فهرست مطالب
Iranian Biomedical Journal
Volume:12 Issue: 2, Apr 2008
- 88 صفحه،
- تاریخ انتشار: 1387/05/15
- تعداد عناوین: 10
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Page 59BackgroundHerpes simplex virus type 2 (HSV-2) is highly prevalent and major cause of genital herpes in humans. The life-long nature of infection and the increasing prevalence of genital herpes imply that vaccination is the best strategy for controlling the spread of infection and limiting HSV disease. HSV glycoprotein D (gD) is one of the most important viral immunogen which has an essential role in virus infectivity and induction of immune responses.MethodsHSV-2 DNA was extracted and used as template in polymerase chain reactions to amplify gD2 gene. The PCR product was confirmed by restriction enzyme analysis, cloned into a cloning vector and then sequenced. The Bac-to-Bac expression system was used to express HSV-2 gD in insect cells. The expressed protein was used as subunit vaccine to immunize guinea pigs after confirmation.ResultsThe expressed protein was confirmed with SDS-PAGE and Western-blot analysis. In Western-blot analysis, two major protein bands, with approximate molecular weights of 52-55 and 41-43 kDa corresponding to the glycosylated and non-glycosylated forms of gD2 protein, were observed, respectively. Immunization with the recombinant gD2 could elicit humoral responses in guinea pigs as measured by neutralization test and ELISA, and offered high protection against induced HSV-2 genital disease.ConclusionThe baculovirus expression of heterologous genes permits proper folding, post-translational modification and oligomerization in manners that are often identical to those that occur in mammalian cells. Expression of proteins under the control of the strong polyhedrin promoter, allowing high level protein production, can be used as subunit vaccine
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Page 67BackgroundAnalysis of many proteins produced during the transition into the stationary phase and under stress conditions (including starvation stress) demonstrated that a number of novel proteins were induced in common to each stress and could be the reason for cross-protection in bacterial cells. It is necessary to investigate the synthesis of these proteins during different stress conditions.MethodsThe changes in protein profile of Flexibacter chinensis at various stages of the starvation process and the other stresses were investigated using two-dimensional gel electrophoresis which has proven to be a powerful tool for investigation of the changes in protein profiles under such conditions.ResultsMost starvation proteins were synthesized during the early stationary phase and many of these proteins remained during long-term starvation. Some of these proteins were transiently synthesized. The sequencing result of one of the proteins showed that there was a 62.5% identity in 8 amino acids overlapped with the 5’ residue of a 10 kDa chaperon protein which is known to be involved in the starvation stress response in other organisms.ConclusionThere are many proteins synthesized in common with many stresses in Flexibacter chinensis. Some of these proteins must play a major role in the stability of the cell under different stresses
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Page 77BackgroundSelective phosphodiesterase (PDE3) inhibitors improve cardiac contractility and may use in congestive heart failure. However, their proarrhythmic potential is the most important side effect.MethodsIn this research, we evaluated the potential cardiotonic activity of six new synthesized selective PDE3 inhibitors (6-hydroxy-4-methylquinolin-2(1H)-one derivatives) using the spontaneously beating atria model. In each experiment, atrium of reserpine-treated rat was isolated and the contractile and chronotropic effects of a synthesized compound were assessed. The 3-isobutyl-1-methylxanthine, a non-selective PDE inhibitor, was used for comparison.ResultsThe results showed that, among new compounds, the best pharmacological profile was obtained with the compound 6-[4-(4-methylpiperazine-1-yl)-4-oxobutoxy]-4-methylquinolin-2(1H)-one, C6, which displayed selectivity for increasing the force of contraction (168 ± 5% change over the control) rather than the frequency rate (138 ± 5% change over the control) at 300 μM. However, C6 at concentrations of 10 and 100 μM produced left and upward shift in the positive inotropic concentration-response curve of isoprenaline. The -log EC50 of isoprenaline was 8.843 ± 0.171 in the absence, 9.448 ± 0.138 and 9.456 ± 0.107 in the presence of 10, 100 μM of C6, respectively (P<0.001, n = 6). Also, amrinone, a selective PDE3 inhibitor, shifted the isoprenaline concentration-response curve to the left and upward. The concentration of 10 and 100 μM amrinone decreased -log EC50 of isoprenaline to 9.527 ± 0.287 and 9.423 ± 0.243, respectively (P<0.001, n = 6). Moreover, the positive chronotropic effect of isoprenaline was not affected by amrinone or C6.ConclusionThis study provides functional evidence for the positive inotropic effect of C6. Considering the augmentation of isoprenaline positive inotropic concentration-response with C6 and amrinone, we conclude that C6 produces its effect via potentiation of cAMP-dependent signaling system and possibly by inhibition of PDE3 activity
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Page 85ObjectivesIt was aimed to investigate the effects of different doses of follicle stimulating hormone (FSH) and activin A on the growth and maturation of preantral mouse follicles during the in vitro culture.MethodsPreantral follicles (90-100 µm in diameter) were harvested from 6-8 week-old Syrian mice and cultured in TCM199 culture medium for 6 days to see the effect of FSH and Activin A. Activin A concentrations in the range of 10-200 ng/ml were used, while 10, 50, 100, 150 and 200 mIU/ml FSH were used in the experiment.ResultsActivin A concentration of 100 ng/ml resulted in a significant increase in follicle diameter (170 µm) with the survival rate of 73% as compared to the control (100 µm and 25%, P<0.05). The number of oocytes matured and the percentage of germinal vesicle breakdown (GVBD) was 61 and 70%, respectively as compared to the control (20 and 29%, P<0.05). Follicle diameter (190 µm) and survival rate (85%) increased significantly in the presence of 100 mIU/ml of FSH as compared to the control (P<0.05). But, the administration of activin A+FSH increased the effect of both factors on follicular diameter (205 µm as compared to 100 µm in control, P<0.01). Follicle survival, oocyte maturation and GVBD rates were 91, 81 and 89%, respectively (P<0.01).ConclusionThese results have suggested that exposure to FSH and activin A before the formation of antral cavity had positive effect on follicle survival and oocyte robustness.
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Page 93Background. The major side effect of cisplatin, used in some tumours, is nephrotoxicity. Reactive oxygen species and oxidative damage are the most important factors in cisplatin-induced acute renal failure. The main purpose of this study is to investigate the protective effects of crocin against cisplatin-induced acute renal failure and oxidative stress in rat. Methods. In this study, animals were randomly divided into 5 groups (6 each). Group one received normal saline (2 ml/day, i.p.). Group two received a single dose of cisplatin (5mg/kg, i.p.). Groups 3 to 5 received crocin (100, 200 and 400 mg/kg, i.p., respectively, for 4 consecutive days one hour before a single dose of cisplatin (5 mg/kg) only at the first day. Blood samples were taken out (on the fifth day) for measuring the level of urea and creatinine. The kidneys were removed for histopathological and biochemical examinations. Furthermore, 24-hour urinary factors were measured. Results. Blood urea, creatinine and urinary glucose and protein concentrations in crocin-treated groups were significantly lower than those of cisplatin-treated group in a dose-dependent manner. Histopathological studies showed a massive damage in S3 segment of proximal tubules in cisplatin-treated group. No damage was observed in crocin-treated groups. Crocin treatment resulted in a significant and dose-dependent reduction in malondialdehyde concentration as compared to the cisplatin-treated group. Moreover, crocin produced a significant elevation in total thiol and glutathione peroxidase concentrations, as compared with cisplatin-treated group. Conclusion. The results of the present study suggest that crocin has a protective effect against cisplatin-induced acute renal failure and relative oxidative stress. Iran. Biomed.
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Page 101BackgroundPrimary cultures of embryonic neurons have been used to introduce a model of neurons in physiological and pathological conditions. However, age-related cellular events limit this method as an optimal model in adult neurodegenerative diseases studies. Besides, short-interval changing media in previous cultures decreases the effectiveness of this model. As an example of this matter, we can refer to the study on some special neuronal secreted factors or the influence of some experimental materials on neurons. Meanwhile, short-interval changing media could remove the effects of some released factors from the environment. In this study, the method for isolation and culturing adult rat hippocampal neurons with long-intervals medium changing has been described.MethodsThe hippocampal neurons of adult male rats were cultured. We used Neurobasal A/B27 culture medium, papain (2 mg/ml), trypsin 0.25% and collagenase (1 mg/ml) for neuronal isolation, OptiPrep density gradient for separation of neurons from other cell types and also debris and FGF2 (10 ng/ml) for increasing neuronal survival and regeneration.ResultsThe neuronal sprouting and viability were increased by using papain and mild triturating (P<0.05). Adult neuronal culturing and their regeneration were impossible without FGF2. It was shown that adding new fresh medium every 4 days and exchanging half of it every 8 days had no detrimental effect on neuronal viability.ConclusionThis investigation shows the possibility of culturing adult neuronal cells and their maintaining in long-interval media. It could be happened because of adult neurons rely significantly on the neighboring cells secreted factors for living and making synaptic connections. This model is very useful in physiological and pathological studies which need stable conditions of neuronal culture in a long period of time.
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Page 109BackgroundSeveral methods have been developed for detection of sequence variation in genes and each has its advantages and disadvantages. A disadvantage of them is that the simpler, cost-effective methods are commonly perceived as being less sensitive in their detection of sequence variation, whereas those with proven sensitivity have a requirement for complex or expensive laboratory equipment. In this context, we undertook improvements to the conformation sensitive gel electrophoresis (CSGE) method which provides a cost-effective approach to mutation detection and compared the results with scanning carried out using denaturing high performance liquid chromatography (DHPLC) which utilises a dedicated analyser.MethodsWe designed PCR primers to amplify the seven protein-coding exons of the human SPP2 gene which encodes secreted phosphoprotein 24 (spp24) such that the amplified products included the immediately-adjacent intronic regions. Five improvements were made to the CSGE method that was used to the scan the PCR-amplified DNA. The scanning was then repeated using DHPLC and the results were compared.ResultsUsing CSGE, a single nucleotide polymorphism was discovered in exon 2 and another in intron 2 of the gene. Re-scanning of the same regions by DHPLC detected no additional sequence polymorphisms.ConclusionWith modification of the original protocol, CSGE is capable of providing a simple and cost-effective approach to the detection of DNA sequence polymorphisms that appears to be comparable in sensitivity to DHPLC. Iran. Biomed
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Page 115BackgroundPhenylalanine dehydrogenase (PheDH; EC 1.4.1.20) is a NAD+-dependent enzyme that performs the reversible oxidative deamination of L-phenylalanine to phenylpyruvate. It plays an important role in detection and screening of phenylketonuria (PKU) diseases and production of chiral intermediates as well. The main goal of this study was to find a simple and rapid alternative method for purifying PheDH.MethodsThe purification of recombinant Bacillus sphaericus PheDH was investigated in polyethylene glycol (PEG) and ammonium sulfate aqueous two-phase systems (ATPS). The influences of system parameters including PEG molecular weight and concentration, pH and (NH4)2SO4 concentration on enzyme partitioning were also studied. The purity of enzyme was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis.ResultsA single extraction process was developed for separation and purification of recombinant PheDH from E. coli BL21 (DE3). The optimized conditions for partitioning and purification of PheDH were 9% (w/w) PEG-6,000 and 16% (w/w) (NH4)2SO4 at pH 8.0. The partition coefficient, recovery, yield, purification factor and specific activity values were achieved 58.7, 135%, 94.42%, 491.93 and 9828.88 U/mg, respectively. Also, the Km values for L-phenylalanine and NAD+ in oxidative deamination were 0.21 and 0.13 mM, respectively.ConclusionThe data presented in this paper demonstrated the potential of ATPS as a versatile and scaleable process for downstream processing of recombinant PheDH
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Page 123BackgroundThe effect of ovariectomy and accompanying sudden loss of circulating gonad hormones on spatial learning performance in the young adult rats was examined. We hypothesized that spatial learning and memory in a considerable number of women who undergo a surgical menopause and estrogen deprivation before their natural menopause be impaired.MethodsIn this study, we used 26 Wistar rats (approximately five months of age) and divided them into two groups: intact and ovariectomized (OVX). They were tested for spatial reference memory in Morris water maze 6 weeks after OVX.ResultsThe results showed that the performance of OVX group in the water maze was significantly lower than the control group. Although, mean path length decreased across blocks in both groups, OVX rats had significantly longer path length than controls across blocks 2-6 (P<0.05). OVX rats had lower percent of total time spent in target quarter than controls in probe trials (P<0.05). Body weight gain was significant only in OVX group during the experiment (P<0.05). Plasma estrogen significantly decreased after OVX (P<0.05).ConclusionThis finding provides further evidence for the role of estrogen, a gonadal steroid hormone, in the manipulation of functions related to learning and memory. It is suggested that estrogen loss following OVX impaired spatial reference memory in young adult rats. Our results suggest that it is necessary to protect women who undergo a surgical menopause before their natural menopause from cognition impairments.
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Page 129BackgroundAmiodarone is a drug that is used for treatment of cardiac arrhythmia after cardiac ischemia. This drug as b blocker decreases arrhythmia rate but it has many side effects on different tissues. Since there are rare reports about changes of lacrimal glands, this research has been carried out to study the morphological and ultrastructural changes of lacrimal gland cells after amiodarone administration.MethodsMale rabbits (n = 14) were divided into control and experimental groups. Experimental group were intra peritoneally injected with a daily single dose of 80 mg/kg amiodarone for two weeks. The control group only received normal saline. At the end of the injection period, the two groups were anesthetized and perfused with Karnovsky''s fixative. The lacrimal glands were removed, fixed and then prepared for light and electron microscopic studies. Quantitative studies on lacrimal gland cell micrographs were performed by point counting method. The results were statistically compared between the two groups.ResultsLight microscopic observation showed many secretory granules in the cytoplasm of the lacrimal gland cells, which were also seen in the lumen of acini. Ultrastructure study of these cells showed the presence of inclusions in their cytoplasm with homogenous and dense structure. In quantitative analysis, the volume fractions (Vv) of mitochondria and nucleus to the cell showed no differences between the two groups but the Vv of euchromatin to the nucleus was different (P<0.05).ConclusionThe presented results show adverse effects of amiodarone on rabbit lacrimal gland cells