Search PubMed⌕ Search

Biomedical subjects

E Orr

Publications and source records attributed to E Orr.

At least 19 recordsLinked to original sources
PubMed2001

An unusual beta-ketoacyl:acyl carrier protein synthase and acyltransferase motifs in TaK, a putative protein required for biosynthesis of the antibiotic TA in Myxococcus xanthus.

The antibiotic TA of Myxococcus xanthus is produced by a type-I polyketide synthase mechanism. Previous studies have indicated that TA genes are clustered within a 36-kb region. The chemical structure of TA indicates the need for several post-modification steps, which are introduced to form the final bioactive molecule. These include three C-methylations, an O-methylation and a specific hydroxylation. In this study, we describe the genetic analysis of taK, encoding a specific polyketide beta-ketoacyl:acyl carrier protein synthase, which contains an unusual beta-ketoacyl synthase and acyltransferase motifs and is likely to be involved in antibiotic TA post-modification. Functional analysis of this beta-ketoacyl:acyl carrier protein synthase by specific gene disruption suggests that it is essential for the production of an active TA molecule.

3-Oxoacyl-(Acyl-Carrier-Protein) Synthase↗
PubMed2000

Penetration of the coral-bleaching bacterium Vibrio shiloi into Oculina patagonica.

Inoculation of the coral-bleaching bacterium Vibrio shiloi into seawater containing its host Oculina patagonica led to adhesion of the bacteria to the coral surface via a beta-D-galactose receptor, followed by penetration of the bacteria into the coral tissue. The internalized V. shiloi cells were observed inside the exodermal layer of the coral by electron microscopy and fluorescence microscopy using specific anti-V. shiloi antibodies to stain the intracellular bacteria. At 29 degrees C, 80% of the bacteria bound to the coral within 8 h. Penetration, measured by the viable count (gentamicin invasion assay) inside the coral tissue, was 5.6, 20.9, and 21.7% of the initial inoculum at 8, 12, and 24 h, respectively. The viable count in the coral tissue decreased to 5.3% at 48 h, and none could be detected at 72 h. Determination of V. shiloi total counts (using the anti-V. shiloi antibodies) in the coral tissue showed results similar to viable counts for the first 12 h of infection. After 12 h, however, the total count more than doubled from 12 to 24 h and continued to rise, reaching a value 6 times that of the initial inoculum at 72 h. Thus, the intracellular V. shiloi organisms were transformed into a form that could multiply inside the coral tissue but did not form colonies on agar medium. Internalization of the bacteria was accompanied by the production of high concentrations of V. shiloi toxin P activity in the coral tissue. Internalization and multiplication of V. shiloi are discussed in terms of the mechanism of bacterial bleaching of corals.

Animals↗
PubMed1999

Aromatic and basic residues within the EVH1 domain of VASP specify its interaction with proline-rich ligands.

Short contiguous peptides harboring proline-rich motifs are frequently involved in protein-protein interactions, such as associations with Src homology 3 (SH3) and WW domains. Although patches of aromatic residues present in either domain interact with polyprolines, their overall structures are distinct, suggesting that additional protein families exist that use stacked aromatic amino acids (AA domains) to bind polyproline motifs [1] [2] [3]. A polyproline motif (E/DFPPPPTD/E in the single-letter amino-acid code), present in the ActA protein of the intracellular bacterial pathogen Listeria monocytogenes, serves as a ligand for the Ena/VASP protein family --the vasodilator-stimulated phosphoprotein (VASP), the murine protein Mena, Drosophila Enabled (Ena) and the Ena/VASP-like protein Evl [4] [5] [6] [7]. These share a similar overall structure characterized by the two highly conserved Ena/VASP homology domains (EVH1 and EVH2) [5]. Here, using three independent assays, we have delineated the minimal EVH1 domain. Mutations of aromatic and basic residues within two conserved hydrophilic regions of the EVH1 domain abolished binding to ActA. Binding of an EVH1 mutant with reversed charges could partially be rescued by introducing complementary mutations within the ligand. Like SH3 domains, aromatic residues within the EVH1 domain interacted with polyprolines, whereas the ligand specificity of either domain was determined by reciprocally charged residues. The EVH1 domain is therefore a new addition to the AA domain superfamily, which includes SH3 and WW domains.

Amino Acid Sequence↗
PubMed1999

Cloning and characterization of a Myxococcus xanthus cytochrome P-450 hydroxylase required for biosynthesis of the polyketide antibiotic TA.

The antibiotic TA, a complex macrocyclic polyketide of Myxococcus xanthus, is produced, like many other polyketides, through successive condensations of acetate by a type I polyketide synthase (PKS) mechanism. The chemical structure of this antibiotic and the mechanism by which it is synthesized indicate the need for several post-modification steps, such as a specific hydroxylation at C-20. Previous studies have shown that several genes, essential for TA biosynthesis, are clustered in a region of at least 36kb, which was subsequently cloned and analyzed. In this study, we report the analysis of a DNA fragment, containing a specific cytochrome P-450 hydroxylase, presumably responsible for the sole non-PKS hydroxylation at position C-20. Functional analysis of the cytochrome P-450 hydroxylase gene through specific gene disruption confirms that it is essential for the production of an active TA molecule.

Amino Acid Sequence↗
PubMed1999

The first gene in the biosynthesis of the polyketide antibiotic TA of Myxococcus xanthus codes for a unique PKS module coupled to a peptide synthetase.

The polyketide antibiotic TA is synthesized by the Gram negative bacterium Myxococcus xanthus in a multi-step process in which a unique glycine-derived molecule is used as a starter unit and elongated through the condensation of 11 acetate molecules by polyketide synthases (PKSs). Analysis of a 7.2 kb DNA fragment, encoding the protein that carries out the first condensation step, revealed that the fragment constitutes a single open reading frame, referred to as Ta1, which lacks the 5' and 3' ends and displays two regions of similarity to other proteins. The first 1020 amino acid residues at the N terminus of the polypeptide are similar to sequences of the large family of enzymes encoding peptide synthetases. They are followed by a second region displaying a high degree of similarity to type I PKS genes. The genetic analysis of this open reading frame is compatible with the proposed chemical structure of TA. The data indicate that the genes encoding TA have a modular gene organization, typical of a type I PKS system. The unusual feature of Ta1 is that the first PKS module of TA resides on the same polypeptide as the peptide synthetase functional unit.

Acetates↗
PubMed1999

A NusG-like transcription anti-terminator is involved in the biosynthesis of the polyketide antibiotic TA of Myxococcus xanthus.

The antibiotic TA of Myxococcus xanthus is synthesized through a type I polyketide synthase mechanism. Previous studies have indicated that several genes essential for TA production are clustered within a 40-kb region and are transcriptionally co-regulated. In this study, we report the genetic analysis of the first gene in the TA gene cluster, identified as a NusG-like transcription anti-terminator. Functional analysis of this NusG-like anti-terminator gene by specific gene disruption confirms that it is essential for TA production but not for normal growth and development.

Amino Acid Sequence↗
PubMed1999

Mutations in the Saccharomyces cerevisiae gene SAC1 cause multiple drug sensitivity.

Wild-type yeast Saccharomyces cerevisiae are surprisingly resistant to a wide range of drugs and agents. We had previously isolated novobiocin-sensitive mutants to aid the study of the intracellular target for this drug. Characterization of one of these mutants, mds1, revealed that it was sensitive not only to novobiocin but also to a wide range of drugs. The nature of this multiple drug-sensitive phenotype was shown to be different from that of previously isolated multiple drug-sensitive mutants. We have shown that the multiple drug-sensitivity of mds1 is due to mutations within the gene SAC1 and have identified a variety of mutations within the gene from the Mds1 strain. SAC1 encodes a protein which has been previously implicated in the correct function of the actin cytoskeleton, in inositol metabolism, in ATP transport in the endoplasmic reticulum and in Sec14p (PI-TP) function. We have shown that multiple drug-sensitivity is a new phenotype seen in some, but not all, of the previously characterized sac1 mutants. Based on our findings, we propose a mechanism by which Sac1p could affect drug resistance and also mediate other effects on cell growth.

Anti-Bacterial Agents↗
PubMed1999

A nonessential signal peptidase II (Lsp) of Myxococcus xanthus might be involved in biosynthesis of the polyketide antibiotic TA.

Myxococcus xanthus is a gram-negative soil bacterium that produces the polyketide antibiotic TA. In this study, we describe the analysis of an M. xanthus gene which encodes a homologue of the prolipoprotein signal peptidase II (SPase II; lsp). Overexpression of the M. xanthus SPase II in Escherichia coli confers high levels of globomycin resistance, confirming its function as an SPase II. The M. xanthus gene encoding the lsp homologue is nonessential for growth, as determined by specific gene disruption. It has been mapped to the antibiotic TA gene cluster, and the disrupted mutants do not produce the antibiotic, indicating a probable involvement in TA production. These results suggest the existence of more than one SPase II protein in M. xanthus, where one is a system-specific SPase II (for TA biosynthesis).

Amino Acid Sequence↗
PubMed1995

Orthopedic disability, conformity, and social support.

The relation between physical disability, social support, and conformist behavior was tested in two studies. The first compared the conforming responses of persons who had orthopedic disabilities with those of able-bodied individuals and correlated perceived social support of those with the disability with their tendency to conform. The second was an experimental study in which university students made choices between actors who had disabilities and actors who were able bodied; orthopedic disability was signified by a person sitting in a wheelchair and social support by the choice of a teammate. Results showed that persons with disabilities (compared with those who were able bodied) reported a significantly higher tendency to conform and that this tendency was negatively related to reported levels of perceived social support. In the experimental study, conforming behaviors of both disabled and able-bodied actors elicited more social support than did assertive behaviors. The actors who had "disabilities" received less social support than those who were "able bodied," but the former were considered more original when they did not conform. The findings imply that individuals with orthopedic disabilities are expected to conform but, although their conforming behavior elicits social support within specific encounters, it does not affect overall social support across encounters.

Activities of Daily Living↗
PubMed1995

Actual and perceived parental social status: effects on adolescent self-concept.

This paper proposes a model in which parental social status (PSS) and perceived parental social status (PEPSS) affect adolescent self-concept in distinct ways: PSS affects school achievement and consequently self-concept in the academic domain, whereas PEPSS affects self-concept in the social domain. Two studies of Israeli high school students (N = 569), one on kibbutz and one on urban youth, were carried out. PSS was measured by father's education and occupation, and PEPSS by a new scale. Academic and social self-concepts were tapped by Marsh's SDQ II in the urban sample, and by a combination of the latter and Harter's SPPA in the kibbutz sample. The findings supported the model with one exception: in the urban setting, PSS was not related to academic self-concept. The findings rejected a model underlying the theory of Rosenberg and Pearlin, in which perceived parental social status mediates the effect of parental social status on adolescent self-concept.

Adolescent↗
PubMed1994

Novobiocin activates the mating response in yeast through the alpha-pheromone receptor, Ste2p.

In a previous publication we reported the isolation of several yeast mutants sensitive to the coumarin drug novobiocin. Here we characterise the wild-type gene (NBS5), obtained by rescuing a novobiocin-sensitive nbs5 strain to natural novobiocin resistance. We show that nbs5 is allelic to the previously characterised gene sst2, conferring super-sensitivity to the mating pheromones. Although nbs5/sst2 cells of both mating types are super-sensitive to the opposite mating pheromone, only cells of mating-type a are sensitive to novobiocin. We show that the entire effect of the drug is mediated through Ste2p, the alpha-pheromone receptor. Thus, novobiocin is a functional agonist of Ste2p, and may identify a potentially useful interaction between coumarin drugs and the family of G-protein-coupled receptors.

Genetic Complementation Test↗
PubMed1994

Cloning of an intracellular receptor for protein kinase C: a homolog of the beta subunit of G proteins.

Protein kinase C (PKC) translocates from the soluble to the cell particulate fraction on activation. Intracellular receptors that bind activated PKC in the particulate fraction have been implicated by a number of studies. Previous work identified 30- to 36-kDa proteins in the particulate fraction of heart and brain that bound activated PKC in a specific and saturable manner. These proteins were termed receptors for activated C-kinase, or RACKs. In the following study, we describe the cloning of a cDNA encoding a 36-kDa protein (RACK1) that fulfills the criteria for RACKs. (i) RACK1 bound PKC in the presence of PKC activators, but not in their absence. (ii) PKC binding to the recombinant RACK1 was not inhibited by a pseudosubstrate peptide or by a substrate peptide derived from the pseudosubstrate sequence, indicating that the binding did not reflect simply PKC association with its substrate. (iii) Binding of PKC to RACK1 was saturable and specific; two other protein kinases did not bind to RACK1. (iv) RACK1 contains two short sequences homologous to a PKC binding sequence previously identified in annexin I and in the brain PKC inhibitor KCIP. Peptides derived from these sequences inhibited PKC binding to RACK1. Finally, RACK1 is a homolog of the beta subunit of G proteins, which were recently implicated in membrane anchorage of the beta-adrenergic receptor kinase [Pitcher, J., Inglese, L., Higgins, J. B., Arriza, J. A., Casey, P. J., Kim, C., Benovic, J. L., Kwatra, M. M., Caron, M. G. & Lefkowitz, R. J. (1992) Science 257, 1264-1267]. Our in vitro data suggest a role for RACK1 in PKC-mediated signaling.

Amino Acid Sequence↗
PubMed1994

Isolation and complete sequence of CBR, a gene encoding a putative cytochrome b reductase in Saccharomyces cerevisiae.

We have isolated and characterised a novel yeast gene, CBR (cytochrome b reductase), encoding a 35-kDa yeast novobiocin-binding protein. The predicted protein sequence of CBR displays considerable similarity to both plant nitrate reductases and mammalian cytochrome b5 reductases indicating that it is a putative member of the flavoprotein pyridine-nucleotide-cytochrome-reductase family. Disruption of CBR is not lethal under various growth conditions, suggesting the presence of some functional overlap with other reductases, possibly with the cytochrome P-450 reductase.

Amino Acid Sequence↗
PubMed1994

Identification, partial sequence and genetic analysis of mlpA, a novel gene encoding a myosin-related protein in Physarum polycephalum.

Studies of motility in Physarum polycephalum have concentrated on the well-defined actomyosin system in plasmodia. It is clear from recent genetic studies in lower eukaryotes that myosin is involved in a number of physiological processes in addition to the contractile functions previously ascribed to the classical type II myosins. Moreover, the myosin protein family has proved to be more complex than anticipated, with an increasing number of reported specialized isoforms. Although a myosin type II activity has been identified in both amoebae and plasmodia of P. polycephalum, and it has been inferred that these proteins undergo a phase-specific isoform switch during development, this phenomenon has not been analysed genetically. In an effort to understand the putative developmental expression of actomyosin-associated proteins, we isolated a 180-kDa protein from amoebae which is highly enriched, along with actin and myosin, in actomyosin preparations in the presence of mM concentrations of Mg++ ions and 10 mM of ATP. Using polyclonal antisera raised against pl80 we have cloned and sequenced a partial cDNA encoding a protein whose predicted amino-acid sequence indicates some similarity with the Dictyostelium discoideum myosin heavy-chain tail domain. Southern-blot and RFLP analyses indicate that the gene involved, designated mlpA (myosin-like protein), occurs in a single copy in the genome, is a novel Physarum gene and is expressed during amoebal and plasmodial growth and in the dormant forms of both these cell types.

Amino Acid Sequence↗
PubMed1993

A proline-rich protein, verprolin, involved in cytoskeletal organization and cellular growth in the yeast Saccharomyces cerevisiae.

A gene (VRP1) encoding a novel proline-rich protein (verprolin) has been isolated from the yeast Saccharomyces cerevisiae as a result of its hybridization to a chick vinculin cDNA probe. The deduced protein sequence contains 24% proline residues present as proline-rich motifs throughout the verprolin sequence. Several of these motifs resemble recently identified sequences shown to bind Src homology 3 (SH3) domains in vitro. Replacement of the wild-type VRP1 allele with a mutant allele results in strains that grow slower than wild-type strains and are temperature sensitive. The vrp1 mutants are impaired in both cell shape and size and display aberrant chitin and actin localization. We propose that verporlin is involved in the maintenance of the yeast actin cytoskeleton, through interactions with other proteins, possibly containing SH3 domains.

Actins↗
PubMed1991

The identification and purification of a mammalian-like protein kinase C in the yeast Saccharomyces cerevisiae.

We have purified a yeast protein kinase that is phospholipid-dependent and activated by Diacylglycerol (DAG) in the presence of Ca2+ or by the tumour-promoting agent tetradecanoyl-phorbol acetate (TPA). The properties of this enzyme are similar to those of the mammalian protein kinase C (PKC). The enzyme was purified using chromatography on DEAE-cellulose followed by hydroxylapatite. The latter chromatography separated the activity to three distinguishable sub-species, analogous to the mammalian PKC isoenzymes. The fractions enriched in PKC activity contain proteins that specifically bind TPA, are specifically phosphorylated in the presence of DAG and recognized by anti-mammalian PKC antibodies.

Animals↗
PubMed1991

The yeast type II myosin heavy chain: analysis of its predicted polypeptide sequence.

We have completed the nucleotide sequence of the yeast MYO1 gene and deduced its amino acid sequence. The gene is 5553 bp long and contains no introns. Analysis of the sequence, as well as its comparison with other myosins, demonstrate that the yeast protein is a type II myosin heavy chain with characteristic head and tail regions. The latter domain contains six proline residues in two clusters of three, at approximately two thirds from the start of the gene.

Acanthamoeba↗