Journal of Cell and Molecular Research

Antioxidant activities protect the cell against oxidative agents that are constant metabolic by-products. The aim of this study was to investigate the relationship between harvesting time of Thymus transcaspicus and its antioxidant activities. The plant samples were harvested 5 times in different growth phases from 17 April to 22 July 2008, and its antioxidant activity was studied using the ferric reducing antioxidant power (FRAP), 1,1- diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity, and β-carotene bleaching (BCB) assays. The results of FRAP assay indicated that the reduction activity of the plant was in the highest level in stage 5 of sampling. The result of DPPH assay showed that the crude extract of the plant was more capable of DPPH radical scavenging in stage 2. The highest level of gallic acid and quercetin in the crude extract of T. transcaspicus was determined as 85.29 ± 6.22 mg and 18.88 ± 0.9 mg in stage 2, respectively. Therefore, stage 2 was the optimum time to harvest the T. transcaspicus.

Trehalose is the alpha, alpha-1,1-linked glucose disaccharide. Its metabolism is found in a wide variety of organisms and is seen as evolutionary old. Trehalose metabolites are however present at only very low concentrations and their role in plants are not understood. The physiological effects of 100 mM trehalose on growth and carbon allocation in seedlings are characterized in this paper. Trehalose feeding to Arabidopsis thaliana elicits strong responses. On 100 mM trehalose, seedlings germinate and extend cotyledons but fail to develop primary leaves. The primary roots do not grow beyond 2-3 mm and there is not any starch in root tips. In light, growth arrest on 100 mM trehalose can be rescued by exogenous supply of metabolisable sugar. Trehalose feeding results in anthocyanin accumulation and chlorophyll reduction. Trehalose causes cells of the root extension zone to swell and lysis. Trehalase expression analysis showed, WT seedlings grown on trehalose have 10-fold induced AtTRE1 expression compared to sorbitol treatment.

Asafoetida is the dried latex exuded from the living underground rhizome or tap root of Ferula assafoetida. Antibacterial characteristic of asafoetida was shown using the circular zone diameter of bacterial growth inhibition by disk-diffusion method on two gram positive and three gram negative bacteria. Then, the bacterial genomic DNA damage, induced by F. assafoetida latex, was demonstrated using the comparison of random amplification of polymorphic DNA profiles generated by polymerase chain reaction of control and treated bacterial genomes. The results showed that the number of primers that produced bands in each bacterium were higher in control samples compared to those treated with asafoetida. This and the absence or presence of bands between controls and treatments confirm rearrangements and DNA damage in the priming binding sites of bacterial genome.

Identification of genes affecting energy balance, milk yield and feed intake is an interesting area of researches in animal breeding. Leptin gene polymorphism is associated with key economic affair. Considering rich resources for animals, in our country, accomplishing a few assays to identify a gene that controls her traits with molecular genetics, and identifying the candidate genes in sheep breeds using DNA test can greatly help to her breeding progress. For analyzing Leptin gene polymorphism and its association with growth traits in Kermani sheep, blood samples of 120 sheep of both genes rearing at breeding centre of Shahre Babak were taken. In addition growth traits were measured. PCR was performed to amplify 275 bp fragments of exon 3 from Leptin gene. Then Single Strand Conformation Polymorphism (SSCP) of PCR product was performed and Leptin band patterns (genotypes) were obtained using acrylamid gel and silver staining. For Leptin gene 10 genotypes including A/A, C/C, A/B, A/C, A/B/C, A/B/E, A/B/F, A/C/F, A/B/D/E and A/B/C/F were obtained. The results of this study showed that the growth traits are significantly affected by the genotypes. Accordingly, A/B/E, A/C, A/B/C/F and A/B/C/F genotypes had higher body weight at 3, 6, 9 and 12 months of ages respectively. The animals with A/B, A/B, A/B/F and A/B/D/E genotypes had the smallest body weight at 3, 6, 9 and 12 months of ages respectively. It is suggested that polymorphism in Leptin gene loci can be used as a selective criterion to improve growth traits in Kermani sheep.

Regeneration is a biological phenomenon, which takes place via two main mechanisms: first, dedifferentiation of mature cells followed by their differentiation into functional new cells and second, activation of endogenous somatic stem cells for regeneration of damaged or lost tissues. One of the best examples of healing process in mammals is the regeneration of damaged pinna in rabbits by blastema tissue. The aim of present study was to investigate culture requirements, proliferative properties and expression of some stemness factors in cells derived from regenerating blastema tissue obtained from rabbit pinna in vitro. The regenerating tissues were obtained from male New Zealand white rabbits by double punching of the pinna and cell culture conditions were set to derive and enrich the self renewing cells for further characterisation. The cells were subjected to survival and growth examinations in vitro, and expression of several stemness factors was studied in these cells using reverse transcription polymerase chain reaction (RT-PCR). Results revealed that the derived cells are rather immortal, as they have been growing for more than 120 passages in culture up until this report. Furthermore, RT-PCR and flow cytometry analyses showed that these cells express a number of stemness related genes including Oct4 and Sox2. In conclusion, in this study, stem like cells were derived from blastema tissue of rabbit ears for the first time, showing great self renewing capacity, which provides a suitable in vitro model for regeneration studies. Moreover, they could be considered as a good source of stem like cells for future applications.

Microglia, the sentries of the brain, is highly implicated in neurodegeneration as in neuroprotection. Chronic microglial activation endangers neuronal survival through the release of various potentially neurotoxic mediators including Nitric Oxide (NO). Thus, negative regulators of microglial activation have been considered as potential therapeutic candidates to target neurodegeneration, such as those in Alzheimer's and Parkinson's diseases and even in chronic epileptic syndromes. Bromelain, a mixture of cysteine proteases, derived from pineapple stem, has been reported to have anti-inflammatory and immunomodulatory effects. Neonatal rat primary microglial cells were isolated from the brain according to the Floden's method. The purity of the cultures was determined by immunostaining with an OX-42 antibody which showed a purity greater than 95%.The activation profile of microglia was investigated by determining the effects of Bromelain (1, 10, 20, 30, 40 and 50 µg/ml) on the level of neurotoxin, NO, mitochondrial activity and morphological changes in treated microglia with lipopolysaccharide (LPS) (1µg/ml), as an endotoxin. Our results showed that pretreatment of primary rat microglia with bomelain (30 µg/ml), decreased the production of NO induced by LPS (1µg/ml) treatment in a dose-dependent manner, which prevented the deramification of microglia and its phagocytic morphology. Moreover, bromelain does not show cytotoxicity at any of the applied doses, suggesting that the anti-inflammatory effects of bromelain are not due to the cell death. In conclusion, Bromelain reduces the NO synthesis in vitro by potentially exerting its anti-inflammatory effects. Bromelain naturally found in pineapple stem, can be considered as a useful agent for neuroprotection and alleviation of symptoms in neurodegenerative diseases.

Transgenic and non transgenic Nicotiana tabacum L (cultivar Wisconsin) containing Ri T-DNA were treated with 0, 0.2 and 0.4 mgL -1 GA 3 in Murashig and Skoog medium. Some physiological parameters including shoot length, leaf area, number of auxiliary bud, fresh and dry weight, number and length of trichomes were measured. Shoot length, fresh weight and dry weight were increased but number of trichome did not change by GA 3 treatment. Chlorophyll, carotenoid and anthocyanin pigments of leaf were decreased. Auxin and gibberellic acid content of leaf and root were also measured. Exogenous GA 3 increased root auxin in the transgenic plants while it did not change in shoot. GA 3 treatment increased gibberellin content in both of root and shoot.

Saffron (Crocus sativus) is the most valuable and indigenous crop in Iran. The stigmas of flower are used as a popular natural flavouring, colouring and medicinal agent. However, the market suffers from frauds in this plant such as mixing with safflower petals due to high profit. Identification of these frauds with conventional and biochemical methods is difficult and low sensitive. Therefore, application of molecular markers such as random amplified polymorphic DNA (RAPD)/sequence characterized amplified regions (SCAR) is being considered as an alternative. In this study, DNA was extracted from dry stigmas of 5 Saffron accessions and dry petals of 7 safflower cultivars. RAPD reactions with ten 15-mer random primers resulted in two specific monomorphic bands (500 and 700 bp) for safflower, while they were absent in saffron accessions. PCR analysis with specific SCAR primers amplified two specific bands (414 and 589 bp) for safflowers in different combinations of saffron stigmas and safflower petals. This was the case with very low rates or 1% of safflower. Therefore, this method seems to be suitable for fraud identification of safflower petals in commercial saffron samples.

Equisetum telmateia (Equisetaceae) seems to have anti inflammatory and antioxidant properties. In the present study, the neuroprotective effects of organic and inorganic silica were investigated on spinal cord alpha motoneuron of rats after injury of sciatic nerve. After highly compression of sciatic nerve in 42 Wistar rats, the injured rats were divided into sham (n= 6) and two experimental groups which each were divided into 3 subgroups (n= 6). The first subgroups received 3, 6 or 9 injections (15 mg/kg/injection, ip) of horse tail extract and the second subgroups received 3, 6 or 9 injections (6 mg/kg/injection, ip) of sodium meta silicate, respectively. The first injection was made after sciatic nerve injury and the others by 72 hours intervals. After a month, the rats were sacrificed and their spinal cord lumber segment sampled, processed for histological preparation and analyzed stereologicaly (the dissector technique) for estimation of numerical density of alpha motoneurons. The results showed significant decrease in the numerical density of alpha motoneurons in shams (p< 0.05) and no significant differences between experimental and control groups. This may suggest the neuroprotective effects of silica on the survival of alpha motoneurons.

Due to the wide range of applications for ozone and its increasing use for medical and industrial purposes, studying its effects has become a very important line of research. The ozone has been suspected to be a carsinogen. Because of the increasing use of ozone, the human could be more and more exposed to this gas. In this study the effects of ozone inhalation on chromosomes and its clastogenic consequences have been investigated using in vivo micronucleus assay in bone marrow cells of treated rats. Animals were treated for 6 hours a day at 3 ppm of ozone during 10 consecutive days. The micronucleus assay was performed immediately and 11 days after the last exposure. The frequency of micronucleated polychromatic erythrocyte of bone marrow (MNPCE) increased in both groups compared to the control. Such increase confirmed the clastogenic effects of ozone. The elevated frequency of MNPCE did not decrease after 11 days of the last ozone exposure. Results indicate that ozone inhalation could induce persistent chromosomal damages even to bone marrow cells which were not in direct contact to it. Also, once more, the results confirmed the usefulness of the micronucleus assay in toxicological studies.

Study of the different aspects of protection against the exposure of ionizing radiation has always been an active area of research. High cost and toxicity of radioprotective drugs have limited their use. So, search for new drugs with a high degree of protection and lower cost and side effects seem a necessity. In this study radioprotective effect of aqueous as well as alcoholic extracts of the Mann of Cotoneaster nummularia( Shirkhesht), regarding their high accessibility and possibly low side effects, against 2 Gy Gamma irradiation, was analyzed using micronucleus assay on bone marrow cells of male mice (Balb/c). Different doses of 250, 500, 1000 mg/kg/BW for aqueous and 3750, 7500, 15000 mg/kg/BW for alcoholic extract of Shirkhesht were administered IP for five constitutive days prior to 2 Gy gamma irradiation. The result compared with the known radioprotective effect of vitamin E after the same treatment schedule. High frequency of micronucleus was observed in non treated gamma-exposed mice, which represented the clastogenic effect of irradiation. Vitamin E, aqueous and alcoholic extracts of Shirkhesht treated mice represented a 5.56, 3.32 and 2.1 times decrease in the gamma-induced micronucleus frequency respectively. The data suggest a radioprotective effect of shirhkesht compared to vitamin E.

Phosphoenolpyruvate Carboxykinase, encoded by the pepck gene, plays an important role in gluconeogenesis. It also seems to be important in metabolism of nitrogenous compounds in developing seeds of legumes, including amides and ureides which are then transformed into amino acids, necessary for the synthesis of storage proteins. In this research, pepck gene expression in mRNA level, in different genotypes of chickpea (Cicer arietinum L.), was determined. Two low protein genotypes (MCC291 and MCC373) and two high protein genotypes (MCC458 and MCC053) out of 20 chickpea genotypes were selected. Total RNA were extracted through different stages of seed development, and the expression of the pepck gene was estimated by semi-quantitative RT-PCR. The results of the RT-PCR showed that two isoforms of this gene are expressed in high protein genotypes, whereas in the low protein genotypes, the expression of these isoforms was not obvious. Also this method showed a differential expression of pepck gene in different stages of flowering and seed development. pepck gene is expressed in higher levels during the sheet formation and developing seeds compared to the flowering and seed formation stages. Probably, the differential expression of pepck gene is related to its possible role in metabolism of seed components, particularly in determination of the protein content of chickpea seeds.

Basement membrane of glomerular mesangium (BMG) is a thin membrane which helps to support the capillary loops in a renal glomerulus and type IV collagen is require for complete BM formation during glomerulogenesis. In this investigation specific antibody of type IV collagen has been used in light microscopy to study development of BMG of the embryonic and postnatal mouse glomerular mesangium. In this study, 20 female Balb/C mice were selected randomly and finding vaginal plug was assumed as day zero of pregnancy. 12 pregnant mice were sacrified by cervical dislocation in one of gestational days 13-18, their fetuses were fixed and serially sectioned. Immunohistochemical Study for tracing of collagen type IV in BMG was carried out. The same processes were carried out for kidneys preparation of pups on 5, 10, 15 and 20 days after birth (2 mothers for each day). The result of the present study revealed that collagen IV reaction was weak on day 15 of gestation. The amount of collagen increased continuously until next days of fetal life and primary of 10 days postnatal in BMG. After this period, collagen IV showed no significant change in newborns. These data indicate that collagen IV appears just during the glomerular vasculogenesis and because of continuity and glomerular endothelial cell differentiation, type IV collagen, is the major structural protein in BMG, have been implicated in these processes. Keywords: collagen IV, glomerular basement membrane, kidney, mouse

Osmotic stress is one of the major factors that significantly reduce yields in dry areas. Plants respond to this abiotic stress at physiological and molecular levels. Many genes are induced under stress conditions by transcription factors. Dehydration responsive element binding (DREB) protein is a subfamily of AP2/ERF transcription factors which control expression of many osmotic stress-inducible genes. In this study, 21 days old seedlings of Sardari cultivar, dry farming bread wheat transferred into hydroponics culture using Hoagland solution. Osmotic stress treatments performed with adding 100, 200 and 400 g/l poly-ethylene glycol 6000 to hydroponics culture to obtain –0.15, –0.49, and –1.76 MPa water potential, respectively. After the seedlings were withered and colorless, relative water content, dry weight, and photosynthesis measured. In addition, RTPCR, and cDNA sequencing carried out. Molecular analysis of DREB translated protein sequence performed by DNAMAN, BLASTN, Pfam and PROSITE software. Results showed that osmotic stress decreased relative water content, root and shoot dry weight and net photosynthesis rate in comparison to control, significantly (P < 0.05). Sequence alignment indicated 98% homology with other Triticum aestivum DREB protein mRNA. There was an AP2 domain in the translated protein with three -sheets and one -helix and contains the Val14 and Glu19 amino acids. An EST Sequence deposited in NCBI GenBank database with the accession number of ES466900.

Schizophrenia is a sophisticated mental disability which has affected nearly1.1% of people all over the world. According to recent researches, the key proteins triggered in the immune system are cytokines which might also be taking part in the pathogenesis of schizophrenia. The aim of this study was to evaluate the relationship between the-1082G/A and +874T/A polymorphisms of IL-10 and IFN-γ genes, respectively, in patients with schizophrenia. Total of 94 schizophrenic patients and 97 individuals as control samples were enrolled in this study. All samples were genotyped by amplification mutation refractory system-polymerase chain reaction (ARMS PCR) for candidate SNPs in IFN-γ and IL-10 genes. No significant association was found among various genotypes of IFN-γ and IL-10 in selected SNPs with risk of schizophrenia. As well as there was no significant variation in allelic frequency of IFN-γ and IL-10 genes with the risk of disease. These data suggest that the-1082G/A of IL-10 and +874T/A IFNγ genes are not involved in the development of schizophrenia risk. To validate of this data, suggesting more studies in diverse populations with larger sample size.

Wound healing is a complex biological process in which many molecules, including microRNA molecules, play an essential role in its regulation. It is well-established that reducing miR-155 expression can accelerate wound healing. This study investigated the effect of using nanoparticles loaded with vitamin C on miR-133, collagen I, and III expressions. In this study, first, nanoparticles of albumin protein were produced and then loaded with vitamin C. 3T3 mouse fibroblast cells were affected by these nanoparticles, and cell behavior was investigated to evaluate the toxicity and appropriate doses. In addition, the expression of collagen I and III genes was studied. The results showed that nanoparticles containing vitamin C in 20 µg/ml concentration had a positive effect on collagen I and III expressions compared to the control group. Moreover, we observed a decrease in the expression of miR-133 in comparison to the control group. Therefore, according to the results of this study, it can be argued that nanoparticles containing vitamin C can significantly decrease the expression of the miR-133 gene and lead to collagen I and III gene overexpression in fibroblasts cells, which is directly effective in wound healing.

Applying microorganism in oil recovery has attracted attentions recently. Surfactin produced by Bacillus subtilis is widely used industrially in a range of industrial applications in pharmecutical and environmental sectors. Little information about molecular mechanism of suffactin compound is available. In this study, we performed promoter and network analysis of surfactin production genes in Bacillus subtilis subsp. MJ01 (isolated from oil contaminated soil in South of Iran), spizizenii and 168. Our analysis revealed that comQ and comX are the genes with sequence alterations among these three strains of Bacillus subtilis and are involved in surfactin production. Promoter analysis indicated that lrp, argR, rpoD, purr and ihf are overrepresented and have the highest number of transcription factor binding sites (TFBs) on the key surfactin production genes in all 3 strains. Also the pattern of TFBs among these three strains was completely different. Interestingly, there is distinct difference between 168, spizizenii and MJ01 in their frequency of TFs that activate genes involve in surfactin production. Attribute weighting algorithms and decision tree analysis revealed ihf, rpoD and flHCD as the most important TF among surfactin production. Network analysis identified two significant network modules. The first one consists of key genes involved in surfactin production and the second module includes key TFs, involved in regulation of surfactin production. Our findings enhance understanding the molecular mechanism of surfactin production through systems biology analysis.

Cytotaxonomy is a branch of cytogenetics, devoted to the comparative study of karyological features for systematic and evolutionary purposes. Surely, awareness of chromosomal characters increases our knowledge in different fields of studies. In this study, cytogenetic analyses were performed in 92 Mus musculus specimens from 26 localities in Iran. Cytogenetic characteristics of the house mouse, Mus musculus, in Iran show that the chromosome number is 2n=40 and the arm number is NF=40. The karyotyping results indicated the presence of 20 Acrocentric (A) chromosome pairs. The L/S (r ratio) was between 2.0621 and 4.5862. The length of shortest chromosome, length of longest chromosome and mean of chromosomal length in different populations were between 2-3.58, 6.07-7.01 and 3.43-5.05 (μm), respectively. The results showed two distinct karyotypic formulae, namely cytotype B and cytotype C. Asymmetry indexes (AI, DI, As%, A, A2, A1 and Syi%) in all population except Birjand and Khash showed symmetry in chromosomes. In clustering methods using the matrix of symmetrical indexes similarities, four clusters were revealed, one for specimens of central and east of Iran, the second cluster for specimens from south and west of Iran, the third cluster was related to the eight specimens of Birjand and finally, the fourth cluster for two specimens of Khash locality.

Genetic diversity is one of the three levels of biodiversity. The aim of present study was to compare levels of genetic polymorphism between wild Bream populations using seven microsatellite loci. Genetic diversity was investigated by studying samples collected from two regions, the coast of Chamkhale and Bandaranzali of Gilan province. A total of seven microsatellite loci (MFW7, MFW26, Mcs1EH, Rser10, Bl1-153, Bl2-114 and IC654) were used. The average number of alleles in Chamkhale and Bandaranzali coast were 10 and 10.71 alleles, respectively. The numbers of effective alleles were 7.05 and 7.74 alleles in each population.Allele frequency was found to have declined in wild fish due to inbreeding and genetic drift. The mean of observed heterozygosity values were 0.66 and 0.70 in Chamkhale and Bandaranzali coast, respectively.Approximately all of loci showed deviation from Hardy-Weinberg equilibrium. The genetic similarity and distance between the two populations were 0.316 and 0.684, respectively.The Fst value was 0.024 that indicates the low genetic differentiation between the two locations which could be explained by the low number of alleles in two populations. Also, the Natural Migration (Nm) between two stations was obtained 16.30. According to the analysis, it seems that Abramis brama hasn᾽t a desirable genetic diversity in the investigated regions.

The prevalence of type 2 diabetes mellitus (T2DM) is rising dramatically in the Middle East, especially in the Islamic Republic of Iran, but the genetic basis of type 2 diabetes in Iran is poorly understood. Polymorphisms of hepatocyte nuclear factor-1α (HNF-1α) and glucagon-like peptide-1 receptor (GLP-1R) genes showed association with type 2 diabetes in several ethnic groups. In this study, we evaluated whether these markers confer susceptibility to T2DM in a diabetic population living in Mashhad (northeast of Iran). Genotyping of Ala98Val (HNF-1α) and Thr149Met (GLP-1R) was done by the restriction fragment length polymorphism-PCR (RFLP-PCR) method in the following groups: 1) early-onset diabetes (age at onset ≤ 35 years); 2) late-onset diabetes (age at onset > 35 years); and 3) control. Our results showed that CT (Ala/Val) genotype of HNF-1α was higher in the early-onset type 2 diabetic group compared to the controls but difference was not significant. We did not find the GLP-1R Thr149Met mutation in all participants. The prevalence of the HNF-1α (Ala98Val) and (GLP-1R) Thr149Met mutations has not been previously reported in Iranian participants. We conclude that these mutations are not a common cause of T2DM in our studied population.

An Agrobacterium-mediated transient gene expression assay was carried out in alfalfa (Medicago sativa) leaves for expression of a chimeric gene encoding a part of capsid protein of Foot and Mouth Disease virus called VP1. The plant leaves were transformed via agroinfiltration procedure. The presence of the foreign gene and its expression in transformed plants were evaluated by polymerase chain reaction (PCR), real time PCR, protein Dot blot and ELISA. Moreover, gene expression in the transformed leaves was quantified by ELISA method. The results obtained in this investigation indicated high level of gene expression in alfalfa leaves, showing that transient gene expression can be applied as an effective and time-saving procedure for the production of recombinant proteins. The procedures for transformation, detection of recombinant protein and its application for molecular experiments are described in the study.

There are different subtypes of brain tumors, classified according to the origin of the abnormally proliferated glial cells. Glioblastoma multiforma (GBM) is the grade 4 of brain tumors, gliomas, with the least life expectancy. microRNAs (miRNAs) are small, single stranded, non-coding RNAs with 20-25 nt length with post-transcriptional gene regulatory activities. An altered expression of miRNAs is linked to developmental disorders and some diseases, most importantly cancers. miR-21 is a well-known microRNA, overexpressed in almost all cancer types, including brain tumors. It targets several genes with vital roles in cellular pathways involve in proliferation, invasion and metastatic behaviors. Exosomes are 30-100 nm extracellular vesicles which are packed with various molecules, including miRNAs. Here, we suppressed miR-21 expression level in HEK-293T cells by transfecting them with the miRZip-21 vector. However, when U87-MG cells were cultured in the presence of exosomes isolated from conditioned medium of engineered HEK-293T cells derived exosomes, we did not observe any suppressing effect on host cells' miR-21 expression level. Moreover, by analyzing the effects of miRZip-21-enriched cell's conditioned media on three other brain cell lines including 1321N1, A-172 and DAOY, cell type-specific effects of exocrine miRZip-21 were revealed. These data suggested that cell lines from different brain tumor subtypes could exert different responses to microRNA-based therapies, based on their cellular origin and clinical behaviors.

MicroRNAs (miRNAs) are a group of short non-coding RNAs implicated in numerous fundamental cellular processes, and their disregulations have been linked to several pathologic conditions, mainly cancers. Determining tissue distribution of miRNAs is a prerequisite for understanding their exact functions during development, tissue homeostasis and abnormality. In situ hybridization is a powerful technique to delineate the sub-cellular localization and tissue distribution patterns of mRNAs as well as miRNAs. Due to the important role of miRNAs in tumorigenesis, we optimized an ISH technique for detection of two well-known miRNAs (miR-302 and miR-21) in formalin-fixed paraffin-embedded (FFPE) tumor samples along with a pluripotent embryonal carcinoma cell line, NTERA-2 (NT2). After fixation of cells on slides/sectioning of FFPE blocks, proteinase K digestion, probe concentration, antibody development and light sensitive color reaction were optimized for both the FFPE samples and cell line. Signals for U6 snRNA, as an internal control, were detected in the nuclei of the cells. MiR-21 and miR-302 expression was detected in the cytoplasm of FFPE samples of seminoma carcinoma and in NT2 cell line, respectively. In this study, we optimized ISH for miRNA detection in FFPE samples and NT2 cell line.

Environmental stresses affect plant growth and cause losses worth hundreds of million dollars to agricultural industry each year. Many genes are induced in response to environmental stresses. The DREB1A gene is a stress-inducible transcription factor which its ectopic over-expression improves plant tolerance to environmental stresses. To produce environmental stress tolerant plants carrying the DREB1A gene, the full length cDNA of the DREB1A gene was amplified from Arabidopsis thaliana Col-0 plants by gene specific primers and cloned into pGEMT-Easy vector, and transformed into E.coli. Presence of the DREB1A gene was confirmed by restriction analysis as well as DNA sequencing. A 668-bp XbaI/BamHI digested fragment of DREB1A gene from the pGEMT::DREB1A construct was sub-cloned into the pBI121 binary vector. The recombinant plasmids were transferred into Agrobacterium tumefaciens cells (strain LBA4404) and screened on LB medium supplied with kanamycin/rifampicin (50 mg/l). Positive bacterial colonies were selected based on colony-PCR analysis and saved for further application in plant materials.

Connexin-43 (Cx-43) plays axial roles in the propagation of action potentials and contractile coupling in the heart. Down-regulation of Cx-43 in the heart is associated with arrhythmia, dilated cardiomyopathy, and heart failure. To date, no studies have examined the effects of androgen deprivation therapy (ADT)-induced hypogonadism on the expression of Cx-43 in the heart. This study investigated the effects of testosterone deprivation and its replacement with testosterone enanthate on the expression of Cx-43 mRNA and the muscle-specific miRNAs miR-206 and miR-1, as two potential regulators of the Cx-43 protein expression in the ventricular tissue. Accordingly, 21 male Wistar rats were divided into three groups: Ι) Normal control, П) ORX-S: castrated rats serving as animal models for ADT and receiving the sesame oil as a solvent of testosterone enanthate for ten weeks, and Ш) ORX-T: these animals were castrated, receiving testosterone enanthate (25 mg/kg) for ten weeks. The relative expression of Cx-43 mRNA, miR-206, and miR-1 was determined by qRT-PCR. Cx-43 mRNA was found to be decreased in the ORX-S group. The Cx-43 mRNA was up-regulated after the administration of testosterone enanthate. There were no significant changes in miR-206, and miR-1 levels in the ORX-S and ORX-T groups compared to the controls. Our results indicated that testosterone should be regarded as an important factor in the regulation of Cx-43 mRNA expression in the heart, and testosterone deprivation may down-regulate the Cx-43 mRNA expression; however, it doesn't alter miR-1 and miR-206 levels. These results suggest that ADT-induced hypogonadism may put males at risk for cardiac dysfunctions.