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Coronary Artery Disease and Diabetes Mellitus

Introduction The coronary artery supplies the blood to the heart muscles to enrich it with oxygen and other nutrients. It also carries deoxygenated blood away from the heart. The coronary artery consists of 2 main arteries; the right coronary artery which supplies blood to the right ventricles and right atrium and the left coronary artery which supplies blood to the left ventricles and left atrium. The two many arteries further divide into two; the left coronary artery divide into the circumflex artery which supplies blood to the back of the heart, the left anterior descending artery supplies blood to the front of the heart; the right coronary artery is divided into right posterior descending artery and large marginal arteries and supply blood to the sinoatrial nodes that control the heart rhythmic rate. The coronary arteries have 3 layers of tissues; the tunica adventitia which covers the outside, the tunica media which is the middle layer and the tunica intima endothelium which is the inner layer. The diameters of the coronary arteries range from 0.6mm-4.4mm, any blockage to any of these arteries that stops blood flow to the affected area lead to coronary artery disease (CAD).
The normal blood glucose range is 4-6 mmol/L and 7.8mmol/L 2 hours after meal. This range is controlled by insulin which causes cells to absorb excess glucose in blood and glucagon which causes cells to release glucose from stores. Insulin is produced by the beta cells of the islets of Langerhans of the pancreas. When blood glucose level rises above its normal range insulin binds to the extracellular subunits of its receptors (IRS-1 and IRS-2) on the cell surface which sends signals into the cell causing the intracellular proteins to alter their activity which in turn initiates the movement of glucose transporters (GLU1-4, depending on the cell/tissues involved) to the cell membrane which then transports glucose into the cell where it may be further be converted glycogen, the storage form of glucose. Any impairment to the function of insulin, or its receptors lead to hyperglycemia and when excess of this glucose in bloodstream is passed in urine it results to diabetes mellitus. Diabetes mellitus can be classified into two main types; Diabetes mellitus type 1 (DM 1) and diabetes mellitus type 2 (DM 2). High sugar levels in blood (hyperglycemia) maybe due to insulin resistance in cases of type 2 diabetes or destruction of beta cells of the pancreas in cases of type 1 diabetes, which downstream leads to CAD.
Atherosclerosis which can occur in any part of the body result from endothelia damage which can be caused by high blood pressure, smoking, genetics, age, gender, high blood glucose, weight gain etc. when atherosclerosis occurs in any of the coronary arteries it leads to coronary artery disease. Events leading to atherosclerosis include; Endothelial damage which leads to inflammatory responses such as accumulation of white blood cells , low density lipoprotein (LDL)and high density lipoprotein (HDL), oxidation of LDL induced by free radicals (reactive oxygen species), platelet aggregation, chemotaxis of macrophages, formation of foam cells, proliferation of smooth muscle cells (atheroma occurs), fibrous tissue and calcium salts cause the atheroma to harden this results in less elasticity of the artery (atherosclerosis). All of these events narrow the coronary artery from the normal physiological range of 0.6-4.4mm (including small coronary arteries branching from the main arteries) to very smaller diameter depending on the level of narrowing and then eventual blockage preventing or limiting blood and nutrient supply to heart tissues leading to death of affected heart tissues, heart attack or even death of the patient.
According to statistics, diabetes and coronary artery disease are closely related, this is because 50% of patients with diabetes are at risk of suffering CAD alongside. In the United States 77% cause of death is diabetic CAD. The prevalence of diabetes globally is increasing and it’s the major risk factor of other health conditions. The National Institute of Health reported that 65% of diabetic patients are more at risk of developing stroke, high blood pressure, obesity, kidney failure, and heart diseases such as cardiac arrest, myocardial infarction, and atherosclerosis in the heart (CAD) which when not optimally managed could lead to death.
CAD and Diabetes Mellitus The cause of DM1 is unknown but studies say it could be genetic or viral infection which leads to an autoimmune condition where the body defense mechanism destroys its cells, in this case the pancreatic beta cells where insulin is produced. When the beta cells of the pancreas are destroyed, the pancreas will no longer be able to make insulin which downstream causes the bloodstream to be glucose logged due to inability of the body cells to move glucose out of the bloodstream, leading to impaired insulin secretion, decreased signalling in the hypothalamus, increased food intake, weight gain and hyperglycemia, which downstream leads to atherosclerosis.
DM 2 is due to the body cells’ inability to respond to insulin stimulation. Insulin resistance is due to obesity, age and sedentary life style (irregular body activities), Age and sedentary lifestyle both lead up to increase in body weight (accumulation of adipose tissue). With or without hyperglycemia, insulin resistance can cause atherosclerosis, this results from increased lipolysis of adipocytes leading to increased nonesterified fatty acid secretion (NEFA), pro-inflammatory cytokines such as tumour
P1521598x necrosis factor-a (TNF-a), interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1). NEFA can be deposited in and cause dysfunction of pancreatic beta cells, liver and skeletal muscles, all of which enhance insulin resistance and reduce production of insulin. Accumulation of NEFA in skeletal muscle leads to competition with glucose for substrate oxidation thereby increasing the intracellular content of fatty acid metabolites such as diacylglycerol (DAG), fatty acyl coenzyme A and ceremide which together activate serine/threonine kinase processes leading to insulin receptor substrate 1and 2 (IRS 1

Gene chlR in Streptomyces Venezuelae

PCR amplification and overexpression of the positive regulatory gene chlR in Streptomyces venezuelae
J. L. CLAYTON – BROWN
Introduction The polymerase chain reaction (PCR) is a technique used in the amplification of DNA which utilises thermal stable polymerase, Thermus aquaticus (Taq) and primers which aid in the annealing of the chosen DNA strand, producing numerous replications through a cycle of appropriate temperature changes (Lorenz, 2012). Developed in 1983 by Dr. Kary Banks Mullis, PCRs ability to quantitate transcription levels of specific genes has revolutionised research and the understanding of gene function (Bustin, 2000) in its many applications, including the ability to: detect DNA polymorphs and point mutations (Orita et al., 1989), amplify specific genes for the construction of overexpression vectors (Liang et al., 2015), and recognising bacterial (Hill, 1996) and viral (Holodniy, 1994) pathogens. Advances within PCR have only broadened the spectrum of its implementations, with new techniques such as Quantative PCR, and Inverse PCR yielding new insights into once misunderstood areas of molecular biology (Jain and Varadarajan, 2013).
ChlR is a cluster-associated transcriptional activator consisting of 987 base pairs within the putative CHL biosynthetic operon, predicated to encode the only positive regulator responsible for the initiation of production of chloramphenicol (CHL) (Fernández-Martínez, et al., 2014); with the usance of PCRs cloning capacity, it is predicted that the introduction of a plasmid capable of overexpression of the chlR gene will result in amplified activity of the CHL biosynthetic gene cluster.
Method DNA amplification by means of PCR often requires a high fidelity taq polymerase within the PCR mixture to minimise mutations (McInerney et al., 2014). The chlR DNA fragment was inserted into the vector pIJ10257 [1] prior to PCR. The final reaction mixture consisted of the following: 10μl 5X Colorless GoTaq® Reaction Buffer, 2μl PCR Nucleotide Mix (10mM each dNTP), 5μl Apra_BamHI_F primer, 5μl Apra_BamHI_R primer, 2μl purified chlR chromosomal DNA, 5μl Dimethyl sulfoxide (D MSO), 20μl Nuclease-Free water, and 1μl GoTaq® G2 DNA Polymerase, equating to a total reaction volume of 50μl within a sterile, nuclease-free PCR tube, labelled as group 2.
The appropriate annealing temperature was set accordingly with the melting temperature (Tm) of the hybridising portion of the primer. The extension temperature was calculated upon the approximation of 1 minute per every 1kb of required amplified DNA. The resulting thermal cycle was applied as such: an initial cycle at 95°C for 5 min (denaturation), 95°C for 30 secs (secondary denaturation), 56°C for 30 secs (annealing), 72°C for 90 secs (extension), repeated for 30 cycles from secondary denaturation. The final extension temperature was 72°C for a period of 5 mins (holding temperature 10°C). Gel electrophoresis (GE) was preformed using a 0.8% polysaccharide agarose gel within a Tris/Borate/EDTA (TBE) buffer and inserted into the 2nd column.
Results Gels were removed from the gel box and inspected underneath a UV light. No band of DNA was visible within column 2; the molecular weight ladder and chromosomal DNA within columns 3, 13, and 16 from other accompanying PCR (run simultaneously under the same conditions previously described) were observable. When compared to the molecular ladder, successful DNA fragments indicated a base pair (bp) length of approximately 1000bp, in correlation with the 987bp of the chlR regulatory sequence, an indication that the inserted DNA is present.

Fig.1 0.08% agarose gel exhibiting the DNA ladder and S. veneuzlae chromosomal DNA within column 3, 13, and 16. Column 3 shows an excessive quanity of DNA, an indication overproportionate amount of template DNA were added during procedure. The absence of DNA within column 2 (indiciated in red) evinces the failed PCR described in this paper.
Discussion The absence of DNA within column 2 demonstrated the failure to obtain a PCR product. As each component was correctly incorporated, other aspects must be adjusted to result in an adequate amount of DNA cloning. There are several alterations implementable to increase the likelihood of success within the reaction, firstly being the redesign of appropriate primers as the most crucial component for successful amplification of the reaction (Dieffenbach et al., 1993); analysis based software for enhancing the specificity of the primers without compromising their sensitivity can be implemented, with programs such as Primer3 and QuantPrime offering the possibilities of designing internal oligonucleotides alongside primer pairs, and the optimisation of these primer pair designs enabling specificity evaluation, respectively (Noguera et al., 2014).
If the primers present correctly, changes to the temperature cycle should next be ensured. A decrease in the annealing temperature has previously shown to reduce the risk of unspecific binding and preferential amplification (Sipos et al., 2007). A final modification to the protocol is to adjust the number of PCR cycles, as this change can influence aspects of the reaction; a low PCR cycle number may provide accurate estimation of bacterial richness and a decrease of PCR errors (Ahn et al., 2012), whilst an increase in cycles can improve fluorescent intensity of some dyes (SYBR® Green I) (Ramakers et al., 2003).
Electroporation is a common method of transformation concerning plasmids, involving a brief high-voltage pulse which renders the membrane pores to transiently open and allow the subsequent uptake of DNA into the host cell (Pigac and Schrempf, 1995); an associated example is an electrotransformed Escherichia coli bacteriumwith a cloned, overexpressed chlR gene.
In order to clarify correct insertion and amplification of the correct sequence, the DNA must be sequenced. The most common method of DNA sequencing for cloned PCR products is the Sanger sequence, which technique lies in the use of chain-terminating nucleotides (Sanger, et al., 1977). Once clarified, the replicative vector can then be transferred to S. venezuelae via coagulation from the E. coli, transferrable due to the origin of transfer (oriT) within the vector (Mazodier et al., 1989).
It Is expected that an overexpression of the chlR gene would result in elevated levels of the encoded transcription factor protein, initiating increased transcription of the CHL biosynthetic cluster and producing a higher chloramphenicol yield; this would be observable through analysation via High Performance Liquid Chromatography, a sensitive method appropriate for gene expression analysis (Sivakumaran, et al., 2003).Recent research [2] has strongly indicated that the constitutive expression of chlR effects the overall expression of the speculated, proceeding genes within the cluster, confirming chlR’s role as a transcriptional activator (Fernández-Martínez et al., 2014).

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