Introduction

In this report the two distinct experiments will be performed. Experiment 1 is the simulation of
drug effects on the guinea pig ileum and for experiment 2 the separation of proteins in the human
leukemic cell homogenates using 1D SDS-PAGE gel is performed.
Experiment 1

The guinea pig ileum is a

classic pharmacological
preparation and is widely used
in practical classes
for several reasons. Enough
ileum can be isolated from one
guinea pig to supply an entire
practical
class and the pieces of ileum
are robust enough to
withstand handling by
students with little
experience of organ bath
experimentation. Most
importantly, it is primarily
composed of
extensively innervated smooth
muscle, and so can be used
to study many different
aspects of
smooth muscle and enteric
nervous system function, as
well as investigating how drugs
interact
with these systems.
In this experiment we will
investigate the effects of
acetylcholine (ACh) and
histamine on the
ileum, and then determine how
these effects change in the
presence of antagonists of
ACh and
histamine receptors. ACh is
the main neurotransmitter of
the parasympathetic nervous
system,
which innervates the gut, so
it is reasonable to expect that
ACh will mimic the effects
of
parasympathetic nerve
activation (i.e. act as a
parasympathomimetic) and
cause ileum
contraction. Histamine is a local
hormone that plays an important
role in inflammation. It is
usually
released by mast cells and
contracts most non-vascular
smooth muscle preparations,
although its
effectiveness varies
considerably between different
muscles and in different
species. This suggests
that histamine is also likely to
contract the ileum.
We expect that the actions of
ACh on the ileum will be
mediated by activation of ACh
receptors, in
this case postsynaptic
muscarinic receptors on the
smooth muscle cells. Similarly,
we expect that
histamine also activates specific
histamine receptors on the
smooth muscle to cause
contraction.
If this is the case, then a
muscarinic antagonist (e.g.
atropine) would be expected to
inhibit ACh-
induced contractions without
affecting histamine
contractions, and a histamine
antagonist (e.g.
mepyramine) would be
expected to inhibit histamine-
induced contractions without
affecting ACh
contractions.
The ileum of a guinea pig is a well-known pharmacological preparation. It is made up of smooth
muscle that is well-innervated. Many different facets of smooth muscle and enteric nervous
system activity can be explored with it. In this experiment, we'll look at how the guinea-pig
ileum responds to two agonists, carbachol and Atropine, at three different doses on the ileum,
and then see how these responses change in the presence of receptor antagonists. Atropine is a
competitive reversible muscarinic antagonist of ACH as well as a parasympathetic nerve
stimulation antagonist, which means that sympatholytic nerve stimulation is prevented. Atropine
has a variety of effects when it functions on a variety of muscle forms, including competitively
binding to M3 receptors, which prevents ACH from binding. While atropine binding is poor and
reversible, it causes smooth muscle contractile responses to be diminished, allowing the smooth
muscle to relax instead. Carbachol is a complete agonist (direct acting acetylcholine analogue)
that activates muscarinic receptors to cause contractions. z has a longer period of action than
acetylcholine because it is resistant to cholinesterase hydrolysis. This makes it more capable of
increasing tone and contractile function in the intestine than acetylcholine (Johnson, 2002). This
material can cause tachycardia, secretion inhibition, and smooth muscle relaxation. Atropine's
activities on the ileum are thought to be regulated by acetylcholine receptor activation,
specifically postsynaptic muscarinic receptors on smooth muscle cells. If this is the case, ACh-
induced contractions can be blocked by a muscarinic antagonist (e.g., atropine).
The goal of this experiment was to measure contractile reaction in the guinea pig ileum in
response to various agonists in different circumstances, such as in contrast to another agonist,
regarding a non-competitive antagonist, and concerning to a competitive antagonist, as well as
assessing the strength of the established agonists and antagonists.

Materials and Methods

The tissue sample for the organ bath experiment in this laboratory was guinea pig ileum. A
laboratory technician separated the tissue, and pieces were suspended in an organ wash. To keep
the tissue alive until it was suspended in the organ wash, it was reserved in a petri dish which
contains Krebs solution in it. After that, the tissue was suspended in the organ pan. The tissue
was equilibrated for 30 minutes before the procedure began. The organ bath was drained and
refilled (washing the tissue) per 5-minute interval, and the resting stress on the ileum was set to
0.5 g. The LabChart reader was already starting when the equilibration began, and it was not
halted until all of the tests were finished. We used a force transducer to bind LabChart to the
organ bath, which enabled us to record any force produced by the tissue.
Reagents are compounds that are used to make chemicals.
Bio-Rad (Hercules, CA, USA) provided the protein molecular mass marker for SDS-PAGE
Precision Plus Dual Color, as well as the SDS molecular weight (MW) Scale Standard for SDS-
capillary gel electrophoresis (CGE). Acrylamide (30%), 1.5 M Tris HCL Amonium per sulphate
(25%), and acetic acid (10%) were used in the gel preparation.

Results
Experiments on laboratory animals are fundamental to pharmacology as a discipline. For the
biological assay, mice, guinea pigs, rabbits, and other laboratory animals are used. The
laboratory animal species must be slaughtered only for a piece of tissue in order to instruct using
isolated strip preparations from different organs. The aim of this thesis was to use a simulation
model for pharmacological experiments that would replicate the real laboratory conditions
without putting experimental animals at risk. As seen in figure 1, the dose reaction curves of
carbachol alone and in the presence of various concentrations of atropine were plotted and their
EC50 values were compared.

Dose Response
120

100

80

60

40

20

0
-9 -8 -7 -6 -5 -4 -3 -2

Control
Atropine 2E -8 atropine 2E-7
atroine 2E-6

Figure 1. The dose response graph of three different concentrations of atropine and control.
Based on the Schild plot, the consequence of atropine in terms of form of antagonism was
projected, and pA2 values were obtained. The findings indicated that atropine provided a dose-
dependent increase in response to an increase in atropine concentration, but that the increase in
dose response was very minor. In the case of higher concentrations, the dose response measured
is 60 mM, although it stays similar to 63 nM. Atropine the concentration response curve shifted
toward right with a change in EC50 value. The value calculated for 100nM atropine is 7.9 which
is almost similar to the previous values. Schild plots indicated that antagonism formed by
atropine was regarded as competitive in nature as shown in figure 2. The pA2 values of atropine
were found significantly high.

Figure 2. Schild plots for three different conc of atropine.

Discussion
The aim of this study was to create a pharmacological testing simulation that would replicate the
real laboratory conditions without compromising the experimental animals. The lab was planned
to create a concentration reaction curve of cabochal separately and also in the presence of
varying concentrations of atropine to determine the suitability of pig ileum. The same technique
was used to determine the EC50 and pA2 values as well as the influence of atropine on the
concentration response curve of atropine in pig ileum. In the case of pig ileum, atropine given
alone in the tissue bath had an EC50 value that was virtually equivalent to that of rat ileum. In a
related way, atropine counteracted carbochal's effect on isolated muscle preparation.
However, the pattern of carbochal reaction and essence of atropine antagonism is close to that of
rat ileum preparation.
These observations indicate that isolated pig ileum and preparations should be used in routine
Pharmacology topic studies. The preparation can also be used to perform tests on isolated ileum,
such as determining the pA2 value, three-point/four-point bioassays, determining the effect of an
antagonist on a drug's DRC, and comparing the actions of various agonists such as acetylcholine
and barium chloride.

Hypotheses
In this experiment we will test
two hypotheses:
1. ACh and histamine will
cause a concentration-
dependent contraction of the
guinea pig
ileum sections
2. The muscarinic antagonist
atropine will inhibit
contractions to ACh but not
histamine,
whereas the histamine
antagonist mepyramine will
inhibit contractions to
histamine but
not ACh.
Experiment 2
Leukemia, especially acute myeloid leukemia, is the most prevalent cancers in the world (AML).
The unchecked development of dysfunctional white blood cells in the bone marrow causes AML,
which prevents the formation of regular blood cells (Lindsley et al., 2015). Chemotherapeutic
agents are used in most modern treatment plans to destroy cancerous cells, but they have a host
of serious toxic side effects (Bahar et al., 2013). As a result, the quest for novel anti-leukemia
drugs has intensified, and natural products alone or with mixtures have emerged as a valuable
source of information due to their varied functions (Buchi et al., 2011). Oxylipins, which are
oxidized metabolites of long-chain polyunsaturated fatty acids, play essential roles in cell
signaling and defense in animals and plants as they are attacked by microbes or pathogens
(Mosblech, Feussner and Heilmann, 2009). The anti-cancer effects seem to be mediated by a
variety of cellular and molecular pathways. Furthermore, using a 1D-gel dependent proteomics
technique, proteins from leukemia cells were analyzed, and differentially expressed proteins
were detected using mass spectrometry. (Li et al., 2016). Human leukemic cells are used in this
experiment for separation of protein, including control cells (that are untreated( and treated cells
with 10 uM SAHA and 10uM Panobinostat.
Material and Methods
Polyacrylamide is used to make polyacrylamide gels, as it is evident from its name. This type of
gel uses bis-acrylamide as its ingredient because it forms a polymer and also solidifies rapidly at
room temperature when it is mixed with reagents and buffers. Furthermore, it can be beneficial
for its efficient the gel's sieving properties which can easily varied by adding the different
concentration of acrylamide. This gel is formed with two layers, a stacking and resolving layer.
The resolving layer is the part of get where the bands separate from one another on the gel.
Resolving layer separates the polypeptides by creating a resistance in DNA movement due to
higher acrylamide concentration. As compare to resolving layer the stacking layers has less
concentration of acrylamide and thus facilitates protein stacking. In the casting tray where gel is
hardening we poured the resolving gel first between two slabs of glass.
The alcohol is cleaned out repeatedly with water until the resolving coating has polymerized, and
the piling gel is transferred on top of the resolving gel. The gel will incorporate protein samples
if the stacking layer has polymerized. Users may also order precast gels, which ensure that the
outcomes of the tests are consistent.
Buffers are important in gel making. There are several buffers available for SDS-PAGE which
are necessary to add conductivity properties during electrophoresis. It is known that SDS-PAGE
generally has a higher number of buffer structures than DNA gel electrophoresis.
For this experiment we use Tris-HCl in the resolving layer, and add another buffer Tris-Glycine
in the tank. Glycine may have two different charge states in this form. During electrophoresis,
positively charged glycine in the tank buffer enters the stacking gel and takes on a neutral
charge, eventually flowing across the polar field created by the electrodes.
The chlorine in the gel buffer, on the other hand, migrates much faster than the glycine and
forms a negative charge front in front of it. These opposing fronts bookend the protein samples
as they approach the stacking/resolving layer interface, pinching them into a thin line.
By following the standard procedure mentioned above 1 D gel was prepared to separate the
protein in AML.

Results
Many experiments have looked for AML-specific protein signatures by associating the
proteomes of AML and normal healthy samples. Kwak et al. found eight proteins that were
expressed uniquely in 12 AML patients and 12 healthy subjects. When the proteomes of AML
and normal white blood cells were compared, 31 proteins with significantly altered expression
were discovered.
S100 proteins have been shown to be biomarkers for disease development and prognosis in a
variety of cancers. Acute myeloid leukemia (AML) is a condition in which immature
myeloblasts replace normal hematopoietic cells in the bone marrow. It is an extremely
heterogeneous and violent disease. Using spectrometry, we were able to identify smear at several
sites. Any of these could be oncogene products, while others could play a role in cell cycle
modulation and signal transduction. But a clear band in case of treatment were shown in figure 3.
From the 1 D gel electrophoresis technique two visible bands in lane 3 and lane 4 can be seen
near 14 kDa. This protein can be one of the markers for protein.

Figure 3. SDS-page gel electrophoresis of human leukemic cells.

(left to right) Lane 1: Protein molecular weight marker (~10 to ~170 kDa (image in above
page) Lane2: Control cells Lane 3: 10uM SAHA treated cells Lane 4: 10uM Panobinostat cell

Discussion
Proteomics is a post-genomic research area whose aim is to explain the distribution and roles of
all proteins in cells, tissues, and animals. Proteomic analysis helps one to have a deeper
understanding of important processes. (González-Díaz et al., 2008)(Hanash et al., 2002). In
proteomics, there are three main technologies to consider: 1-DE, bioinformatics, and mass
spectrometric research (Wittmann-Liebold, Graack and Pohl, 2006). As a result, the analytical
process is normally divided into three steps: first, 1-DE isolation of proteins from samples;
second, mass spectrometry labeling of the extracted proteins; and third, bioinformatic research
storage, handling, and comparing of data relating to proteins.
In IEF, the size of the protein sample has a significant impact on the 1-DE spectrum resolution
and reproducibility. (Oh-Ishi and Maeda, 2007; Wiśniewski et al., 2009). Some of the protein
spots appeared undefined or missing when the sample size was too small, while some of the
spots appeared agglutinated or sedimented when the sample size was too high. We obtained high
resolution, reproducible 1-DE maps of the leukemia cells and successfully detected one
differentially expressed protein spot using these protein sample processing, 1-DE, gel staining,
screening, and image analysis techniques, as well as a series of other systematic operations,
establishing a research platform for the leukemia cell proteome. Furthermore, this experiment not
only provides valuable evidence for further study and the development of a similar protein
expression database, but it also establishes a foundation for future research.

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