Association of ATP-binding cassette transporter A1 [ABCA1]-565 C/T gene polymorphism with hypoalphalipoproteinemia and serum lipids, IL-6 and CRP levels

Background: ATP-binding cassette transporter A1 [ABCA1] is a membrane integral protein which plays a vital role in High Density Lipoprotein [HDL] metabolism and exerts a protective effect against Hypoalphalipoproteinemia [HA] by mediation of rate-limiting step in HDL biogenesis. In addition, this protein possesses anti-inflammatory effects by inhibiting the production of some inflammatory cytokines in macrophages. This study investigated the association of ABCA1-565 C/T gene polymorphism with HA and serum lipids, IL-6 and CRP levels

Methods: A population which consisted of 101 HA and 95 normal subjects were genotyped for ABCA1-565C/T polymorphism by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism [PCR-RFLP]. The serum concentrations of lipids, IL-6 and high sensitive-CRP [hs-CRP] were measured by the relevant methods

Results: The frequency of T allele was significantly higher in the HA group than the controls [31.7 vs. 19.5%, p=0.002]. Thus, carriers of the T allele [CT and TT genotypes] had a higher risk for HA [p=0.016, OR=2.04, 95% CI=1.14-3.63]. T allele carriers demonstrated decreased HDL-C and increased triglyceride, IL-6 and CRP levels than those with the CC genotype

Conclusion: This study suggests that the-565 C/T polymorphism of ABCA1 gene is associated with an increased risk of HA, decreased HDL-C and increased TG, IL-6 and CRP

Cloning and Optimization of Soluble Vascular Endothelial Growth Factor [165] Expression in Escherichia coli

Background: Vascular Endothelial Growth Factor [VEGF] is a coordinate regulator of physiological angiogenesis during embryogenesis, skeletal growth and reproductive functions. There are several types of VEGF, including VEGF [165] . VEGFs stimulate endothelial cell growth, angiogenesis, and capillary permeability. Low induction temperature is a major factor for expression of the recombinant VEGF [165] in soluble form. The purpose of this study was cloning and optimization of soluble vascular endothelial growth factor [165] expression in Escherichia coli [E. coli]

Methods: In this study, total RNA of HeLa cell [cervix epithelium] was extracted. The VEGF [165] gene was amplified by reverse transcription polymerase chain reaction [RTPCR], and then VEGF [165] was subcloned into prokaryotic expression vectors pET 32a[+] and transformed into BL21 [DE3] E. coli strain. VEGF [165] expression was optimized by fine adjustments such as induction time and incubation temperature. VEGF [165] was analyzed by DNA sequencing prior to expression and the protein was further characterized by SDS-PAGE and immunoblotting using Hisotag specific polyclonal antibody

Results: Our results demonstrated that VEGF [165] was successfully cloned and expressed in pET-32a[+] vector. Optimization of the expression procedure showed that, induction by 1 mM IPTG at OD600=0.7 and overnight incubation at 22 degree C resulted in the highest expression levels of soluble VEGF [165]

Conclusion: In this study, the expression of VEGF [165] in a high soluble level was successfully cloned and optimized

Development of an immunoaffinity method for purification of streptokinase

Streptokinase is a potent activator of plasminogen to plasmin, the enzyme that can solubilize the fibrin network in blood clots. Streptokinase is currently used in clinical medicine as a thrombolytic agent. It is naturally secreted by beta-hemolytic streptococci. To reach an efficient method of purification, an immunoaffinity chromatography method was developed that could purify the streptokinase in a single step with high yield. At the first stage, a CNBr-Ac-tivated sepharose 4B-Lysine column was made to purify the human blood plasminogen. The purified plasminogen was utilized to construct a column that could purify the streptokinase. The rabbit was immunized with the purified streptokinase and the anti-streptokinase [IgG] purified on another streptokinase substituted sepharose-4B column. The immunoaffinity column was developed by coupling the purified anti-Streptokinase [IgG] to sepharose 6MB-Protein A. The Escherichia coli [E.coli] BL21 [DE3] pLysS strain was transformed by the recombinant construct [cloned streptokinase gene in pGEX-4T-2 vector] and gene expression was induced by IPTG. The expressed protein was purified by immunoaffinity chromatography in a single step. The immunoaffinity column could purify the recombinant fusion GST-SK to homogeneity. The purity of streptokinase was confirmed by SDS-PAGE as a single band of about 71 kD and its biological activity determined in a specific streptokinase assay. The yield of the purification was about 94%. This method of streptokinase purification is superior to the previous conventional methods.

C-reactive protein level and pregnancy rate in patients undergoing IVF/ICSI

C-reactive protein [CRP] can be increased after hormonal stimulations. The changes of CRP might affect the success of in-vitro fertilization [IVF]. The aim of this study was to determine the possible relationship between the serum CRP level and outcome of controlled ovarian stimulation, and pregnancy rate in patients undergoing IVF or intra cytoplasmic sperm injection [ICSI]. This prospective cross sectional study was performed in Avicenna Infertility Clinic on 70 consecutive infertile patients [Jan 2008-Aug 2009] who were candidate for IVF/ICSI, using standard long GnRH agonist protocol. Blood was drawn 4 times during the cycle, on first day of stimulation, the day of HCG injection, the day of ovum pick up, and the day of embryo transfer. In 82.2% of cases, the serum CRP level was higher in day of HCG injection than first day of stimulation and also the day of ovum pick up the day of HCG injection. The ratio of CRP level in the day of transfer to the day of ovum pick up, was significantly higher [ratio >/= 1.23] in patients who became pregnant after ICSI [p=0001]. All patients with less than this Ratio have not been pregnant. Controlled ovarian hyper stimulation and puncture of ovaries can potentiate systemic stimulation. Increasing serum CRP level in day of embryo transfer rather than ovum pick up can predict the success in patients undergoing IVF/ICSI

Production and purification of streptokinase by protected affinity chromatography

Streptokinase is an extracellular protein, extracted from certain strains of beta hemolytic streptococcus. It is a non-protease plasminogen activator that activates plasminogen to plasmin, the enzyme that degrades fibrin cloth through its specific lysine binding site; it is used therefore as a drug in thrombolytic therapy. The rate of bacterial growth and streptokinase production was studied in condition of excess glucose addition to culture media and its pH maintenance. The Streptokinase product of the bacterial culture was preliminary extracted by salt precipitation and then purified by affinity chromatography on plasminogen substituted sepharose-4B in a condition that the plasminogen active site was protected from streptokinase-induced activation. The purity of streptokinase was confirmed by SDS-PAGE and its biological activity determined in a specific Streptokinase assay. The results showed that in the fed-batch culture, the rate of Streptokinase production increased over two times as compared with the batch culture while at the same time, shortening the Streptokinase purification to a single step increased the yield over 95% at the chromatography stage