Human neurotrophin-3: A one-step peptide mapping method and complete disulfide characterization of the recombinant protein

Human neurotrophin-3 (NT-3) is a member of the nerve growth factor (NGF) family of neurotrophic factors, and the recombinant protein is being developed as a therapeutic for neurodegenerative diseases. The final product purity and lot-to-lot variation are monitored routinely by peptide mapping. However, only the N-terminal region of NT-3 was susceptible to proteolysis under native conditions. Complete digestion required that the protein be chemically modified by reduction and S-alkylation prior to proteolysis. Complete proteolytic degradation of the protein was achieved simply by an intial denaturation of NT-3 in 6 M guanidinium chloride (pH 6) for 2 hr at 37°C, followed by a tenfold dilution with the digestion buffer (0.1 M Tris-HCl, 1 mM CaCl₂ at pH 7.0) and immediate addition of chymotrypsin at 1% by weight. Direct comparison of the peptide map with an identical aliquot that had been reduced and alkylated also allowed the establishment of the cystine linkages present in NT-3: Cys¹⁴ to Cys⁷⁹, Cys⁵⁷ to Cys¹⁰⁸, and Cys⁶⁷ to Cys¹¹⁰. This disulfide structure is homologous to the NGF family of neurotrophic factors.     

3-Aryl/Heteryl-5-Phenylindeno[1,2-d]thiazolo[3,2-a]pyrimidin-6(5H)-ones: Synthesis,Characterization, and Antimicrobial Investigation

Russian Journal of Bioorganic Chemistry - Discovery towards the potent antimicrobial agents is indispensable for the treatment of infections caused by resistant microbes. Thus, we prepared a novel...     

Multi-unit Recording Methods to Characterize Neural Activity in the Locust (Schistocerca Americana) Olfactory Circuits

Detection and interpretation of olfactory cues are critical for the survival of many organisms. Remarkably, species across phyla have strikingly similar olfactory systems suggesting that the biological approach to chemical sensing has been optimized over evolutionary time¹. In the insect olfactory system, odorants are transduced by olfactory receptor neurons (ORN) in the antenna, which convert chemical stimuli into trains of action potentials. Sensory input from the ORNs is then relayed to the antennal lobe (AL; a structure analogous to the vertebrate olfactory bulb). In the AL, neural representations for odors take the form of spatiotemporal firing patterns distributed across ensembles of principal neurons (PNs; also referred to as projection neurons)^2,3. The AL output is subsequently processed by Kenyon cells (KCs) in the downstream mushroom body (MB), a structure associated with olfactory memory and learning^4,5. Here, we present electrophysiological recording techniques to monitor odor-evoked neural responses in these olfactory circuits.First, we present a single sensillum recording method to study odor-evoked responses at the level of populations of ORNs^6,7. We discuss the use of saline filled sharpened glass pipettes as electrodes to extracellularly monitor ORN responses. Next, we present a method to extracellularly monitor PN responses using a commercial 16-channel electrode³. A similar approach using a custom-made 8-channel twisted wire tetrode is demonstrated for Kenyon cell recordings⁸. We provide details of our experimental setup and present representative recording traces for each of these techniques.     

Multiplex ligation-dependant probe amplification study of children with idiopathic mental retardation in South India

BACKGROUND:

Mental retardation (MR) is a heterogeneous dysfunction of the central nervous system exhibiting complex phenotypes and has an estimated prevalence of 1-3% in the general population. However, in about 50% of the children diagnosed with any form of intellectual disability or developmental delay the cause goes undetected contributing to idiopathic intellectual disability.

MATERIALS AND METHODS:

A total of 122 children with developmental delay/MR were studied to identify the microscopic and submicroscopic chromosome rearrangements by using the conventional cytogenetics and multiplex ligation dependent probe amplification (MLPA) analysis using SALSA MLPA kits from Microbiology Research Centre Holland [MRC] Holland.

RESULTS:

All the recruited children were selected for this study, after thorough clinical assessment and metaphases prepared were analyzed by using automated karyotyping system. None was found to have chromosomal abnormality; MLPA analysis was carried out in all subjects and identified in 11 (9%) patients.

CONCLUSION:

Karyotype analysis in combination with MLPA assays for submicroscopic micro-deletions may be recommended for children with idiopathic MR.     

Reduced Expression of EXTL2, a Member of the Exostosin (EXT) Family of Glycosyltransferases,in Human Embryonic Kidney 293 Cells Results in Longer Heparan Sulfate Chains

Heparan sulfate proteoglycans are ubiquitously located on cell surfaces and in the extracellular matrices. The negatively charged heparan sulfate chains interact with a multitude of different proteins, thereby influencing a variety of cellular and developmental processes, for example cell adhesion, migration, tissue morphogenesis, and differentiation. The human exostosin (EXT) family of genes contains five members: the heparan sulfate polymerizing enzymes, EXT1 and EXT2, and three EXT-like genes, EXTL1, EXTL2, and EXTL3. EXTL2 has been ascribed activities related to the initiation and termination of heparan sulfate chains. Here we further investigated the role of EXTL2 in heparan sulfate chain elongation by gene silencing and overexpression strategies. We found that siRNA-mediated knockdown of EXTL2 in human embryonic kidney 293 cells resulted in increased chain length, whereas overexpression of EXTL2 in the same cell line had little or no effect on heparan sulfate chain length. To study in more detail the role of EXTL2 in heparan sulfate chain elongation, we tested the ability of the overexpressed protein to catalyze the in vitro incorporation of N-acetylglucosamine and N-acetylgalactosamine to oligosaccharide acceptors resembling unmodified heparan sulfate and chondroitin sulfate precursor molecules. Analysis of the generated products revealed that recombinant EXTL2 showed weak ability to transfer N-acetylgalactosamine to heparan sulfate precursor molecules but also, that EXTL2 exhibited much stronger in vitro N-acetylglucosamine-transferase activity related to elongation of heparan sulfate chains.     

New clerodane diterpenoids from the aerial parts of Pulicaria wightiana

Two new ent-clerodane-type diterpenoids, compounds 1 and 2, were isolated from the aerial parts of Pulicaria wightiana, together with three known constituents. Their structures were established based on spectroscopic data, and their antibacterial activities were evaluated (Table 2).     

Pod surface exudates of wild relatives of pigeonpea influence the feeding preference of the pod borer, Helicoverpa armigera

Wild relatives of crops are an important source of resistance genes against insect pests. However, it is important to identify the accessions of wild relatives of crops with different mechanisms of resistance to broaden the basis and increase the levels of resistance to insect pests. Therefore, we studied the feeding behavior of pod borer, Helicoverpa armigera, which is the most damaging pest of pigeonpea, in relation to biochemical characteristics of the pod surface exudates in a diverse array of germplasm accessions belonging to 12 species of pigeonpea wild relatives. Feeding by H. armigera larvae was significantly lower on the unwashed or water-, methanol-, or hexane-washed pods of Canajus sericeus, C. scarabaeoides, Flemingia bracteata, F. stricta, and Rhynchosia aurea than those of C. acutifolius, C. albicans, C. cajanifolius, C. lineatus, D. ferruginea, P. scariosa, R. bracteata, and the cultivated pigeonpea, C. cajan genotypes, ICPL 87, and ICPL 332, although there were a few exceptions. The methanol-washed pods of wild relatives were less preferred for feeding by the H. armigera larvae than the unwashed pods, but the hexane-washed pods were preferred more than the unwashed pods. The results suggested that methanol extracted the phagostimulants from the pod surface, while hexane removed the antifeedants. The high-performance liquid chromatography (HPLC) finger printing of methanol and hexane pod surface extracts showed qualitative and quantitative differences in compounds present on the pod surface of different wild relatives of pigeonpea. Some of the peaks in HPLC profiles were associated with feeding preference of the third-instar larvae of H. armigera. There was considerable diversity in wild relatives of pigeonpea as revealed by principal component analysis based on HPLC fingerprints of pod surface extracts in methanol and hexane, and H. armigera feeding on the pods. Wild pigeonpea accessions with low amounts of phagostimulants and high amounts of antifeedants may be used for introgression of resistance genes into the cultivated pigeonpea to develop varieties with broad-based resistance to H. armigera. There is considerable diversity among the wild relatives of pigeonpea, and the accessions with resistance to pod borer. These can be used to broaden the basis and increase the levels of resistance to H. armigera.     

Oxidation of Bovine β-Casein by Hypochlorite

We recently observed two 2,4-dinitrophenylhydrazine (DNPH)-reactive proteins of 40 and 120 kDa in the bronchoalveolar lavage fluids of rats exposed to >95% O₂ for 48 h. The N-terminal sequences of these proteins were both identical over 16 amino acids with rat β-casein, which, in addition to its more common association with milk, is produced by cytotoxic T-lymphocytes, and has been found to have proinflammatory properties. Because of the inflammatory response that accompanies hyperoxic lung injury, we investigated the oxidation of bovine β-casein by HOCl. Following exposure to HOCl at 4°C for 15 min, derivatization with DNPH, washing, and digestion with trypsin, the resultant peptides were separated by reverse-phase HPLC. One peptide isolated from a peak absorbing at 365 nm was identified as AVP(Y*)PQR, corresponding to amino acids 177–183 of bovine β-casein. Analysis of the peptide by both electrospray and matrix assisted laser desorption ionization (MALDI) mass spectrometry identified a molecular ion MH⁺ of 1008.5 Da, which represents an increase of 178 Da from the calculated monoisotopic MH⁺ of the unmodified peptide of 830.45 Da. Daughter ion spectra of the doubly charged parent ion of the peptide further support the oxidation of the tyrosine to the quinone methide, with subsequent conversion to the corresponding hydrazone with DNPH. A second pair of products were identified as arising from oxidation of Y¹⁹³ within the tryptic peptide constituted by amino acids 184–202, and the corresponding chymotryptic cleavage side product, 191–202. Exposure of β-casein to increasing amounts of HOCl revealed that M and Y residues were the most susceptible, although bovine β-casein contains no C, and a single W, which would not be detected by our methods. The approach described in the present report can be used to evaluate the contributions of distinct mechanisms of oxidation in other experimental or pathological models. © 1997 Elsevier Science Inc.     

Studies on SAGO wastewater treatment using anaerobic rotating biological contactor

Experimental studies were done in a laboratory scale Anaerobic Rotating Biological Contactor (RBC), for treatment of Synthetic sago wastewater. This paper describes the development and laboratory testing of an Anaerobic RBC process that couples the advantages of the fixed film horizontal flow RBC process with the high strength, starch degradation capabilities of anaerobic systems. The reactor was operated at ambient temperature and was subjected to organic and hydraulic loading rates. The reactor performance with respect to Chemical Oxygen Demand (COD) removal, alkalinity, volatile acids at each stage and biogas production were evaluated. The Anaerobic RBC reactor liquid volume is 70 litres and total disc surface area is 4.45 m². The reactor was operated with about 100% of the disc area submerged and with a rotational speed held constant at 9?rev/min. The synthetic sago wastewater was started with a COD value of 1087?mg/l at a hydraulic retention time(HRT) of 42?h and it was varied till maximum COD of 9522?mg/l. From the present study, the optimum COD load was found to be 6860?mg/l with a COD removal efficiency of 97.2%.With this optimum COD load, hydraulic loading rate(HLR) study was done at 24?h to 48?h HRT. COD removal efficiencies at hydraulic loading rates were compared with the work of Subrahmanyam &; Sastry (1988). From the present study, the proportionality coefficient was found to be 1.18 with process efficiencies at different hydraulic loading rates.