Journal of Cell and Molecular Research

50-55

Authors: Nazila Nouraee, Mohamad Vasei, Shahriar Semnani, Seyed Javad Mowla

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.

62-67

Authors: Balal Sadeghi, Mohammadreza Nassiri, Ali Masoudi-Nejad, Mojtaba Tahmoorespour, Hesam Dehghani and Hamed Ahmadi

MicroRNAs (miRNA) are a class of noncoding and regulatory RNA molecules about 22 nucleotides in length. MicroRNAs regulate gene expression by an RNA interfering pathway through cleavage or inhibition of the translation of target mRNA. Many miRNAs have been reported for their important roles in developmental processes in various animals, but there is limited information about cattle and sheep miRNAs. The comparative genomics approach due to their conserved nature is a good source for the miRNAs discovery. Cattle and sheep are ideal model organisms for biological and comparative genomics studies. In our study, a computational method based on expressed sequence tag (EST) analysis was used for detection of cattle and sheep miRNAs. In cattle, 25 miRNA candidates found by homology searching frequently clustered at certain chromosomes and 28 miRNAs in sheep had been detected. Our results show that the cattle and sheep miRNA database can be providing useful information for investigating biological functions of miRNAs in cattle and sheep. Furthermore, the bioinformatics approach is a good manner for studying these functions.

76-80

Authors: Eisa Kohan Baghkheirati, Mohammad Bagher Bagherieh-Najjar and Mahnaz Aghdasi

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.

89-96

Authors: Mahnaz Kiani, Homa Zarghami, Farshid Memariani and Ali Tehranifar

Tissue culture methods provide tools to supplement traditional methods for collection, propagation and preservation of endangered plant species. In this study, in vitro propagation of Diaphanoptera khorasanica Rech.f., a rare and threatened plant species with limited distribution range and population was investigated. This species has a potential as an ornamental plant. Single node explants were provided from both adult and seedling sources. Several disinfection treatments were tried to permit selection of a suitable method. Different growth regulators were used for establishment, proliferation and rooting stages. Explants showed the highest establishment percentage after 5 min treatment with 1% sodium hypochlorite (NaOCl), cultured in MS medium containing 2.2 µM 6-benzylaminopurine (BAP) and 2.4 µM indole-3-butyric acid (IBA). The highest proliferation of explants from both adult and seedling source explants was obtained from media supplemented by BA treatment in contrast to TDZ. No significant differences were found between different concentrations of BAP and TDZ. Proliferated shoots in TDZ were longer and had more internode length and less vitrification, in comparison with those in BAP. In vitro rooting of proliferated shoots just induced in liquid half-strength MS medium and rooting was not observed in solid medium. The shoots that originated from adult plants gave rise to the highest rooting rate with 4.8µM α-naphthalene acetic acid (NAA) and 2.4 µM, but NAA rooted plantlets showed higher survival percentage in acclimatization step. This study was aimed towards developing an efficient protocol for in vitro propagation of D. khorasanica and conservation of this vulnerable species.

1-10

Authors: Mahnaz Aghdasi, Fariba Fazli and Mohammad Bagher Bagherieh

TRR14 is a novel protein important in trehalose (α-D-glucosyl-[1,1]-α-D-glucopyranoside) signaling in Arabidopsis. In this research, we provided evidences to demonstrate that TRR14 plays role in Arabidopsis responses to salt and drought stress. Transgenic Arabidopsis plants over-expressing TRR14 under the control of CaMV 35S promoter were generated. The transformed lines showed higher transcript levels of TRR14 than that of the Wild Type (WT) Arabidopsis plants. The RT-PCR results showed that TRR14 transcript level increased markedly by salt and drought stress both in WT and transformed lines. Further experiments indicated that the TRR14 transformed lines had unchanged seed germination, root length and chlorophyll content under stress conditions. In addition, the activity of oxidative enzymes like peroxidase and catalase were significantly induced in transformed lines under salt and drought treatments. Thus, the present data indicate that a novel protein, TRR14, is involved in plant salt and drought tolerance.

18-27

Authors: Hassan Monhemi, Mohammad Reza Housaindokht, Mohammad Reza Bozorgmehr and Ahmad Reza Bahrami

Signaling pathways are not isolated from their surroundings. They are also intervened by other signaling pathways known as “crosstalk mechanism”. One of the most important crosstalk mechanisms is the insulin- EGF network. Although insulin and epidermal growth factor (EGF) networks have some complexity in their isolated forms, their complexities will grow in the crosstalk network. In this study, we used the analytical tools of the systems biology workbench for elucidating some ambiguities of the insulin-EGF crosstalk. Based on sensitivity analysis, we reconstructed an elucidated model with 51 chemical reactions in comparison with the previous model with 111 chemical reactions. Interestingly, this reduced model reproduces the results of the original model in synergy conditions. We noticed two controlling pathways with direct participation of phosphorylated insulin and EGF receptors that involve Insulin Receptor Substrate (IRS) and Src kinase modules. Also, insulin pathway by producing phosphatidylinositol-3, 4, 5-triphosphate (PIP3), and EGF pathway by activation of GAB1, control the downstream events and lead to potentialities in the mitogenic signal. Surprisingly, Shc and phosphatase SHP2-dependent reactions have no significant roles in the synergy conditions and are not involved in the reduced model. Regarding sensitivity analysis, all Ras/ERK cascade reactions are crucial for signal transduction and were kept in the reduced model.

34-42

Authors: Somayeh Panahi Moghadam, Zohreh Hojati and Majid Motovali-Bashi

StrR is a putative pathway specific regulator of streptomycin production in Streptomyces griseus. Because of finding new spo0j domain in strR by bioinformatics methods, the purpose of this study was to suggest another role for strR gene. This domain can be seen in proteins that are involved in initiation of sporulation and normal chromosome partitioning. So, 51 bps of strR in accordance to spo0j domain was deleted to investigate effects of deletion mutation in StrR functions. A unique specific procedure, including three consecutive PCRs, known as SOEing PCR were used here for site directed mutagenesis. Application and feasibility of this PCR was studied here. Bioinformatic studies were carried out for comparison of the sequence similarities between StrR and Spo0J proteins. An exclusive procedure, including three consecutive PCRs, was designed here in order to delete a 51 bp from the native strR. Other PCRs such as Semi-Nested PCR and RFLP PCR were used for strR isolation and structural confirmation of the isolated strR and deleted strR genes. Routine genetic engineering procedures were conducted in order to clone the native and deleted strR genes into E. coli. Obtained sequence information, from Conserved Domains Database (CDD) and Clustal W program, revealed that the StrR is similar to members of ParB family. Here, the strR was initially isolated from Iranian strain of Streptomyces griseus (PTCC1127). It was then confirmed as StrR by Semi-Nested PCR and RFLP-PCR. A 51 base pair region of the strR gene was deleted by specifically designed overlapped primers. A ten nucleotide overlap region was considered for a set of these primers. The recombinant cassette pSPMstrRΔ17 was constructed and cloned in E. coli. The sequencing results showed that a specific deletion is produced in the desired site and region in the strR gene. Therefore the designed three steps PCRs (known as SOing PCR) is a very rapid, cheap, and precise method for introducing such a deletion in any preferred gene.

43-50

Authors: Sasan Mohsenzadeh, Kolsoum Abdolahi and Hassan Mohabatkar

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.

57-65

Authors: Soraya Abbasi Habashi, Farzaneh Sabouni, Ali Moghimi, Saeed Ansari Majd and Azita Parvaneh Tafreshi

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.

75-80

Authors: Jalil Abbaspour, Ali Akbar Ehsanpour and Fariba Amini

Transgenic and non transgenic Nicotiana tabacum L (cultivar Wisconsin) containing Ri T-DNA were treated with 0, 0.2 and 0.4 mgL-1 GA3 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 GA3 treatment. Chlorophyll, carotenoid and anthocyanin pigments of leaf were decreased. Auxin and gibberellic acid content of leaf and root were also measured. Exogenous GA3 increased root auxin in the transgenic plants while it did not change in shoot. GA3 treatment increased gibberellin content in both of root and shoot.

1-11

Authors: Fatemeh Moosavi, Hassan Mohabatkar and Sasan Mohsenzadeh

The family, Flaviviridae includes viruses which cause several diseases including Dengue fever, Japanese encephalitis, Murray Valley encephalitis, Tick-borne encephalitis, West Nile encephalitis, Yellow fever and Hepatitis C virus infection. Members of this family have monopartite, linear, single-stranded RNA genomes of positive polarity with 9.6-12.3 kb in length. Here, we analyzed the codon usage of 13 species of this family by using gene infinity package. Base analysis was performed by CAIcal server and amino acid composition was calculated by PseAAC web-server. The results showed that the highest number of A, G and C bases were seen in the RNA genome of Dengue virus 2, Tick borne encephalitis virus and Hepatitis C virus, respectively. Although the number of U bases used in RNA genomes was very close, the highest U nucleotide amount was 23.77% in Wesselsbron virus. The lowest number of C, G, U and A bases was seen in Bovine viral diarrhea virus, Dengue virus 2, Tick borne encephalitis virus and Hepatitis C virus, respectively. In this study, it was found that the complete genome of classical swine fever virus has a lower GC content and genome of Tick borne encephalitis virus, Hepatitis C virus and Powassan virus have the highest GC content among other examined species. We also classified the amino acids as rare (Phenylalanine, Cysteine, Histidine, Methionine, Asparagine, Glutamine, Tryptophan and Tyrosine), frequent (Alanine, Glutamic acid, Glycine, Leucine, Valine and Threonine), and intermediate (all others). The highest numbers of preferred codons exist in Wesselsbron virus and the lowest in West Nile virus.

19-24

Authors: Massoud Ranjbar, Zahra Hajmoradi and Roya Karamian

In the present paper, the cytogenetic study including meiotic chromosome number and behavior along with pollen viability were performed in 4 populations of Trigonella spruneriana Boiss. This is the first cytogenetic report of the taxon. All populations are diploid and possess 2n = 2x = 16 chromosome number, which is consistent with the proposed basic number of x = 8. In addition, some meiotic irregularities observed in different populations included chromosomes stickiness, B-chromosomes, chromosome bridges resulting from stickiness, the occurrence of laggard chromosomes, formation of micronuclei in tetrad cells and cytomixis. The highest and the lowest percentages of pollen viability were observed in populations SPR 658 and SPR 566, respectively.

31-37

Authors: Najme Javanmardi, Abdolreza Bagheri, Nasrin Moshtaghi, Ahmad Sharifi and Abbas Hemati Kakhki

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.

56-60

Authors: Saber Zahri, Gadir Nouri-Ganbalani and Shahram Agahi

Crustacean hemocyanin represents a unique case of molecular heterogeneity among oxygen-carrying proteins. Two populations of Gammarus lacustris and six populations of Gammarus komareki were identified and isolated from aquatic habitats of Ardabil (northwest of Iran). The populations were nominated based on the name of the isolated region. The Principal component analysis of morphological absolute attributes showed that the populations were relatively dissociated. The Meshkin population of G. komareki was completely different from the other populations. The analysis of relative attributes did not show any dissociation. The Hemolymph profile analysis using native PAGE revealed 5 to 7 hemocyanin subunits for four populations of G. komareki and 8 subunits for two populations of G. lacustris. The Detection of copper ions using bathocuproin sulfonate on the native PAGE surface confirmed the hemocyanin bands. Using SDS PAGE, 1-3 and 1 protein bands were separated for hemolymph samples from G. lacustris and G. komareki, respectively. Molecular weights of the bands were 75 - 80 kDa. Comparison of our results with the previous published data confirms the species specificity of hemocyanin subunit composition. These results imply that the ecological variation may affect the morphological traits, but the hemocyanin profiles are useful as a molecular marker for the taxonomic studies and identifying very closely-related species of Gammarus.

67-73

Authors: Moslem Shojaei, Mohammadreza Mohammad Abadi, Masood Asadi Fozi, Omid Dayani, Amin Khezri and Masoumeh Akhondi

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.

81-85

Authors: Mahnaz Kiani, Homa Zarghami, Ali Tehranifar and Farshid Memariani

In vitro methods provide a variety of tools to supplement traditional methods for collection, propagation and preservation of endangered plant species. In this study, an efficient protocol was developed for in vitro propagation of Colutea gifana, a rare and endangered plant species with limited reproductive capacity that grows in a narrow area of Iran. Single node explants were used for a series of experiments to select the appropriate disinfection method and growth regulators for establishment, proliferation and rooting stages. Explants showed the highest establishment percent after treatment with 2% Sodium hypochlorite (NaOCl) for 15 min, cultured in MS medium containing 2.2 µM 6-benzylaminopurine (BA) and 1 µM indole-3-butyric acid (IBA). In proliferation stage, 8.8 µM of BA was more effective cytokinin than Kinetin (Kin) and Thidiazuron (TDZ) for growth induction of axillary shoots. In vitro rooting of proliferated shoots was induced in half- strength MS medium in all concentrations of both tested auxins i.e. IBA and α-naphthalene acetic acid (NAA). Eighty percent of the plantlets were successfully acclimatized to ex vitro conditions, showed normal development. These plants were used to replenish declining populations in the collection sites and conserve C. gifana from extinction.

93-102

Authors: Reihaneh Sabbaghzadeh, Majid Monajjemi, Fatemeh Mollaamin and Shahrbano Oryan

Insulin-like growth factor (IGF-1) is an anti-apoptosis factor in multiple cell types associated with various cancers. Computational methods allow investigating the systems between 50–100 atoms in the frame of quantum mechanics and up to 50,000 atoms with molecular dynamics. Since there are specific interactions between the residues, the solvent could play an important role in the stability of the native structure. Therefore it is useful to carry out such simulations at atomistic detail. MC, MD and LD simulations of the IGF-1 were performed with the HyperChem7.0 program. The geometries, and the interaction energies, bonds, angles, stretch-bends, electrostatic and the VDW Interactions were carried out in solution and gas phase. We have computed the transition temperature for the IGF-1 molecule. Studying the changes occurred in the potential energy of the three force fields showed that Amber force field is better than MM+ and OPLS force field and also MD simulation, at least in this model, is more effective than MC and LD methods. After equilibration, the MD simulation was very stable, and the difference between the relation coefficients R2=0.8173 in gas and R2=0.7558 in water was compared. The Pearson correlation suggests that there is an inverse relationship (R=- 0.25) between in vitro temperature and stability of the structure.