مجله پژوهش های سلولی مولکولی (زیست شناسی ایران)
سال بیست و نهم شماره 3 (پاییز 1395)

Chitin is a Biopolymer with more than 5000 N- acetyl glucosamine monomer. Its extraction occurs by conventional chemical methods and overall four steps; removal of proteins using alkaline compounds, removal of fat, minerals eliminating and dye remove. Using alkaline materials in the removal of protein, cause changes in the structure of space, De-polymerization, quality decrease and a large number of chemical materials and environmental pollution. This study was done to replace proteolytic enzyme of Bacillus subtilis bacterium instead of sodium hydroxide, a substance that is damaging for the nature. The results showed that the mentioned bacterium is able to digest 90±5 % of protein compounds by photolytic enzymes. Quality of the gained chitin was investigated by using of an infrared spectrometer to analysis the element structure. Also by x-rays radiography and via determination the viscosity, molecular weight, crystallinity percent, color, and molecular structure. The gained chitin have had a profile introduction containing of 49.6 % carbon, 8.2 % nitrogen, 7.5 % hydrogen and 34.5 % oxygen. Their physical characteristics were showed as 4.9×106 Dalton average molecular weight, 74.5 % crystallinity, 32 °poise viscosity oriented at 20 °C, and white color. The chemical structure of each unit of chitin was obtained as C7H12NO4. The results show that replacement of a biological method rather than chemical methods to achieve this production with the right conditions could help to achieve the better quality products and may cause eliminating the use of environmentally harmful chemical environmental pollution such as heavy sodium hydroxide.

According to public concerns about side effects of chemical preservatives tend to use natural products without preservatives has been increased. This study aimed to investigate the antimicrobial extract of saffron stigmas and petals of different species on two types of g bacteria (Staphylococcus aureus, Bacillus subtilis) and two g- bacteria (Pseudomonas aeruginosa and Escherichia coli), causing the infection and the most important food poisoning. In this study, the antibacterial activity of three species of saffron with the scientific names of Crocus caspius, Crocus speciosus and Crocus sativus examined. The extraction of the three species of crocus was performed using methanol acid. The antibacterial activity of the extracts obtained by measuring the diameter of bacterial growth as well as the determination of minimum inhibitory concentration was determined. The results showed that the extract of saffron stigmas and petals of different species, had different antibacterial effects, so that B. subtilis and E. coli were the most sensitive and resistant bacteria to extract, respectively. Moreover, the petal extract of C. sativus and the stigma extract of C. speciosus had the most inhibitory effect on the examined microorganisms. Therefore, the stigma and the petals of the saffron in addition to the antioxidant properties can also be used as antibacterial agents.

Although cyanide is a poisonous and lethal compound, it is used in several industries such as gold extraction. Industrial wastewater contains highly amounts of cyanide. That in order to degrade it, different chemical and physical methods are used. These methods not only include high expenses, but also if they combine with other materials, will produce more hazardous compound as well. On the other hand, utilizing biodegradation method, in addition to decreasing expenses, is environmentally friendly. In this study, bacterium was isolated from Muteh gold mining wastewater. Determination of morphological and biochemical characteristics and 16S rDNA described that the bacterium belong to Enterobacter genus. To achieve the maximum degradation by Enterobacter ZS, culture conditions has been optimized. The variables parameters that effect on the degradation procedure with maximum efficiency was achieved. By optimizing the values of affective factors and consider the interactions between factors, culture conditions were optimized by response surface methodology (RSM).

Previous data have shown that the thiazolidinediones and their derivatives are found to possess divergent and very interesting biological activities such as, antioxidant and antibacterial activities. In this study, we investigated the cytotoxicity and inhibitory effects of synthesized TZD-OCH2CH3 on lipopolysaccharide (LPS)-induced over-expression of inducible nitric oxide synthase (iNOS) using Raw 264.7 cells. To assess whether the tested TZD-OCH2CH3 affected cell viability, RAW 264.7 cells were incubated with LPS (1μg/ml) in the presence of different TZD-OCH2CH3 concentrations (0-300 μg/ml). The results of MTT assay showed that TZD-OCH2CH3 had cytotoxicity at higher concentrations of 50 μg/ml (IC50=120 μg/ml). We then further evaluated the inhibitory activity of different TZD OCH2CH3 concentrations (30 and 60 μg/ml) against NO production in LPS-stimulated RAW 264.7 cells. The synthesized TZD-OCH2CH3 was found to have the most significant inhibitory effect on NO production in LPS-induced RAW 264.7 cells. These findings demonstrated that TZD-OCH2CH3 has good anti-inflammatory activity and thus has great potential for use in the control of various inflammatory diseases.

Gold nanoparticles (GNPs) have different applications in the medicinal field. There are three methods for producing nanoaprticles. Recent study was conducted on the assessment the toxic effects of the biologically produced GNPs. Bacillus cereus (PTCC 1015) was purchased and cultured in Nutrient broth medium. The bacterial cells were harvested by centrifugation and the obtained supernatant was incubated with Chloroauric acid solution at the final concentration of 1mM. Then the color changed supernatant was used for spectrophotometery, X-ray diffraction (XRD) and transmission electron microscopy (TEM) analyses. Two cell lines (HepG2 and CIRC-HLF) were used for 3-(4,5 dimethyl thiazol-2yl)-2,5 diphenyl tetrazolium bromide (MTT) assay. Spectrophotometery and XRD analyses proved the formation of GNPs and TEM images showed that the sizes of the produced GNPs were around 10-70nm. MTT assay has showed that GNPs had low cytotoxic effect and this effect was dose dependent. In the recent study the cell culture assay showed that the produced GNPs had toxic effect when they were used at high concentrations and the Hepatocyte (HepG2) cell line was more resistant than the Fibroblast (CIRC-HLF) one against the higher concentration of GNPs. The biologically produced GNPs can be easily produced, washed, sterilized and can be used in vivo in future studies.

Sugar beet (Beta vulgaris L.) is a dicotyledonous and two years plant from Chenopodiceae family and its economic importance is due to unique ability to produce high amounts of sugar. Sugar beet cyst nematode is the most important disease in the area under cultivation of this product. So this study was performed to assess the genetic diversity of 20 genotypes resistant and susceptible nematode using SSR markers. Following DNA extraction, PCR amplification was performed using 10 pairs of SSR primers and 49 polymorphic bands were selected from 7 pairs. The highest of Polymorphism Information Content (PIC) and highest Marker Index (MI) related to REP primer pairs and 10114 respectively. Cluster analysis using NTSYS software with UPGMA method based on Dice's similarity matrix divided genotypes into 4 clusters. The highest genetic similarity between tolerant and susceptible sugar beet cyst nematode was found. According to the results, it can be concluded that genetic variation does not have a direct relationship with resistant or sensitive genotypes. In fact, it was found that genotypes of sugar beet based resistance spectrum, sensitivity and tolerance to nematode disease were not in separate groups and a significant correlation was not observed.

Colorectal cancer (CRC) is one of the most common malignancies in the world (rate of death: 33%). v-Ki-ras2 Kirsten rat sarcoma (KRAS) mutations represent an early event in the development and progression of CRC. Codons 12 and 13 of exon 1 are the hot spots for occurrence of mutations in KRAS. This study is attempted to define the occurrence of KRAS mutations at codon 12 and 13 in patients from Khuzestan suffering from sporadic colorectal cancer (SCRC) and compare them with patients from other regions of Iran and other countries. Genomic DNA was first extracted from 45 SCRC specimens. KRAS point mutations were genotyped by PCR-RFLP analysis followed by enrichment PCR. Enrichment PCR is an appropriate method which we use for detecting even lower percentage of heterozygous mutations in one specimen. Finally Sanger sequencing was used to confirm mutations identified. 13.33% of (6 out of 45) tumor samples showed mutations at codon 12 or 13 of the KRAS gene. It’s totally different with the frequency of those from many of other countries (33-53%) and alternative studies in Iran (20.3% and 28%); that could be because of various reasons such as less role of serrated pathway in development of CRC among Khuzestani population, probable microsatellite instability-high status of these tumors, insufficient sensitivity of RFLP method, different genetic and environmental factors like high intake of omega-3 fat in the Khuzestani usual diet. Also, use of enrichment method, doubled the frequency of mutations identified in this cohort.

Petroleum pollution is the most important environmental pollution due to increasing usage of oil and oil dependent products. Soil microorganisms are the most important organisms that are affected at the early stage of pollution and also are effective for removing and degradation of the oil. In this research, fungi in agricultural soils were adapted for growing in petroleum-polluted media. Trichoderma fungal species are saprophytes that are living in agricultural soils. The aim of this research was to elucidate the effect of petroleum pollution on the growth of soil fugal species including Trichoderma species. Ten species of Trichoderma were selected and adapted for growing in the presence of oil. The fungi were cultured in the PDA containing of 1% crude oil and then were transferred to the media containing 2, 4 and 6% crude oil, step by step. Growth ability of the fungi was studied in the media containing different crude oil after 11 days based on colony diameter. Results showed that the all fungi were able to adapt to petroleum pollution and are growing in the presence of crude oil but it was less than control media. The highest colony diameter was observed in T. citrinoviride and the lowest one was in T. koningiopsi. These results indicated that the growth ability of T. citrinoviride in the petroleum-polluted media is more than other fungal species and so is more suitable for bioremediation of petroleum-polluted soils.

To identify chemical composition and antibacterial activity of essential oil of Calendula officinalis, the flowers of this plant which grows in a village in Kerman Province in July 2013 were collected. The samples were cleaned and then drying in the shade, making essential oil hydro distillation method was performed. Essential oil was analyzed by capillary gas chromatography (GC) using flame ionization (FID) and capillary gas chromatography coupled mass spectrometry (GC/MS) for detection. Also antibacterial activity of essential oil this plant was determined against gram negative and gram positive bacteria in this study by diameter ofinhibition zone.The main oil content from the plants of Calendula officinalis was 0.25%. The nineteen compounds were identified in the essential oil of Calendula officinalis that concluded 98.56% of the total oil. The major components were δ-cadinene (25.67%), α-cadinol (21.37%), Epi-α-muurolol(12.45%)and γ-cadinene(8.22%). For study of antibacterial activity of the oil sample, the essential oil tested against six bacteria by disc diffusion method. The antibacterial effects of this essential oil was determined against gram positive bacteria Staphylococcus epidermidis (PTCC=1436) and Streptococcus faecalis (PTCC=1237) and gram negative bacteria Pseudomonas aeroginosa (PTCC=11430), Shigella flexneri (PTCC=1716), Kellebsiella pnuomonae (PTCC=1053) and Escherichia coli (PTCC=1533). Effect of essential oil of Calendula officinalis on gram positive bacterium Staphylococcus epidermidis, gram negative bacterium Kellebsiella pnuomonae and Escherichia coli were examined and compared with tetracycline antibiotic. The results showed the essential oil of Calendula officinalis had anti-bacterial effects in comparison to common antibiotics.

Pectinases belong to the pectolytic enzymes which breakdown pectin polysaccharide present in plant tissues. Pectinase enzymes are the most widely used in food industries to produce fruit juice, tea, coffee, and oil. Bacteria are interesting sources in production of industrial enzymes. The aim of the present study was to isolate and molecular identify pectinase enzyme producing bacteria from soil. In this study, soil samples containing citrus peels were collected from some regions of Golestan province. The optimized pectin agar medium was used to isolate pectinase producing bacteria. Pectinase activity was indicated by the diameter of clear, hydrolyzed zones on the medium plates containing citrus pectin by adding Lugol's iodine. Bacterial isolates with higher pectinase activity were identified by 16S rRNA sequencing method. 20 out of 30 bacterial isolates revealed pectinase activity. All of the isolates were identified based on biochemical tests. Among them, 2 isolates had higher pectinase activity with regard to increase in the diameter of clear zone. Molecular studies indicated that the isolates were Bacillus cereus and Bacillus subtilis. In this study, two species of bacteria were introduced with suitable ability in pectinase production. Further studies are needed to assay the enzyme kinetics under a set of several constraints.