Iranian Biomedical Journal
Volume:9 Issue: 3, Jul 2005

A complication in the analysis of the role of ICP34.5 gene in the herpes simplex virus type-1 (HSV-1) lifecycle is the presence of overlapping antisense gene, open reading frame P (ORF P), which is also deleted in HSV-1 ICP34.5 negative mutants. A HSV-1 wild type strain (17+) ICP34.5/ORF P deletion mutant (1716) is totally avirulent in animal models and impaired in a number of in vitro functions: replication in 3T6 cells; and replication and shutoff of cellular protein synthesis in SK-N-SH cells. To attribute characteristics of 1716 to each of these two genes (ICP34.5 or ORF P), a number of HSV-1 recombinant viruses that express ICP34.5 and ORF P independently were constructed, purified and characterized. The parent of these recombinants is 1716 and they are (so called): 1622, expressing ICP34.5; 1624/24.5, expressing ORF P; and 1625, expressing both ICP34.5 and ORF P in separate loci. Using homologous recombination in cell culture, the recombinants were constructed and their DNA were analyzed by Southern-blotting. Expression of ICP34.5 and ORF P from the recombinants was checked by Western-blotting. Using cell culture, titration and plaque assay techniques, replication kinetics of the recombinants were compared with 17+ and 1716 in 3 cell lines, BHK, 3T6, and SK-N-SH. The results showed that (i) ICP34.5 restored the ability to replicate and prevent host shutoff similar to wild type in SK-N-SH cells; (ii) ICP34.5 restored the replication phenotype to near wild type levels in 3T6 cells; and (iii) ORF P expression had no effect on the replication of these mutants. The characteristics of 1716 is obviously due to the lack of ICP34.5 and ORF P has no role in the characteristics studied. Thus, the function of ORF P still has to be determined. Iran. Biomed.

Our previous studies have shown that median and ulnar nerve lesion induced calbindin (CB) immunoreactivity in some injured motoneurons in developing rats. Motoneuron death induced by sciatic nerve transection in neonatal rats has been related to induction of neuronal isoform of nitric oxide synthase (nNOS). The present study investigated whether expression of CB and nicotinomid adenin dinucleotide phosphate-diaphorase (NADPH-d) activity, a marker for nNOS, is related to the death or survival of forelimb motoneurons in response to axotomy. After median and ulnar nerve transection at either P2 or P7, NADPH-d histochemistry was performed on cervical spinal cord sections to analyze the induction of nNOS in motoneurons retrogradely labeled with FB and immunostained for CB. NADPH-d reactivity was not detectable in FB labeled motoneurones up to 2 weeks after nerve lesion at P2. However, following nerve lesion at P7, some FB labeled motoneurons showed NADPH-d activity 2 weeks after nerve lesion. These NADPH-d positive motoneurons were not CB immunoreactive. The results indicate the possible role of nitric oxide (NO) in nerve regeneration and the role of CB in neuroprotection from cell death or in mechanisms of neurodegeneration. Iran. Biomed.

Many investigators have used xenogeneic, especially murine stromal cells and fetal calf serum to maintain and expand human stem cells. The proliferation and expansion of human hematopoietic stem cells in ex vivo culture were examined with the goal of generating a suitable protocol for expanding hematopoietic stem cells for patient transplantation. Using primary fetal liver cells, we established a serum-free culture system to expand human primitive stem/progenitors cells. Non-enriched cord blood CD34+ cells were cultured on a monolayer of mouse primary fetal liver cells in the presence of trombopoietin, flt3/flk2 ligand, and/or stem cell factor, IL-6 and IL-3 under serum-free conditions. After 1 or 2 weeks of culture, cells were examined for clonogenic progenitors and percentage of CD34+ CD38- cells. In the presence of trombopoietin, flt3/flk2 ligand, and stem cell factor, fetal liver cells supported expansion of CD34+ cells more than 10 to 20 fold. In addition, colony forming unit-cell assay was expanded more than 5- and 10-fold after 1 and 2 weeks of culture, respectively. These results strongly suggest that fetal liver cells may be a suitable feeder layer for expansion of hematopoietic progenitors from umbilical cord blood in vitro. Iran. Biomed.

A capture ELISA system was developed for diagnosis of visceral leishmaniasis (VL) using a monoclonal antibody raised against an antigen previously detected in the urine of VL patients. Urine samples from confirmed VL cases from Yemen, Nepal, Spain, Sudan and Brazil were tested in the capture ELISA in comparison with urine samples from endemic and non-endemic areas along with urine samples from patients with malaria, brucellosis, schistosomiasis and patients with non-infectious diseases. All of VL patient samples from different geographical areas (apart from 2 samples from Brazil) gave a positive result, while no cross-reaction was found with the control samples. The results obtained with the capture-ELISA were compared to those obtained with KAtex, a previously described latex agglutination test, showed that the KAtex and the new ELISA are comparable in terms of specificity (100%) but a better sensitivity (94.1%) was found for the capture-ELISA. Moreover, the capture-ELISA adds a useful quantitative dimension to antigen detection. In addition, the boiling of urine samples, which is necessary for KAtex, was not required in the capture-ELISA. These results suggest that the antigen detection in urine by the new capture ELISA system provides a useful method for diagnosis of VL and fulfils the requirements of a non-invasive method for diagnosis of VL. Iran. Biomed.

In the previous studies the relaxant, anti-cholinergic (functional antagonism) and anti-histaminic effects of Nigella sativa have been demonstrated on guinea pig tracheal chains. To elucidate the main substance responsible for the relaxant effect of this plant, the effect of thymoquinone, one of the main constituent of Nigella sativa was examined in this study. The bronchodilatory effects of three cumulative concentrations (40, 80, and 120 µM) of thymoquinone were examined by their relaxant effects on precontracted tracheal chains of guinea pigs using (1) 10 µM methacholine and (2) 60 mM KCl. The results were compared with the effects of saline, theophylline, and extracts (macerated and aqueous) of Nigella sativa (n = 5 for each group). The results showed significant relaxant effects of theophylline and extracts from Nigella sativa as compared to saline in group 1 experiments (P<0.05 and P<0.005). There were no significant differences between the effects of two higher concentrations of extracts with those of theophylline. However, none of the three thymoquinone concentrations showed any relaxant effect in both groups. There were also significant differences between the relaxant effect of theophylline and extracts with those of thymoquinone concentrations (P<0.05 and P<0.001). In group 2, only theophylline showed a significant relaxant effect (P<0.05 and P<0.001). The effects of two higher concentrations of both extracts and thymoquinone were significantly lower than those of theophylline in group 2 (P<0.05 to P<0.001). These results indicated that the relaxant effect of Nigella sativa is not due to its constituent thymoquinone. Iran. Biomed.

The present study was undertaken to determine whether two-month treatment of streptozotocin (STZ)-diabetic rats with aqueous leaf extract of Trigonella foenum-graecum (TFG; 200 mg/kg; i.p.) could improve thoracic aortic responsiveness and to evaluate its endothelium dependency. For this purpose, vascular responses to KCl and noradrenaline (NA) were measured. Diabetic state significantly increased contractile responses to KCl and NA in aortic rings in both endothelium-intact and -denuded rings. Extract-treated diabetic rats showed a significant lower maximal contractile response to KCl only in endothelium-intact rings as compared to diabetic rings. It is concluded that intraperitoneal administration of aqueous leaf extract of TFG for two months could improve some functional indices of the vascular system in diabetic state and the integrity of the endothelium is essential for its beneficial effects. Iran. Biomed.

A multiplex PCR-based assay was developed for detection of Salmonella typhi and identification of other salmonella serotypes. Three primer-sets were selected from different genomic sequences, malo2-F/malo2-Ra primers from invasion gene, Parat-s/Parat-as as well as tyv-s/tyv-as primers from O-antigen gene cluster of the genus Salmonella. This method differentiated Salmonella spp., based on size and number of amplified fragments. The Salmonella para typhi A and B yielded two bands of 373 bp and 285 bp, respectively, and the other species including S. paratyphi C, S. infantis and S. havana yielded only one 373 bp band. The PCR products of S. typhi and S. enteritidis were 373, 285 and 615 bp. In testing the specificity of the assay, no amplification was observed in non-Salmonella species such as Shigella, Kelebsiella, E. coli, Proteus, Staphylococcus and Streptococcus. The sensitivity of the method was evaluated about 2.5 × 102 CFU/ml, that could be detected by the PCR assay. Iran. Biomed.

Familial hypercholesterolemia (FH) is an autosomal co-dominant disorder of lipid metabolism, caused by mutations in LDL receptor gene. The penetrance of FH is almost 100%, meaning that half of the offspring of affected parents born with disease. The patients are at risk of premature coronary heart disease (CHD). There is no report about the molecular basis of FH in Iran. Identification of mutations allows unequivocal diagnosis in potentially affected relatives. To characterize genetic aberrations in Iranian FH patients, after ruling out the most common mutation producing familial defective ApoB-100 (R3500Q), we screened exon 4 in LDL receptor gene in 30 heterozygous FH patients by single strand conformation polymorphism (SSCP). A new missense mutation (445G>T) was found in proband and his mother. This causes a Gly to Cys change in repeat 3 of LDL binding domain. This nucleotide change was not found in 50 normal individuals. Iran. Biomed.