PhyloFacts: an online structural phylogenomic encyclopedia for protein functional and structural classification

The Berkeley Phylogenomics Group presents PhyloFacts, a structural phylogenomic encyclopedia containing almost 10,000 'books' for protein families and domains, with pre-calculated structural, functional and evolutionary analyses. PhyloFacts enables biologists to avoid the systematic errors associated with function prediction by homology through the integration of a variety of experimental data and bioinformatics methods in an evolutionary framework. Users can submit sequences for classification to families and functional subfamilies. PhyloFacts is available as a worldwide web resource from .     

Monoclonal antibody based dot-enzyme linked immunosorbent assay (Dot-ELISA) and agar gel precipitation test (AGPT) for identification of Newcastle disease virus (NDV)

Two mouse monoclonal antibodies (MAbs), viz. 2B7 and 2 D10 raised against haemagglutinin-neuraminidase glycoprotein of Newcastle disease virus (NDV) were used to identify several other field isolates and vaccine strains of NDV. These MAbs reacted specifically with all the NDV strains/isolates in Dot-ELISA whereas, only MAb 2D10 reacted with all the NDV strains/isolates in agar gel precipitation test. These two tests employing the MAbs were standardised for rapid diagnosis and identification of NDV.     

G-protein-dependent cell surface dynamics of the human serotonin1A receptor tagged to yellow fluorescent protein

Serotonergic signaling appears to play a key role in the generation and modulation of various cognitive, behavioral, and developmental processes. The serotonin(1A) receptor is an important member of the superfamily of seven transmembrane domain G-protein-coupled receptors and is the most extensively studied among the serotonin receptors. Several aspects of serotonin(1A) receptor biology such as cellular distribution and signal transduction characteristics are technically difficult to address in living cells on account of the inability to optically track these receptors with fluorescence-based techniques. We describe here the characterization of the serotonin(1A) receptor tagged to the enhanced yellow fluorescent protein (EYFP) stably expressed in Chinese hamster ovary (CHO) cells. These receptors were found to be essentially similar to the native receptor in pharmacological assays and can therefore be used to reliably explore aspects of receptor biology such as cellular distribution and dynamics on account of their intrinsic fluorescent properties. Analysis of the cell surface dynamics of these receptors by fluorescence recovery after photobleaching (FRAP) experiments has provided novel insight into the molecular mechanism of signal transduction of serotonin(1A) receptors in living cells. Interestingly, addition of pharmacologically well-characterized ligands or activators of G-proteins altered the diffusion characteristics of the receptor in a manner consistent with the G-protein activation model. These results demonstrate, for the first time, that membrane dynamics of this receptor is modulated in a G-protein-dependent manner.     

BMP-2 regulates cardiomyocyte contractility in a phosphatidylinositol 3 kinase-dependent manner

Bone morphogenetic protein-2 (BMP-2) regulates development of heart during vertebrate embryogenesis. In vitro BMP-2 induces differentiation of precardiac cells into mature cardiomyocytes by inducing the expression of cardiac-specific genes. However, the role of BMP-2 and its signaling in other cardiac functions have not been studied. We examined the action of phosphatidylinositol (PI) 3 kinase in isolated adult rat cardiomyocytes. Incubation of rat ventricular cardiomyocytes with BMP-2 increased the PI 3 kinase activity. Ly294002, a pharmacological inhibitor of PI 3 kinase, blocked BMP-2-induced PI 3 kinase activity completely. To investigate the contractility of isolated cardiomyocytes, fractional shortening was examined. BMP-2 significantly increased the percent fractional shortening of the cardiomyocytes. Inhibition of PI 3 kinase activity completely abolished this action of BMP-2. These data indicate that PI 3 kinase regulates BMP-2-induced myocyte contractility. To further confirm this observation, we used adenovirus-mediated gene transfer to express a constitutively active myristoylated catalytic subunit of PI 3 kinase in rat cardiomyocytes. Infection of cardiomyocytes with the adenovirus vector increased the expression of constitutively active PI 3 kinase within 24 h. Expression of constitutively active PI 3 kinase significantly increased cardiomyocyte contractility. Together, these data show for the first time that the growth and differentiation factor, BMP-2, stimulates cardiomyocyte contractility. Also we provide the first evidence that BMP-2-induced PI 3 kinase activity regulates this cardiomyocyte function.     

Influence of aluminium on neurotoxicity of lead in adult male albino rats

Influence of aluminium on neurotoxicity of lead was studied in male albino rats. Aluminium enhanced the net deposition of lead in brain. This was further substantiated by higher levels of malondialdehyde (MDA), and lower activities of acetylcholinesterase enzyme in the brain homogenates of the rats treated with both lead and aluminium as compared to those of rats treated with lead only. In lead plus aluminium treated animals, a significant neurological deficit was observed when the animals were subjected to rota-rod, traction performance (TP) and tail immersion tests.     

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.     

COX-2 induction during murine gammaherpesvirus 68 infection leads to enhancement of viral gene expression

The murine gammaherpesvirus 68 (MHV-68 or gammaHV-68) model provides many advantages for studying virus-host interactions involved in gammaherpesvirus replication, including the role of cellular responses to infection. We examined the effects of cellular cyclooxygenase-2 (COX-2) and its by-product prostaglandin E(2) (PGE(2)) on MHV-68 gene expression and protein production following de novo infection of cultured cells. Western blot analyses revealed an induction of COX-2 protein in MHV-68-infected cells but not in cells infected with UV-irradiated MHV-68. Luciferase reporter assays demonstrated activation of the COX-2 promoter during MHV-68 replication. Two nonsteroidal anti-inflammatory drugs, a COX-2-specific inhibitor (NS-398) and a COX-1-COX-2 inhibitor (indomethacin), substantially reduced MHV-68 protein production in infected cells. Inhibition of viral protein expression and virion production by NS-398 was reversed in the presence of exogenous PGE(2). Global gene expression analysis using an MHV-68 DNA array showed that PGE(2) increased production of multiple viral gene products, and NS-398 inhibited production of many of the same genes. These studies suggest that COX-2 activity and PGE(2) production may play significant roles during MHV-68 de novo infection.     

Immunolocalization of detergent-susceptible nucleoplasmic lamin A/C foci by a novel monoclonal antibody

The A-type lamins are localized in the interior of the nucleus as well as on the nuclear periphery. In this study, we have characterized a monoclonal antibody LA-2F9 produced against recombinant rat lamin A which stains a subpopulation of various cell types in a pattern of small nucleoplasmic foci that are unusually susceptible to mild detergent/salt extraction. The specific reactivity of mAb LA-2F9 towards lamins was confirmed by immunoblotting of HeLa and C3H10T(1/2) whole cell lysates and nuclear lysates. The epitope for LA-2F9 was narrowed down to amino acid residues 268-278 (SAKLDNARQSA). To check whether the appearance of lamin foci was cell-cycle-dependent, C3H10T(1/2) cells were serum-starved and then refed to trigger cells to enter the G(1) phase of the cell-cycle. The intensity of staining increased 3.5-fold within 6 h of refeeding, when the maximum number of cells were labeled with LA-2F9. We also checked whether the LA-2F9 foci colocalized with nuclear proteins known to be distributed in small foci such as hnRNPs, snRNPs, SC-35, and p80 coilin, but did not find evidence of colocalization. Our studies suggest that LA-2F9 has a novel and specific reactivity towards detergent-susceptible lower order lamin structures that are likely to be assembly intermediates.     

Identification of a cis-element that determines autonomous DNA replication in eukaryotic cells

A 36-bp human consensus sequence (CCTMDAWKSGBYTSMAAWTWBCMYTTRSCAAATTCC) is capable of supporting autonomous replication of a plasmid after transfection into eukaryotic cells. After transfection and in vitro DNA replication, replicated plasmid DNA containing a mixture of oligonucleotides of this consensus was found to reiterate the consensus. Initiation of DNA replication in vitro occurs within the consensus. One version, A3/4, in pYACneo, could be maintained under selection in HeLa cells, unrearranged and replicating continuously for >170 cell doublings. Stability of plasmid without selection was high (> or =0.9/cell/generation). Homologs of the consensus are found consistently at mammalian chromosomal sites of initiation and within CpG islands. Versions of the consensus function as origins of DNA replication in normal and malignant human cells, immortalized monkey and mouse cells, and normal cow, chicken, and fruit fly cells. Random mutagenesis studies suggest an internal 20-bp consensus sequence of the 36 bp may be sufficient to act as a core origin element. This cis-element consensus sequence is an opportunity for focused analyses of core origin elements and the regulation of initiation of DNA replication.