Expression of recombinant Streptokinase from local Egyptian Streptococcus sp. SalMarEg

Streptokinase (SK) is a therapeutically important thrombolytic agent. Cardiovascular disease is the first cause of adult death worldwide. In Egypt about 13% of the population die every year due to ischemic heart disease. In spite of this fact, there is no local production of cardiovascular therapeutics. We reported for the first time the expression of a recombinant SK from a local Streptococcus strain. When produced on industrial scale this r-SK may substantially contribute to reducing the costs of thrombolytic therapy in developing countries. In this study, a highly purified r-SK from Streptococcus sp. isolated from Egyptian pharyngitis patients was obtained. The isolated strain was partially identified using 16S rDNA sequencing and named Streptococcus sp. SalMarEg. It was found to be phylogenetically related to Streptococcus pyogenes. Analysis of the obtained sequence showed high similarity with other SK genes. The protein expression in a prokaryotic system obtained a 47-kDa SK protein that could be purified using a single-step his-tagged affinity purification chromatography, with nearly 80% recovery. The clot lytic activities of both recombinant and commercial SK were similar, thus giving the basis to scale up this SK product in order to evaluate the possibilities of its commercialization in local and/or regional markets.Key words: Streptokinase, Streptococcus SalMarEg, thrombolytic agent, heterologous expression

Partial Purification and Characterization of Two Endo-ȕ-1, 4-glucanase from Trichoderma sp. (Shmosa tri)

Abstract: Two endoglucanases (EC 3.2.1.4) from Trichoderma sp. (shmosaTri) FJ937359 were purified to homogeneity using ammonium sulfate precipitation and gel filtration. Purity was confirmed by SDS/PAGE. Enzymatic properties and molecular weights were determined. Molecular weights of CMCase I and II were 58 and 34 KDa, respectively. The effect of temperature on the 2 endoglucanase activity was studied and results showed that optimum activity obtained at 50°C for both CMCase I and II. The enzymes withstand 60 min at 50ºC without loss of enzymatic activity. CMCase I and II retained 14.0 and 26.5 % of their original activities at 70°C after 90 min. The optimum pH for CMCase I and II was 5.0. Results also show that CMCase I was active at room temperature after 24 hrs over a broad pH range (3.0-9.0) while CMCase II was relatively stable in pH range (4.0-6.0). Among different kinds of substrates, both enzymes showed a high preference for carboxymethyl cellulose while both CMCase I and II did not show any hydrolytic activity against chitin, starch and cellobiose. On the other hand both CMCase I and II have relatively low hydrolytic activity towards ȕ glucan and xylan. All metallic ions used as well as EDTA and SDS at a concentration of 20 ug/ml of reaction mixture have an inhibitory effect on both CMCase I and II

A simple method to assess the oxidative susceptibility of low density lipoproteins

BACKGROUND: Oxidative modification of low density lipoproteins (LDL) is recognized as one of the major processes involved in atherogenesis. The in vitro standardized measurement of LDL oxidative susceptibility could thus be of clinical significance. The aim of the present study was to establish a method which would allow the evaluation of oxidative susceptibility of LDL in the general clinical laboratory. RESULTS: LDL was isolated from human plasma by selective precipitation with amphipathic polymers. The ability of LDL to form peroxides was assessed by measuring thiobarbituric acid reactive substances (TBARS) after incubation with Cu(2+) and H(2)O(2). Reaction kinetics showed a three-phase pattern (latency, propagation and decomposition phases) which allowed us to select 150 min as the time point to stop the incubation by cooling and EDTA addition. The mixture Cu(2+)/H(2)O(2) yielded more lipoperoxides than each one on its own at the same time end-point. Induced peroxidation was measured in normal subjects and in type 2 diabetic patients. In the control group, results were 21.7 ± 1.5 nmol MDA/mg LDL protein, while in the diabetic group results were significantly increased (39.0 ± 3.0 nmol MDA/mg LDL protein; p < 0.001). CONCLUSION: a simple and useful method is presented for the routine determination of LDL susceptibility to peroxidation in a clinical laboratory

Cysteamine inhibits lysosomal oxidation of low density lipoprotein in human macrophages and reduces atherosclerosis in mice

Background and aims: We have shown previously that low density lipoprotein (LDL) aggregated by vortexing is internalised by macrophages and oxidised by iron in lysosomes to form the advanced lipid/protein oxidation product ceroid. We have now used sphingomyelinase-aggregated LDL, a more pathophysiological form of aggregated LDL, to study lysosomal oxidation of LDL and its inhibition by antioxidants, including cysteamine (2-aminoethanethiol) which concentrates in lysosomes by several orders of magnitude. We have also investigated the effect of cysteamine on atherosclerosis in mice. Methods: LDL was incubated with sphingomyelinase, which increased its average particle diameter from 26 to 170 nm, and was then incubated for up to 7 days with human monocyte-derived macrophages. LDL receptor-deficient mice were fed a Western diet (19-22 per group) and some given cysteamine in their drinking water at a dose equivalent to that used in cystinosis patients. The extent of atherosclerosis in the aortic root and the rest of the aorta was measured. Results: Confocal microscopy revealed lipid accumulation in lysosomes in the cultured macrophages. Large amounts of ceroid were produced, which colocalised with the lysosomal marker LAMP2. The antioxidants cysteamine, butylated hydroxytoluene, amifostine and its active metabolite WR-1065, inhibited the production of ceroid. Cysteamine at concentrations well below those expected to be present in lysosomes inhibited the oxidation of LDL by iron ions at lysosomal pH (pH 4.5) for prolonged periods. Finally, we showed that the extent of atherosclerotic lesions in the aortic root and arch of mice was significantly reduced by cysteamine. Conclusions: These results support our hypothesis that lysosomal oxidation of LDL is important in atherosclerosis and hence antioxidant drugs that concentrate in lysosomes might provide a novel therapy for this disease

Oxidation of Low-Density Lipoprotein by Iron at Lysosomal pH: Implications for Atherosclerosis

Low density lipoprotein (LDL) has recently been shown to be oxidised by iron within the lysosomes of macrophages and this is a novel potential mechanism for LDL oxidation in atherosclerosis. Our aim was to characterise the chemical and physical changes induced in LDL by iron at lysosomal pH and to investigate the effects of iron chelators and α-tocopherol on this process. LDL was oxidised by iron at pH 4.5 and 37°C and its oxidation monitored by spectrophotometry and HPLC. LDL was oxidised effectively by FeSO4 (5-50 µM) and became highly aggregated at pH 4.5, but not at pH 7.4. Cholesteryl esters decreased and after a pronounced lag 7-ketocholesterol increased greatly. Total hydroperoxides (measured by tri-iodide assay) increased up to 24 h and then decreased only slowly. The lipid composition after 12 h at pH 4.5 and 37°C was similar to that of LDL oxidised by copper at pH 7.4 and 4°C, i.e. rich in hydroperoxides but low in oxysterols. Previously oxidised LDL aggregated rapidly and spontaneously at pH 4.5, but not at pH 7.4. Ferrous was much more effective than ferric iron at oxidising LDL when added after the oxidation was already underway. The iron chelators diethylenetriaminepentaacetic acid and, to a lesser extent, desferrioxamine inhibited LDL oxidation when added during its initial stages, but were unable to prevent LDL aggregating after it had been partially oxidised. Surprisingly, desferrioxamine increased the rate of LDL modification when added late in the oxidation process. α-Tocopherol enrichment of LDL initially increased the oxidation of LDL, but inhibited it later. The presence of oxidised and highly aggregated lipid within lysosomes has the potential to perturb the function of these organelles and to promote atherosclerosis

General Anesthetics Inhibit Erythropoietin Induction under Hypoxic Conditions in the Mouse Brain

Background: Erythropoietin (EPO), originally identified as a hematopoietic growth factor produced in the kidney and fetal liver, is also endogenously expressed in the central nervous system (CNS). EPO in the CNS, mainly produced in astrocytes, is induced under hypoxic conditions in a hypoxia-inducible factor (HIF)-dependent manner and plays a dominant role in neuroprotection and neurogenesis. We investigated the effect of general anesthetics on EPO expression in the mouse brain and primary cultured astrocytes. Methodology/Principal Findings: BALB/c mice were exposed to 10 % oxygen with isoflurane at various concentrations (0.10–1.0%). Expression of EPO mRNA in the brain was studied, and the effects of sevoflurane, halothane, nitrous oxide, pentobarbital, ketamine, and propofol were investigated. In addition, expression of HIF-2a protein was studied by immunoblotting. Hypoxia-induced EPO mRNA expression in the brain was significantly suppressed by isoflurane in a concentration-dependent manner. A similar effect was confirmed for all other general anesthetics. Hypoxia-inducible expression of HIF-2a protein was also significantly suppressed with isoflurane. In the experiments using primary cultured astrocytes, isoflurane, pentobarbital, and ketamine suppressed hypoxia-inducible expression of HIF-2a protein and EPO mRNA. Conclusions/Significance: Taken together, our results indicate that general anesthetics suppress activation of HIF-2 an

Publisher: Institute for Animal Husbandry, Belgrade-Zemun UDC 636.38 FINGERPRINTING OF FECB GENE IN FIVE EGYPTIAN SHEEP BREEDS

Original scientific paper Abstract: Recently, many aspects of FecB gene, including reproductive endocrinology, organs development and body mass have been studied. FecB has an additive effect on litter size and ovulation. The present investigation was carried out to study polymorphism by forced PCR-RFLP of FecB gene in five Egyptian local sheep breeds and its comparison with other foreign sheep breeds. Genomic DNA was isolated from a total of 100 animals of Egyptian sheep breeds namely Rahmani, Ossimi, Awassi, Barki and Awassi x Barki crossbred. Forced PCR of the FecB gene 190 base pair (bp) was amplified using specific primer designed to introduc a point mutation in the resulting PCR products with FecB carrier sheep containing an AvaII restriction site (G|GACC), whereas products from noncarriers lacked (of) this site..Digestion of FecB gene 190 base pair with AvaII restriction enzyme resulted in non carrier 190 bp band (wild type) in all the animals belonging to the five Egyptian breeds studied revealing absence of this restriction site in those five breeds