Iranian Biomedical Journal
Volume:14 Issue: 4, Oct 2010

Members of the eukaryotic Hsp90 family function as important molecular chaperones in the assembly, folding and activation of cellular signaling in development. Two hsp90 genes, hsp90 alpha and hsp90 beta, have been identified in fish and homeothermic vertebrates but not in poikilothermic vertebrates. In the present study, the expression of hsp90 alpha and hsp90 beta genes in Xenopus laevis, which is phylogenetically positioned between zebrafish and mammals, has been addressed. Partial Xenopus hsp90 alpha and hsp90 beta cDNA were identified and isolated using RT-PCR, and a full-length Xenopus hsp90 beta cDNA was isolated from an embryonic cDNA library. Northern-blot analysis was used to study the expression of hsp90 alpha and hsp90 beta genes in total RNA of the embryos and in situ hybridization was used to compare the expression of these genes with that of hsp70 and MyoD genes in Xenopus embryogenesis. Northern-blot analysis revealed that the hsp90 beta gene was strongly expressed constitutively at all stages of embryogenesis, but weakly induced following the heat shock. In contrast, the hsp90 alpha gene was weakly expressed in embryos at control temperature, but strongly up-regulated following heat shock. In situ hybridization results showed that hsp90 alpha gene was observed predominantly in cells of the developing somite. Microscopic sections showed that hsp90 alpha and MyoD mRNA are expressed in similar regions in somite and this pattern was distinct from that of hsp70 and hsp90 beta. These data support the hypothesis that the presence of hsp90 alpha and hsp90 beta genes is conserved among vertebrates, and these genes are differentially regulated in a tissue, stress, and development stage-specific manner.

Most cancer cells become resistant to anti-cancer agents. In the last few years, a new approach for targeted therapy of human cancer has been developed using immunotoxins which comprise both the cell targeting and the cell killing moieties. In the present study, the recombinant Shiga toxin A1 subunit fused to human granulocyte-macrophage colony stimulating factor (A1-GM-CSF), previously produced in E. coli, was further characterized. The recombinant protein could cause 50% cytotoxicity and induced apoptosis in cells bearing GM-CSF receptors. The non-specific toxicity of the fusion protein was assessed in C57BL/6 and BALB/c mice. No mortality was observed in either group of mice, with different concentration of fusion protein. The lymphocyte proliferation assay, induction of specific IgG response and a mixed (Th1/Th2) response were observed only in BALB/c mice. The mixed response in BALB/c mice (Th1/Th2) could be explained on the basis of the two components of the fusion protein i.e. A1 and GM-CSF.

Recent clinical studies of treating traumatic brain injury (TBI) with autologous adult stem cells led us to compare effect of intravenous injection of bone marrow mesenchymal stem cells (BMSC) and bone marrow hematopoietic stem cell mobilization, induced by granulocyte colony stimulating factor (G-CSF), in rats with a cortical compact device. Forty adult male Wistar rats were injured with controlled cortical impact device and divided randomly into four groups. The treatment groups were injected with 2 × 106 intravenous bone marrow stromal stem cell (n = 10) and also with subcutaneous G-CSF (n = 10) and sham-operation group (n = 10) received PBS and «bromodeoxyuridine (Brdu)» alone, i. p. All injections were performed 1 day after injury into the tail veins of rats. All cells were labeled with Brdu before injection into the tail veins of rats. Functional neurological evaluation of animals was performed before and after injury using modified neurological severity scores (mNSS). Animals were sacrificed 42 days after TBI and brain sections were stained by Brdu immunohistochemistry. Statistically, significant improvement in functional outcome was observed in treatment groups compared with control group (P<0. 01). mNSS showed no significant difference between the BMSC and G-CSF-treated groups during the study period (end of the trial). Histological analyses showed that Brdu-labeled (MSC) were present in the lesion boundary zone at 42nd day in all injected animals. In our study, we found that administration of a bone marrow-stimulating factor (G-CSF) and BMSC in a TBI model provides functional benefits.

Role of nitric oxide (NO) in morphine-induced conditioned place preference (CPP) has already been proposed in the rat medial septum (MS), but no molecular evidence has been provided to clear this fact. Effects of intraseptal injections of L-arginine and/or NG-nitro-L-arginine methyl ester (L-NAME) on morphine place conditioning in Wistar rats were examined. Morphine (2.5-7.5 mg/kg) was injected s.c. using a three-day schedule of an unbiased place preference. All of the brain samples were examined histochemically by nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d), the main marker for NO activation. Morphine induced a significant CPP in the rats. Single injections of L-arginine or L-NAME (0.3, 1.0 and 3.0 µg/rat) did not induce CPP. In addition, co-administration of morphine (5.0 mg/kg) with L-arginine or L-NAME (0.3, 1.0 and 3.0 µg/rat) did not affect morphine response. However, administration of L-arginine (0.3, 1.0 and 3.0 µg/rat) prior to morphine conditioning testing enhanced the expression of morphine response. Moreover, pre-injection of L-NAME (0.3, 1.0 and 3.0 µg/rat) to L-arginine (0.3 µg/rat) did not reverse the response to the agent. The expression of NADPH-d was observed in the rat brain samples treated by L-arginine. A decreased expression of NADPH-d was also observed in rats pre-injected by L-NAME. This finding strongly suggests that NO system in the rat MS has an impact on the expression of morphine rewarding, and that the NO participates in place conditioning induced of morphine.

This work aimed to show and compare the degradation time of some of cartilage extracellular matrix components using an in vitro model for cartilage degradation induced by interleukin-1α. It is known that elucidation of molecular events under Interleukin-1α induction of bovine nasal cartilage could obtain useful data to understand more about involving mechanisms for tissue breakdown in joint disease. The cartilage was taken from an adult bovine in a local slaughterhouse. After removing the whole perichondrium, the equal 2 mm diameter pieces of bovine nasal cartilage were punched out and cultured in Dulbecco''s modified Eagle''s medium DMEM with or without 10 ng/ml Interleukin-1α for 24 days. Each 3 days, the media were removed and exchanged by fresh media, and the removed media were stored in -20C. Sodium dodecyl sulphate/polyacrylamid-gel electrophoresis SDS-PAGE and Western-blot methods were used for analyzing the samples. The first fragment of fibromodulin (FM) was seen at day 6 and further fragments were appeared at day 18. Cartilage oligomeric matrix protein (COMP) releasing was as a successive pattern during culture period and the first fragment was found at day 6. Collagen IX fragments were seen at day 9 and in a progressive pattern until the end of the study. This study shows that FM and COMP could be considered as the suitable candidates for studying the mechanisms that participate in the cartilage degradations.

Morphine and lidocaine are known to influence the perception of pain. The present study sought to determine the influence of local administration of morphine on lidocaine-induced analgesia in morphine non-dependent (MND), morphine dependent (MD) and morphine withdrawal (MW) animals. Adult male Wistar rats were divided into four groups: Control, MND, MD and MW rats. Lidocaine (0.5, 1 and 2%) and morphine (200, 400 and 800 µg) were injected in the plantar surface of the right paw. MD animals received chronic oral morphine (0.1, 0.2, 0.3 and 0.4 mg/ml in their drinking water) for 20 days. Twenty four hours before experiment, the animals in the MW group were deprived of morphine in their drinking water (physical dependence was observed by precipitating an abstinence syndrome with naloxone 2 mg/kg i.p.). Analgesia was assessed using hot-plate apparatus. Morphine (400 µg) and lidocaine (2%) produce local analgesia in MND group. In MND rats, non-analgesic doses of each drug (200 µg morphine and 1% lidocaine) were used in combination and produced analgesia. In MD animals, all doses of lidocaine produced analgesia, while in MW animals, it failed to produce analgesia. In this situation, local administration of morphine could eventually influence the analgesic effect of lidocaine. Opioid withdrawal is one of the most common problems in clinic. This study determined the analgesic effect of lidocaine in MW animals in which lidocaine had no analgesic effect. In this regard, local administration of morphine with combination of lidocaine could probably produce an effective analgesia.

Apoptosis is a process that plays an important role during early stage of implantation. The aim of this study was to investigate the incidence of apoptosis in mice endometrium after ovarian stimulation at implantation period. NMRI female mice were divided into two groups: 1) control group, which were rendered pseudopregnant by vaginal stimulation and 2) experimental group, which were stimulated using an intrapritoneal injection of 10 IU hMG followed by another injection of 10 IU hCG after 48 h. In the evening of the second injection, the mice were rendered pseudopregnant the same as control group. Samples were obtained from 1/3 middle part of uterine horns during implantation period. Apoptosis was assessed in two groups at implantation period using light and electron microscopic studies, TUNEL staining and semiquantitative RT-PCR. Our morphological and ultrastructural results showed apoptosis in both groups, while TUNEL analysis showed that the percentage of TUNEL-positive cells was higher in stimulated group than in the control group (P≤0.05). The expression of P53, Fas and FasL mRNA was similar in two groups but Bax and Bcl2 were much higher in control group than in the stimulated group (P≤0.05). The ratio of Bax/Bcl2 expression was much higher in stimulated group than in the control group (P≤0.05). The ovarian stimulation could change the expression of some apoptosis-related genes and enhance the incidence of endometrial apoptosis at implantation period; thus, it could affect on the implantation rate and endometrial receptivity.

“Teucrium polium L.” (TP) has been long recommended in Iranian folk medicine for its anti-diabetic activities. We attempt here to evaluate the effect of TP extract on insulin secretion in rat pancreas. Rat pancreas was isolated in situ and perfused with Krebs solution containing low glucose (LG, 2.8 mM) or high glucose (HG, 16.7 mM) as perfusate. The aqueous extract (Aq. E) and methanolic extract (Met. E) of TP aerial parts and two partition fractions of Met. E were added to perfusate to evaluate insulin release. Diazoxide (DZX) and verapamil (VPM) were also used for assessing the probable mechanism of the effects. In each experimental group, the peak and baseline of insulin levels in effluent samples were compared. The GC/MS analysis was carried out to detect active ingredients in the extracts. Adding Met. E to the LG caused a significant increase (P<0.05) in insulin release from the basal level of 0.17 ± 0.05 µg/l to a peak value of 3.94 ± 1.29 µg/l. when Met. E was introduced to the HG, there was a further protracted stimulation of insulin release from 2.15 ± 1.35 µg/l to 6.16 ± 0.52 µg/l. Both DZX and VPM when added separately to the LG, led to inhibition of Met. E induced insulin secretion. The Aq. E and fractions had no significant effect on insulin secretion. Only in the Met. E, the component 5-hydroxy-4'',7-dimethoxyflavone (apigenin-4'',7-dimethylether) was detected. It can be concluded that the insulinotropic properties of TP extracts can be attributed to the presence of apigenin existing only in Met. E, but not in Aq. E and fractions. Moreover, certain types of K+ and Ca2+ channels take part in this effect.