Identification,cDNA Cloning,and Characterization of Luteinizing Hormone Beta Subunit (lhb) Gene in Catla catla

Reproductive hormones play a significant role in the gonadal development and gametogenesis process of animals. In the present study luteinizing hormone beta, (lhb) subunit gene was cloned and characterized from the brain of Catla catla. The lhb full-length of cDNA sequence is 629 bp which consists of 43bp 5'-UTR (untranslated region) 447bp, ORF(open reading frame) and 139 bp of 3'-UTR respectively. The coding region of lhb gene encoded a peptide of 148 amino acids. The coding sequence of lhb gene consist of a single N-linked glycosylation site (NET) and 12 cysteine knot residues. Phylogenetic analysis of C. catla Lhβ deduced amino acid sequence showed high similarity with Carassius auratus followed by Gobiocypris rarus. 3D structure Lhβ protein comprises of five β-sheets and six coils/loops. The qPCR results revealed lhb mRNA is mainly expressed in the pituitary, ovary while moderate expression was observed in brain and testis. To best our knowledge, this is the first report on the identification, molecular characterization and structural information regarding luteinizing hormone in Indian major carp.     

Purification,characterization and kinetics of thiol protease inhibitor from goat (<Emphasis Type="Italic">Capra hircus</Emphasis>) lung

In the present study, two molecular forms of goat lung cystatin (GLC), I and II, were purified to homogeneity by a two-step procedure including ammonium sulfate precipitation (40–60%) and ion exchange chromatography. The inhibitor forms migrated as single bands under native and SDS-PAGE with and without reducing agent giving molecular mass of 66.4 and 76.4 kDa, respectively. GLC-I possesses 0.07% and GLC-II 2.3% carbohydrate content and no -SH groups. GLC-I showed greater affinity for papain than for ficin and bromelain. Immunological studies showed that the inhibitor was pure and there was cross reactivity between anti-GLC-I serum and goat brain cystatin. Both inhibitor forms were stable in the pH range of 3–10 and up to 75°C. GLC-I was found to possess 49% α-helical structure by CD spectroscopy. The inhibitor-papain complexes showed conformational changes as invoked by UV and fluorescence spectroscopic studies. Published in Russian in Biokhimiya, 2009, Vol. 74, No. 7, pp. 963–971.     

Methotrexate binding causes structural and functional changes in lung cystatin

Regulation of cysteine proteinases and their inhibitors is of utmost importance in diseases like lung cancer, chronic inflammatory conditions such as asthma, emphysema, and idiopathic pulmonary fibrosis. Protease-antiprotease imbalance accelerates disease progression. In the present study, the effect of antineoplastic and antirheumatic drug methotrexate (MTX) on lung cystatin (a cysteine protease inhibitor) was studied to explore drug induced changes in functional and structural integrity of the protein. The basic binding interaction was studied by UV-absorption, FT-IR and fluorescence spectroscopy. The quenching of protein fluorescence confirmed the binding of MTX with goat lung cystatin (GLC-I). Stern-Volmer analysis of MTX-GLC-I system at different temperatures indicates the presence of static component in the quenching mechanism. The thermodynamic parameters ΔH? and ΔS? were -3.8 kJ/mol and 94.97 J?mol?1?K?1, respectively, indicating that both hydrogen bonds and hydrophobic interactions played a major role in the binding of MTX to GLC-I. Methotrexate (7 μM) caused complete inactivation of lung cystatin after 6 hours. The results of FT-IR spectroscopy reflect perturbation of the goat lung cystatin on interaction with MTX. Methotrexate induced loss of function change in the inhibitor could provide a rationale for the off target tissue injury caused by the drug and for the design of agents against such an injury.     

In-vitro diagnosis of single and poly microbial species targeted for diabetic foot infection using e-nose technology

Background

Effective management of patients with diabetic foot infection is a crucial concern. A delay in prescribing appropriate antimicrobial agent can lead to amputation or life threatening complications. Thus, this electronic nose (e-nose) technique will provide a diagnostic tool that will allow for rapid and accurate identification of a pathogen.

Results

This study investigates the performance of e-nose technique performing direct measurement of static headspace with algorithm and data interpretations which was validated by Headspace SPME-GC-MS, to determine the causative bacteria responsible for diabetic foot infection. The study was proposed to complement the wound swabbing method for bacterial culture and to serve as a rapid screening tool for bacteria species identification. The investigation focused on both single and poly microbial subjected to different agar media cultures. A multi-class technique was applied including statistical approaches such as Support Vector Machine (SVM), K Nearest Neighbor (KNN), Linear Discriminant Analysis (LDA) as well as neural networks called Probability Neural Network (PNN). Most of classifiers successfully identified poly and single microbial species with up to 90% accuracy.

Conclusions

The results obtained from this study showed that the e-nose was able to identify and differentiate between poly and single microbial species comparable to the conventional clinical technique. It also indicates that even though poly and single bacterial species in different agar solution emit different headspace volatiles, they can still be discriminated and identified using multivariate techniques.     

Isolation and screening of potential actinobacteria for rapid composting of rice straw

Rice straw is produced as a by-product from rice cultivation, which is composed largely of lignocellulosic materials amenable to general biodegradation. Lignocellulolytic actinobacteria can be used as a potential agent for rapid composting of bulky rice straw. Twenty-five actinobacteria isolates were isolated from various in situ and in vitro rice straw compost sources. Isolates A2, A4, A7, A9 and A24 were selected through enzymatic degradation of starch, cellulose and lignin followed by the screening for their adaptability on rice straw powder amended media. The best adapted isolate (A7) was identified as Micromonospora carbonacea. It was able to degrade cellulose, hemicelluloses and carbon significantly (P ≤ 0.05) over the control. C/N ratio was reduced to 18.1 from an initial value of 29.3 in 6 weeks of composting thus having the potential to be used in large scale composting of rice straw.     

Amelioration of altered antioxidant status and membrane linked functions by vanadium andTrigonella in alloxan diabetic rat brains

Trigonella foenum graecum seed powder (TSP) and sodium orthovanadate (SOV) have been reported to have antidiabetic effects. However, SOV exerts hypoglycemic effects at relatively high doses with several toxic effects. We used low doses of vanadate in combination with TSP and evaluated their antidiabetic effects on antioxidant enzymes and membrane-linked functions in diabetic rat brains. In rats, diabetes was induced by alloxan monohydrate (15 mg/100 g body wt.) and they were treated with 2 IU insulin, 0.6 mg/ml SOV, 5% TSP and a combination of 0.2 mg/ml SOV with 5% TSP for 21 days. Blood glucose levels, activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), Na⁺/K⁺ ATPase, membrane lipid peroxidation and fluidity were determined in different fractions of whole brain after 21 days of treatment. Diabetic rats showed high blood glucose (P < 0.001), decreased activities of SOD, catalase and Na⁺/K⁺ ATPase (P < 0.01,P < 0.001 andP < 0.01), increased levels of GPx and MDA (P < 0.01 andP < 0.001) and decreased membrane fluidity (P < 0.01). Treatment with different antidiabetic compounds restored the above-altered parameters. Combined dose ofTrigonella and vanadate was found to be the most effective treatment in normalizing these alterations. Lower doses of vanadate could be used in combination with TSP to effectively counter diabetic alterations without any toxic effects.