose to risk of precipitating Wernicke encephalopathy.
Diagnosis made by in RBC transketolase activity following vitamin B1 administration.
DISORDER CHARACTERISTICS
Wernicke Acute, reversible, life-threatening neurologic condition. Symptoms: Confusion, Ophthalmoplegia/
encephalopathy Nystagmus, Ataxia (CorONA beer).
Korsakoff syndrome Amnestic disorder due to chronic alcohol overuse; presents with confabulation, personality
changes, memory loss (permanent).
Wernicke-Korsakoff Damage to medial dorsal nucleus of thalamus, mammillary bodies. Presentation is combination of
syndrome Wernicke encephalopathy and Korsakoff syndrome.
Dry beriberi Polyneuropathy, symmetric muscle wasting. Spell beriberi as Ber1Ber1 to remember
Wet beriberi High-output cardiac failure (due to systemic vitamin B1.
vasodilation).

Vitamin B2 Also called riboflavin.

FUNCTION Component of flavins FAD and FMN, used as FAD and FMN are derived from riboFlavin
cofactors in redox reactions, eg, the succinate (B2 ≈ 2 ATP).
dehydrogenase reaction in the TCA cycle.
DEFICIENCY Cheilosis (inflammation of lips, scaling The 2 C’s of B2.
and fissures at the corners of the mouth),
“magenta” tongue, corneal vascularization.
Vitamin B3 Also called niacin, nicotinic acid.

FUNCTION Constituent of NAD+, NADP+ (used in redox NAD derived from Niacin (B3 ≈ 3 ATP).
reactions and as cofactor by dehydrogenases).
Derived from tryptophan. Synthesis requires
vitamins B2 and B6. Used to treat dyslipidemia
( VLDL, HDL).
DEFICIENCY Glossitis. Severe deficiency of B3 leads to Hartnup disease—autosomal recessive.
A
pellagra, which can also be caused by Hartnup Deficiency of neutral amino acid (eg,
disease, malignant carcinoid syndrome tryptophan) transporters in proximal renal
( tryptophan metabolism serotonin tubular cells and on enterocytes neutral
synthesis), and isoniazid ( vitamin B6). aminoaciduria and absorption from the
Symptoms of B3 deficiency (pellagra) (the 3 gut tryptophan for conversion to niacin
D’s): diarrhea, dementia (also hallucinations), pellagra-like symptoms. Treat with high-
dermatitis (C3/C4 dermatome circumferential protein diet and nicotinic acid.
“broad collar” rash [Casal necklace], Pellagra = vitamin B3 levels fell.
hyperpigmentation of sun-exposed limbs A ).
EXCESS Facial flushing (induced by prostaglandin, not Podagra = vitamin B3 OD (overdose).

histamine; can avoid by taking aspirin with
niacin), hyperglycemia, hyperuricemia.
Vitamin B5 Also called pantothenic acid. B5 is “pento”thenic acid.

FUNCTION Component of coenzyme A (CoA, a cofactor for acyl transfers) and fatty acid synthase.
DEFICIENCY Dermatitis, enteritis, alopecia, adrenal insufficiency may lead to burning sensation of feet
(“burning feet syndrome”; distal paresthesias, dysesthesia).
Vitamin B6 Also called pyridoxine.

FUNCTION Converted to pyridoxal phosphate (PLP), a cofactor used in transamination (eg, ALT and AST),
decarboxylation reactions, glycogen phosphorylase. Synthesis of glutathione, cystathionine, heme,
niacin, histamine, and neurotransmitters including serotonin, epinephrine, norepinephrine (NE),
dopamine, and GABA.
DEFICIENCY Convulsions, hyperirritability, peripheral neuropathy (deficiency inducible by isoniazid and oral
contraceptives), sideroblastic anemia (due to impaired hemoglobin synthesis and iron excess).

Vitamin B7 Also called biotin.

FUNCTION Cofactor for carboxylation enzymes (which add a 1-carbon group):
Pyruvate carboxylase (gluconeogenesis): pyruvate (3C) oxaloacetate (4C)
Acetyl-CoA carboxylase (fatty acid synthesis): acetyl-CoA (2C) malonyl-CoA (3C)
Propionyl-CoA carboxylase (fatty acid oxidation and branched-chain amino acid breakdown):
propionyl-CoA (3C) methylmalonyl-CoA (4C)
DEFICIENCY Relatively rare. Dermatitis, enteritis, alopecia. Caused by long-term antibiotic use or excessive
ingestion of raw egg whites.
“Avidin in egg whites avidly binds biotin.”
Vitamin B9 Also called folate.

FUNCTION Converted to tetrahydrofolic acid (THF), a Found in leafy green vegetables. Also produced
coenzyme for 1-carbon transfer/methylation by gut microbiota. Folate absorbed in jejunum
reactions. (think foliage in the “jejun”gle).
Important for the synthesis of nitrogenous bases Small reserve pool stored primarily in the liver.
in DNA and RNA.
DEFICIENCY Macrocytic, megaloblastic anemia; Deficiency can be caused by several drugs (eg,
hypersegmented polymorphonuclear cells phenytoin, trimethoprim, methotrexate).
(PMNs); glossitis; no neurologic symptoms (as Supplemental folic acid at least 1 month prior
opposed to vitamin B12 deficiency). to conception and during pregnancy to risk
Labs: homocysteine, normal methylmalonic of neural tube defects. Give vitamin B9 for the
acid levels. Seen in chronic alcohol overuse 9 months of pregnancy, and 1 month prior to
and in pregnancy. conception.

Vitamin B12 Also called cobalamin.

FUNCTION Cofactor for methionine synthase (transfers Found in animal products. Synthesized only
CH3 groups as methylcobalamin) and by intestinal microbiota. Site of synthesis in
methylmalonyl-CoA mutase. Important for humans is distal to site of absorption; thus B12
DNA synthesis. must be consumed via animal products.
DEFICIENCY Macrocytic, megaloblastic anemia; Very large reserve pool (several years) stored
hypersegmented PMNs; paresthesias primarily in the liver. Deficiency caused
and subacute combined degeneration by malabsorption (eg, sprue, enteritis,
(degeneration of dorsal columns, lateral Diphyllobothrium latum, achlorhydria,
corticospinal tracts, and spinocerebellar tracts) bacterial overgrowth, alcohol overuse), lack of
due to abnormal myelin. Associated with intrinsic factor (eg, pernicious anemia, gastric
serum homocysteine and methylmalonic bypass surgery), absence of terminal ileum
acid levels, along with 2° folate deficiency. (surgical resection, eg, for Crohn disease),
Prolonged deficiency irreversible nerve certain drugs (eg, metformin), or insufficient
damage. intake (eg, veganism).
B9 (folate) supplementation can mask the
hematologic symptoms of B12 deficiency, but
not the neurologic symptoms.
Protein Fatty acids with odd number of
carbons, branched-chain amino acids
THF Methionine SAM
CH3 to anabolic
pathways Methylmalonyl-CoA
Methylmalonyl-CoA
B12 Methionine synthase
S-adenosyl B12 mutase
homocysteine
Succinyl-CoA
B6
THF–CH3 Homocysteine
Heme TCA cycle
B6 Adenosine
Cysteine
Vitamin C Also called ascorbic acid.

FUNCTION Antioxidant; also facilitates iron absorption Found in fruits and vegetables.
by reducing it to Fe2+ state. Necessary Pronounce “absorbic” acid.
for hydroxylation of proline and lysine in Ancillary treatment for methemoglobinemia by
collagen synthesis. Necessary for dopamine reducing Fe3+ to Fe2+.
β-hydroxylase (converts dopamine to NE).
DEFICIENCY Scurvy—swollen gums, easy bruising, Deficiency may be precipitated by tea and toast
petechiae, hemarthrosis, anemia, poor wound diet.
healing, perifollicular and subperiosteal Vitamin C deficiency causes sCurvy due to a
hemorrhages, “corkscrew” hair. Collagen hydroCylation defect.
Weakened immune response.
EXCESS Nausea, vomiting, diarrhea, fatigue, calcium
oxalate nephrolithiasis (excess oxalate from
vitamin C metabolism). Can iron toxicity in
predisposed individuals by increasing dietary
iron absorption (ie, can worsen hemochromatosis
or transfusion-related iron overload).

Vitamin D D3 (cholecalciferol) from exposure of skin (stratum basale) to sun, ingestion of fish, milk, plants.
D2 (ergocalciferol) from ingestion of plants, fungi, yeasts.
Both converted to 25-OH D3 (storage form) in liver and to the active form 1,25-(OH)2 D3 (calcitriol)
in kidney.
FUNCTION intestinal absorption of Ca2+ and Cholesterol →
PO43–. Diet 7-dehydrocholesterol
bone mineralization at low levels. Sun/UV exposure
bone resorption at higher levels. D2 D3
2+ (Ergocalciferol) (Cholecalciferol)
REGULATION PTH, Ca , PO43–
1,25-(OH)2D3 production.
1,25-(OH)2D3 feedback inhibits its own
production. 25-hydroxylase
PTH Ca2+ reabsorption and
PO43– reabsorption in the kidney. 25-OH D 3
DEFICIENCY Rickets in children (deformity, such ↑ ↑
as genu varum “bowlegs” A ), Ca2+, PO43–
A
osteomalacia in adults (bone pain
and muscle weakness), hypocalcemic
tetany. 1α-hydroxylase
Caused by malabsorption, sun
exposure, poor diet, chronic kidney 1,25-(OH)2 D3
disease (CKD), advanced liver disease.
Give oral vitamin D to breastfed infants. Bone Intestines Renal tubular cells
Darker skin and prematurity predispose
to deficiency.
↑ Ca2+ and ↑ PO43– ↑ absorption of Reabsorption: ↑ Ca2+, ↑ PO43–
EXCESS Hypercalcemia, hypercalciuria, loss of
released from bone Ca2+ and PO43– Urine: Ca2+, PO43–
↑ ↑
appetite, stupor. Seen in granulomatous
diseases ( activation of vitamin D by
epithelioid macrophages). ↑ Ca2+ and ↑ PO43–
Vitamin E Includes tocopherol, tocotrienol.

FUNCTION Antioxidant (protects RBCs and
neuronal membranes from free radical damage).
DEFICIENCY Hemolytic anemia, acanthocytosis, muscle Neurologic presentation may appear similar
weakness, demyelination of posterior columns to vitamin B12 deficiency, but without
( proprioception and vibration sensation) and megaloblastic anemia, hypersegmented
spinocerebellar tract (ataxia). neutrophils, or serum methylmalonic acid
levels.
EXCESS Risk of enterocolitis in enfants (infants) with High-dose supplementation may alter metabolism
excess of vitamin E. of vitamin K enhanced anticoagulant effects
of warfarin.

Vitamin K Includes phytomenadione, phylloquinone, phytonadione, menaquinone.

FUNCTION Activated by epoxide reductase to the K is for Koagulation. Necessary for the
reduced form, which is a cofactor for the maturation of clotting factors II, VII, IX,
γ-carboxylation of glutamic acid residues on X, and proteins C and S. Warfarin inhibits
various proteins required for blood clotting. vitamin K–dependent synthesis of these factors
Synthesized by intestinal microbiota. and proteins.
DEFICIENCY Neonatal hemorrhage with PT and aPTT Not in breast milk; “breast-fed infants Don’t
but normal bleeding time (neonates have Know about vitamins D and K”. Neonates are
sterile intestines and are unable to synthesize given vitamin K injection at birth to prevent
vitamin K). Can also occur after prolonged use hemorrhagic disease of the newborn.
of broad-spectrum antibiotics.
Zinc
FUNCTION Mineral essential for the activity of 100+ enzymes. Important in the formation of zinc fingers
(transcription factor motif).
DEFICIENCY Delayed wound healing, suppressed immunity, male hypogonadism, adult hair (axillary, facial,
A pubic), dysgeusia, anosmia. Associated with acrodermatitis enteropathica ( A , defect in intestinal
zinc absorption). May predispose to alcoholic cirrhosis.
Protein-energy malnutrition
Kwashiorkor Protein malnutrition resulting in skin lesions, A B
edema due to plasma oncotic pressure (due
to low serum albumin), liver malfunction
(fatty change due to apolipoprotein synthesis
and deposition). Clinical picture is small child
with swollen abdomen A .
Kwashiorkor results from protein-
deficient MEALS:
Malnutrition
Edema
Anemia
Liver (fatty)
Skin lesions (eg, hyperkeratosis,
dyspigmentation)
Marasmus Malnutrition not causing edema. Diet is
deficient in calories but no nutrients are
entirely absent.
Marasmus results in muscle wasting B .

Ethanol metabolism
NADH/NAD+ ratio inhibits
NADPH NADP+
CYP2E1
ROS
TCA cycle acetyl-CoA used
Microsome
in ketogenesis ( ketoacidosis),
Fomepizole Disulfiram lipogenesis ( hepatosteatosis).
– – Females are more susceptible than
Alcohol dehydrogenase Acetaldehyde dehydrogenase males to effects of alcohol due
Ethanol Acetaldehyde Acetate
to  activity of gastric alcohol
NAD+ NADH NAD+ NADH
Cytosol
Mitochondria dehydrogenase,  body size,
 percentage of water in body
weight.
Catalase NAD+ is the limiting reagent.
H2O2 H2O Peroxisome Alcohol dehydrogenase operates via
zero-order kinetics.
Ethanol metabolism NADH/
Gluconeogenesis Glycolysis NAD+ ratio in liver, causing:
Glucose
NADH NAD+ Lactic acidosis— pyruvate
conversion to lactate
Glyceraldehyde-3-P DHAP ↑ Glycerol-3-P
4A Fasting hypoglycemia—
gluconeogenesis due to
PEP
(fasting NADH NAD+ conversion of OAA to malate
hypoglycemia)
Pyruvate ↑ Lactate ↑ Triglycerides
Ketoacidosis—diversion of
Q (anion gap metabolic acidosis)
(hepatic acetyl-CoA into ketogenesis
steatosis) rather than TCA cycle
↑ Ketoacids
S Hepatosteatosis— conversion
Ketogenesis
↑
OAA Acetyl-CoA of DHAP to glycerol-3-P
4A ; acetyl-CoA diverges into
4B
NADH ↑ Fatty acids fatty acid synthesis 4B , which
R
OAA Isocitrate NAD+ Lipogenesis combines with glycerol-3-P to
synthesize triglycerides
NADH Fomepizole—competitive inhibitor
NAD+
TCA cycle of alcohol dehydrogenase;
↑ Malate α-KG
Pathways stimulated by ↑ NADH/NAD+ ratio preferred antidote for overdoses
NAD+
Pathways inhibited by ↑ NADH/NAD+ ratio of methanol or ethylene glycol.
Succinyl-
Alcohol dehydrogenase has
CoA NADH higher affinity for ethanol
than for methanol or ethylene
glycol ethanol can be used as
competitive inhibitor of alcohol
dehydrogenase to treat methanol
or ethylene glycol poisoning.
Disulfiram—blocks acetaldehyde
dehydrogenase acetaldehyde
hangover symptoms
discouraging drinking.

Biochemistry  BIOCHEMISTRY—Metabolism SEC TION II 71
` BIOCHEMISTRY—METABOLISM
Enzyme terminology An enzyme’s name often describes its function. For example, glucokinase is an enzyme that
catalyzes the phosphorylation of glucose using a molecule of ATP. The following are commonly
used enzyme descriptors.
Kinase Catalyzes transfer of a phosphate group from a high-energy molecule (usually ATP) to a substrate
(eg, phosphofructokinase).
Phosphorylase Adds inorganic phosphate onto substrate without using ATP (eg, glycogen phosphorylase).
Phosphatase Removes phosphate group from substrate (eg, fructose-1,6-bisphosphatase 1).
Dehydrogenase Catalyzes oxidation-reduction reactions (eg, pyruvate dehydrogenase).
Hydroxylase Adds hydroxyl group (−OH) onto substrate (eg, tyrosine hydroxylase).
Carboxylase Transfers CO2 groups with the help of biotin (eg, pyruvate carboxylase).
Mutase Relocates a functional group within a molecule (eg, vitamin B12–dependent methylmalonyl-CoA
mutase).
Synthase/synthetase Joins two molecules together using a source of energy (eg, ATP, acetyl-CoA, nucleotide sugar).
Rate-determining enzymes of metabolic processes

PROCESS ENZYME REGULATORS
Glycolysis Phosphofructokinase-1 (PFK-1) AMP ⊕, fructose-2,6-bisphosphate ⊕
ATP ⊝, citrate ⊝
Gluconeogenesis Fructose-1,6-bisphosphatase 1 AMP ⊝, fructose-2,6-bisphosphate ⊝
TCA cycle Isocitrate dehydrogenase ADP ⊕
ATP ⊝, NADH ⊝
Glycogenesis Glycogen synthase Glucose-6-phosphate ⊕, insulin ⊕, cortisol ⊕
Epinephrine ⊝, glucagon ⊝
Glycogenolysis Glycogen phosphorylase Epinephrine ⊕, glucagon ⊕, AMP ⊕
Glucose-6-phosphate ⊝, insulin ⊝, ATP ⊝
HMP shunt Glucose-6-phosphate dehydrogenase (G6PD) NADP+ ⊕
NADPH ⊝
De novo pyrimidine Carbamoyl phosphate synthetase II ATP ⊕, PRPP ⊕
synthesis UTP ⊝
De novo purine Glutamine-phosphoribosylpyrophosphate AMP ⊝, inosine monophosphate (IMP) ⊝,
synthesis (PRPP) amidotransferase GMP ⊝
Urea cycle Carbamoyl phosphate synthetase I N-acetylglutamate ⊕
Fatty acid synthesis Acetyl-CoA carboxylase (ACC) Insulin ⊕, citrate ⊕
Glucagon ⊝, palmitoyl-CoA ⊝
Fatty acid oxidation Carnitine acyltransferase I Malonyl-CoA ⊝
Ketogenesis HMG-CoA synthase
Cholesterol synthesis HMG-CoA reductase Insulin ⊕, thyroxine ⊕, estrogen ⊕
Glucagon ⊝, cholesterol ⊝

72 SEC TION II Biochemistry  BIOCHEMISTRY—Metabolism
Metabolism sites
Mitochondria Fatty acid oxidation (β-oxidation), acetyl-CoA production, TCA cycle, oxidative phosphorylation,
ketogenesis.
Cytoplasm Glycolysis, HMP shunt, and synthesis of cholesterol (SER), proteins (ribosomes, RER), fatty acids,
and nucleotides.
Both Heme synthesis, urea cycle, gluconeogenesis. Hugs take two (both).
Summary of pathways
Galactokinase (mild galactosemia) Galactose metabolism

Galactose-1-phosphate Galactose B Requires biotin cofactor
uridyltransferase
(severe galactosemia) T Requires thiamine cofactor (TPP)
Glycolysis # Irreversible, important point of regulation
Hexokinase/glucokinase Galactose-1-phosphate Glucose
Glycogen
Glucose-6-phosphatase HMP shunt
(von Gierke disease)
UDP-glucose Glucose-1-phosphate Glucose-6-phosphate 6-phosphogluconolactone
Glucose-6-phosphate
dehydrogenase Glycogenesis / glycogenolysis
Transketolase Fructose-6-phosphate Ribulose-5-phosphate
T
Phosphofructokinase-1 Gluconeogenesis
Fructose-1,6-bisphosphatase 1 Fructose-1,6-bisphosphate Fructose metabolism
Fructokinase (essential fructosuria)
Aldolase B (fructose intolerance) Glyceraldehyde-3-P DHAP Fructose-1-phosphate Fructose
Aldolase B (liver), A (muscle)
Triose phosphate isomerase 1,3-bisphosphoglycerate Glyceraldehyde
Pyruvate kinase
3-phosphoglycerate Glycerol Lipid metabolism
Pyruvate dehydrogenase
Pyruvate carboxylase
2-phosphoglycerate Triglycerides
PEP carboxykinase
Citrate synthase
Phosphoenolpyruvate (PEP) Fatty acids
Isocitrate dehydrogenase
α-ketoglutarate dehydrogenase Alanine Pyruvate Lactate Cholesterol
Malonyl-CoA
Carbamoyl phosphate synthetase I T
B
Ornithine transcarbamylase B Acetyl-CoA Mevalonate
Propionyl-CoA carboxylase Acetoacetyl-CoA HMG-CoA
HMG-CoA reductase
Citrate Acetoacetate
Oxaloacetate
NH3 + CO2 Aspartate
Citrulline Isocitrate β-hydroxybutyrate
Carbamoyl phosphate Argininosuccinate

Ketogenesis
Malate TCA cycle
Ornithine Odd-chain fatty acids,
Urea cycle α-ketoglutarate isoleucine, valine, methionine,
Fumarate threonine, pyrimidines
T
Arginine
Succinate Succinyl-CoA Methylmalonyl-CoA Propionyl-CoA
Urea B12 B
H2O Protein metabolism

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held it close for inspection.
“M’ name’s James B. Lee,” he announced thickly, “but ev’ybody
calls me Lonesome Lee. Now, what in —— do you reckon anybody’d
write a letter to me for? This’n jist come on the stage.”
He handed the letter to Hashknife, or rather he started to; but the
flashily-dressed person had moved nearer and secured it. For a
moment nobody spoke. Lonesome swallowed with great difficulty
and tried to clear his throat.
“Right sudden, ain’t you?” said Sleepy.
The man ignored his question and spoke directly to Lonesome
Lee.
“Nobody ever wrote to you, Lonesome.”
“Yeah, they did, Spot. I—I—” whined Lonesome.
“The envelop will show who it’s for,” said Hashknife easily.
Spot Easton turned to the bartender.
“‘Windy,’ how many times do I have to tell you not to let Lonesome
have any more whisky?”
“Lay off the bartender,” advised Hashknife. “I paid for the old man’s
drink, if you care to know.”
Spot Easton seemed to see Hashknife for the first time, and the
discovery did not please him.
“Who in —— are you?” he growled.
“Me?” Hashknife grinned. “I’m the li’l jasper that’s goin’ to make
you give the letter back to Lonesome Lee.”
“Yeah?”
Easton’s brows lifted in surprize, as he looked Hashknife over
appraisingly.
“How are you goin’ to do it, if I may ask?”
Hashknife turned his body toward the bar. It was a disarming
move. Easton stepped in closer to Hashknife; stepped in just in time
to be in reach of the right swing that Hashknife pivoted to
accomplish.
It caught Mr. Easton flush on the left ear and the force of the
smash knocked the gentleman’s feet loose from the floor. The thud
of his fall had barely sounded, when Hashknife leaned over him and
took away the letter.
Easton did not move. The piano crashed a discord and stopped.
One of the girls gave a throaty little squeak and stopped dancing.
Hashknife turned to hand the letter to Lonesome Lee, but that worthy
was going out of the front door as fast as his unsteady legs would
carry him.
“Well, that kinda beats ——!” grunted Hashknife.
The bartender had dropped the glass he was polishing, but
continued the action on the bunched fingers of his left hand. He
breathed on the fingers and polished harder.
Spot Easton sat up, holding his left ear. He looked around as if
wondering what had happened. His eyes strayed to the ceiling, as if
wondering that it was still intact. Then he got slowly to his feet and
brushed the dust off his broadcloth raiment.
“You asked a question,” reminded Hashknife seriously, “but I don’t
reckon you need an answer—not now.”
Spot Easton did not express any opinion. He wadded a silk
handkerchief against his bruised ear, turned, and went to the back of
the room.
“I’ve got the letter and nobody to give it to,” chuckled Hashknife,
and then to the bartender—
“Whatcha polishin’ your fingers for, pardner?”
The bartender, suddenly realizing that he did not have a glass in
his hand, recovered the one from the floor.
“What’sa matter with everybody around here?” asked Sleepy. “The
old man hummed out of here like a spike, and you got absent-
minded. Ain’t the War-Bonnet used to seein’ trouble, or is all this
honkatonk only a blind for a Sunday school?”
“That—that was Spot Easton,” stammered the bartender.
“Who’s he—the king?” asked Hashknife.
The bartender glanced keenly toward the rear of the place, where
Easton had entered one of the built-in rooms. He leaned across the
bar and whispered:
“You better look out for him, gents. Spot Easton’s a ——winder,
y’betcha. He’s quicker’n a flash with a gun, and he used to be a
middle-weight prize-fighter. Glad it ain’t me he’s sore at.”
“You don’t reckon he’s sore at me, do you?” Hashknife seemed
penitent.
“Huh?” Such a foolish question amazed the bartender.
“Gee cripes! He must be touchy if he is,” observed Sleepy. “Some
folks wears their feelin’ on their sleeves.”
“Well, for ——’s sake!” wailed the bartender. “I dunno whatcha
mean by that. If you got hit in the ear——”
“Aw, come on, Sleepy,” said Hashknife. “Never seen a bartender
or a sheepherder yet that had any sense.”
As they started to cross the street, a rider on a mouse-colored
horse passed in front of them, going down toward the sheriff’s office.
The man was almost as tall as Hashknife; his features were hidden
by the shadow of his low-pulled Stetson.
Bliz Skelton and the sheriff were coming away from the office, and
the sheriff hailed this rider, who swung over to the board sidewalk
beside them.
“Wears bat-wing chaps, beaded vest and a polky-dot shirt,”
observed Hashknife aloud, “rides with his stirrups a notch too short;
all of which makes me feel that I know that hombre, Sleepy.”
“Let’s look him over,” suggested Sleepy. “Looks a li’l gaudy to me,
but mebbe he’s all right.”
The stranger was talking earnestly to the sheriff, as they walked
up, and the conversation seemed to interest Skelton. The stranger
turned and looked at Hashknife, but continued to talk.
“I dunno,” said Skelton, shaking his head. “I’m much obliged to
you, but I ain’t made up my mind yet jist what ’m goin’ t’ do. ’F I sell
out I won’t need no hired help.”
“And if you don’t, you do.”
The sheriff was a trifle ungrammatical, but sincere.
“Yeah,” admitted Skelton.
“It don’t make me no never mind,” stated the stranger. “I’m just
open f’r engagement. Jake’ll tell you that I’m a top-hand, y’betcha.”
“All of which makes it so,” stated Hashknife.
Jake Blue squinted at Hashknife and up at the cowboy. The latter
seemed surprized that any one might doubt his ability.
“Who ’re you?” asked the cowboy.
“Names don’t mean nothin’,” replied Hashknife. “I don’t know your
name, but I ain’t inquisitive. ’Pears to me that I’ve knowed you
some’ers.”
“Huh!”
The cowboy’s eyebrows lifted slightly, but no sign of recognition
crossed his features. He was not at all handsome—due partly to a
crooked nose, a split lip and a week’s growth of downy, blond
whiskers.
“What are you cuttin’ in here fer?” asked Blue angrily. “Mister
Hagen’s goin’ t’ work for Skelton.”
“Zasso?” Skelton seemed surprized. “I ain’t hired nobody yet,
Blue.”
“You ain’t goin’ to have work for three men, are you?” asked
Hashknife, turning to Skelton.
“Three men?” queried Blue quickly.
“T-h-r-e-e,” spelled Hashknife. “Me and Sleepy’s done hired out to
him.”
The sheriff spat explosively and looked at Skelton.
“Zasso, Skelton?”
“Well, yuh—uh—might say it was,” faltered Skelton.
“I’m goin’ to be the foreman,” stated Hashknife, “and if you got any
top-hands, you might send ’em to me, sheriff.”
“——!”
Mr. Hagen spoke very peevishly, turned his horse and rode back
to the War-Bonnet hitch-rack. There he dismounted, kicked his horse
in the belly, and went into the saloon.
“There ain’t no question but what he’s a top-hand,” agreed
Hashknife. “All top-hands kick their broncs in the belly thataway.
Kinda makes the bronc respect you.”
“Where’s the Swede?” asked Sleepy.
Jake Blue had been staring toward the War-Bonnet, deep in
thought, and Sleepy’s question seemed to jar him awake.
“The Swede? He’s in jail. Where’d you think he was?”
“In jail,” said Sleepy.
“Then what in —— did you ask fer?” Mr. Blue growled.
“You can’t hook that killin’ onto the Swede.” This from Hashknife.
“Can’t I?” The sheriff grew very indignant. “Well, mebbe I ain’t goin’
t’ try very hard.”
He stepped off the sidewalk as if to leave, but turned and added—
“’F I was you I’d be hopin’ that it was hooked onto the Swede.”
With this parting shot, the sheriff crossed the street and went into
the Paris restaurant, banging the door behind him.
“You made him mad,” observed Skelton seriously. “He was only
tryin’ to git a job for this Hagen feller.”
“Who’s this Hagen?” asked Hashknife.
“I dunno him. He’s been with the 88 f’r a while, but he quit, or got
fired or somethin’.”
“Who owns the 88?”
“Lonesome Lee used t’ own it, but he drank it mostly all up, I
reckon. Mebbe Spot Easton owns it by now. Lonesome got t’ drinkin’
and playin’ poker, and I reckon he’s lost all the money he ever had.
He stays at the ranch—when he ain’t drunk—which ain’t often.”
“Big outfit?” asked Hashknife.
“Bigger’n mine,” answered Skelton.
“With two top-hands your ranch ought to grow,” stated Hashknife
seriously. “You don’t mind us hirin’ out to you?”
“I dunno where your pay’s comin’ from, but I don’t mind, if you
don’t. Want to go back to the ranch now?”
Hashknife shook his head.
“No-o-o. You see I knocked Spot Easton loose from the floor a
while ago, and if we left now it would look like I was runnin’ away.”
“You did!” gasped Skelton. “Spot Easton? Well——”
Skelton scratched his head and squinted at Hashknife’s serious
face.
“Well, I—I reckon yo’re a top-hand, Hartley. Come out to the ranch
any ol’ time you git ready. Whoo-ee!”
The old man slapped his hat back on his head and bow-legged his
way back to the sheriff’s office.
Hashknife took the letter from his pocket and looked at it.
“She sure belongs to Lonesome Lee, Sleepy. The epitaph
proclaims it to be for James B. Lee, Caldwell, Montana, and the little
doohicky in a circle says that she was sent from Boston.”
“Now, whatcha reckon Mister Easton wanted this here letter for,
Sleepy?”
“Don’t glare at me!” complained Sleepy. “You act like it was my
letter. How’d I know what Easton wants?”
“Where did the old man go?” asked Hashknife, paying no heed to
Sleepy’s question.
“There you go ag’in! Think I’m a fortune-teller? You saw him the
last time I did.”
“Well, I reckon the only way to find him is to look for him. Come
on.”
They went up the sidewalk, past the Hole-in-the-Wall feed-corral,
and almost bumped into Lonesome Lee, who was coming out from
the narrow alley between the feed-corral and general store. The old
man’s cheeks were streaked with tears and dust, and he was half-
sobbing—drunkenly. He gawped at Hashknife and Sleepy and tried
to avoid them, but Hashknife took him by the arm and drew him
back.
“What’s the matter with you?” growled Hashknife. “Ain’t nobody
goin’ to hurt you, old-timer. Here’s your letter.”
Lonesome Lee stared at the letter, but made no effort to take it. In
fact he seemed afraid of it.
“You ain’t scared of Spot Easton, are you?” asked Sleepy.
Lonesome did not say, but his actions spoke volumes.
“Has that tin-horn got you buffaloed, old-timer? Snap yourself
together! You’ve blotted up so much hooch that your nerves are
dancin’, but you’re a —— good man yet.” Hashknife’s voice was
encouraging.
“Th-think so?”
Lonesome wiped his lips with shaking fingers and moved his feet
uncertainly.
“Better read the letter,” urged Sleepy. “It might be good news; you
never can tell.”
“Wh-where’s it from?” he stammered. “My eyes ain’t worth a ——
no more.”
“She’s from Boston.”
Lonesome licked his lips and stared into space.
“Bub-Boston! ——!”
He staggered off the sidewalk, almost fell in the dust, and weaved
a crooked trail straight for the doorway of the War-Bonnet.
“’F that don’t beat ——, I’m a pigeon-toed fool!” grunted Hashknife
foolishly.
“His ear-drums kinda shrink from Boston,” observed Sleepy, as
Lonesome seemed to carom from one side of the door to the other.
“Scared plumb to death,” declared Hashknife. “It’s a danged
shame for a man to get in that shape. Somethin’ has sure put the
Injun sign on the old gent, Sleepy. This Easton’s a bass-drummer
among these canary-birds, ’cordin’ to what I can get in my loop; so
he must be somethin’ besides a card shark.”
“Let’s go over and talk to the blacksmith,” suggested Sleepy. “I’ve
got to have some shoes put on my bronc’ pretty soon, and maybe I
can save about four-bits by gettin’ real friendly. I have done it, by
cripes.”
Barney Stout was inserting a new felly into a wagon-wheel, and
swearing mournfully over the fitting. He rubbed his nose with the
back of a very dirty hand and nodded to Hashknife and Sleepy. They
had squatted down against the wall and were rolling cigarets.
“How’s tricks?” asked Sleepy.
“Tricks?”
Barney squinted at the rim of the wheel, as he felt of the joint with
a thumb.
“There ain’t no tricks in this trade; it’s all —— hard work and
disappointment. Hear they put the Swede in jail.”
Barney rubbed his hands on his hips and, reaching for Sleepy’s
sack of tobacco, squatted down beside them.
“I dunno who killed Quinin Quinn, but it’s a dead immortal cinch
that Swede Sam never did.”
“Where’d that .30-30 rifle come from, do you figure?” asked
Hashknife.
Barney shook his head and puffed violently.
“I never seen the gun,” he said. “Quinn tol’ me that he’d been shot
at three or four times in the last year. ’S far as that’s concerned, so
has old Skelton.”
“Any idea why?” asked Sleepy.
“Nope. I heard Jake Blue say that it was likely that folks hadn’t
forgot what happened to their graveyard—but Quinin didn’t have
nothin’ to do with that. He came here quite a while after that.”
“Folks got kinda sore about it, eh?” queried Hashknife.
“Yeap. Can you blame ’em? They sure as —— lost track of their
ancestors. Ev’body tried to relocate their dead, but it was no use. M’
wife had one of them Kodiak things that you take pictures with, and
she photygraphed the graveyard one day; but it’s kinda blurred-like.
They took that along to try and figure out things, but it didn’t help ’em
a danged bit.”
“Lots of folks buried there, eh?” queried Hashknife.
Barney nodded.
“All them markers in Skelton’s yard indicates a body. The first
corpse was old Billy Meek, who was some sanctimonious old
whippoorwill; and the last one was a gambler by the name of ‘Faro’. I
never did know his name.
“Spot Easton shot him over a poker-table. Folks kicked about him
bein’ buried in the cemetery, and old ‘Peg-leg’ Smith refused to dig
the grave; but Spot and Doc Clevis dug the grave, and I reckon Jake
Blue performed the funeral oration—I dunno. Anyway, Faro never
got a headstone, ’cause his grave-mound was lost with the rest.”
“Did they bury this here Faro person right in with the rest?” asked
Hashknife.
“Danged if I know for sure. Seems to me that somebody said they
planted him off to one side; kinda between the others and the creek.
I never seen the grave.”
Came the sound of a boot on gravel, and they turned to see
Doctor Clevis coming in the front door. He peered at Hashknife and
Sleepy.
“Wanted to tell you that the inquest’ll be held t’morrow afternoon
’bout two o’clock,” he announced. “Likely need your testimony.”
“We’ll be here,” nodded Hashknife.
The doctor walked out, and Barney got to his feet.
“Gotta git that —— felly fixed up, I suppose,” he groaned. “Hope I
never git sick and have to call Doc Clevis. Him and Jake Blue are
thicker’n two drunks in one bunk. Besides, I never like to have any
truck with a doctor who is the undertaker, too.”
“Gets ’em goin’ and comin’, eh?”
“Gotta cinch,” agreed Barney. “And Jake Blue ain’t as particular as
he might be, especially when the reward notice don’t specify ‘dead or
alive.’”
“We’ll see you again, pardner,” said Hashknife, as he and Sleepy
walked out the front door.
“Come in any ol’ time,” yelled Barney. “Mostly always I’ve got time
to talk.”
Hashknife led the way past the War-Bonnet and up to the hitch-
rack where they got on their horses and rode back toward the
Tombstone Ranch. Hashknife looked back and saw Hagen standing
in the doorway, looking in their direction; but there was no sign of
Lonesome Lee nor of Spot Easton.
“Do you reckon they’ll hang the killin’ onto Swede Sam?” asked
Sleepy, as they poked off down the dusty road.
Hashknife eased himself in the saddle and reached for his cigaret
material.
“I’ll do everythin’ I can to hook it onto him,” he said.
“You will?” Sleepy’s surprize was genuine.
“Sure will. Me and you and Skelton have got to lie like —— to
keep out of jail ourselves.”
“But the Swede never killed him.”
“Neither did we, Sleepy. I reckon Mrs. Frosty Snow can get along
without Swede Sam for a while—and Swede Sam ain’t got brains
enough to mind bein’ locked up.”
“But they couldn’t put me and you in jail,” protested Sleepy.
“Thasso? They put Swede Sam in.”
Which left Sleepy without an argument worth while.
Hashknife locked a long forefinger around his spoon and fended it
away from his right eye, while he sipped thoughtfully at his cup of
coffee. Finally he nodded slowly.
“Yeah, that’s true, Skelton. Whisky does pe-culiar things to a
man’s nerves; but why does ol’ Lonesome go hippety-hoppin’ like a
scared rabbit when he sees that danged letter?”
Skelton helped himself to more coffee from the old battered pot
and reached for Sleepy’s cup.
“Not any more, Bliz,” said Sleepy. “You ought to grind that coffee
before and after makin’, ’cause she’s sure hard to chew.”
“Lonesome Lee’s sure in tough shape,” admitted Skelton, ignoring
Sleepy’s insult to his ability as a coffee maker.
Hashknife took the letter from his pocket and studied it closely.
“Steam,” said Sleepy slowly. “Steam’ll cut the stickum on an
envelop.”
Hashknife squinted hard at Sleepy.
“That’s a crooked thought, Mister Stevens. Sometimes you
surprize me.”
“You say that Spot Easton wanted the letter?” asked Skelton.
Hashknife yawned widely and glanced around the room.
“Skelton, you ain’t got anythin’ like mucilage, have you?”
“Y’betcha, I have. Li’l bottle, with a brush attached. I dunno what it
was used fer, and she’s been here since before Heck’s father went
wooin’. Whatcha want it fer?”
“To make this danged letter look like it never was opened.”
Sleepy grinned joyously.
“Gimme credit——”
“Fer nothin’,” finished Hashknife. “That was a common thing
before your great, great-grandfather was lynched for tryin’ to tell folks
what to do.”
Hashknife held the envelop over the steam from the tea-kettle,
until the flap was softened, and removed the letter. He spread out
the single sheet on the table, and the three of them read it together.
Dear Dad:
I will arrive nearly as soon as this letter, but am sending it anyway. I hope that
your injured arm is better now. It was very kind of your foreman, Mr. Easton, to
write in your stead, and I shall thank him personally for his offer to meet me at
Gunsight.
I can hardly wait to see you. Just to think that I have never seen you since I was
old enough to remember, but we will make that all up, daddy. I have just money
enough to take me to Caldwell, and I am coming as fast as I can travel.
Since mother passed out I have felt entirely alone in the world, and even if you
and mother could not be happy together, I am sure we can. Loads of love and a
big hug very soon.
Your loving daughter,
Jane.
P. S.—I will be with you in time to celebrate my eighteenth birthday.
Along the margin of the paper was written—

I am glad you liked the picture of myself, which I sent you, daddy.
Hashknife lifted his eyes from the paper and looked at Skelton,
who was moving his lips slowly over the written words. Skelton
straightened up and shook his head.
“I don’t sabe that foreman stuff. Spot Easton never was foreman of
the 88.”
“Has Lonesome Lee been nursin’ a sore arm?” asked Sleepy.
Skelton laughed shortly.
“’F he has, I never knowed it. That letter’s sure got me pawin’ m’
head. I never knowed that Lonesome Lee had a wife or daughter.”
“And,” added Hashknife meaningly, “Spot Easton was kind enough
to want to meet her in Gunsight. He was also doin’ the writin’ for
Lonesome, ’cause Lonesome had a sore arm.”
“Whatcha make of it, Hashknife?” asked Sleepy.
Hashknife pondered over the manufacture of a cigaret, and read
the letter again before he spoke.
“’Pears to me that the lady done sent her picture, previous. Mebbe
she’s pretty, which would attract Mister Easton. It also appears that
Mister Easton has got old Lonesome Lee where the hair’s short and
tender, and he’s kinda runnin’ Lonesome’s business.
“Accordin’ to signs, Mister Easton has lied to said lady, who thinks
her paw is somebody. Paw ain’t got no nerve left to object, and
Mister Easton has likely told him that he has invited this here
daughter to live at the 88. Paw ain’t got the guts to howl against such
things, and when he finds that the letter is from Boston he’s plumb
shaky that it tells about daughter’s de-parture. Mister Easton
naturally is wishful to know how his invite has worked out; which is
the reason he grabbed at the letter. That’s how she looks to me.”
“’F that’s a fact, I sure as —— feel sorry fer her,” stated Skelton
sadly.
“Yuh might feel sorry for Lonesome, too,” said Hashknife. “He’s all
shot to pieces with hooch, and he likely knows that she’s comin’ to
find him.”
“Figurin’ she’s goin’ to be happy with him,” added Sleepy
mournfully. “Comin’ to celebrate her eighteenth birthday. ——!”
Hashknife got to his feet and walked over to the open door, where
he leaned against the casing and contemplated deeply. The sun had
already dropped behind the hills, which looked like blue silhouettes,
with silver trimmings. Far away on the skyline drifted a herd of cattle;
their outlines blurred from the back-light of the sunset.
From below the long sheds came a string of cattle, heading for the
water-hole opening on the brushy stream; bawling softly, as they
followed the deeply-worn trail. Magpies chattered sleepily in the
cottonwoods.
“Makes a feller wonder how a man can live in a land like this and
hate anybody,” muttered Hashknife.
He turned to come back to the table, when—
Ping-g-g—Whop!
Skelton fell backward out of his chair, clawing at the coffee, which
sprayed all over him. Sleepy threw himself sidewise out of line with
the door, and from somewhere came the thin, whip-like report of a
high-powered rifle.
Hashknife kicked the door shut and gawped at Skelton, who got to
his feet, shook the coffee out of his eyes, and picked up the coffee-
pot—or what remained of it.
The soft-nose bullet had hit it near the bottom and there was
nothing much left to identify it as a coffee-pot, except the color and
odor. Even the ceiling was dotted with coffee-grounds.
“Anybody hurt?” asked Hashknife.
Skelton gazed ruefully at the remains of the pot, and dug inside his
collar after more grounds.
“——!” he snapped. “They didn’t miss us very far that time.”
“Common occurrence?” asked Hashknife.
“Periodical. Last week I was shakin’ some stuff out of a fry-pan
outside, and they nailed the ol’ pan, dead-center. Wrenched —— out
of m’ wrist, too. Never even saw where the bullet came from. I dunno
whether they’re hintin’ fer me to move, or missin’ their target.”
“Got —— good eyes, if they shot at that pot,” grunted Hashknife,
“’cause that rifle wasn’t closer than five hundred yards.”
“Cat-eyes,” added Sleepy. “Nobody could see into a house at this
time of the day. That hombre wasn’t aimin’ to spill our coffee, y’
betcha.”
“Got a rifle, Skelton?” This from Hashknife.
“Dang right I have.”
He walked over to one of the bunks and threw back the blankets.
He ran his hand over them, dug under the straw-tick, and stepped
back, looking curiously around.
“What do you know about that?” he grunted. “It ain’t there!”
“Are you sure?” asked Hashknife.
“Lemme think. It was there yeste’day, ’cause I took it out when I
made the bed. I know danged well—no, I ’member leanin’ it agin’ the
wall.”
He glanced around the room and shook his head.
“Don’t make a —— bit of difference; it’s gone.”
“What kind was she?” asked Sleepy.
“Winchester.30-30.”
“That wasn’t it we found near the Swede, was it?”
“No-o-o—I’m —— ’f I know whether it was or not. I never looked at
it. Fact is, I never used it. I’m not worth a —— with a rifle, but I sure
do sabe the old shotgun and buckshot, or a six-gun. Never liked that
idea of shootin’ a man with a mushroom bullet.”
“Does kinda unravel a man,” Hashknife agreed. “When did you
buy that .30-30?”
“I acquired it with this —— ranch, along with the rest of the
misery.”
Hashknife nodded slowly and considered the ceiling. A question
had suddenly popped into his head and he wanted to consider it
before speaking. The coffee-grounds were beginning to loosen from
the ceiling, and some of them drifted into his eye. He dug them out
thoughtfully and turning to Skelton said—
“You got any relations, Skelton?”
“Not a danged kin,” grinned Skelton. “One of my kind is e-nough,
ain’t it?”
“’F you got killed,” suggested Hashkinfe, “who’d get this ranch?”
Skelton scratched his head violently.
“Never thought of that, Hartley. Why, I reckon the sheriff would sell
it to the highest bidder. But who would bid on it—I dunno.
“Shucks!” Skelton added. “It must be somethin’ pers’nal. Nobody’d
kill me to get a chance to buy this —— ranch. That ain’t reasonable.”
“Human nature is a queer thing,” said Hashknife. “I knowed a feller
who was sent to the penitentiary for stealin’ Christmas presents,
which were goin’ to be given to him.”
“Why didn’t you add the fact that he knowed it?”
“I know when to quit lyin’,” said Hashknife gravely.
He got to his feet, went to the door, and peered out.
“Gets dark quick around here,” he said. “I reckon it’s plumb safe to
saddle up now. That bushwhacker likely went away as soon as he
fired that one shot.”
“Saddle up? What for, f’r gosh sake?”
Sleepy settled back comfortably in his chair.
“Me and you are goin’ to Caldwell.”
“What fer?”
“That inquest is tomorrow afternoon, Sleepy.”
“Oh, I see,” said Sleepy sarcastically. “’Fraid you’ll be late if you
don’t start now?”
“You might put it thataway,” admitted Hashknife. “We’ll be back
kinda late, Skelton, I reckon; so I’ll call m’ name when we come
home.”
Skelton nodded dubiously and said:
“’S your own business, Hartley, and I reckon you can take care of
yourself. I dunno what you got on your mind, but I wish you well.”
Hashknife grinned at Sleepy’s disgruntled way of pulling on his
chaps, and went out of the door. Sleepy swore softly as he followed
him.
Spot Easton was not in a happy frame of mind at all. His ear had
swollen to twice its normal size and had assumed the shade of a
pickled beet. It not only pained him, but it hurt his pride; he was not
in the habit of getting the worst of a personal encounter.
The evening business of the War-Bonnet was beginning to be
audible to Spot, who was sequestered in his little private room in the
rear. A half-empty whisky bottle decorated the table beside him, and
his jaws were clamped tightly over a badly frayed cigar, which
smoked much from the wrong end. He jerked it out of his mouth,
cursed and hurled it across the room where it continued to throw up
a streamer of smoke.
Just then, without any warning, the door swung open and
Lonesome Lee staggered in. The old man was gloriously drunk, but
tried to brace up when he faced Easton.
“Sus-somebody said you wanted to shee me,” he muttered thickly.
“Yes; you lousy old bum!” snapped Easton, kicking a chair away
from the table.
Lonesome eased himself shakily into the chair and sprawled
weakly.
“Where’s that letter?” demanded Easton.
“Tha’ letter?” Lonesome grinned foolishly. “Wha’ letter?”
“The one you got today. The letter—oh, ——!”
Lonesome had emitted a long-drawn snore and his head sank
slowly until his chin was buried in his collar.
Spot Easton shoved away from the table and, going over to
Lonesome, proceeded to go through the old man’s pockets. He
shook Lonesome, but the old man continued to snore loudly.
Spot caressed his aching ear, while he reviled Lonesome with
every foul epithet his tongue could command. Tiring of that, he drank
half of the remaining liquor, threw the bottle across the room, and sat
down again.
Then came Jack Blue. He too was a privileged character and did
not wait to knock on the door. He squinted at Lonesome and sat on
the edge of the table.
“Why don’t you have Doc Clevis fix up yore ear?” he asked,
noticing that Easton was fingering the sore organ.
“That —— veterinary!” exploded Easton.
“Doc could take out the soreness.”
“I’m —— if he could!” rasped Easton. “Only one thing’d take the
soreness out of that ear, and that’s to notch a sight on that long-
geared misfit that hit me.”
“He’s a fresh whippoorwill, all right,” admitted Blue. “Never seen
anybody with the gall he’s got. Somebody’s due to make jerky out of
his tongue.”
“Y’betcha,” agreed Easton, “and I’m him.”
Blue jerked his head toward the sleeping Lonesome——
“Did he have that letter, Spot?”
“Naw!”
“That puncher still got it?”
Spot looked very disconsolate, but did not answer.
“What was in it, do you reckon?”
“How’d I know?”
Blue gnawed off an enormous chew of tobacco and moved to a
chair.
“’F he’s still got the letter I’ll git it for you tomorrow, Spot.”
“How?”
“Law requires that I search all prisoners, tha’s why.”
“Thasso?” Spot Easton grew interested. “You goin’ to put him in
jail?”
“I sure as —— am. More’n that, I’m goin’ to put the both of ’em in
jail, along with old man Skelton.”
“How you goin’ to make it look right?”
Blue spat copiously and grinned at the ceiling.
“That was old Skelton’s rifle which they found beside the drunk
Swede.”
“Skelton’s rifle? And he brought it to you?”
“Nope. I went past there yesterday and I dropped in to call on
Skelton—knowin’ he was in town.”
“And swiped his rifle?”
“Uh-huh. Belonged to old Bill Wheeler, and she’s got a li’l 33 cut
into the forearm. She’s a cinch to hang it onto Skelton, and I can
hold them other two—easy.”
Easton laughed and got to his feet.
“You’re clever, Jake. Let’s go and get a drink.”
“I sure am.”
Blue was not adverse to applauding himself. Being a sheriff in
Lodge-Pole county entailed too much danger for the remuneration;
so nobody cared much about a sheriff’s morals—or methods.
Easton gazed approvingly upon the amount of activity within the
four walls of the War-Bonnet, as he led the sheriff to the bar. The
click of dice, the rattle of poker chips and the droning voices of
dealers was sweet music to Easton’s ears.
A number of men were standing at the bar, but Easton and Blue
ignored them. Two cowboys were shaking dice on the bar-top at
Easton’s right hand.
“’At’s horse ’n horse,” declared one of them. “One flop, Sleepy.”
Easton shot a sidewise look at the speaker. It was the tall cowboy,
who had hit him on the ear, standing elbow to elbow with him; intent
on his dice shaking.
Easton slowly turned his head and looked at Blue, who was toying
with his glass of liquor. The dice rattled.
“You’re stuck!” exclaimed Hashknife.
Easton jerked his head around and looked square into Hashknife’s
face.
“How’s the ear?” asked Hashknife.
The question placed Easton in an embarrassing position. He could
not see Hashknife’s right hand, and his own hands were on the bar.
Blue squinted past Easton’s shoulder at Hashknife, and Hashknife
grinned at him.
Sleepy leaned forward on the bar and craned his neck around
Hashknife.
“I hope to die, if I ain’t terror-stricken!” he gasped. “We’ve been
told that it’s fash’nable to be plumb scared of Mister Easton; so we
turns pale, politely.”
Easton tore his eyes away from Hashknife’s grinning face and
looked straight into the back-bar. His mind worked swiftly, but got
nowhere. He was being insulted in his own house. Jake Blue leaned
away from the bar, as if to move into the crowd, but Sleepy stepped
around behind Hashknife and Blue leaned back against the bar.
“Where’s the old man—old Lonesome Lee?” asked Hashknife.
Easton turned quickly.
“What do you want of him?”
“Want to give him that letter,” explained Hashknife.
“Oh!” Easton’s grunt seemed to relieve him.
“’F he ain’t around here, mebbe you could take care of it for him,
eh?”
“Sheriff’s nervous,” interrupted Sleepy. “’Pears to have a itch on
his hip. Likely comes from a callous caused by packin’ such a heavy
gun.”
Jake Blue scowled, but said nothing.
“I’ll give him the letter,” nodded Easton, trying to not appear too
eager to be of service.
Hashknife’s concealed right hand flipped the letter to the bar in
front of Easton and dropped back. Easton picked up the letter and
started to put it in his vest-pocket, but Hashknife stopped him.
“Whoa, Blaze!”
Easton stared at him wonderingly, as Hashknife motioned for him
to stop.
“Not in a vest-pocket, pardner. Put it in your side pants-pocket, if
you don’t mind. That’s the only pocket where a tin-horn gambler
don’t pack a derringer.”
Easton scowled and shoved the letter into the designated pocket.
He wondered if this tall cowpuncher was a mind reader, and knew
that he was going to use the letter as an excuse to get at the two-
barreled derringer in his vest-pocket.
“’F you don’t stop hankerin’ t’ scratch—” Sleepy’s voice held a note
of menace—“’f you don’t, I’m goin’ to get a piece of sandpaper and
give you one good curryin’, Mister Sheriff. Ain’tcha ashamed to
scratch thataway in comp’ny?”
“By ——, I’m tired of this!” wailed the exasperated Mr. Blue.
“Who’re you, anyway, I’d like to know? What right you got to tell me
when I can scratch and when I can’t?”
“I’m just teachin’ you how to act polite, ain’t I?” complained Sleepy.
“Gee cripes, you sure do act peevish over learnin’ things. ’F I was
you——”
“Don’t tease the li’l gent, Sleepy,” Hashknife said, chuckling. “His
chilblains has likely extended up to his hips. You know how cold feet
makes you itch.”
Hashknife kept his eyes on Easton, while talking direct to Sleepy,
and he saw a flash of relief come over Easton’s face. A man had
stepped in behind him, brushing against Hashknife’s right elbow, and
Easton’s eyes had followed this man.
The conversation had been even lower than ordinary and had
attracted no attention.
It all happened in a few seconds. As the man brushed Hashknife’s

arm, Hashknife stepped quickly away from the bar; stepped away
just in time to let Hagen, the ex-88 cowboy, crash into Easton.
Hagen had intended to bump Hashknife hard enough to knock him
off his balance, but he had not expected Hashknife to move so
quickly.
Easton whirled half-around and jammed his heels on to Jake
Blue’s toes, while Hagen half-fell to his knees. Like a flash, Easton
struck at Hashknife, and his bare knuckles came in contact with
Hashknife’s heavy six-shooter.
Sleepy sprang in to prevent Blue from drawing a gun, and his knee
caught Hagen just under the chin, knocking his head against the
solid bar with a dull tunk! Easton’s right hand went out of commission
and he stumbled awkwardly over Hagen’s legs, falling flat on the
floor, while Sleepy pinned Blue’s arms in a bear-like hug, swung him
up bodily and backed to the door. Hashknife backed swiftly out with
him, covering the surprized crowd, which had no idea of what had
been going on.
Once outside they went swiftly to the hitch-rack, with Sleepy still
carrying the cursing sheriff.
“What’ll I do with him?” panted Sleepy. “I don’t want him.”
“Got his gun?” asked Hashknife.
“It’s back in the War-Bonnet.”
“Let him loose,” laughed Hashknife. “We ain’t collectin’ knick-
knacks.”
Sheriff Blue sat down so heavily in the hard street that his tongue,
for once, refused to function. Hashknife and Sleepy mounted swiftly
and whirled back past the War-Bonnet, where men were crowding
the doorway.
Spot Easton cursed bitterly as he saw them flash past the beams
of yellow light, then he turned back to “Blondy” Hagen, who was still
sitting in front of the bar, holding his head in his hands.
Easton’s right hand was deeply cut and swelling rapidly. He cursed
it fluently and turned to see Jake Blue coming in, covered with dust,
his face badly scratched.
Blue had nothing to say. Men crowded around them, wondering
what had been the reason for the fight, but none of the three victims
seemed inclined to explain things. Hagen got to his feet and started
for the door.
“You!” gritted Easton bitterly.
Hagen scowled blackly and shouldered his way out of the door,
where he turned and glared back at Easton.
“Aw! You be ——!” he snorted, and went away.
“It’s a large night,” said Blue inanely.
The coroner’s inquest over the remains of Quinin Quinn caused little
excitement in Caldwell. The fact that Quinin was dead was enough in
itself; who killed him, was merely conjectured and Lodge-Pole county
felt that it would remain so, according to precedent.
The jury listened patiently to Hashknife, Sleepy and Skelton, while
Doc Clevis, puffing with his own importance, crossquestioned them.

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First Aid for the USMLE Step 1 2022,

First Aid For The USMLE Step 1 2018 Tao Le

First Aid for the USMLE Step 1 2019, Twenty-Ninth

First Aid Cases For The USMLE Step 1 4th Edition

First Aid for the USMLE Step 1 2020, 30th Anniversary

First Aid for the USMLE Step 1 2022 - A Student-to-

First Aid For the USMLE Step 1 2020, Thirtieth Edition

First Aid for the USMLE Step 2 CK 11th Edition Tao Le

Contributing Authors vii General Acknowledgments xiii

Introduction 2 Test-Taking Strategies 19

` SECTION I SUPPLEMENT S P E C I A L S I T UAT I O N S 25

` SECTION II HIGH-YIELD GENERAL PRINCIPLES 27

How to Use the Database 28 Pathology 203

FAS1_2022_00_Frontmatter.indd 5 11/10/21 10:50 AM

How to Use the Database 738 Biochemistry 742

FAS1_2022_00_Frontmatter.indd 6 11/10/21 10:50 AM

DNA was the first three-dimensional Xerox machine.

This high-yield material includes molecular biology, genetics, cell

Do not spend time learning details of organic chemistry, mechanisms, or

FAS1_2022_01-Biochem.indd 31 11/4/21 11:59 AM

Chromatin structure DNA exists in the condensed, chromatin form to

Heterochromatin Condensed, appears darker on EM (labeled H Heterochromatin = highly condensed.

Euchromatin Less condensed, appears lighter on EM (labeled Eu = true, “truly transcribed.”

FAS1_2022_01-Biochem.indd 32 11/4/21 11:59 AM

Nucleotides Nucleoside = base + (deoxy)ribose (sugar).

Purines (A,G)—2 rings. Pure As Gold.

N10–Formyl- Nitrogenous base CH₂

FAS1_2022_01-Biochem.indd 33 11/4/21 11:59 AM

Hydroxyurea AMP GMP monophosphate dehydrogenase

Purine and pyrimidine synthesis:

5-FU, and pyrimethamine: inhibit dihydrofolate

CPS1 = m1tochondria, urea cycle, found in liver

FAS1_2022_01-Biochem.indd 34 11/4/21 11:59 AM

Purine salvage deficiencies

Nucleic acids Ribose 5-phosphate Nucleic acids

Genetic code features

FAS1_2022_01-Biochem.indd 35 11/4/21 11:59 AM

FAS1_2022_01-Biochem.indd 36 11/4/21 11:59 AM

joining repair double-stranded breaks. 3´ 5´ 3´

homologous dsDNA as a template. Homologous recombination

UV exposure Pyrimidine dimer Deaminated C

Newly replaced segment

Nucleotide excision repair Base excision repair Mismatch repair

FAS1_2022_01-Biochem.indd 37 11/4/21 11:59 AM

Altered amino acids

AUG AUG AUG

FAS1_2022_01-Biochem.indd 38 11/4/21 11:59 AM

DNA 5´ CAAT TATAAA GT AG GT AG AATAAA 3´

Regulation of gene expression

FAS1_2022_01-Biochem.indd 39 11/4/21 11:59 AM

Cleavage at 5′ splice site; lariat-

FAS1_2022_01-Biochem.indd 40 11/4/21 11:59 AM

Exon 1 Exon 2 Exon 3 Exon 4 Exon 5 Exon 6

FAS1_2022_01-Biochem.indd 41 11/4/21 11:59 AM

Start and stop codons

FAS1_2022_01-Biochem.indd 42 11/4/21 11:59 AM

FAS1_2022_01-Biochem.indd 43 11/4/21 11:59 AM

Cell cycle arrest

Fibroblast Preprocollagen S ynthesis—translation of collagen α

enaturation—DNA template, DNA primers, a heat-stable DNA polymerase, and