Abstract

        Background and Objective: Hirudin is an anticoagulant polypeptide secreted from the salivary glands of leeches. Recombinant hirudin is a strong anticoagulant agent in arterial and venous thrombosis. The aim of this study was to evaluate the effect of inserting protein A signal peptide sequence of pEZZ18 plasmid on expression and secretion of the recombinant hirudin in E.coli.

       Methods: the synthetic hirudin gene was amplified by PCR using specific primers. First, the gene was purified and cloned into PTG19-T cloning vector, and then it was subcloned into pEZZ18 expression vector by SalI / SacI enzymatic digestion and finally transformed into E.coli JM107. After the expression of recombinant hirudin protein, different cellular fractions were isolated and analyzed on SDS-PAGE and further confirmed by Western blotting.

         Results: PCR product (522 bp) was first subcloned into the T-Vector (replicating vector) and then successfully subcloned into the pEZZ18 (expression vector). Cloning and subclonig were confirmed by enzymatic digestion and Colony PCR. After the expression and isolation of fractions, the presence of hirudin (about 29 kDa) in different cell fractions due to the effects of signal peptide was observed in SDS-PAGE and finally confirmed by Western blotting.

       Conclusion: The gene of anticoagulant hirudin protein (desirudin) was cloned into the pEZZ18 vector containing Protein A signal peptide sequence and later transformed into E.coli JM107. The recombinant hirudin protein expression in the extracellular space was approved.

        Keywords: Hirudin; Desirudin; Protein Sorting Signals.

ABSTRACT
             Background and Objectives: Apolipoprotein O (apoO) is a 198 amino acids protein that exists predominantly in high-density lipoprotein (HDL). It may exert cardioprotective effects via decreasing fat accumulation and increasing removal of cholesterol from macrophages. Although the health benefits of exercise are well documented, no study has yet investigated the effects of various types of training, including resistance training on apoO level. Therefore, we aimed to determine effects of five weeks of circuit resistance training with different intensities on plasma levels of apoO, HDL, total cholesterol (TC) and triglyceride (TG) in young untrained men.
             Methods: Forty-five age- and weight-matched healthy untrained men were randomly assigned to a control group (n=10) and four training groups: training at 20% intensity (n=9), training at 40% intensity (n=8), training at 60% intensity (n=7) and training at 80% intensity (n=8). The subjects performed circuit resistance training at 10 stations (30 seconds at each station) with three repetitions, without rest between stations and with 3-minute active rest between the repetitions. The training was carried out for 45 minutes per session, three sessions a week, for five weeks. Venous blood samples were taken 48 hours before the first exercise session and 48 hours after the last training session. Plasma levels of apoO, HDL, TC and TG were measured using commercial kits. Data were analyzed using repeated measures ANOVA and Tukey's post hoc test at significance level of 0.05.
             Results: After the training intervention, mean plasma level of TC and TG did not differ significantly between the study groups (P>0.05). Training at 80% of one-repetition maximum (1RM) caused a slight decrease in the apoO concentrations. Moreover, apoO concentration was significantly higher in the 20% 1RM training group compared to other study groups (F=11.599, P<0.002).
             Conclusion: Our results indicate that circuit resistance training at 80% of 1RM can decrease HDL-associated apoO level but does not significantly alter other parameters.
             KEYWORDS: Circuit resistance training, HDL-O, TC, TG, Young men.

ABSTRACT
            Background and Objectives: Coronary artery disease (CAD) is the leading cause of death worldwide. It is well established that low level of high-density lipoprotein-cholesterol (HDL-C) is a strong and independent risk factor for CAD. Apolipoprotein M (apoM) is a component of HDL, which is involved in pre-β-HDL formation and cholesterol efflux to HDL. It is believed that resistance and aerobic exercise can significantly reduce risk of cardiovascular disease, especially by increasing serum levels of HDL-C. However, little is known about effects of these activities on HDL-apoM levels. The aim of this study was to investigate effects of circuit resistance training at different intensities on HDL-associated apoM levels in young untrained men.
            Methods: Forty-five age- and weight-matched healthy untrained men were randomly assigned to a control group (n=10) and four training groups: 20% 1-repetition maximum (1RM) (n=9), 40% 1RM (n=8), 60% 1RM (n=7) and 80% 1RM (n=8). The subjects performed circuit resistance training consisting of barbell bench press, underarm flab, seated barbell curl, triceps exercise with chains, lying leg curl, squats, hyperextension, abs workout, sit-ups and quadriceps workouts (30 seconds each) in three bouts without rest between stations and with active rest (3 minutes) between sets or bouts. The training protocol was carried out for 45 minutes per session, three sessions a week, for five weeks. Venous blood samples were taken 48 hours before the first exercise session and 48 hours after the last training session. After separating plasma, HDL-associated apoM was measured using commercial ELISA kits. SPSS 16 was used for analysis of data using two-way ANOVA and Tukey's post hoc test at significant level of 0.05.
            Results: After the training intervention, the exercise groups had higher apoM levels in total HDL and HDL-2 compared to the control group (P>0.05). However, no significant difference in HDL-associated apoM level was observed between the study groups.
            Conclusion: The results of this study indicate that various intensities of circuit resistance training can alter HDL-associated apoM levels. The decreased HDL-3-associated apoM level could indicate increased rate of apoM transfer to HDL-2, which could potentially prevent development of atherosclerosis and CAD by enhancing the antioxidant effects of HDL.
            Keywords: Circuit Resistance Training, Total HDL-M, HDL3-M, HDL2-M.