Iranian Biomedical Journal
Volume:8 Issue: 4, Oct 2004

Sequence analysis and phylogenetic study of hemagglutinin (HA) gene of H9N2 subtype of avian influenza virus isolates (outbreaks of 1998-2002) in Tehran province (Iran) were studied. Two sets of forward and reverse primers in highly conserved regions, based on sequences of HA gene in Genbank, were designed. PCR products of a 430-bp fragment of 16 isolates were sequenced and then were aligned with the reported sequences in Genbank. Nucleotide sequence comparisons of HA gene from Iranian isolates showed 97-99% identity within the group, and 98% homology with the two isolates [A/Parakeet/Narita/92A/98 (H9N2)] and [A/Parakeet/Chiba/1/97 (H9N2)] from Pakistani parakeets imported to Japan. On the basis of phylogenetic evidence, it is proposed that the emergence of H9N2 avian influenza infection in Iran originated in Pakistan, and it was due to low quarantine measures in the international boundaries. Due to the high percentage of H9N2 homology isolates of Iran with other isolates, namley A/quail/HongKong/G1, in Genbank and based on published reports for high similarity with infecting human H5N1 isolates, it seems that the potential of Iranian avian influenza isolates to infect human should be considered.

There is a growing concern about the application of molecular methods in epidemiological studies of infectious diseases. The objective of this study was to determine the genetic stability of methicillin resistance genes in Staphylococcus aureus for the evaluation of resistance strain distribution. One hundred and fifteen S. aureus isolates from patients with staphylococcal infection were collected. The isolates were screened for methicillin resistance by minimum inhibitory concentration (MIC) examination. The stability of methcillin resistance genes was examined by physical curing and PCR screening methods. The results showed that methicillin resistance Staphylococcus aureus (MRSA) had risen up to 43% in Nemazi Hospital (Shiraz, Iran). Indeed, the incidence of MRSA in our hospital was 10% during the last four years. The ability to lose (curability) of methicillin resistance genes (mecA) was examined by physical curing method in 49 isolates with MIC ³ 16 μg ml-1. No sign of curability of mecA gene was observed where 500 colonies from each strain have been studied and exhibited by the same MIC values before and after curing test. Positive PCR results for isolates with MIC ³ 16 μg ml-1 before and after curing experiment have been achieved. These data confirm the results of curing method, indicating that stable genetic determinants confer methicillin resistance. These results support the hypothesis that resistance isolates may be selected due to clonal selection under antibiotic pressure used in clinics rather than transmission of mobile genetic determinant. The high prevalence of MRSA immerged in our Hospital could be originated due to antibiotic pressure and poor control measures.

Early in the development of many animals, before transcription begins, any change in the pattern of protein synthesis is attributed to a change in the translational activity or stability of mRNA in the egg and early embryo. As a result, translational control is critical for a variety of developmental decisions, including oocyte maturation and initiation of preimplantation development. In this study, using real-time RT-PCR method, we defined the time course of degradation and deadenylation of an oocyte specific gene (c-mos) more precisely and a gene that is re-synthesized after ZGA (cyclin A2). Our data indicate that oocyte-specific transcript, c-mos, degrades rapidly while cyclin A2 mRNA does not and the deadenylation of c-mos mRNA precedes the process of degradation. Our findings suggest that time-dependent elimination of different maternal mRNA is a way for regulation of translation in early development of mouse embryos.

The link between IL-13 and bronchial hyper-responsiveness has brought this cytokine as a potential therapeutic target for asthma and allergic diseases. At the present study, we address the role of B cell derived IL-13 in the IgE and other immunoglobulin development. Antisense oligo for human IL-13 m-RNA was used to study IgE down regulation. Human B-lymphocytes were purified by positive selection using magnetic cell sorting and were cultured in the complete medium plus anti-CD40 monoclonal antibody and recombinant human IL-4. Immunoglobulin assay was performed by ELISA in the presence and absence of antisense oligonucleotide. We demonstrated that IL-13 antisense causes the decrease of IgE and increase of IgA significantly and no significant changes in IgM and IgG levels (p<0.01). We also demonstrated that both IL-13 inhibition and IL-4 removal cause the complete blocking of IgE and significant decrease of IgM and IgG levels. Our IL-13 antisense oligo can block B-cell IL-13 productions and consequently inhibits IgE production followed by IgA class switching in vitro. We suggest that in contrast to the IL-4, IL-13 is apparently more potent in the IgE switching and has no significant role in IgG and IgM levels

Angiotensin II is a major endocrine hormone that affects directly both vascular smooth muscle and endothelial cells. Since vascular reactivity to angiotensin II changes in more physiological and pathophysiological conditions, the present study was performed to investigate the effect of intraperitoneal administration of angiotensin-converting enzyme inhibitor and captopril (30 and 50 mg kg-1, once daily for 8 weeks) on contractile response of rat aorta. After 8 weeks, the treated rats were anesthetized, their thoracic aortas were excised and placed in a Petri dish filled with Krebs solution for recording of contraction and relaxation response. The obtained results showed that captopril did not modify body weight gain and food or water intake but contractile response of aortic rings to phenylephrine in treated rats with 30 and 50 mg kg-1 captopril, in the presence of endothelium, decreases about 11-22% and 29-32% (P<0.05-P<0.01), respectively, when compared to the controls. Denuded aortic rings from 30 and 50 mg kg-1 captopril-treated rats showed 11-21% and 7-11% decrease in contractile response, respectively. There was a marked endothelium-dependent relaxation response to acetylcholine in 50 mg kg-1 captopril-treated rats compared to the controls (P<0.05). Endothelium-independent relaxation response to isosorbide dinitrate showed no significant difference in all groups. According to these results, it is suggested that captopril exerts its relaxant effect directly and/or indirectly through endothelium by production and releasing of endothelium-derived relaxing factors.

Aeromonas hydrophila secretes several extracellular proteins including enterotoxin, hemolysin and aerolysin that are associated with the bacterial virulence. Previous studies have shown that two hemolytic toxins, hemolysin A and aerolysin A contribute to the virulence of Aeromonas hydrophila. In the current study, a total of 50 strains of Aeromonas hydrophila, including 28 (56%) strains isolated from diarrheal cases and 22 (44%) strains isolated from healthy asymptomatic controls, were used. These strains were tested for cytochrome oxidase activity based on Kovac’s method and confirmed as A. hydrophila with API 20E multi-test systems. To determine hemolysin, cytotoxin and enterotoxin activities, horse blood agar, Vero cells and the suckling mouse model were used, respectively. The presence of two hemolytic toxin genes: hlyA and aerA was determined by PCR assay. About 89% of diarrheal strains tested were positive for hemolysins, 68% for cytotoxin and 89% for enterotoxin activity. These figures for healthy asymptomatic isolates were 72%, 23% and 14%, respectively. A significant association was found between cytotoxin and enterotoxin activity in diarrheal disease, whereas such association was not found in case of hemolysin. Almost 93% of diarrheal isolates and 73% of healthy person strains were PCR positive for hlyA gene. The corresponding figures for aerA gene were 86% and 45.5% respectively. The aerA+ hlyA+ genotype and in some cases aerA+ genotype could be considered as reasonable predictors of human diarrheal disease. However, the role of other unknown hemolytic and cytolytic factors cannot be discounted

In order to improve simultaneous detection and identification of Helicobacter genus in general and Helicobacter pylori specifically and reduce the number of amplifications needed, we established a multiplex PCR. In this study, two pairs of primers: Hcom1 and Hcom2 specific for Helicobacter genus, Hicd1 and Hicd2 specific for Helicobacter pylori species were used. To determine the sensitivity of our multiplex PCR, the lower limits of DNA detection from pure culture were established using phenol-chloroform method. To evaluate the specificity of our protocol, we tested DNA extracted from various Gram-negative and -positive bacteria. A study was subsequently undertaken on stomach tissue samples from 18 patients to evaluate this protocol for detection of Helicobacter in tissue samples. In our optimized PCR, two fragments of 389- and 1200-bp were produced using Hcom1-Hcom2 and Hicd1-Hicd2 primers, respectively. Amplification of Helicobacter pylori genomic DNA was achieved at concentration as low as 0.03 pg equivalent to 150 bacteria. No DNA amplification of other Gram- positive and -negative bacteria was seen. Twelve specimens, positive in culture and rapid urease test for Helicobacter pylori were positive for DNA of this organism using this multiplex PCR. Our results demonstrate that this protocol represents a specific and sensitive assay for simultaneous detection of Helicobacter genus members, in general, and Helicobacter pylori species, specifically, in clinical samples yielding no false-positive

Gamma irradiation is routinely used for suppression of lymphocyte function in transfusion and transplantation procedures. In recent years, some investigators focused on the effects of ionizing radiation on special aspects of lymphocyte function and considered the possibility of its clinical application for treatment of some immunological disorders. In this study, we evaluated the effects of five different doses of γ-ray on proliferation and IL-5 production of peripheral blood lymphocytes. Lymphocytes were separated from blood and were treated with 5, 10, 20, 30 and 40 Gy irradiation (using a 137 Cs source) and then were cultured for 72 h in the presence of phytohemagglutinin (PHA). The proliferative response of samples was evaluated by MTT assay, and the supernatant of the cells was collected for IL-5 detection. The results showed that the ionizing radiation had a suppressive effect on lymphocyte proliferation. IL-5 production was affected in a dose-response manner, augmented in response to 5 and 10 Gy and reached to its peak value at 20 Gy. At 30 Gy, IL-5 production was diminished lower than peak value, but still remained higher than control baseline, and 40 Gy led to IL-5 values lower than baseline.