[Expression of recombinant protein B subunit pili from vibrio Cholera]

Vibrio cholerae is a gram-negative bacterial pathogen that causes cholera disease. Following ingestion by a host and entry into the upper intestine, V. cholera colonizes and begins to emit enterotoxin. One of the most pathogenic factors of Vibrio cholera is toxin-coregulated pili [TCP]. Toxin-Coregulated pili is as the primary factor requiered for the colonization and insistence of bacteria in the small intestine. The toxin-coregulated pili are bundle-forming pili that are coordinately regulated with cholerae toxin [CT]. The CT operon is part of the genome of the cholera toxin bacteriophage [CTXQ] which utilizes TCP as its receptor. The aim of this study is to produce a recombinant vaccine for V. cholerae in the future. The tcpB gene was amplified by Polymerase chain reaction [PCR] method and subcloned into pET32a expression vector. Escherichia coli BL21 [DE3] plysS competent cells were transformed by pET32a - tcpB recombinant plasmid. In different media with changing the parameters of nutrient content like glucose as carbon source and yeast extract as nitrogen source, protein expression was induced by using IPTG. Recombinant protein were purified by affinity chromatography [Ni-NTA]. The concentration of Recombinant proteins measured according to Bradford assay. The sequencing results by Sanger method showed a similar sequence as tcpB gene. Escherichia coli BL21 plysS was transformed with TCPB-pET32a and gene expression was induced by IPTG. The expressed protein was purified by affinity chromatography and Ni-NTA kit. Recombinant protein tcpB was produced in the cytoplasm of Escherichia coli BL21 plysS, by pET32a expression vector. Therefore, utilization of this protein in Escherichia coli BL21 plysS by expression vectors such as pET32a is possible

[Expression of recombinant streptokinase [SKA] of group A streptococci in E. coli]

Background: Streptokinase A is an antigenic protein secreted by Streptococcus pyogenes. This protein can be used to liquefy pus in pneumonia and the purulent joint swelling and also as an antigen for detection of group A streptococcal infections

Objective: The purpose of this study was expression and production of recombinant streptokinase A of group A streptococci in Escherichia coli in line with diagnostic and therapeutic purposes

Methods: Streptokinase A gene was initially amplified by polymerase chain reaction [PCR] method and sub-cloned into prokaryotic expression vector pET32a. Later, the pET32a vector was transformed into Escherichia coli BL21-DE3-plySs. Gene expression product, induced by IPTG, was purified by Ni-NTA purification kit, and measured by Bradford method. Recombinant SKA was further analyzed by Western Blot. Gene was amplified and sequenced using the Sanger method and the amplified gene in plasmid pTZ57R / T were identical to Recorded sequence in gene bank for streptokinase gene A

Findings: The nucleotide sequence of the gene amplified by PCR was determined by Sanger method. Sequencing results showed similarity in nucleotide sequence of the cloned gene in E. coli with that of group A streptococci available in GeneBank database. The amount of protein product obtained by Bradford method was 3 mg/ml. Recombinant streptokinase protein reacted with mouse sera containing anti-streptokinase A

Conclusion: Our data showed that expression of recombinant SKA protein is possible in Escherichia coli host. The protein product had an approximate molecular weight of 67 kDa with its antigenic properties unchanged. Thus, it can be substituted for ASO and SLO tests used in diagnosis of patients with group A streptococcal infections

[Efficacy of RT-PCR for the detection of E. coli in drinking water]

Escherichia coli [E. coli] is an indicator of potential human fecal contamination. Polymerase Chain Reaction [PCR] as an ideal detection method for detecting E. coli has some advantages like rapidity, high sensitivity and accuracy, easy performance, ability to run in high numbers and inexpensiveness. On the other hand, the disadvantage of PCR is possibility of its false positive results. In this study, reverse transcriptase polymerase chain reaction [RT-PCR] method was used to overcome the problem, and the results were compared to most probable number [MPN]. 16srRNA forward [SF] and 16srRNA reverse [SR] primers were designed using E. coli 16srRNA sequence. After preparing different diluted samples of E. coli in distilled water, the bacteria were separated by FHLP and HAWP filters and its 16srRNA was propagated using mentioned primers. To confirm the sensitivity of the RTPCR method compared to MPN one, samples obtained from 15 water sources in Arak city were examined. The number of bacteria in dilutions were confirmed with culture. RT-PCR data showed that FHLP compared to HAWP filters have a higher capability in separating of bacteria in different dilutions. Also there was a higher sensitivity of RT-PCR compared to RT-PCR and MPN. RT-PCR can detect the bacteria in lower dilutions of bacterial suspension. Hydrophobic filters [e.g. FHLP] compared to hydrophilic filters [e.g. HAWP] have higher capability in separating bacteria. To detect all coliform bacteria RT-PCR amplifications achieved by cells concentrated with hydrophobic filters are recommended

Improvement of PCR in detection of coliform in water pollution

In molecular diagnosis of microbial agent, purification of chromosome is very important step. In this study, after cell destruction, DNA replication was done by increasing the denaturation time, without DNA purification. In this experimental study eight different dilution of E.coli [8/100, 4/100, 2/100, 1/100, 1/200, 1/400, 1/800 and 1/1600] solution were madce in D.W, Bacteria were separated by filtration. Polymerase chain reaction method was used to propagate 162 rRNA gene by design primers without DNA Purification. In order to confirme sensitivity of PCR, contamination of 15 different sources of Arak well water wafer was compared by MPN method. For confirmed sensitivity of PCR, 15 sources of water in Arak were examined and compared with MPN method. Present of bacteria in diution soution were confirmed by culture. Polymerale Chain reaction [PCR] data were shown this method is able to recognize bacteria in above dilutions after filtration. This study showed high sensitivity of PCR method in compare to MPN method. Results were shown without stages of extraction of DNA, PCR were done without losing chromosome. Therefore false negative results were decrease and avoided from difficult phases

Early detection of noise induced hearing loss by extended high frequency audiometery

Surrounding noise, especially in industrial environments, is one of the most common etiologic factors of sensory-neural hearing loss [SNHL], which is not curable, but preventable. By industrialization of communities, the prevalence of the disease and its unfavorable socioeconomic outcome is growing up. Since the preventable defect, early diagnosis has the utmost importance. In recent years, the role of extended high frequency audiometry [EHFA] as a sensitive diagnostic tool for noise induced hearing loss has received much attention. This study deals with the role of EHFA in early diagnosis of this disorder. This was a retrospective cohort study during 2003-4 in Isfahan, Iran. A total number of 30 male labors, aged 20-50 years working in a noisy industrial environment, were compared with an equal number of controls with normal conventional audiometry, and no risk factor for other causes of SNHL according to their history and otoscopic examination. All 60 individuals underwent both types of conventional and extended high-frequency audiometry. Data were analyzed with SPSS software using t-test, paired t-test and correlation tests of Pearson and Spearman. There was no difference between the right and left ears. The exposed subjects had significantly worse hearing than the non exposed group, at all tested frequencies [especially at 16 KHz]. The age effect was notable in both groups. The correlation between high frequency threshold and duration of noise-exposure was significant only at 16, 18 and 20 KHz. The higher mean frequency threshold of the cases was predictable; the normal findings of conventional audiometry, reveal that EHFA can be useful in early diagnosis of acoustic injuries

Evaluating serum levels of IgM, IgA, IgG, changes after adenotonsillectomy

Adenoids and tonsils, which are major components of secondary lymphoid organs, appear to function as the host's first line of defense against exogenous microorganisms. The purpose of this study was to investigate possible impacts of Adenotonsillectomy in serum levels of IgA, IgG, 1gM as indicators of humoral immunity, but there is a controversy about immunologic effect of tonsillectomy and adenoidectomy. Pre and post operative serum samples were collected from 40 patients [7-11 years old] referred for adenotonsillectomy. Serum samples were analyzed, using Radial Immune Diffusion [RID] method to determine levels of IgA, IgG, IgM, before and 3 week after surgery. The level of IgA and IgM were reduced in post operative period. But this was not statistically significant. However, it was found that the level of IgG was reduced in the post operative period and this reduction was statistically significant [P<0.01]. The results of this study indicate that adenotonsillectomy had not a significant effect on two major parts of humoral immunity [IgA and IgM] but it reduced IgG levels

[Expression of recombinant streptolysin O by escherichia coli]

Streptolysin O is an antigenic protein that is secreted by Streptococcus pyogenes. Streptococcal infections are diagnosed with anti streptolysin O. At present, streptolysin O is produced by vectors that have fusion protein. In this study we produced streptolysin O without fusion protein vectors. In this experimental study, we amplified Streptolysin O gene by Polymerase chain reaction [PCR] method and subcloned it to prokaryotic expression vector pET28a. Escherichia coli BL21-DE3-plySs were transformed with pET28a-SLO and gene expression was induced by IPTG. Then it was purified by Ni-NTA kit. The concentration of SLO was assayed by Bradford method. To confirm recombinant SLO Western Blot was used. The sequencing result was confirmed by Sanger method and was the same as SLO gene. Escherichia coli BL21 [DE3] pLysS was transformed with pET28a-SLO and gene expression was induced by IPTG. The expressed protein was purified by affinity chromatography by Ni-NTA resin. The concentration of purified protein was 100microg/ml. The integrity of product was confirmed by Western Blot analysis using a mouse anti streptolysin O. Our data showed that recombinant SLO protein can be produced by pET28a in Escherichia coli. This protein maintains its antigenic effect very well. Therefore, recombinant SLO has same epitopes with natural form of this antigen

[Specificity of recombinant P39 protein in detection of anti-brucella antibody in patients with brucellosis]

In many studies, immunogenicity of Brucella proteins such as P39 in animals is investigated. In this study, we evaluated antigenicity of recombinant P39 from Brucella abortus in patients with Brucellosis. In this experimental study, at first recombinant P39 was produced in Escherichia coli. Sera reactivity of six infected individuals against the recombinant P39 protein was analysed by Western Blot. Data indicated that P39 protein from Brucella abortus was recognized by patients' sera antibodies. Our data showed that recombinant P39 protein can be detected as an antigen by sera in infected human. Therefore, recombinant P39 have same epitopes with natural form of this antigen

Blood superoxide dismutase and catalase activities in women affected with breast cancer

Experimental and epidemiological evidences implicate the involvement of oxygen derived radicals in the pathogenesis of cancer development. Oxygen derived radicals are able to cause damage to membranes, mitochondria and macromolecules including proteins, lipids and DNA. Accumulation of DNA damages has been suggested to contribute to carcinogenesis. It would, therefore, be advantageous to pinpoint the effects of oxygen derived radicals in cancer development. We investigated superoxide dismutase [SOD] and Catalase [CAT] activities in the whole blood of 50 breast cancer [BC] patients and 50 healthy and age matched women. The rate of SOD and CAT activities in BC patients was significantly lower [P<0.001] than controls. No effect of stage on SOD and CAT activities was observed. The results of our study have shown a higher reactive oxygen species [ROS] production and decreased SOD and CAT activities, which support the oxidative stress hypothesis in carcinogenesis. The relative lower SOD and CAT activities may not be adequate to detoxify high levels of H[2]O[2] into H[2]O leading to the formation of the most dangerous OH radical. Therefore, administration of antioxidants may be helpful in the management of BC patients. However, elaborate clinical studies are required to evaluate the role of such antioxidant enzymes [AOE] in BC management

new method for the purification of Cu-Zn superoxide dismutase from human erythrocytes

The human erythrocyte is a rich raw material for the purification of Cu-Zn superoxide dismutase [SOD]. We applied a simple and rapid procedure for the purification of SOD from human erythrocytes by ion exchange chromatography. The purified SOD had a specific activity of 2285.6 u/mg protein and gave a single band on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate [SDS] and each of its to subunit has a molecular weight about 18600 daltons [SOD molecular weight is 37200 daltons].The physicochemical properties of the enzyme obtained by this method are identical to those of the native protein.This procedure appears, therefore, to be a convenient and easily method for isolating this enzyme

Role of social and cultural factors in aids pandemic and their effect on health promotion programs

In most countries, educational programs and condom use is recommended as the most important approaches in controlling HIV infection. We examine the role of different approaches in controlling AIDS epidemics in our region. We find a high failure rate and probably undesirable effects for condom recommendation in epidemiological scale especially considering the low prevalence of HIV infection in our region. Past experience suggests that educational programs in isolation have insufficient effect on health-related behavior in high-risk groups. Present picture of spread of HIV infection discloses the role of religious beliefs, moral values, social nonacceptance of unhealthy lifestyles and illegality of homosexuality, prostitution and drug abuse in controlling AIDS epidemics. In order for health promotion programs to be successful these social and cultural factors should be regarded

Cystic meningioma: a case report

A patient with cystic meningioma is reported. The computerized tomography scan of this meningioma may mimic that of a glial or metastatic tumor with cystic or necrotic changes and lead to an incorrect presumptive diagnosis. Radiological evaluation and recognition are important for surgical removal of these potentially curable neoplasms