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At least 829 records · Page 46Linked to original sources
PubMed1999

Elapid venom toxins: multiple recruitments of ancient scaffolds.

Nigroxins A and B, two myotoxic phospholipases A2 (PLA2s) from the venom of the American elapid Micrurus nigrocinctus, belong to a new PLA2 subclass. Their primary structures were established and compared with those of PLA2s that have already been studied with respect to myotoxic activity. The combination of amino acid residues Arg15, Ala100, Asn108 and a hydrophobic residue at position 109 is present exclusively in class I PLA2s that display myotoxic activity. These residues cluster within a surface region rich in positive charges and are suggested to play a role in the interaction with the target membrane of the muscle fibers. It is concluded that the myotoxic PLA2s resulted from recruitment of an ancient scaffold. Dendrotoxins and alpha-neurotoxins are similarly derived from other old structures, which are, however, now also present in nontoxic proteins that are widely distributed throughout the animal kingdom. The evolutionary pathways by which elapid PLA2s acquired myotoxicity and dendrotoxins acquired K+-channel blocker activity are traced. They demonstrate how existing scaffolds were adapted stepwise to serve toxic functions by exchange of a few surface-exposed residues.

Amino Acid Sequence↗
PubMed2000

Evidence for expression of heteromeric serotonin 5-HT(3) receptors in rodents.

The gene and cDNAs that encode a novel subunit of rodent serotonin 5-HT(3) receptors were isolated from mouse and rat tissues. Each of the new rodent subunits shares 40% amino acid identity with the rat 5-HT(3A) subunit and 73% identity with the human 5-HT(3B) subunit. Despite a relatively low level of structural conservation, sequence analysis and functional studies suggest that the new rodent subunits are orthologues of the human 5-HT(3B) subunit. In common with homologous human receptors, rat heteromeric 5-HT(3) receptors displayed a substantially larger single-channel conductance than homomeric 5-HT(3A) receptors. In addition, the rat heteromeric receptors were less sensitive to antagonism by tubocurarine. However, in contrast to human heteromeric receptors, those of the rat displayed pronounced inward rectification of both the whole-cell and single-channel current amplitudes. Transcripts of the mouse 5-HT(3A) and 5-HT(3B) subunits are coexpressed in several cell lines that possess endogenous 5-HT(3) receptors. In addition, treatment of rat PC12 cells with nerve growth factor induced expression of both subunit mRNAs, with a similar time course for accumulation of each transcript. The combination of functional data and expression patterns is consistent with the existence of heteromeric 5-HT(3) receptors in rodent neurons.

Amino Acid Sequence↗
PubMed1998

Mutations in the connexin 26 gene (GJB2) among Ashkenazi Jews with nonsyndromic recessive deafness.

BACKGROUND: Mutations in the GJB2 gene cause one form of nonsyndromic recessive deafness. Among Mediterranean Europeans, more than 80 percent of cases of nonsyndromic recessive deafness result from inheritance of the 30delG mutant allele of GJB2. We assessed the contribution of mutations in GJB2 to the prevalence of the condition among Ashkenazi Jews. METHODS: We tested for mutations in GJB2 in DNA samples from three Ashkenazi Jewish families with nonsyndromic recessive deafness, from Ashkenazi Jewish persons seeking carrier testing for other conditions, and from members of other ethnic groups. The hearing of persons who were heterozygous for mutations in GJB2 was assessed by means of pure-tone audiometry, measurement of middle-ear immittance, and recording of otoacoustic emissions. RESULTS: Two frame-shift mutations in GJB2, 167delT and 30delG, were observed in the families with nonsyndromic recessive deafness. In the Ashkenazi Jewish population the prevalence of heterozygosity for 167delT, which is rare in the general population, was 4.03 percent (95 percent confidence interval, 2.5 to 6.0 percent), and for 30delG the prevalence was 0.73 percent (95 percent confidence interval, 0.2 to 1.8 percent). Genetic-linkage analysis showed conservation of the haplotype for 167delT but the existence of several haplotypes for 30delG. Audiologic examination of carriers of the mutant alleles who had normal hearing revealed subtle differences in their otoacoustic emissions, suggesting that the expression of mutations in GJB2 may be semidominant. CONCLUSIONS: The high frequency of carriers of mutations in GJB2 (4.76 percent) predicts a prevalence of 1 deaf person among 1765 people, which may account for the majority of cases of nonsyndromic recessive deafness in the Ashkenazi Jewish population. Conservation of the haplotype flanking the 167delT mutation suggests that this allele has a single origin, whereas the multiple haplotypes with the 30delG mutation suggest that this site is a hot spot for recurrent mutations.

Connexin 26↗
PubMed1983

Identification by transfection of transforming sequences in DNA of human T-cell leukemias.

DNA from human T-cell leukemia cell lines was tested for focus-inducing activity on cultures of NIH 3T3 cells. Three leukemias yielded DNA active in this assay; restriction enzyme sensitivity of this activity indicated that similar, relatively large DNA sequences were involved. Southern blot analysis revealed conserved size classes of restriction fragments containing human repetitive (Alu) sequences in serially transfected foci derived from the active DNAs. Similar blot hybridizations with a probe specific for the human N-ras oncogene detected a 9-kilobase EcoRI fragment in all cases. DNA containing this fragment from one of the leukemias, molecularly cloned in bacteriophage lambda, displayed highly amplified focus-inducing activity in transfection assays. Thus, the N-ras oncogene appears to be active in these three human leukemias of T-cell origin.

Animals↗
PubMed1995

Geographic and climatic control of primate diversity.

Although the comparative ecology of primates has been relatively well studied and there have been a number of outstanding studies of individual primate communities, the factors determining primate species diversity on either a local or regional level are largely unexplored. Understanding the determinants of species abundance is an important aspect of biodiversity and is critical for interpreting the comparative ecology of these different communities and for designing effective strategies of conservation. Comparative analysis of species diversity in more than 70 primate communities from South America, Africa, Madagascar, and Asia shows that on major continental areas and large tropical islands, there is a high positive correlation between the number of primate species and the area of tropical forest. Within major continental areas, the species diversity at individual sites is highly correlated with mean annual rainfall for South America, Africa, and Madagascar, but not Asia.

Africa↗
PubMed1998

Vascular endothelial growth factor B (VEGF-B) binds to VEGF receptor-1 and regulates plasminogen activator activity in endothelial cells.

The vascular endothelial growth factor (VEGF) family has recently expanded by the identification and cloning of three additional members, namely VEGF-B, VEGF-C, and VEGF-D. In this study we demonstrate that VEGF-B binds selectively to VEGF receptor-1/Flt-1. This binding can be blocked by excess VEGF, indicating that the interaction sites on the receptor are at least partially overlapping. Mutating the putative VEGF receptor-1/Flt-1 binding determinants Asp63, Asp64, and Glu67 to alanine residues in VEGF-B reduced the affinity to VEGF receptor-1 but did not abolish binding. Mutational analysis of conserved cysteines contributing to VEGF-B dimer formation suggest a structural conservation with VEGF and platelet-derived growth factor. Proteolytic processing of the 60-kDa VEGF-B186 dimer results in a 34-kDa dimer containing the receptor-binding epitopes. The binding of VEGF-B to its receptor on endothelial cells leads to increased expression and activity of urokinase type plasminogen activator and plasminogen activator inhibitor 1, suggesting a role for VEGF-B in the regulation of extracellular matrix degradation, cell adhesion, and migration.

3T3 Cells↗
PubMed1998

Characterization of the glycosyltransferase enzyme from the Escherichia coli K5 capsule gene cluster and identification and characterization of the glucuronyl active site.

Bacterial capsular polysaccharides play an important role in virulence and survival. The Escherichia coli K5 capsule consists of a repeat structure of -4)GlcA-beta(1,4)-GlcNAc alpha(1-, identical to N-acetylheparosan. A 60-kDa protein, KfiC, has been identified as a bifunctional glycosyltransferase, responsible for the alternating alpha and beta addition of each UDP-sugar to the nonreducing end of the polysaccharide chain. Using hydrophobic cluster analysis, a conserved secondary structure motif characteristic of beta-glycosyltransferases was identified along with two highly conserved aspartic acid residues at positions 301 and 352 within the KfiC protein. Site-directed mutagenesis was used to identify catalytically active amino acids within domain A of the KfiC protein. The conserved aspartic acid residues at 301 and 352 were shown to be critical for the beta addition of UDP-GlcA (uridine diphosphoglucuronic acid) to defined nonreducing end oligosaccharide acceptors, suggesting that these conserved aspartic acid residues are catalytically important for beta-glycosyltransferase activity. A deleted derivative of the kfiC gene was generated, which encoded for a truncated KfiC (kfiC') protein. This protein lacked 139 amino acids at the C terminus. This enzyme had no UDP-GlcA transferase activity but still retained UDP-GlcNAc transferase activity, indicating that two separate active sites are present within the KfiC protein.

Amino Acid Sequence↗
PubMed2001

Analysis of murine Brca2 reveals conservation of protein-protein interactions but differences in nuclear localization signals.

In this report, we have analyzed the protein encoded by the murine Brca2 locus. We find that murine Brca2 shares multiple properties with human BRCA2 including its regulation during the cell cycle, localization to nuclear foci, and interaction with Brca1 and Rad51. Murine Brca2 stably interacts with human BRCA1, and the amino terminus of Brca2 is sufficient for this interaction. Exon 11 of murine Brca2 is required for its stable association with RAD51, whereas the carboxyl terminus of Brca2 is dispensable for this interaction. Finally, in contrast to human BRCA2, we demonstrate that carboxyl-terminal truncations of murine Brca2 localize to the nucleus. This finding may explain the apparent inconsistency between the cytoplasmic localization of carboxyl-terminal truncations of human BRCA2 and the hypomorphic phenotype of mice homozygous for similar carboxyl-terminal truncating mutations.

Animals↗
PubMed2002

Promoter use by sigma 38 (rpoS) RNA polymerase. Amino acid clusters for DNA binding and isomerization.

Sigma(38) is a non-essential but highly homologous member of the sigma(70) family of transcription factors. In vitro mutagenesis and in vivo screening were used to identify 22 critical amino acids in the promoter interaction domain of Escherichia coli sigma(38). Electrophoretic mobility shift assay studies showed that residues involved in duplex DNA binding largely segregated into distinct regions that coincided with those of sigma(70). However, the majority of these amino acids were in non-conserved positions. Analysis indicates that this region of the two sigma(s) probably has a common overall organization but differs in how its amino acids are used to form functional open complexes. Placement of the mutations on the known sigma(70) holoenzyme structure shows two clusters; one appears to be used for duplex DNA recognition and the other for the subsequent isomerization events. Permanganate assays for DNA melting support this view.

Amino Acid Sequence↗
PubMed2003

Interaction of L2 with beta-actin directs intracellular transport of papillomavirus and infection.

Viruses that replicate in the nucleus, including the primary causative agent of cervical cancer, human papillomavirus type 16 (HPV16), must first cross the cytoplasm. We compared the uptake of HPV16 virus-like particles (VLPs) either with or without the minor capsid protein L2. Whereas VLPs containing only the major capsid protein L1 were diffusely distributed within the cytoplasm even 6 h post-infection, VLPs comprising both L1 and L2 exhibited a radial distribution in the cytoplasm and accumulated in the perinuclear region of BPHE-1 cells within 2 h. L2 of HPV16 or bovine papillomavirus was shown to bind to a 43-kDa cellular protein that was subsequently identified as beta-actin by matrix-assisted laser desorption ionization time-of-flight analysis. A conserved domain comprising residues 25-45 of HPV16 L2 was sufficient for interaction with beta-actin. HPV16 L2 residues 25-45 fused to green fluorescent protein, but not green fluorescent protein alone, colocalized with actin and caused cell retraction and disruption of the microfilament network. Finally, wild-type L2, but not L2 with residues 25-45 deleted, facilitated HPV16 pseudovirion infection. Thus, binding of beta-actin by L2 residues 25-45 facilitates transport of HPV16 across the cytoplasm during infection, and blockade of this novel interaction may be useful for prophylaxis.

Actins↗
PubMed2004

Structural and functional characterization of a novel phosphodiesterase from Methanococcus jannaschii.

Methanococcus jannaschii MJ0936 is a hypothetical protein of unknown function with over 50 homologs found in many bacteria and Archaea. To help define the molecular (biochemical and biophysical) function of MJ0936, we determined its crystal structure at 2.4-A resolution and performed a series of biochemical screens for catalytic activity. The overall fold of this single domain protein consists of a four-layered structure formed by two beta-sheets flanked by alpha-helices on both sides. The crystal structure suggested its biochemical function to be a nuclease, phosphatase, or nucleotidase, with a requirement for some metal ions. Crystallization in the presence of Ni(2+) or Mn(2+) produced a protein containing a binuclear metal center in the putative active site formed by a cluster of conserved residues. Analysis of MJ0936 against a panel of general enzymatic assays revealed catalytic activity toward bis-p-nitrophenyl phosphate, an indicator substrate for phosphodiesterases and nucleases. Significant activity was also found with two other phosphodiesterase substrates, thymidine 5'-monophosphate p-nitrophenyl ester and p-nitrophenylphosphorylcholine, but no activity was found for cAMP or cGMP. Phosphodiesterase activity of MJ0936 had an absolute requirement for divalent metal ions with Ni(2+) and Mn(2+) being most effective. Thus, our structural and enzymatic studies have identified the biochemical function of MJ0936 as that of a novel phosphodiesterase.

Amino Acid Sequence↗
PubMed2001

Immunochemical control of the species origin of intestinal mucosa used for heparin purification.

Species specific antisera against bovine, ovine, and porcine serum albumin were produced in order to control the absence of bovine, ovine, or caprine tissues in the porcine intestinal mucosa used for heparin production. Two immunoassays were developed. An enzyme linked immunosorbent assay (ELISA) was very sensitive down to 1 ng/mL bovine albumin or 10 ppm bovine intestinal mucosa in porcine intestinal mucosa. For routine control, a more convenient single radial immunodiffusion assay (SRID) was found suitable to detect 2 microg/mL albumin or 3 p 1000 bovine, ovine, or caprine intestinal mucosa in porcine intestinal mucosa. Conditions of extraction of albumin from intestinal mucosa were optimized and a CV % of 4.1 was obtained for its quantitation. Due to higher albumin concentrations, detection of bovine hashed gut and lung was more sensitive (1.5 and 0.9 p 1000, respectively). Using antisera raised against porcine albumin the SRID can be applied to certify the porcine origin of the intestinal mucosa used for heparin purification and to control its adequate conservation before analysis.

Albumins↗
PubMed1990

Analysis of the molecular basis of calmodulin defects that affect ion channel-mediated cellular responses: site-specific mutagenesis and microinjection.

The ability of microinjected calmodulin to temporarily restore an ion channel-mediated behavioral phenotype of a calmodulin mutant in Paramecium tetraurelia (cam1) is dependent on the amino acid side chain that is present at residue 101, even when there is extensive variation in the rest of the amino acid sequence. Analysis of conservation of serine-101 in calmodulin suggests that the ability of calmodulin to regulate this ion channel-associated cell function may be a biological role of calmodulin that is widely distributed phylogenetically. A series of mutant calmodulins that differ only at residue-101 were produced by in vitro site-specific mutagenesis and expression in Escherichia coli, purified to chemical homogeneity, and tested for their ability to temporarily restore a wild-type behavioral phenotype to cam1 (pantophobiacA1) Paramecium. Calmodulins with glycine-101 or tyrosine-101 had minimal activity; calmodulins with phenylalanine-101 or alanine-101 had no detectable activity. However, as a standard of comparison, all of the calmodulins were able to activate a calmodulin-regulated enzyme, myosin light chain kinase, that is sensitive to point mutations elsewhere in the calmodulin molecule. Overall, these results support the hypothesis that the structural features of calmodulin required for the transduction of calcium signals varies with the particular pathway that is being regulated and provide insight into why inherited mutations of calmodulin at residue 101 are nonlethal and selective in their phenotypic effects.

Amino Acid Sequence↗
PubMed2000

Genetic variability of human immunodeficiency virus type 2 C2V3 region within and between individuals from Bissau, Guinea-Bissau, West Africa.

The V3 loop of both HIV-1 and HIV-2 is characterized by a high degree of genetic variation. To investigate the spectrum of HIV-2 variability in nature we have focused on the C2V3 region of Env and analyzed 108 viral sequences obtained from uncultured peripheral blood mononuclear cells obtained from 16 HIV-2-seropositive individuals from Bissau (Guinea-Bissau). The estimated values of genetic divergence between individuals were higher than those calculated from sequence information collected in a single individual. We have also found that the sequences surrounding the V3 loop contribute significantly to the overall genetic diversity of the C2V3 region of HIV-2 gp105, while the V3 loop itself seems to be rather conserved. Phylogenetic analysis demonstrated that all the individuals enrolled in this study were infected with HIV-2 subtype A viruses.

Adolescent↗
PubMed1995

Using a measure of structural variation to define a core for the globins.

As the database of three-dimensional protein structures expands, it becomes possible to classify related structures into families. Some of these families, such as the globins, have enough members to allow statistical analysis of conserved features. Previously, we have shown that a probabilistic representation based on means and variances can be useful for defining structural cores for large families. These cores contain the subset of atoms that are in essentially the same relative positions in all members of the family. In addition to defining a core, our method creates an ordered list of atoms, ranked by their structural variation. In applying our core-finding procedure to the globins, we find that helices A, B, G and H form a structural core with low variance. These helices fold early in the folding pathway, and superimpose well with helices in the helix-turn-helix repressor protein family. The non-core helices (F and the parts of other helices that interact with it) are associated with the functional differences among the globins, and are encoded within a separate exon. We have also compared the variability measure implicit in our core structures with measures of sequence variability, using a procedure for measuring sequence variability that helps correct for the biased sampling in the databanks. We find, somewhat surprisingly, that sequence variation does not appear to correlate with structural variation.

Algorithms↗
PubMed2003

EPPS: mining the COG database by an extended phylogenetic patterns search.

SUMMARY: EPPS runs under Microsoft Windows. It is an extended version of the phylogenetic patterns search (PPS). The output condition of PPS is the exact match of a user defined phylogenetic pattern with the pattern represented by the respective cluster of orthologous groups (COG). In contrast, the software described here is less restrictive. The user may define the accuracy of the search by the number of genomes that are allowed not to match the predefined phylogenetic pattern. Thus, EPPS has the advantage to detect COGs even if organisms defined to be included are not or organisms defined to be excluded are present in the output COGs.

Amino Acid Sequence↗
PubMed1997

Comparison between the Escherichia coli and Bacillus subtilis genomes suggests that a major function of polynucleotide phosphorylase is to synthesize CDP.

Genome comparison permits identification of chromosome regions conserved during evolution. Bacillus subtilis and Escherichia coli are so distant that there exists very few conserved landmarks in their genome organisation. Analysis of the conserved cmk rpsA cluster pinpointed the importance of cytosine nucleotide metabolism. In these bacteria, mRNA turnover provides an efficient means to fulfil the need for CDP as a precursor of DNA synthesis. The cmk rpsA operon is responsible for CDP synthesis. This function is self-explained in the case of the cmk gene (which codes for cytidylate kinase). The case of rpsA, that codes for ribosomal protein S1, is more subtle. It is suggested here that S1 is a RNA-binding protein helping polynucleotide phosphorylase (PNPase, known to be phylogenetically related to S1) to degrade mRNA, or helper molecule involved in other RNase activities. This provides an explanation for the elusive function of PNPase, which generates nucleoside diphosphates (not monophosphates) when degrading RNA. This also accounts for the discovery that the B. subtilis comR gene product is PNPase. This article briefly discusses the availability of cytosine nucleotides in eukaryotes, and suggests that they are derived from phospholipids turnover. Finally, the GC content of genomes is discussed in this new light.

Bacillus subtilis↗