204 Textbook of Biochemistry— Case Studies

RECENT ADVANCES IN MEDICINE AND BIOCHEMISTRY

Changing Scenario of Medicine and Biochemistry

¾¾ Traditionally, concerned with metabolic pathways, nutrition
¾¾ Molecular biology – Technology and scope of biochemistry expanded
¾¾ Signal transduction, genetic disorders, ion channelopathies, neurochemistry
¾¾ Changing scenario in diagnostics and therapeutics – MRI, RNAi.

Alfred Nobel - The Man Behind the Nobel Prize

T’is then the mind, which care no more absorbs, In search of God ascends his glowing orbs, Or grapples with the
mysteries which surround Creation’s work within a narrower bound; For clearer than in distant worlds we find The hand
of Nature and her magic mind Remirrored here: the structure of a grain Has tasked the wisdom of th’Almighty’s brain,

2001
Cell Cycle Regulation

Leland H Hartwell R Timothy (Tim) Hunt Sir Paul M Nurse

Cell Cycle Regulation

¾¾ Two key classes of regulatory molecules that determine a cell’s progress through the cell cycle: cyclins and cyclin-
dependent kinases.
¾¾ Leland H. Hartwell, R. Timothy Hunt, and Paul M. Nurse won the 2001 Nobel Prize in Physiology or Medicine -
Discovery of these central molecules in the regulation of the cell cycle.
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¾¾ Cyclins form the regulatory subunits and CDKs the catalytic subunits of an activated heterodimer; cyclins have no
catalytic activity and CDKs are inactive in the absence of a partner cyclin.
¾¾ When activated by a bound cyclin, CDKs phosphorylate target proteins, that activate or inactivate them to orchestrate
coordinated entry into the next phase of the cell cycle.

Cell Cycle

A. Howard, S.R. Pelc 1950s

Arthur Pardee – Restriction points

`
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Molecules Involved…
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Phase Transitions…

Cell Cycle and Cancer

¾¾ A fundamental aspect of cancer is dysregulated cell cycle control.
¾¾ Normal cells - Proliferate when compelled to do so by developmental or other mitogenic signals in response to tissue
growth needs. Proliferation of cancer cells proceeds essentially unchecked.
¾¾ They are no longer subject to proliferation-inhibitory influences, nor do they necessarily require extrinsic growth
factors to recruit them into or maintain their proliferative state.
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2002
Apoptosis (Programmed Cell Death)

Sydney Brenner H Robert Horvitz John E Sulston

¾¾ One of the main types of programmed cell death (PCD), and involves an orchestrated series of biochemical events
leading to a characteristic cell morphology and death.
¾¾ In contrast to necrosis, which is a form of cell death that results from acute cellular injury, apoptosis is carried out in
an orderly process that generally confers advantages during an organism’s life cycle.

¾¾ Excessive apoptosis causes cells to lose, such as in ischemic damage, whereas an insufficient amount results in
uncontrolled cell proliferation, such as cancer.
¾¾ Defective apoptotic processes - Implicated in an extensive variety of diseases.
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¾¾ Between 50 billion and 70 billion cells die each day due to apoptosis in the average human adult.
¾¾ In a year, this amounts to the proliferation and subsequent destruction of a mass of cells equal to an individual’s body
weight.
¾¾ Sydney Brenner, Robert Horvitz and John Sulston shared the Nobel Prize for Medicine or Physiology in 2002 for
their work on apoptosis in C elegans.

¾¾ Can occur when a cell is damaged beyond repair, infected with a virus, or undergoing stress conditions such as
starvation.
¾¾ DNA damage from ionizing radiation or toxic chemicals can also induce apoptosis via the actions of the tumour-
suppressing gene p53.
¾¾ The “decision” for apoptosis can come from the cell itself, from the surrounding tissue, or from a cell that is part of
the immune system.

¾¾ Plays a role in preventing cancer; if a cell is unable to undergo apoptosis, due to mutation or biochemical inhibition,
it can continue dividing and develop into a tumor.
¾¾ For example, infection by papillomaviruses causes a viral gene to interfere with the cell’s p53 protein, an important
member of the apoptotic pathway.
¾¾ This interference in the apoptotic capability of the cell plays a critical role in the development of cervical cancer in
women.
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Role of PCD (Apoptosis)

¾¾ Homeostasis
¾¾ Development
¾¾ Cell damage
¾¾ Lymphocyte interaction
¾¾ Defective apoptotic pathway has been linked to many diseases including cancers and in HIV progression.

¾¾ Controlled by a diverse range of cell signals which may originate either extracellularly (extrinsic inducers) or
intracellularly (intrinsic inducers).
¾¾ Extracellular signals may include hormones, growth factors, nitric oxide or cytokines.
¾¾ Signals may positively or negatively induce apoptosis.
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¾¾ Intracellular apoptotic signalling is a response initiated by a cell in response to stress, and may ultimately result in
cell suicide.
¾¾ Binding of nuclear receptors by glucocorticoids, heat, radiation, nutrient deprivation, viral infection and hypoxia are
examples of intrinsic factors.

¾¾ Apoptotic signals must be connected to the actual death pathway by regulatory proteins.
¾¾ This step allows apoptotic signals to either culminate in cell death, or be aborted should the cell no longer need to die.
¾¾ Two main kinds of regulations are present – Mitochondrial & via adaptor proteins (FADD).

Mitochondrial Regulation
¾¾ Apoptotic proteins which target mitochondria affect
them in many ways –
(1) Cause mitochondrial swelling through the
formation of membrane pores, or
(2) Increase the permeability of the mitochondrial
membrane and cause apoptotic effectors to leak
out.
¾¾ Many pathways and signals lead to apoptosis, there is
only one mechanism which actually causes the death of
the cell in this process.
¾¾ After the appropriate stimulus has been received by the cell and the necessary controls exerted, a cell will undergo the
organized degradation of cellular organelles by activated proteolytic caspases.
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Microscopy
1. Cell shrinkage and rounding due to the breakdown of the proteinaceous cytoskeleton by caspases.
2. The cytoplasm appears dense, and the organelles appear tightly packed.
3. Chromatin undergoes condensation into compact patches against the nuclear envelope in a process known as
pyknosis, a hallmark of apoptosis.

4. The nuclear envelope becomes discontinuous and the DNA inside it is fragmented in a process referred to as
karyorrhexis. The nucleus breaks into several discrete chromatin bodies or nucleosomal units due to the degradation
of DNA.
5. The cell membrane shows irregular buds known as blebs.
6. The cell breaks apart into several vesicles called apoptotic bodies, which are then phagocytosed.

¾¾ Dying cells that undergo the final stages of apoptosis display phagocytotic molecules, such as phosphatidylserine,
on their cell surface.
¾¾ Phosphatidylserine is normally found on the cytosolic surface of the plasma membrane, but is redistributed during
apoptosis to the extracellular surface by a hypothetical protein known as scramblase.
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¾¾ These molecules mark the cell for phagocytosis by cells possessing the appropriate receptors, such as macrophages.
¾¾ Upon recognition, the phagocyte reorganizes its cytoskeleton for engulfment of the cell.
¾¾ Removal of dying cells by phagocytes occurs in an orderly manner without eliciting an inflammatory response.

2003
Magnetic Resonance Imaging (MRI)

Paul C Lauterbur Sir Peter Mansfield

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¾¾ Reflecting the fundamental importance and applicability of MRI in the medical field, Paul Lauterbur and Sir Peter
Mansfield were awarded the 2003 Nobel Prize in Medicine or Physiology for their “discoveries concerning magnetic
resonance imaging”.
¾¾ The 2003 Nobel Prize in Medicine award was vigorously protested by Raymond Vahan Damadian, who claimed that
he was the inventor of MRI.

¾¾ Magnetic resonance imaging (MRI), formerly referred to as magnetic resonance tomography (MRT) or, in chemistry
nuclear magnetic resonance (NMR), is a non-invasive method used to render images of the inside of an object.
¾¾ It is primarily used in medical imaging to demonstrate pathological or other physiological alterations of living tissues.

¾¾ MRI also has uses outside of the medical field, such as detecting rock permeability to hydrocarbons and as a non-
destructive testing method to characterize the quality of products such as produce and timber.
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¾¾ In its early years MRI was referred to as nuclear magnetic resonance imaging (NMRI); the word nuclear has been
associated with ionizing radiation exposure, which is not used in an MRI, so to prevent patients from making a
negative association between MRI and ionizing radiation, the word has been almost universally removed.

¾¾ Medical MRI most frequently relies on the relaxation properties of excited hydrogen nuclei in water and lipids.
¾¾ When the object to be imaged is placed in a powerful, uniform magnetic field, the spins of atomic nuclei with a
resulting nonzero spin have to arrange in a particular manner with the applied magnetic field according to quantum
mechanics.

Physics of MRI
¾¾ The magnetic dipole moment of the nuclei then precesses around the axial
field.
¾¾ While the proportion is nearly equal, slightly more are oriented at the low
energy angle.
¾¾ The frequency with which the dipole moments precess is called the Larmor
frequency.
¾¾ The tissue is then briefly exposed to pulses of electromagnetic energy (RF
pulses) in a plane perpendicular to the magnetic field, causing some of the
magnetically aligned hydrogen nuclei to assume a temporary non-aligned
high-energy state.
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¾¾ In other words, the steady-state equilibrium established in the static magnetic field becomes perturbed and the
population difference of the two energy levels is altered.
¾¾ The frequency of the pulses is governed by the Larmor equation to match the required energy difference between the
two spin states.
¾¾ Image formation
¾¾ Scanner construction and operation
¾¾ Contrast enhancement

Applications
¾¾ In clinical practice, MRI is used to distinguish pathologic tissue (such as a brain tumor) from normal tissue.
¾¾ One advantage of an MRI scan is that it is harmless to the patient.
¾¾ Uses strong magnetic fields and non-ionizing radiation in the radiofrequency range. Compare this to CT scans and
traditional X-rays which involve doses of ionizing radiation and may increase the risk of malignancy, especially in a
fetus.

¾¾ While CT provides good spatial resolution (the ability to distinguish two structures an arbitrarily small distance
from each other as separate), MRI provides comparable resolution with far better contrast resolution (the ability to
distinguish the differences between two arbitrarily similar but not identical tissues).
¾¾ Basis of this ability is the complex library of pulse sequences that the modern medical MRI scanner includes, each of
which is optimized to provide image contrast (based on the chemical sensitivity of MRI).
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Specialized MRI Scans

1. Diffusion MRI
2. Magnetic resonance angiography

3. Magnetic resonance spectroscopy

4. Functional MRI
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5. Interventional MRI
6. Radiation therapy simulation
7. Current density imaging
8. Magnetic resonance guided focused ultrasound
9. Multinuclear imaging

2004
Odorant Receptors and Olfactory System

Richard Axel Linda B Buck

Olfactory Receptors
¾¾ In 2004 Linda B. Buck and Richard Axel won the Nobel Prize in Physiology or Medicine for their work on olfactory
receptors.
¾¾ Olfactory receptors are a type of G protein-coupled receptor in olfactory receptor neurons.
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¾¾ In vertebrates, the olfactory receptors are located in the olfactory epithelium. _ In insects, olfactory receptors are
located on the antennae.
¾¾ Sperm cells also express odor receptors, which are thought to be involved in chemotaxis to find the egg cell.
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¾¾ Olfactory receptors bind to structures on odor molecules.

¾¾ Once the odorant has bound to the odor receptor, the receptor activates the attached G protein on the inside of the
olfactory receptor neuron.
¾¾ G protein in turn activates adenylate cyclase which converts ATP to cyclic AMP (cAMP).

¾¾ cAMP opens ion channels which allow sodium ions into the cell, depolarizing the olfactory receptor neuron and
beginning an action potential which carries the information to the brain.

¾¾ Wide range of odor receptors, with as many as 1000 in the mammalian genome (347 functional genes code for
olfactory receptors).
¾¾ Olfactory receptors may make up as much as 3% of the genome.
¾¾ Only a portion of these potential genes form functional odor receptors.

 
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¾¾ Many odors activate more than one type of odor receptor; hence limitless number of different molecules are involved.
¾¾ In 2006, it was shown that another class of odorant receptors exists for volatile amines.
¾¾ This class of receptors consists of the trace amine-associated receptors (TAAR) with the exception of TAAR1 which
is a receptor for thyronamines.

¾¾ Olfactory system is the sensory system used for olfaction.

¾¾ Accessory olfactory system senses pheromones.
¾¾ Olfactory system is often spoken of along with the gustatory system as the “chemosensory senses” because both
transduce chemical signals into perception.
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2005
Helicobacter Pylori and Gastritis, Peptic Ulcer

Barry J Marshall J Robin Warren

Helicobacter Pylori
¾¾ Helicobacter pylori - A bacterium that infects the mucus lining of the stomach and duodenum.
¾¾ Many cases of peptic ulcers, gastritis, and duodenitis are caused by H. pylori infection.
¾¾ Only known microorganism that can thrive in the highly acidic environment of the stomach.

¾¾ In 1994, NIH (USA) published an opinion stating that most recurrent gastric ulcers were caused by H. pylori, and
recommended that antibiotics be included in the treatment regimen.
¾¾ In 2005, Warren and Marshall were awarded the Nobel Prize in Medicine for their work on H. pylori.
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History
¾¾ 1875 – Spiral bacteria in stomach
¾¾ 1892 – Spiral bacteria in dogs’ stomach (Giulio Bizzozero)
¾¾ 1899 – Stomach washings (Prof. Jarowski, Published in Polish)
¾¾ 1979 – Study restarted by Marshall & Warren. Cultured from stomach with ulcer and gastritis.

¾¾ H. pylori is a spiral-shaped gram-negative bacterium.

¾¾ With its flagella and its helical shape, the bacterium drills into the mucus layer of the stomach, and then can be found
in a number of locations: in the mucus, attached to epithelial cells, or inside vacuoles in epithelial cells.
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¾¾ It produces adhesins which bind to membrane-associated lipids and carbohydrates and help its adhesion to epithelial
cells.
¾¾ It excretes the enzyme urease, which converts urea into ammonia and bicarbonate.

¾¾ The release of ammonia is beneficial to the bacterium since it partially neutralizes the very acidic environment of the
stomach (whose very purpose is to kill bacteria).
¾¾ Ammonia is, however, toxic to the epithelial cells, and with other products of H. pylori, including protease, catalase,
and phospholipases, causes damage to those cells.
¾¾ Under conditions of environmental stress, Helicobacter will convert from the spiral to a coccoid form.
¾¾ The coccoid form has also been found to be able to adhere to gastric epithelial cells in vitro.
¾¾ Coccoid form of the organism has not been cultured.

¾¾ Infection may be symptomatic or asymptomatic (without visible ill effects). It is estimated that up to 70% of infection
is asymptomatic.
¾¾ The bacteria have been isolated from feces, saliva and dental plaque of infected patients, which suggests gastro-oral
or fecal-oral as possible transmission routes.
¾¾ In patients who are asymptomatic, treatment is not usually recommended.
¾¾ In gastric ulcer patients where H. pylori is detected, normal procedure is eradication to allow the ulcer to heal.
¾¾ It is widely believed that in the absence of treatment, H. pylori infection—once established in its gastric niche—
persists for life.
¾¾ The Sydney gastroenterolgist Thomas Borody invented the first triple therapy in 1987.
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¾¾ Standard triple therapy is amoxicillin, clarithromycin and a proton pump inhibitor such as omeprazole.
¾¾ Unfortunately, an increasing number of infected individuals is found to harbour bacteria resistant to first-line
antibiotics.
¾¾ Gastric cancer and gastric MALT lymphoma (lymphoma of the mucosa-associated lymphoid tissue) have been
associated with H. pylori.
¾¾ In 1996, Martin J Blaser put forward the theory that H. pylori might also have a beneficial effect: by regulating the
acidity of the stomach contents, it lowers the impact of regurgitation of stomach acids into the esophagus.

¾¾ Two related mechanisms by which H. pylori could promote cancer are under investigation.
¾¾ One mechanism involves the enhanced production of free radicals near H. pylori and an increased rate of host cell
mutation.
¾¾ The other proposed mechanism has been called a “perigenetic pathway“- Enhancement of the transformed host cell
phenotype by means of alterations in cell proteins such as adhesion proteins (TNF a, IL-6).

Diagnosis
¾¾ Blood antibody or stool antigen tests.
¾¾ Carbon urea breath test (in which the patient drinks 14C- or 13C-labelled urea, which the bacterium metabolizes
producing labelled carbon dioxide that can be detected in the breath).
¾¾ Endoscopy to provide a biopsy sample for testing for the presence of urease “rapid urease test”.
¾¾ Histology.
¾¾ Microbial culture.
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2006
RNA Interference (RNAi)

Andrew Z Fire Craig C Mello

¾¾ Mechanism of gene regulation mediated by double stranded RNA.

¾¾ Post-transcriptional gene silencing involves an antisense RNA marking mRNA for degradation by ribonuclease.
¾¾ Involves RISC complex and Dicer; both of which are highly conserved molecules.

¾¾ Synthetic dsRNA can be incorporated into a system to produce RNAi.

¾¾ In 2006, Andrew Fire and Craig C Mello shared the Nobel Prize in Physiology or Medicine for their work on RNA
interference in the nematode worm C. elegans (Nature, 1998).
¾¾ Both endogenous and exogenous examples of RNAi are known.
¾¾ Known in many plant species.
¾¾ Known to protect against viruses and transposons.
¾¾ Heterochromatin formation and histone modifications may involve RNAi mechanisms.
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Applications
¾¾ Gene knockdown studies
¾¾ Direct therapeutic applications
• Macular degeneration
• Liver failure
• Anti-viral therapies (Cancer, influenza, hepatitis, measles, RSV)
• Neuro-degeneration (Huntington’s disease)
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¾¾ RNAi-based drugs – Under trials.

• “Off-target effect” is estimated at 10%.
• High death rate, due to “over-saturation” of dsRNA pathway.

History
¾¾ The discovery of RNAi was preceded by reports of unexpected outcomes in experiments performed by plant scientists
in the USA and the Netherlands.
¾¾ In an attempt to alter flower colors in petunias, researchers introduced additional copies of a gene encoding a key
enzyme for flower pigmentation into petunia plants.
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¾¾ Surprisingly, many of the petunia plants carrying additional copies of this gene did not show the expected deep purple
or deep red flowers but carried fully or partially white flowers.
¾¾ Both types of genes, the endogenous and the newly introduced transgenes, had been turned off !!!

¾¾ _ Evidence was obtained for posttranscriptional inhibition of gene expression that involved an increased rate of
mRNA degradation.
¾¾ This phenomenon was called co-suppression of gene expression, but the molecular mechanism remained unknown.
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¾¾ A few years later plant virologists, in their research aimed towards improvement of resistance of plants against plant
viruses made a similar observation.
¾¾ At that time it was known that plants expressing virus-specific proteins show enhanced tolerance or even resistance
against virus infection.

¾¾ However, they also made the surprising observation that plants carrying only short regions of viral RNA sequences
not coding for any viral protein showed the same effect.
¾¾ They concluded that viral RNA produced by transgenes can also attack incoming viruses and stop them from
multiplying and spreading throughout the plant.
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¾¾ They did the reverse experiment and put short pieces of plant gene sequences into plant viruses. Indeed, after infection
of plants with these modified viruses the expression of the targeted plant gene was suppressed.
¾¾ They called this phenomenon “virus-induced gene silencing” or simply “VIGS”.

Mechanism
¾¾ Dicer, ribonuclease protein, binds and cleaves dsRNA to 20-25 nt ds fragments, called siRNA.
¾¾ siRNA are converted to single strands and incorporated into active RISC complex.
¾¾ RISC brings about active cleavage of mRNA.

Future…
¾¾ Genomics, proteomics, systeomics and other –omics
¾¾ Better diagnostic agents
¾¾ Single molecule inhibitors
¾¾ Targeted therapy, gene therapy
¾¾ Individualized therapy- SNP-based
¾¾ Stem cell applications