Purinergic receptor expression and potential association with human embryonic stem cell-derived oligodendrocyte progenitor cell development

Objective: Due to recent progress in production of human embryonic stem cell-derived oligodendrocyte progenitor cells [hESC-OPCs] for ameliorating myelin disease such as multiple sclerosis [MS] and the role of purinergic signaling in OPCs development, we avaluated the profile of purinergic receptors expression during development of OPCs from hESC

Materials and Methods: In this experimental study, we used reverse transcription and quantitative polymerase chain reaction [RT-qPCR] to obtain more information about potential roles of purinergic receptors during in vitro production of hESC-OPCs. We first determined the expression level of different subtypes of purinergic receptors in hESCs, embryoid bodies [EBs], and hESC-OPCs. The effects of A1adenosine receptor [A1AR] activation on hESC-OPCs development were subsequently examined

Results: hESCs and OPCs had different mRNA expression levels of the AR subtypes. ARs mRNA were expressed in the EB stage, except for A2AAR. We observed expressions of several P2X [P2X1, 2, 3, 4, 5, 7] and P2Y [P2Y1, 2, 4, 6, 11-14] genes in hESCs. hESC-OPCs expressed different subtypes of P2X [P2X1, 2, 3,4,5,7] and P2Y [P2Y1, 2, 4, 6, 11-14]. Except for P2X1 and P2X6, all other P2X and P2Y purinergic receptor subtypes expressed in EBs. We also indicate that A1AR might be involved in modulating gene expression levels of cell cycle regulators in an agonist and/or dose-dependent manner

Conclusion: Elucidation of the expression pattern of purinergic receptors and the effects of different subtypes of these receptors in hESC-OPCs may have a promising role in future cell-based therapy or drug design for demyelinating disease

Focal injection of Ethidium bromide as a simple model to study cognitive deficit and its improvement

Introduction: Memory and cognitive impairments are some of devastating outcomes of Multiple Sclerosis [MS] plaques in hippocampus, the gray matter part of the brain. The present study aimed to evaluate the intrahippocampal injection of Ethidium Bromide [EB] as a simple and focal model to assess cognition and gray matter demyelination

Methods: Thirty Wistar rats were divided into three groups: control group, which received saline, as solvent of EB, into the hippocampus; and two experimental groups, which received 3 microL of EB into the hippocampus, and then, were evaluated 7 and 28 days after EB injection [n=10 in each group], using a 5-day protocol of Morris Water Maze [MWM] task as well as Transmission Electron Microscopy [TEM] assay

Results: Seven days after EB injection, the behavioral study revealed a significance increase in travelled distance for platform finding in the experimental group compared to the control group. In addition, the nucleus of oligodendrocyte showed the typical clumped chromatin, probably attributed to apoptosis, and the myelin sheaths of some axons were unwrapped and disintegrated. Twenty-eight days after EB injection, the traveled distance and the time spent in target quadrant significantly decreased and increased, respectively in experimental groups compared to the control group. Also, TEM micrographs revealed a thin layer of remyelination around the axons in 28 days lesion group

Discussion: While intracerebral or intraventricular injection of EB is disseminated in different parts of the brain and can affect the other motor and sensory systems, this model is confined locally and facilitates behavioral study. Also, this project could show improvement of memory function subsequent to the physiological repair of the gray matter of the hippocampus

Fibroblast growth factor-2 enhanced the recruitment of progenitor cells and myelin repair in experimental Demyelination of rat Hippocampal formations

Objective: Hippocampal insults have been observed in multiple sclerosis [MS] patients. Fibroblast growth factor-2 [FGF2] induces neurogenesis in the hippocampus and enhances the proliferation, migration and differentiation of oligodendrocyte progenitor cells [OPCs]. In the current study, we have investigated the effect of FGF2 on the processes of gliotoxin induced demyelination and subsequent remyelination in the hippocampus

Materials and Methods: In this experimental study adult male Sprague-Dawley rats received either saline or lysolecithin [LPC] injections to the right hippocampi. Animals received intraperitoneal [i.p.] injections of FGF2 [5 ng/g] on days 0, 5, 12 and 26 post-LPC. Expressions of myelin basic protein [Mbp] as a marker of myelination, Olig2 as a marker of OPC proliferation, Nestin as a marker of neural progenitor cells, and glial fibrillary acidic protein [Gfap] as a marker of reactive astrocytes were investigated in the right hippocampi by reverse transcriptase-polymerase chain reaction [RT-PCR]

Results: There was reduced Mbp expression at seven days after LPC injection, increased expressions of Olig2 and Nestin, and the level of Gfap did not change. FGF2 treatment reversed the expression level of Mbp to the control, significantly enhanced the levels of Olig2 and Nestin, but did not change the level of Gfap. At day-28 post- LPC, the expression level of Mbp was higher than the control in LPC-treated animals that received FGF2. The levels of Olig2, Nestin and Gfap were at the control level in the non-treated LPC group but significantly higher in the FGF2-t reated LPC group

Conclusion: FGF2 enhanced hippocampal myelination and potentiated the recruitment of OPCs and neural stem cells [NSCs] to the lesion area. Long-term application of FGF2 might also enhance astrogliosis in the lesion site

Comparing the effects of small molecules BIX-01294, Bay K8644, RG-108 and valproic acid, and their different combinations on induction of pluripotency marker-genes by Oct4 in the mouse brain

Every cell type is characterized by a specific transcriptional profile together with a unique epigenetic landscape. Reprogramming factors such as Oct4, Klf4, Sox2 and c-Myc enable somatic cells to change their transcriptional profile and convert them to pluripotent cells. Small molecules such as BIX-01294, Bay K8644, RG-108 and valproic acid [VPA] are reported as effective molecules for enhancing induction of pluripotency in vitro, however, their effects during in vivo reprogramming are addressed in this experimental study. In this experimental study, Oct4 expressing lentiviral particles and small molecules BIX-01294, Bay K8644 and RG-108 were injected into the right ventricle of mice brain and VPA was systematically administered as oral gavages. Animals treated with different combinations of small molecules for 7 or 14 days in concomitant with Oct4 exogenous expression were compared for expression of pluripotency markers. Total RNA was isolated from the rims of the injected ventricle and quantitative polymerase chain reaction [PCR] was performed to evaluate the expression of endogenous Oct4, Nanog, c-Myc, klf4 and Sox2 as pluripotency markers, and Pax6 and Sox1 as neural stem cell [NSC] markers. Results showed that Oct4 exogenous expression for 7 days induced pluripotency slightly as it was detected by significant enhancement in expression of Nanog [p<0.05]. Combinatorial administration of Oct4 expressing vector and BIX-01294, Bay K8644 and RG-108 did not affect the expression of pluripotency and NSC markers, but VPA treatment along with Oct4 exogenous expression induced Nanog, Klf4 and c-Myc [p<0.001]. VPA treatment before the induction of exogenous Oct4 was more effective and significantly increased the expression of endogenous Oct4, Nanog, Klf4, c-Myc [p<0.01], Pax6 and Sox1 [p<0.001]. These results suggest VPA as the best enhancer of pluripotency among the chemicals tested, especially when applied prior to pluripotency induction by Oct4

Characterization and phylogenetic analysis of Magnaporthe spp. strains on various hosts in Iran

Populations of Magnaporthe, the causal agent of rice blast disease, are pathotypically and genetically diverse and therefore their interaction with different rice cultivars and also antagonistic microorganisms are very complicated. The objectives of the present study were to characterize phylogenetic relationships of 114 native Magnaporthe strains, isolated from rice and different weeds in the North region of Iran and to study their interaction with the fungal and bacterial antagonists. Phylogenetic studies [lineage structure, cluster analysis and gene flow] were performed using AFLP DNA fingerprinting. Antagonistic effects of the native fungal [Trichoderma harzianum] and bacterial [Bacillus subtilis and Pseudomonas fluorescens] against Magnaporthe strains were assayed at In vitro levels using factorial experiments based on completely randomized designs [CRD] and mean comparison tests. In total, 39 clonal lineages including 48 haplotypes were identified among the strains of M. grisea and designated here as A-Z. AFLP marker could finely differentiate the strains isolated from various hosts. The strains isolated from Setaria sp. were much close to those from rice [Oryza sativa L.]. Magnaporthe strains isolated from Digitaria sp. showed higher genetic variation than other strains. Genetic distances revealed by the AFLP markers could be finely differentiated M. grisea and M. salvinii. The rate of gene flow was an evidence of low gene transferring among Magnaporthe populations and the existence of a complex species for Magnaporthe strains. The fungal and bacterial antagonists showed different reactions against different Magnaporthe strains. These results confirmed high genetic diversity between the Magnaporthe strains which was also previously determined by the AFLP experiments. It was concluded that the Magnaporthe populations in Iran have a complex genetic diversity, and therefore, to achieve an efficient control of the different strains and pathotypes of Magnaporthe sp, it is necessary to use different bacterial and fungal biocontrol agents as a dynamic and integrated control system

[Tolerance to anti-nociceptive effects of sodium-salicylate and morphine decreases adenosine deaminase activity in the rat hippocampus]

Adenosine has been considered as a fine-tuner of the neurotransmitters in the nerve system. Adaptive changes in the brain adenosine system occur in some patho-physiological situations such as chronic exposure to morphine. In this study, the adaptive changes in the adenosine deaminase activity as a key enzyme in the adenosine metabolism that converts adenosine to inosine and ammonia, irreversibly, due to morphine dependence and tolerance to anti-nociceptive effects of sodium-salicylate were investigated. Morphine dependence was induced by morphine administration in tap water [0.4mg/ml for 24 days]. Tolerance to sodium-salicylate was induced by 6 i.p. injection [1 injection/day] of sodium-salicylate. Tolerance to antinociceptive effects of sodium-salicylate was measured by tail flick [TF] and hot plate [HP] tests. Right hippocampus was dissected, homogenized at phosphate buffer, centrifuged and then the supernatant fraction was isolated. Protein content of the samples was measured by the Bradford method. Hippocampus adenosine deaminase activity was measured by a calorimetric method of enzyme assay which is based on the direct measurement of the produced ammonia from excessive adenosine degradation by adenosine deaminase. Daily injection of Sodium-salicylate produced antinociception in early days by latency increase rather than saline injection [P<0.0001] but in the following days this antinociceptive effect progressively decreased so at the day 5 and 6 following injection it was similar to saline [P>0.05]. Injection of morphine [5mg/Kg] at the day 7 showed more increase in the latency of saline injected rather than sadium-salicylate injected [P<0.001]. Adenosine deaminase activity was significantly higher in sucrose administrated rather than chronic morhine administrated [P<0.05] and in saline injected rather than sodium-salicylate injected [P<0.05]. Single injection of sodium-salicylate and its control shows the same activity. There was no significant difference in enzyme activity of morphine dependent with sadium-salicylate tolerance [P>0.05]. This decline in the adenosine deaminase activity may be related with adaptation in brain adenosine system subsequent of dependent to morphine or tolerance to sodium-salicylate

Identification of some molecular traits in fluorescent pseudomonads with antifungal activity

We assessed a collection of 47 fluorescent Pseudomonas spp., some with known biological control activity against certain soil-borne phytopathogenic fungi such as, Macrophomina phaseolina, Rhizoctonia solani, Phytophthora nicotianae var. parasitica, Pythium sp. and Fusarium sp. in vitro and the potential to produce known secondary metabolites such as, siderophore, HCN and protease. The results indicated that 66%, 40.42%, 63.82%, 48.94% and 27.65% of strains revealed antagonistic activity against R. solani, M. phaseolina, Pythium sp., P. nicotianae and Fusarium sp., respectively. Among the 47 strains, 76.59%, 97.87% and 17% produced protease, siderophore and HCN, respectively. In this survey, the detection of phlD and phlA genes was evaluated with a PCR-based assay. We detected phlD in strains P-5, P-32, P-47, and phlA in strains P-5, P-18, P-34 and P-35. Strain CHA0 was used as positive control for the detection of both genes. Overall, there was no obvious link between inhibition of fungal growth in vitro and production of the antifungal metabolites or existence of phlD and phlA genes. Characterization of fluorescent pseudomonads with potential to produce of 2, 4-diacetylphloroglucinol will further enhance our knowledge of their function in the suppression of root diseases

effect of swim stress on morphine tolerance development and the possible role of nitric oxide in this process

It has been shown that stress and chronic pain could prevent the development of tolerance to morphine analgesia, which appears to be related to the activation of hypothalamus-pitutitary-adrenal [HPA] axis, activation of neuroendocrine systems and changes in neurochemical levels. Moreover, the involvement of nitric oxide [NO] in the development of tolerance to morphine analgesia has been implicated. In the present study, we have tried to investigate the effect of swim stress, as a painless kind of stress, on the development of tolerance to find out whether the inhibition of tolerance is mediated by the direct effect of pain on the pain conduction pathway, or by its stress aspect. Besides, we evaluated the probable interactions between swim stress, nitric oxide level and the development of morphine tolerance. Adult male Wistar rats, weighing 180-220 g, were used in all these experiments. The experimental groups received chronic morphine [20 mg/kg, i.p], swim stress in 20°C water bath [4 min], or a combination of swim stress and chronic morphine [20 mg/kg, i.p], each for 4 days, while the first control group received saline [1 ml/kg, i.p] for 4 days. On the 5th day, all the experimental and control groups received a single dose of morphine [10 mg/kg i.p]. The second control group received saline for 5 days. The intact group received only one single dose of morphine [10 mg/kg, i.p]. All the mentioned groups were subjected to tail-flick and formalin tests on the 5th day. Other experimental groups were subjected to the assay for measuring nitrite as an indicator of NO, using the Griess method. Our results showed that co-administration of swim stress with chronic morphine prevented the development of morphine tolerance and the level of NO increased in the presence of swim stress [p<0001]. The combination of morphine and swim stress significantly decreased NO production in comparison with the chronic morphine administered group [p<0.001]. These data suggest that the activation of HPA axis and consequently the suppression of [NO] production induced by chronic morphine, lead to the inhibition of morphine tolerance