Activity and structural comparisons of solution associating and monomeric channel-forming peptides derived from the glycine receptor m2 segment

A number of channel-forming peptides derived from the second transmembrane (TM) segment (M2) of the glycine receptor alpha(1) subunit (M2GlyR), including the 22-residue sequence NK(4)-M2GlyR p22 wild type (WT) (KKKKPARVGLGITTVLTMTTQS), induce anion permeation across epithelial cell monolayers. In vitro assays suggest that this peptide or related sequences might function as a candidate for ion channel replacement therapy in treating channelopathies such as cystic fibrosis (CF). The wild-type sequence forms soluble associations in water that diminish its efficacy. Introduction of a single substitution S22W at the C-terminus, NK(4)-M2GlyR p22 S22W, eliminates the formation of higher molecular weight associations in solution. The S22W peptide also reduces the concentration of peptide required for half-maximal anion transport induced across Madin-Darby canine kidney cells (MDCK) monolayers. A combination of 2D double quantum filtered correlation spectroscopy (DQF-COSY), total correlation spectroscopy (TOCSY), nuclear Overhauser effect spectroscopy (NOESY), and rotating frame nuclear Overhauser effect spectroscopy (ROESY) data were recorded for both the associating WT and nonassociating S22W peptides and used to compare the primary structures and to assign the secondary structures. High-resolution structural studies were recorded in the solvent system (40% 2,2,2-Trifluoroethanol (TFE)/water), which gave the largest structural difference between the two peptides. Nuclear Overhauser effect crosspeak intensity provided interproton distances and the torsion angles were measured by spin-spin coupling constants. These constraints were put into the DYANA modeling program to generate a group of structures. These studies yielded energy-minimized structures for this mixed solvent environment. Structure for both peptides is confined to the 15-residue transmembrane segments. The energy-minimized structure for the WT peptide shows a partially helical extended structure. The S22W peptide adopts a bent conformation forming a hydrophobic pocket by hydrophobic interactions.     

Amino acid residues involved in autophosphorylation and phosphotransfer activities are distinct in nucleoside diphosphate kinase from Mycobacterium tuberculosis

Nucleoside diphosphate kinase (NdK) is a ubiquitous enzyme in both prokaryotes and eukaryotes and is primarily involved in the maintenance of cellular nucleotide pools. We have cloned ndk from Mycobacterium tuberculosis strain H37Ra and expressed it in Escherichia coli as a fusion protein with glutathione S-transferase. The purified protein, following thrombin cleavage and gel permeation chromatography, was found to be hexameric with a monomeric unit molecular mass of approximately 16.5 kDa. The protein exhibited nucleotide binding, divalent cation-dependent autophosphorylation, and phosphate transfer ability from nucleoside triphosphate to nucleoside diphosphate. Although UDP inhibited the catalytic activity of the recombinant protein, the classic inhibitors, like cromoglycate, 5'-adenosine 3'-phosphate, and adenosine 3'-phosphate 5'-phosphosulfate, had no effect on the activity. Among three histidine residues in the protein, His-117 was found to be essential for autophosphorylation. However, in subsequent phosphate transfer, we observed that His-53 had a significant contribution. Consistent with this observation, substitution of His-53 with either Ala or Gln affected the ability of the recombinant protein to complement NdK function in Pseudomonas aeruginosa. Furthermore, mutational analysis established critical roles for Tyr-50 and Arg-86 of the M. tuberculosis protein in maintaining phosphotransfer ability.     

Structural requirements at the catalytic site of the heteroduplex substrate for human RNase H1 catalysis

Human RNase H1 cleaves RNA exclusively in an RNA/DNA duplex; neither double-strand DNA nor double-strand RNA is a viable substrate. Previous studies suggest that the helical geometry and sugar conformation of the DNA and RNA may play a role in the selective recognition of the heteroduplex substrate by the enzyme. We systematically evaluated the influence of sugar conformation, minor groove bulk, and conformational flexibility of the heteroduplex on enzyme efficiency. Modified nucleotides were introduced into the oligodeoxyribonucleotide at the catalytic site of the heteroduplex and consisted of southern, northern, and eastern biased sugars with and without 2'-substituents, non-hydrogen bonding base modifications, abasic deoxyribonucleotides, intranucleotide hydrocarbon linkers, and a ganciclovir-modified deoxyribonucleotide. Heteroduplexes containing modifications exhibiting strong northern or southern conformational biases with and without a bulky 2'-substituent were cleaved at a significantly slower rate than the unmodified substrate. Modifications imparting the greatest degree of conformational flexibility were the poorest substrates, resulting in dramatically slower cleavage rates for the ribonucleotide opposing the modification and the surrounding ribonucleotides. Finally, modified heteroduplexes containing modifications predicted to mimic the sugar pucker and conformational flexibility of the deoxyribonucleotide exhibited cleavage rates comparable with those of the unmodified substrate. These data suggest that sugar conformation, minor groove width, and the relative positions of the intra- and internucleotide phosphates are the crucial determinants in the selective recognition of the heteroduplex substrate by human RNase H1 and offer immediate steps to improve the performance of DNA-like antisense oligonucleotides.     

Spectroscopic investigation on gel-forming beta-sheet assemblage of peptide derivatives

The conformational studies of peptide derivatives A and B in a gel state were studied by using circular dichroism (CD), Fourier transformed infrared (FTIR), and fluorescence spectroscopic techniques. Birefringence and electron microscopic studies were carried out to characterize the morphological aspects of the fibrils in the gel. The FTIR spectra of the peptides show the absence of free NH in the gel state, implying that the intermolecular hydrogen-bond formation is the driving force for the aggregation. The CD spectrum of the peptide gels shows the presence of antiparallel and parallel beta-sheet conformation for peptide derivatives A and B, respectively. Electron microscopic studies (EM) of the peptide derivatives A and B reveal that peptide A formed rigid, rod-like structures without cross-linking and peptide B formed loose fibrils organized into highly noncovalently cross-linked mesh-like structural aggregates. Peptide A was much more soluble in alcoholic solvents than peptide B, and no birefringence was observed with Congo red (CR) staining in the temperature range of 0-80 degrees C. The spectroscopic studies indicate that peptide B consists of domains having a significant amount of beta-sheet structure and exhibiting golden yellow birefringence between 53 and 56 degrees C when stained with Congo red. On the other hand, peptide A gives no evidence of birefringence under polarized light. Fluorescence probe binding studies with pyrene in gel state with peptides A and B indicates the polarity in the interior of the aggregates. The data presented in the present work indicate that peptide B forms fibrils, which is similar to amyloid aggregates that are present in biological systems.     

Nypa megafossils from the Tertiary sediments of Northeast India

Nypa megafossils dominated by fruits are described from Oligocene and Lower Miocene sediments of Assam and Mizoram, respectively. The origin, past distribution and migration of Nypa are discussed in the light of recent findings. This study indicates that India might be the place of its origin. Its presence, along with some other coastal elements, reinforces the concept that the Bay of Bengal was extending considerably northward during these epochs than its present day boundary.     

Conservation of wild animals by assisted reproduction and molecular marker technology

Wild animals are an integral component of the ecosystem. Their decimation due to abrupt natural calamities or due to gradual human intervention would be disastrous to the ecosystem and would alter the balance in nature between various biotic components. Such an imbalance could have an adverse effect on the ecosystem. Therefore, there is an urgent need to put an end to the ever increasing list of endangered species by undertaking both in situ and ex situ conservation using tools of modern biology, to ascertain the degree of genetic variation and reproductive competence in these animals. This review highlights the development and use of molecular markers such as microsatellites, minisatellites, mitochondrial control region, cytochrome b and MHC loci to assess the genetic variation in various Indian wild animals such as the lion, tiger, leopard and deer. The review also presents data on the semen profile of the big cats of India. Reproductive technologies such as cryopreservation of semen and artificial insemination in big cats are also highlighted.     

Site specific integration of FLP recombinase in BHK-21 cell line

A binary system for gene activation and site specific integration based on conditional recombination of transfected sequences mediated by FLP recombinase from yeast was implemented in mammalian cells. In several cell lines, FLP rapidly and precisely recombined copies of its specific target sequences to activate an otherwise silent beta-galactosidase reporter gene. Clones of marked cells were generated by excisional recombination within a chromosomally integrated copy of the silent reporters. These clones exhibited intense blue colour with X-Gal staining solution.     

Characterisation of fowl adenoviruses from chickens affected with infectious hydropericardium during 1994-1998 in India

In the present study characterisation has been done for six group I fowl adenoviruses (FAV) isolated from outbreaks of infectious hydropericardium (IHP) of chickens that occurred in different states/regions of India during the years 1994-98. These six viruses were identified as FAV serotype 4 by virus neutralisation and restriction endonuclease analyses. Antigenic analyses of the viruses revealed close relationship (R-values 0.93-0.96). Under the experimental conditions, we have been able to induce IHP using FAV serotype 4 isolate AD: 411 and were also able detect FAV antigens in myocardial tissues by immunofluorescence assay (a new observation), an indication that IHP causing FAV serotype 4 strain replicate in myocardial tissue. Restriction endonuclease analysis of the viral genomes (approximately 46 Kb), using Hind III, Sma I, Xba I, Bam HI, Pst I and Dra I produced identical genetic profiles. Pst I and Bam HI profiles for these six vitus isolates were identical to those published earlier for an IHP causing Pakistani FAV serotype 4 isolate KR31. The identical genetic profiles of viruses, chronology of the outbreaks of IHP in Pakistan during 1989 onward and later in Jammu and Kashmir, India (1994), suggest that FAV serotype 4 isolates involved in outbreaks of IHP in India had probably spread from Pakistan. In order to prevent further spread and economic losses due to IHP in India, based on the antigenic relatedness data in this paper, any one of the six studied FAV serotype 4 isolates can be used as a candidate for mass production of CEH culture based killed vaccine.     

Disruption of distal interactions of Arg 262 and of substrate binding to Ser 52 affect catalysis of sheep liver cytosolic serine hydroxymethyltransferase

The crystal structure of human liver cytosolic recombinant serine hydroxymethyltransferase (hcSHMT) suggested that Ser53 and Arg 263 could participate in the reaction catalyzed by SHMT. The mutation of Arg262 (corresponding to Arg263 in hcSHMT) to "A" in sheep liver cytosolic SHMT (scSHMT) resulted in a 5-fold increase in Km for L-Ser and a 5-fold decrease in kcat compared to scSHMT. Further, in R262A SHMT-glycine complex, the peak at 343 nm (geminal diamine) was more pronounced, compared to wild-type enzyme. Stopped-flow studies showed that the rate constant for the formation of glycine-geminal diamine for R262A SHMT was also decreased. The rate of reaction, concentration of spectral intermediates, fluorescence excitation maximum of glycine geminal diamine and interaction with methoxyamine were altered in R262A SHMT. Although Arg263 in hcSHMT is located outside the PLP binding pocket, it positions Tyr73 for interaction with PLP, by forked H-bonding with the carbonyl groups of main chain residues, Asn71 and Lys72 of the other subunit of the tight dimer. Mutation of Arg262 to Ala and the consequent alteration in orientation of PLP leads to decreased catalytic efficiency. Ser53 (in hcSHMT) is in hydrogen bonding distance to one of the carboxylate oxygens of the amino acid substrate, which also interacts with Tyr83 and Arg402. Replacement of Ser53 with Cys (using 'O' software program) in the structure of hcSHMT resulted in disruption of these interactions, whereas replacement with Ala (S53A) only weakened the substrate interactions. There was a 10-fold increase in Km and 20-fold decrease in catalytic activity efficiency for S52C SHMT, whereas S52A SHMT retained 20% of the activity without change in Km for serine. These results suggest that S52 affects substrate binding and catalysis.