The Effect of Chronic Administration of Buspirone on 6-Hydroxydopamine-Induced Catalepsy in Rats

Purpose: Several evidences show that serotonergic neurons play a role in the regulation of movements executed by the basal ganglia. Recently we have reported that single dose of buspirone improved 6-hydroxydopamine (6-OHDA) and haloperidol-induced catalepsy. This study is aimed to investigate effect of chronic intraperitoneal (i.p.) administration of buspirone on 6-OHDA-induced catalepsy in male Wistar rats. Methods: Catalepsy was induced by unilateral infusion of 6-OHDA (8 μg/2 μl/rat) into the central region of the SNc and was assayed by the bar-test method 5, 60, 120 and 180 min after drugs administration in 10th day. The effect of buspirone (0.5, 1 and 2 mg/kg, i.p. for 10 days) was assessed in 6-OHDA-lesioned rats. Results: The results showed that chronic injection of buspirone (0.5, 1 and 2 mg/kg, i.p. for 10 days) decreased catalepsy when compared with the control group. The best anticataleptic effect was observed at the dose of 1 mg/kg. The catalepsy-improving effect of buspirone was reversed by 1-(2-methoxyphenyl)- 4-[4-(2-phthalimido) butyl]piperazine hydrobromide (NAN-190), 0.5 mg/kg, i.p.,as a 5-HT1A receptor antagonist. Conclusion: Our study indicates that chronic administration of buspirone improves catalepsy in a 6-OHDA-induced animal model of parkinson's disease (PD). We also suggest that buspirone may be used as an adjuvant therapy to increase effectiveness of antiparkinsonian drugs. In order to prove this hypothesis, further clinical studies should be done

Causative agents and antimicrobial susceptibilities of urinary tract infections in the northwest of Iran

SummaryBackgroundThe empirical therapy of urinary tract infections (UTI) relies on the predictability of the agents causing UTI and knowledge of their antimicrobial susceptibility patterns.MethodsIn a prospective study undertaken over a 14-month period, 5136 samples from patients suspected of having a UTI were analyzed, of which 676 were culture-positive. Isolated bacteria were identified by standard tests, and antibiotic susceptibility was determined by disk diffusion method.ResultsAccording to our results, Escherichia coli was the most common etiological agent of UTI (74.6%), followed by Klebsiella spp (11.7%), Staphylococcus saprophyticus (6.4%), and Pseudomonas aeruginosa (2.2%). Analysis of the frequency of isolated bacteria according to the age of the patients revealed that Klebsiella infections are more prevalent in the older age groups (>10 years) and Pseudomonas infections are more prevalent in children and the elderly (<9 years and >60 years). Results of antimicrobial susceptibility analysis for E. coli, as the most prevalent cause of UTI, to commonly used antibiotics are as follows: amikacin (97.8%), gentamicin (97%), ciprofloxacin (94%), nitrofurantoin (87.1%), nalidixic acid (93.7%), trimethoprim–sulfamethoxazole (48.2%), cephalexin (76%), and ampicillin (6.9%).ConclusionsThe results show that the antimicrobial resistance patterns of the causes of UTI are highly variable and continuous surveillance of trends in resistance patterns of uropathogens is important

The Effect of Dried Glycyrrhiza Glabra L. Extract on Obesity Management with Regard to PPAR-γ2 (Pro12Ala) Gene Polymorphism in Obese Subjects Following an Energy Restricted Diet

Purpose: Obesity is a multi-factorial health problem which results from the interaction of environmental and genetic factors. The aim of the present study was to determine the effects of dried licorice extract with a calorie restricted diet on anthropometric indices and insulin resistance with nutrigenetic approach. Methods: For this pilot, double-blind, placebo-controlled randomized clinical trial, 72 eligible subjects were randomly allocated to Licorice or placebo group. They received a low-calorie diet either with a 1.5 g/day of Licorice extract or placebo for 8 weeks. Results: There were no significant differences in anthropometric indices and dietary intake in genotype subgroups at the baseline. Findings indicated that supplementation with Licorice extract did not change anthropometric indices and biochemical parameters significantly compared to a hypocaloric diet alone. However, from the nutrigenetic point of view, significant changes in anthropometric indices and QUICKI were observed in the Pro12Pro genotypes compared to the Pro12Ala at the end of the study (p<0.05 in all variables). Moreover, no interactive effect of the Licorice supplement and Pro12Ala genotype was found. Conclusion: In obese subjects, the Pro/Pro polymorphism of the PPAR-γ2 gene seems to induce favourable effects on obesity management. Further studies are needed to clarify whether PPAR-γ2 gene polymorphisms or other obesity genes can affect responses to obesity treatment

Recombinant expression and purification of L2 domain of human epidermal growth factor receptor

Epidermal growth factor receptor (EGFR) is one of the key molecules in cell growth and multiplication and plays an important role in some malignant processes. L2 domain of extra-cellular part of this receptor involved in ligand binding and its inhibition can prevent activation of related signaling pathways. The aim of the present study was cloning and expressing the fragment coding for L2 region of human EGFR for the production of recombinant L2 protein. The total RNA from A431 cells line was extracted and used for amplification of the sequence coding for L2 domain of EGFR by reverse transcriptase-polymerase chain reaction (RT-PCR) technique. The product was cloned into the PGEM-T vector and used for sequencing. In the next step, the insert was removed from the PGEM-T vector and subcloned into the PET22 expression vector. The expression construct was transformed into the Escherichia coli BL21 (DE3) and recombinant protein expression was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Analyzing the expression of produced recombinant protein showed that the recombinant L2 can be highly expressed by this expression system. This recombinant protein can be used for the production of specific mAb, screening for specific ligands and competitive inhibition of the EGFR.Keywords: Human epidermal growth factor receptor, domain L2, recombinant expressionAfrican Journal of Biotechnology Vol. 9(33), pp. 5292-5296, 16 August, 201

Rapid and simultaneous genotypic detection of Rifampin-Isoniazid and Ethambutol resistant Mycobacterium tuberculosis by use of MAS-PCR

AbstractAims and objectivesThis study aims to identify common mutations leading to Isoniazid (INH), Rifampin (RMP) and Etambutol (EMB) resistance using Multiplex Allele-Specific Polymerase Chain Reaction (MAS-PCR).MethodIn a cross-sectional study during 2012–2013, 257 patients with smear-positive pulmonary tuberculosis residing in five frontier west and north-west provinces of Iran were evaluated in respect of common point mutations leading to resistance to tree first-line drugs.ResultsThe overall frequency of mutations was 37 out of which 8 mutations were related to katG 315, 26 mutations pertained to rpoB 516, 526 and 531 and 3 mutations related to emb B. The rpoB single, double and triple mutations were found in 45.3%, 42.3% and 15.4% of rpoB, respectively. Frequency of patients with mutation to katG and at least one rpoB codon was 7cases (2.7%) at the same time. In this study 60.0% of INH-resistant and 83.3% of RMP-resistant isolates were detected by MAS-PCR technique. Mutation odds were higher in females and in patients with a history of anti-TB drug use.ConclusionThe MAS-PCR is a relatively rapid, sustainable, efficient and accurate technique for detection of drug resistance in tuberculosis. This highlights also the role of mutation at inhA, ahp and oxy R genes in the creation of IHN resistance which may be the causative factor in the remainder of cases

Recombinant expression and purification of L2 domain of human epidermal growth factor receptor

Epidermal growth factor receptor (EGFR) is one of the key molecules in cell growth and multiplication and plays an important role in some malignant processes. L2 domain of extra-cellular part of this receptor involved in ligand binding and its inhibition can prevent activation of related signaling pathways. The aim of the present study was cloning and expressing the fragment coding for L2 region of human EGFR for the production of recombinant L2 protein. The total RNA from A431 cells line was extracted and used for amplification of the sequence coding for L2 domain of EGFR by reverse transcriptase-polymerase chain reaction (RT-PCR) technique. The product was cloned into the PGEM-T vector and used for sequencing. In the next step, the insert was removed from the PGEM-T vector and subcloned into the PET22 expression vector. The expression construct was transformed into the Escherichia coli BL21 (DE3) and recombinant protein expression was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Analyzing the expression of produced recombinant protein showed that the recombinant L2 can be highly expressed by this expression system. This recombinant protein can be used for the production of specific mAb, screening for specific ligands and competitive inhibition of the EGFR

Molecular Cloning and Characterization of P4 Nuclease from Leishmania infantum

Parasite of the genus Leishmania is reliant on the salvage pathway for recycling of ribonucleotides. A class I nuclease enzyme also known as P4 nuclease is involved in salvage of purines in cutaneous Leishmania species but the relevant enzymes have not been characterized in Leishmania infantum (L. infantum). The aim of this study was to clone and characterize the gene encoding class I nuclease in L. infantum. DNA extracted from L. infantum was used for amplification of P4 nuclease gene (Li-P4) by PCR. The product was cloned, sequenced, and expressed in E. coli for further characterization. Analysis of the sequence of Li-P4 revealed that the gene consists of an ORF of 951 bp. Sequence similarity analysis indicated that Li-P4 has a high homology to relevant enzymes of other kintoplastids with the highest homology (88%) to p1/s1 class I nuclease from L. donovani. Western blotting of antirecombinant Li-P4 with promastigote and amastigote stages of L. infantum showed that this nuclease is present in both stages of parasite with higher expression in amastigote stage. The highly conserved nature of this essential enzyme in Leishmania parasites suggests it as a promising drug target for leishmaniasis

Optimized Signal Peptide for Secretory Expression of Human Recombinant Somatropin in E. coli

Purpose: The human somatropin is a single-chain polypeptide with a pivotal role in various biological processes. Although Escherichia coli is considered as a preferred host for the production of human somatropin, the high expression of this protein in E. coli results in the accumulation of protein as inclusion bodies. Periplasmic expression using signal peptides could be used to overcome the formation of inclusion bodies; still, the efficiency of each of the signal peptides in periplasmic transportation is varied and often is protein specific. The present study aimed to use in silico analysis to identify an appropriate signal peptide for the periplasmic expression of human somatropin in E. coli. Methods: A library containing 90 prokaryotic and eukaryotic signal peptides were collected from the signal peptide database, and each signal’s characteristics and efficiency in connection with the target protein were analyzed by different software. The prediction of the secretory pathway and the cleavage position was determined by the signalP5 server. Physicochemical properties, including molecular weight, instability index, gravity, and aliphatic index, were investigated by ProtParam software. Results: The results of the present study showed that among all the signal peptides studied, five signal peptides ynfB, sfaS, lolA, glnH, and malE displayed high scores for periplasmic expression of human somatropin in E. coli, respectively. Conclusion: In conclusion, the results indicated that in-silico analysis could be used for the identification of suitable signal peptides for the periplasmic expression of proteins. Further laboratory studies can evaluate the accuracy of the results of in silico analysis

Chaperone-Assisted Soluble Expression of a Humanized Anti-EGFR ScFv Antibody in E. Coli

Purpose: Formation of inclusion bodies is a considerable obstacle threatening the advantages of E. coli expression system to serve as the most common and easiest system in recombinant protein production. To solve this problem, several strategies have been proposed among which application of molecular chaperones is of remarkable consideration. The aim of this study was to evaluate the effects of molecular chaperones on soluble expression of aggregation-prone humanized single chain antibody. Methods: To increase the solubility of a humanized single chain antibody (hscFv), different chaperone plasmids including PG-tf2 (GroES- GroEL- tig), ptf16 (tig) and pGro7 (GroES- GroEL) were co-expressed in BL21 cells containing pET-22b- hscFv construct. The solubility of recombinant hscFv was analyzed by SDS-PAGE. After purification of soluble hscFv by Ni-NTA column, the biological activity and cytotoxicity of the recombinant protein were tested by ELISA and MTT assay, respectively. Results: SDS-PAGE analysis of the hscFv revealed that chaperone utility remarkably increased (up to 50%) the solubility of the protein. ELISA test and MTT assay analyses also confirmed the biological activity of the gained hscFv in reaction with A431 cells (OD value: 2.6) and inhibition of their proliferation, respectively. Conclusion: The results of this study revealed that co-expression of chaperones with hscFv leads to remarkable increase in the solubility of the recombinant hscFv, which could be of great consideration for large scale production of recombinant single chain antibodies