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D R Hillyard

Publications and source records attributed to D R Hillyard.

At least 37 records · Page 2Linked to original sources
PubMed1996

Rapid detection of the staphylococcal mecA gene from BACTEC blood culture bottles by the polymerase chain reaction.

A rapid polymerase chain reaction (PCR) method for the direct detection of the staphylococcal mecA gene from BACTEC blood culture bottles (Becton Dickinson, Sparks, MD) was developed. Published primer sequences and sample preparation using Achromopeptidase for cell lysis were adapted to the use of the Idaho Technology Air Thermocycler 1605 (Idaho Technologies, Idaho Falls, ID). The method was validated with 80 strains of coagulase-positive and coagulase-negative geographically diverse methicillin-resistant and susceptible isolates of staphylococci. There was a 100% correlation between the PCR results and the results of standard susceptibility testing methods. From BACTEC 9240 blood cultures, mixed aliquots of blood and broth containing gram-positive cocci in clusters were centrifuged at low speed to sediment red blood cells. After additional centrifugation and wash steps, PCR was performed on the resuspended pellet. The turnaround time from initial Gram stain detection of positive BACTEC bottles to PCR amplicon detection by agarose gel electrophoresis is less than 3 hours. In a clinical evaluation of 181 blood culture isolates, there was a 99% correlation with standard susceptibility results for Staphylococcus aureus. Discrepant results for Staphylococcus aureus isolates were verified by a Mueller Hinton plate supplemented with 6 microg/mL of oxacillin and 2% sodium chloride. For coagulase-negative staphylococci, the PCR method detected an additional seven resistant isolates that were reported by the Vitek as susceptible. Coagulase-negative staphylococcal susceptibility results that were in disagreement with the PCR assay were confirmed by the disk-diffusion method. This procedure is accurate, rapid and fits well into laboratory work flow. Rapid detection of the mecA gene on positive blood culture vials has become a routine test in the authors' clinical microbiology laboratory.

Bacterial Proteins↗
PubMed1995

A new family of conotoxins that blocks voltage-gated sodium channels.

Conus peptides, including omega-conotoxins and alpha-conotoxins (targeting calcium channels and nicotinic acetylcholine receptors, respectively) have been useful ligands in neuroscience. In this report, we describe a new family of sodium channel ligands, the mu-O-conotoxins. The two peptides characterized, mu-O-conotoxins MrVIA and MrVIB from Conus marmoreus potently block the sodium conductance in Aplysia neurons. This is in marked contrast to standard sodium channel blockers that are relatively ineffective in this system. The sequences of the peptides are as follows. mu-O-conotoxin MrVIA: ACRKKWEYCIVPIIGFIYCCPGLICGPFVCV mu-O-conotoxin MrVIB: ACSKKWEYCIVPILGFVYCCPGLICGPFVCV mu-O-conotoxin MrVIA was chemically synthesized and proved indistinguishable from the natural product. Surprisingly, the mu-O-conotoxins show no sequence similarity to the mu-O-conotoxins. However, ananalysis of cDNA clones encoding the mu-O-conotoxin MrVIB demonstrated striking sequence similarity to omega- and delta-conotoxin precursors. Together, the omega-, delta-, and mu-O-conotoxins define the O-superfamily of Conus peptides. The probable biological role and evolutionary affinities of these peptides are discussed.

Amino Acid Sequence↗
PubMed1995

Purification, characterization, synthesis, and cloning of the lockjaw peptide from Conus purpurascens venom.

The major groups of Conus peptides previously characterized from fish-hunting cone snail venoms (the alpha-, mu-, and omega-conotoxins) all blocked neuromuscular transmission. A novel activity, the "lockjaw peptide", from the fish-hunting Conus purpurascens, caused a rigid (instead of flaccid) paralysis in fish and increased excitability at the neuromuscular junction (instead of a block). We report the purification, biological activity, biochemical and preliminary physiological characterization, and chemical synthesis of the lockjaw peptide and the sequence of a cDNA clone encoding its precursor. Taken together, the data lead us to conclude that the lockjaw peptide is a vertebrate-specific delta-conotoxin, which targets voltage-sensitive sodium channels. The sequence of the peptide, which we designate delta-conotoxin PVIA, is (O = 4-trans-hydroxyproline) EACYAOGTFCGIKOGLCCSEFCLPGVCFG-NH2. This is the first of a diverse spectrum of Conus peptides which are excitotoxins in vertebrate systems.

Action Potentials↗
PubMed1995

Combinatorial peptide libraries in drug design: lessons from venomous cone snails.

Many present-day drugs are derived from compounds that are natural products, a traditional source of which is fermentation broths of microorganisms. The venoms of cone snails are a new natural resource of peptides that may have a pharmaceutical potential equivalent to those from traditional sources, particularly for developing drugs that target cell-surface receptors or ion channels. In effect, cone snails have used a combinatorial library strategy to evolve their small, highly bioactive venom peptides. The methods by which the snails have generated thousands of peptides with remarkable specificity and high affinity for their targets may provide important lessons in designing combinatorial libraries for drug development.

Amino Acid Sequence↗
PubMed1994

The molecular approach to microbial diagnosis.

Nucleic acid technology is reshaping the laboratory approach to infectious diseases. The intrinsic advantages of the molecular diagnostic approach, coupled with rapid technical progress in this field, ensure its expanding role in clinical microbiology and virology. Interpretation of molecular tests requires a thorough understanding of their theoretical foundations, and laboratory professionals should renew their commitment to education in this area.

DNA Probes↗
PubMed1993

A new Conus peptide ligand for Ca channel subtypes.

A cDNA clone encoding a new omega-conotoxin was identified from Conus magus. The predicted peptide was chemically synthesized using a novel strategy that efficiently yielded the biologically active disulfide-bonded isomer. This peptide, omega-conotoxin MVIID, targets other voltage-gated calcium channels besides the N-subtype and exhibits greater discrimination against the N-channel subtype than any other omega-conotoxin variant to date. Consequently, omega-conotoxin MVIID may be a particularly useful ligand for calcium channel subtypes that are not of the L- or N-subclasses. Of the eight major sequence variants of omega-conotoxins that have been elucidated, four come from Conus magus venom. We suggest that sequence variants from the same venom may be designed to optimally interact with different molecular variants of calcium channels; such omega-conotoxin sets from a single venom may therefore be useful for helping to identify novel calcium channel subtypes.

Amino Acid Sequence↗
PubMed1992

Heterodimeric structure of the spider toxin omega-agatoxin IA revealed by precursor analysis and mass spectrometry.

We report the first molecular characterization of a precursor sequence for a small, Ca2+ channel blocking, peptide spider toxin, omega-agatoxin IA. By integrating information generated from a molecular genetic approach using agatoxin cDNAs with data provided from mass spectrometry of the mature toxin, we were able to deduce the likely mechanisms by which the toxin precursor peptide is processed to its mature heterodimeric form. A particularly interesting feature of the prepropeptide is the occurrence of two glutamate-rich sequences interposed between the signal sequences, the major peptide toxin, and the minor toxin peptide. Excision of the more distal glutamate-rich region appears to be signaled by flanking arginine residues but likely occurs only after a disulfide linkage has formed between the major and minor chains of the mature toxin. Our molecular genetic approach toward characterizing this toxin will allow us to quickly generate a series of spider sequences from which mature toxin structures can be deduced and eventually expressed. Additionally, this approach will provide insights into the evolutionary divergence observed among spider peptide toxins.

Agatoxins↗
PubMed1992

Precursor structure of omega-conotoxin GVIA determined from a cDNA clone.

The Ca2+ channel blocking neurotoxin, omega-conotoxin GVIA, is a 27-amino acid peptide with three disulfide bonds. We have determined the precursor structure of this peptide by analyzing a cDNA clone obtained from a Conus geographus venom duct library. The omega-conotoxin GVIA prepropeptide is 73 amino acids in length comprising a 22 amino acid signal sequence, an intervening region of 23 amino acids, and finally, a 27 amino acid toxin region. A C-terminal glycine residue is later processed to a C-terminal amide moiety. The omega-conotoxin GVIA precursor exhibits regions of strong homology to the previously characterized King-Kong peptide precursor, but shows surprising divergence as well. The possible significance of the precursor organization is discussed.

Amino Acid Sequence↗
PubMed1992

A new Conus peptide ligand for mammalian presynaptic Ca2+ channels.

Voltage-sensitive Ca2+ channels that control neurotransmitter release are blocked by omega-conotoxin (omega-CgTx) GVIA from the marine snail Conus geographus, the most widely used inhibitor of neurotransmitter release. However, many mammalian synapses are omega-CgTx-GVIA insensitive. We describe a new Conus peptide, omega-CgTx-MVIIC, that is an effective inhibitor of omega-CgTx-GVIA-resistant synaptic transmission. Ca2+ channel targets that are inhibited by omega-CgTx-MVIIC but not by omega-CgTx-GVIA include those mediating depolarization-induced 45Ca2+ uptake in rat synaptosome preparations, "P" currents in cerebellar Purkinje cells, and a subset of omega-CgTx-GVIA-resistant currents in CA1 hippocampal pyramidal cells. The characterization of omega-CgTx-MVIIC by a combination of molecular genetics and chemical synthesis defines a general approach for obtaining ligands with novel receptor subtype specificity from Conus.

Amino Acid Sequence↗
PubMed1991

Conotoxins.

Explore the source record for details and available documents.

Amino Acid Sequence↗
PubMed1990

Diversity of Conus neuropeptides.

Conus venoms contain a remarkable diversity of pharmacologically active small peptides. Their targets are ion channels and receptors in the neuromuscular system. The venom of Conus geographus contains high-affinity peptides that act on voltage-sensitive calcium channels, sodium channels, N-methyl-D-aspartate (NMDA) receptors, acetylcholine receptors, and vasopressin receptors; many more peptides with still uncharacterized receptor targets are present in this venom. It now seems that the Conus species (approximately 500 in number) will each use a distinctive assortment of peptides and that the pharmacological diversity in Conus venoms may be ultimately comparable to that of plant alkaloids or secondary metabolites of microorganisms. The cone snails may generate this diverse spectrum of venom peptides by a "fold-lock-cut" synthetic pathway. These peptides are specific enough to discriminate effectively between closely related receptor subtypes and can be used for structure-function correlations.

Amino Acid Sequence↗
PubMed1990

Constant and hypervariable regions in conotoxin propeptides.

Conotoxins are small cysteine rich peptides found in the venom of the predatory cone snails (Conus) which have prove to be useful high affinity ligands for various receptors and ion channels. The first cloning data for conotoxins, reported here, were obtained for the King-Kong peptide, a 27 amino acid conotoxin found in the venom of the cloth-of-gold cone, Conus textile. Analysis of cDNA clones of the King-Kong peptide revealed a family of related toxin transcripts. Three different propeptide cDNA sequences were obtained; only one of these encoded sequence for the King-Kong peptide. The other cDNA sequences encoded two different peptides (KK-1 and KK-2). When the predicted propeptide sequences are compared, well defined conserved and hypervariable regions can be identified. The hypervariable regions comprise four regions between Cys residues in the final peptide toxins; the remainder of the propeptide sequences, i.e. the excised N-terminal regions and the disulfide bonded Cys residues, are highly conserved. We suggest that the conserved regions may direct the formation of a specific disulfide configuration in the King-Kong family of conotoxins.

Amino Acid Sequence↗
PubMed1990

Subunit-specific phenotypes of Salmonella typhimurium HU mutants.

Salmonella hupA and hupB mutants were studied to determine the reasons for the high degree of conservation in HU structure in bacteria. We found one HU-1-specific effect; the F'128 plasmid was 25-fold less stable in hupB compared with hupA or wild-type cells. F' plasmids were 120-fold more unstable in hupA hupB double mutants compared with wild-type cells, and the double mutant also had a significant alteration in plasmid DNA structure. pBR322 DNA isolated from hupA hupB strains was deficient in supercoiling by 10 to 15% compared with wild-type cells, and the topoisomer distribution was significantly more heterogeneous than in wild-type or single-mutant strains. Other systems altered by HU inactivation included flagellar phase variation and phage Mu transposition. However, Mu transposition rates were only about fourfold lower in Salmonella HU double mutants. One reason that Salmonella HU double mutants may be less defective for Mu transposition than E. coli is the synthesis in double mutants of a new, small, basic heat-stable protein, which might partially compensate for the loss of HU. The results indicate that although either HU-1 or HU-2 subunit alone may accommodate the cellular need for general chromosomal organization, the selective pressure to conserve HU-1 and HU-2 structure during evolution could involve specialized roles of the individual subunits.

Bacterial Proteins↗
PubMed1989

Automated polymerase chain reaction in capillary tubes with hot air.

We describe a simple, compact, inexpensive thermal cycler that can be used for the polymerase chain reaction. Based on heat transfer with air to samples in sealed capillary tubes, the apparatus resembles a recirculating hair dryer. The temperature is regulated via thermocouple input to a programmable set-point process controller that provides proportional output to a solid state relay controlling a heating coil. For efficient cooling after the denaturation step, the controller activates a solenoid that opens a door to vent hot air and allows cool air to enter. Temperature-time profiles and amplification results approximate those obtained using water baths and microfuge tubes.

Air↗
PubMed1989

A molluscivorous Conus toxin: conserved frameworks in conotoxins.

We purified and characterized a 27 amino acid toxin from a snail-hunting Conus venom, Conus textile. This toxin causes convulsive-like activity in snails and causes subordinate lobsters to assume an exaggerated dominant posture. The sequence of this peptide is Trp-Cys-Lys-Gln-Ser-Gly-Glu-Met-Cys-Asn-Leu-Leu-Asp-Gln-Asn-Cys-Cys-Asp- Gly-Tyr-Cys-Ile-Val-Leu-Val-Cys-Thr. The sequence was confirmed by determining the nucleotide sequence of a cDNA clone coding for the peptide. The conservation of Cys residues compared to the omega-conotoxins from piscivorous Conus venom suggests that toxins from different cone venoms may use only a few "Cys-motifs" as conserved structural backbones for targeting to a variety of receptors in different animals.

Amino Acid Sequence↗
PubMed1989

DNA ligase and the pyridine nucleotide cycle in Salmonella typhimurium.

Bacterial DNA ligases use NAD as an energy source. In this study we addressed two questions about these enzymes. First, what is the physiological consequence of completely removing the NAD-dependent enzyme and replacing it with an ATP-dependent DNA ligase? We constructed Salmonella typhimurium strains in which the endogenous NAD-dependent DNA ligase activity was inactivated by an insertion mutation and the ATP-dependent enzyme from bacteriophage T4 was provided by a cloned phage gene. Such strains were physiologically indistinguishable from the wild type, even under conditions of UV irradiation or treatment with alkylating agents. These results suggest that specific functional interactions between DNA ligase and other replication and repair enzymes may be unimportant under the conditions tested. Second, the importance of DNA ligation as the initiating event of the bacterial pyridine nucleotide cycle was critically assessed in these mutant strains. Surprisingly, our results indicate that DNA ligation makes a minimal contribution to the pyridine nucleotide cycle; the Salmonella strains with only an ATP-dependent ligase had the same NAD turnover rates as the wild-type strain with an NAD-dependent ligase. However, we found that NAD turnover was significantly decreased under anaerobic conditions. We suggest that most intracellular pyridine nucleotide breakdown occurs in a process that protects the cell against oxygen damage but involves a biochemical mechanism other than DNA ligation.

Adenosine Triphosphate↗