International Journal of Molecular and Cellular Medicine
Volume:3 Issue: 11, Summer 2014

Actinomycetes were isolated from marine sediment samples collected from the east coast of Chennai, Tamil Nadu, India. Well diffusion and agar plug methods were used for the evaluation of antibiotic production by these isolates against drug resistant Methicillin- resistant Staphylococcus aureus (MRSA) and vancomycin resistant Enterococci (VRE). The potential isolate VITBRK2 was mass cultured for morphological and physiological characterization. The culturing conditions of the isolate were optimized and the recommendations of International Streptomyces Project were followed for the assimilation of carbon and nitrogen sources. The isolate was identified by comparing the properties with representative species in the key of Nonomura and Bergey’s Manual of Determinative Bacteriology. Ethyl acetate extract prepared from the cell free culture broth of the isolate was analyzed using HPLC- diode array technique to characterize the metabolites and identify the antibiotics. VITBRK2 was found to be Gram-positive rod grey color aerial mycelium production. It was also non motile in nature with spiral spore chain morphology. VITBRK2 was identified as Streptomyces and designated as Streptomyces sp. VITBRK2. HPLC-DAD analysis showed the presence of indolo compounds (3- methyl-indole and 2-methyl- indole) along with amicoumacin antibiotic. The observed activity of Streptomyces sp. VITBRK2 against MRSA and VRE strains may be due to the presence of indolo compounds in the isolate. The results of this study suggested that secondary metabolites produced by Streptomyces sp. VITBRK2 could be used as a lead to control drug resistant bacterial pathogens.

Diagnosis of subclinical and early stage clinical periodontal dysfunction could prevent from further socioeconomic burden. The aim of this study was to assess the diagnostic applicability of nitric oxide and its end-metabolites in periodontal tissue health and disease. Forty-two patients were enrolled and divided into three groups according to gingivitis (GI) and clinical attachment level (CAL) indices: a healthy group (GI<1, CAL<1), b: gingivitis (GI>1, CAL>1) and c: periodontitis (CAL>1) with 14 patients in each group. Unstimulated saliva and gingival crevicular fluid (GCF) were collected. Samples were evaluated for nitrite, nitrate and total nitric oxide contents with the ELISA method. In addition, CAL, GI, plaque index (PI), decay, missing, filling (DMFT) and bleeding index (BI) scores were also recorded. Except for GCF nitrite content (P= 0.89), there was an increasing trend for measured biomarkers in both saliva and GCF (Periodontitis> gingivitis> healthy periodontium, P< 0.05). Data remained stable after simultaneous adjustment for DMFT and BI scores as confounding factors. Sensitivity, specificity, positive predictive value, negative predictive value, cut point and p- value were as the followings: GCF nitrate (0.71, 0.11, 0.29,0.43, 4.97, P= 0.04), nitric oxide GCF (0.64, 0.18, 0.28, 0.5, 10.12, P= 0.04), nitrite saliva (0.93, 0.96,0.93,0.96,123.48, P< 0.001), salivary nitrate (0.93, 0.96, 0.93, 0.96, 123.6, P< 0.001), salivary nitric oxide (0.93, 0.96, 0.93, 0.96, 246.65, P <0.001). Our results revealed that NO plays an important role in the process of destruction of periodontal tissues. Within the limitation of our study, detecting NO biomarker and its end metabolites in saliva is of more value to assess the periodontal health comparing to GCF.

The optimization of cell culture conditions for growth and productivity of recombinant Chinese hamster ovary (CHO) cells is a critical step in biopharmaceutical manufacturing. In the present study, the effects of the timing and amount of peptone feeding of a recombinant CHO cell line grown in a basal medium in serum-free suspension culture were determined for eight peptones of different origin (plant and casein). The amino acid content and the average molecular weight of the peptones chosen were available. In optimized feeding strategies with single peptones, increase 100 % volumetric productivity and 40 % in cell number were achieved. In feeding strategies with two peptones, several combinations stimulated protein productivity more than either peptone alone, depending on the peptone concentration and time of feeding. Some peptones, which did not stimulate productivity when added alone proved to be effective when used in combination. The combined peptones feeding strategies were more effective with peptones of different origin. Our data support the notion that the origin of peptones provides some guidance in identifying the most effective feeding strategies for recombinant CHO cells.

Cardiovascular disease has become the main factor of death and birth defects in the world and also in Iran. New clinical studies have shown that early diagnosis of patients with coronary artery disease (CAD) can contribute to effective prevention or therapeutic structures, which reduce mortality or the next chance of cardiovascular events, and increase the quality of life. Most studies on CAD disease and its genetic risk factors so far, have been done excluding the Iranian population. PubMed was used to search for all relevant studies published on or before 2013 and rs3184504 was selected for association study for CAD. A total of 200 subjects with 100 cases and 100 controls were ultimately included in the analysis. Blood samples were collected and after DNA extraction the DNA analysis was performed by TaqMan Probe Real Time PCR to evaluate the association between candidate variant with the disease and some blood biochemical factors. Our study demonstrated that there was not a direct association between rs3184504 C>T variant with risk of CAD in Iranian population, whereas, there is a significant association between this variant with increased blood LDL and diastolic blood pressure. Further molecular analysis and other disease association studies are necessary in the Iranian population.

Neisseria meningitidis is a major causative agent of bacterial septicemia and meningitis in humans. Currently, there are no vaccines to prevent disease caused by strains of N.meningitidis serogroup B. The Class 1 Outer Membrane Protein (OMP) has been named porA which is a cation selective transmembrane protein of 45 KDa that forms trimeric pore in the meningococcal outer membrane. PorA from serogroup B N. meningitidis was cloned into prokaryotic expression vector pBAD-gIIIA. Recombinant protein was expressed with arabinose and affinity purified by Ni-NTA agarose, SDS-PAGE and western blotting were performed for protein determination and verification. BALB/c mice were immunized subcutaneously with purified rPorA together with alum adjuvant. Serum antibody responses to serogroups B N.meningitidis were determined by ELISA. Serum IgG response significantly increased in the group immunized with rPorA together with alum adjuvant in comparison with control groups. These results suggest that rPorA can be a potential vaccine candidate for serogroup B N.meningitidis.

Sensorineural non-syndromic hearing loss is the most common disorder which affects 1 in 500 newborns. Hearing loss is an extremely heterogeneous defect with more than 100 loci identified to date. According to the studies, mutations in GJB2 are estimated to be involved in 50- 80% of autosomal recessive non-syndromic hearing loss cases, but contribution of other loci in this disorder is yet ambiguous. With regard to studies, DFNB4 locus (SLC26A4) can be classified as the second cause of hearing loss. So, this study aimed to determine the contribution of this locus in hearing loss as well as the frequency of SLC26A4 gene mutations in a population in the west of Iran. In this descriptive laboratory study, we included 30 families from the west of Iran with no mutation in GJB2 gene. Linkage analysis was performed by DFNB4 (SLC26A4) molecular markers (STR). The families with hearing loss linked to this locus were further analyzed for mutation detection. SLC26A4 gene exons were amplified and analyzed using direct DNA sequencing. In studied families, 2 families displayed linkage to DFNB4 locus. Identified mutations include mutation in exon 5 (c.416 G>T) and in splicing site of exon 7 (IVS-2 A>G or c.919-2 A>G).

Melanocortin- 4 receptor (MC4R) and agouti- related peptide (AgRP) are involved in energy homeostasis in rats. According to MC4R and AgRP effects on luteinizing hormone (LH) secretion, they may influence the estrous cycle of rats. Therefore, the aim of this study was to investigate the expression of MC4R and AgRP mRNAs at different stages of estrous cycle in the rat’s hypothalamus. The estrous cycle stages (proestrus, estrus, metestrus and diestrus) were determined in 20 adult female rats using vaginal smears. The rats were divided into four equal groups (n=5). Four ovariectomized rats were selected as controls two weeks after surgery. Using real- time PCR, relative expressions (compared to controls) of MC4R and AgRP mRNAs in the hypothalamus of rats were compared in four different groups of estrous cycle. The relative expression of MC4R mRNA in the hypothalamus of female rats during proestrus stage was higher than those in other stages (P=0.001). Despite a lower mean of relative expression of AgRP mRNA at proestrus stage, the relative expression of AgRP mRNA of the four stages of estrous cycle did not differ (P>0.05). In conclusion, changes in the relative expression of MC4R and AgRP mRNAs in four stages of rat estrous cycle indicated a stimulatory role of MC4R in the proestrus and preovulatory stages and an inhibitory role of AgRP in gonadotropin releasing hormone (GnRH) and LH secretions.

Drug resistance (especially multiple drug resistance) in Mycobacterium tuberculosis makes global concerns in treatment and control of tuberculosis. Rapid diagnosis of drug resistant strains of the bacteria has vital importance in the prognosis of the disease. The aim of this study was to identify the mutations responsible for drug resistance in Mycobacterium tuberculosis strains derived from patients with tuberculosis using line probe assay (LPA) method which rapidly detect drug resistant strains and respective mutations. Sputum samples from tuberculosis patients were collected and cultured on Lowenstein– Jensen medium, and then the colonies of Mycobacterium tuberculosis from cultures of 54 bacterial positive cases were randomly chosen for DNA extraction. Bacterial DNA was extracted using standard Cetyl Trimethyl Ammonium Bromide (CTAB) method. In order to identify drug resistant strains and related mutations, LPA method was applied. Three subjects out of 54 investigated cases were resistant to quinolone (5.5%), and resistance to kanamycin/ amikacin, streptomycin, rifampin, and isoniazid were observed in 3 (5.5%), 4 (7.4%), 3 (5.5%), and 2 (3.7%) of the Mycobacterium tuberculosis strains, respectively. In the present study, 4 cases (7.4%) were detected to be resistant to more than one drug. Since LPA is a rapid method that simultaneously detects mutations involved in drug resistance, applying this method in the prediction of drug resistance and selecting appropriate treatment in tuberculosis patients is recommended.

Familial adenomatous polyposis (FAP) is responsible for <1% of colorectal cancer (CRC) cases and is inherited as an autosomal dominant trait. Patients generally present hundreds to thousands of adenomas and develop colorectal cancer by age 35- 40 if left untreated. Here we report four patients with germline frameshift mutation (small deletion) at exon 15 of adenomatous polyposis coli (APC) tumor suppressor gene. Peripheral blood samples were collected from patients and Exon 15 of the APC gene was studied by direct sequencing after genomic DNA extraction. Four frameshift mutations were detected. Two patients had 5 bp deletion, c.3927_3931delAAAGA and two siblings presented deletion at codon 849 (c.2547_2548delTA p.Asp849fsX62). This study was the first report of genetic screening in Iranian FAP patients. In contrast to other studies we revealed that one patient with mutation at codon 1309 had an attenuated phenotype.