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Biomedical subjects

J L Sherwood

Publications and source records attributed to J L Sherwood.

10 recordsLinked to original sources
PubMed2006

Plant pathogen forensics: capabilities, needs, and recommendations.

A biological attack on U.S. crops, rangelands, or forests could reduce yield and quality, erode consumer confidence, affect economic health and the environment, and possibly impact human nutrition and international relations. Preparedness for a crop bioterror event requires a strong national security plan that includes steps for microbial forensics and criminal attribution. However, U.S. crop producers, consultants, and agricultural scientists have traditionally focused primarily on strategies for prevention and management of diseases introduced naturally or unintentionally rather than on responding appropriately to an intentional pathogen introduction. We assess currently available information, technologies, and resources that were developed originally to ensure plant health but also could be utilized for postintroduction plant pathogen forensics. Recommendations for prioritization of efforts and resource expenditures needed to enhance our plant pathogen forensics capabilities are presented.

Bioterrorism↗
PubMed2002

Symptom induction by Cowpea chlorotic mottle virus on Vigna unguiculata is determined by amino acid residue 151 in the coat protein.

The type strain of Cowpea chlorotic mottle virus (CCMV-T) produces a bright chlorosis in cowpea (Vigna unguiculata cv. California Blackeye). The attenuated variant (CCMV-M) induces mild green mottle symptoms that were previously mapped to RNA 3. Restriction fragment exchanges between RNA 3 cDNA clones of CCMV-T and CCMV-M that generate infectious transcripts and site-directed mutagenesis indicated that the codon encoding amino acid residue 151 of the coat protein determines the symptom phenotypes of CCMV-T and CCMV-M. Amino acid 151 is within an alpha-helical structure required for calcium ion binding and virus particle stability. No differences in virion stability or accumulation were detected between CCMV-T and CCMV-M. Mutational analysis suggested that the amino acid at position 151 and not the nucleotide sequence induce the symptom phenotype. Thus, it is likely that subtle influences by amino acid residue 151 in coat protein-host interactions result in chlorotic and mild green mottle symptoms.

Bromovirus↗
PubMed2000

Immunoprecipitation of a 50-kDa protein: a candidate receptor component for tomato spotted wilt tospovirus (Bunyaviridae) in its main vector, Frankliniella occidentalis.

A 50-kDa protein that binds to viral particles in solid-phase assays and that is recognized by anti-idiotypic antibodies made against anti-viral glycoproteins G1/G2 (anti-Ids) has been proposed as a receptor candidate for tomato spotted wilt tospovirus (TSWV) in its main thrips vector, Frankliniella occidentalis Pergande (Bandla et al., 1998. Phytopathology 88, 98-104). Here we show the immunoprecipitation of the 50-kDa protein by anti-Ids and by an anti-G1/G2-TSWV conjugate - a new immunoprecipitation method. In addition, we show that anti-Ids made against anti-G1 (anti-IdG1) block virus replication in an insect tissue replication assay. The results indicate that (a) the TSWV-50-kDa protein interaction occurs in solution, as it must do in vivo; (b) G1 is a viral attachment protein; and (c) the 50-kDa protein is a candidate host factor essential for TSWV entry. These results provide additional support for the role of the 50-kDa thrips protein as a viral receptor. Additionally these experiments provide the basis for testing saturable binding and represent an important step toward the first cloning and identification of a cellular receptor for a plant virus.

Animals↗
PubMed1998

Characterization of the nucleic acid binding properties of tomato spotted wilt virus nucleocapsid protein.

Tomato spotted wilt tospovirus (TSWV) is the type member of the plant-infecting viruses of the genus Tospovirus in the family Bunyaviridae. The three TSWV RNAs are encapsidated with nucleocapsid (N) protein to form ribonucleoprotein (RNP) which serves as the template for viral transcription and replication. Regions of the open reading frame coding for the N protein on the small (S) RNA were subcloned into pET protein expression vectors and expressed in Escherichia coli BL21 (DE3) cells. Full-length N, N amino and carboxy halves, and two N carboxy-terminal regions were expressed and isolated by metal chelate affinity chromatography. The N protein, both of its halves and the extreme carboxy-terminal region, bound cooperatively and irrespective of sequence to radiolabeled single-stranded RNA produced by runoff transcription of clones of either TSWV S RNA or cowpea chlorotic mottle virus RNA3. N protein did not bind to radiolabeled double-stranded TSWV RNA. The density of the synthetic RNase-sensitive N protein-RNA complexes was 1.32 g/ml, similar to the density of authentic Bunyaviridae RNPs. These studies are the first to indicate differences in the nucleic acid binding abilities of Tospovirus and Hantavirus nucleocapsid proteins, the only characterized nucleocapsid proteins of the family Bunyaviridae.

Binding Sites↗
PubMed1994

Stimulation of phospholipase D in rabbit platelet membranes by nucleoside triphosphates and by phosphocreatine: roles of membrane-bound GDP, nucleoside diphosphate kinase and creatine kinase.

Previous work has shown that guanosine 5'-[gamma-thio]triphosphate (GTP[S]) and GTP stimulate phospholipase D (PLD) in rabbit platelet membranes and that these effects are greatly enhanced by pretreatment of platelets with phorbol esters that activate protein kinase C [Van der Meulen and Haslam (1990), Biochem. J. 271, 693-700]. In the present study, the effects of Mg2+, various nucleoside triphosphates and phosphocreatine (PCr) were investigated. Platelet membranes containing phospholipids labelled with [3H]glycerol were assayed for PLD in the presence of an optimal Mg2+ concentration (10 mM) by measuring [3H]phosphatidylethanol formation in incubations that included 300 mM ethanol. In membranes from phorbolester-treated platelets, the same maximal increases in PLD activity (5-fold) were seen with 1 microM GTP[S]), and 100 microM GTP. Addition of adenosine 5'-[gamma-thio]triphosphate (ATP[S]), ITP, XTP, UTP and CTP had similar stimulatory effects, but only at > or = 1 mM. In contrast, ATP had a biphasic action, causing a maximal (2-fold) stimulation at 10 microM and smaller effects at higher concentrations; the inhibitory component of the action of ATP was blocked by 2 microM staurosporine. Guanosine 5'-[beta-thio]diphosphate decreased the stimulatory effects of ATP and ATP[S]. UDP, which can inhibit nucleoside diphosphate kinase (NDPK), decreased the activation of PLD by ATP[S], ATP, XTP, CTP and to a lesser extent ITP, but had no effect on the actions of GTP[S] and GTP. Rabbit platelet membranes contained NDPK and addition of [gamma-32P]ATP led to the formation of [32P]GTP in amounts sufficient to explain most or all of the activation of PLD; UDP prevented GTP formation. PCr (0.04-1 mM) also stimulated membrane PLD activity, an effect that was dependent on endogenous membrane-bound creatine kinase (CK). UDP and guanosine 5'-[beta-thio]diphosphate each inhibited this effect of PCr. The results show that in rabbit platelet membranes, CK, NDPK and the GTP-binding protein that activates PLD can be functionally coupled. However, assay of membrane preparations at increasing dilutions showed that stimulation of PLD by the compounds studied, with the partial exception of ATP[S], involved diffusible rather than protein-bound intermediates.

Animals↗
PubMed1994

Enhanced recovery of transformants of Agrobacterium tumefaciens after freeze-thaw transformation and drug selection.

Freeze-thaw transformation provides a simple and rapid method to transform Agrobacterium tumefaciens directly with plasmid DNA. Competent A. tumefaciens cells of strains LBA4404, GV3850 and EHA101 were transformed with four to nine plasmids differing in size, size of insert and in some cases sensitivity to antibiotics. A threefold to fourfold increase in transformed colonies per microgram of DNA was obtained by freezing cells with liquid nitrogen vs. dry ice/ethanol. Freezing cells in liquid nitrogen followed by incubation of transformed cells in a low concentration of appropriate antibiotics prior to plating resulted in a ninefold increase in colonies obtained compared with the procedure of freezing cells in dry ice/ethanol without the incubation period in the low concentration of antibiotics prior to plating. Restriction fragments of the expected sizes from the plasmids indicated that the procedural modifications did not cause apparent recombinations in the region of the inserts.

Agrobacterium tumefaciens↗
PubMed1993

Photoaffinity labelling of cyclic GMP-binding proteins in human platelets.

The photoaffinity labelling of platelet cyclic GMP (cGMP)-binding proteins by [32P]cGMP was studied; at least five labelled proteins (110, 80, 55, 49 and 38 kDa) were detected in platelet cytosol and four (80, 65, 49 and 38 kDa) in platelet membranes. The 110 kDa species was identified as cGMP-inhibited cyclic AMP (cAMP) phosphodiesterase (PDE III) by immunoprecipitation and by the inhibition of photolabelling by specific inhibitors of this enzyme. Similarly, the 80 kDa species was identified as cGMP-dependent protein kinase by immunoprecipitation and by the effects of cGMP analogues on photolabelling. Addition of cAMP greatly enhanced the labelling of this 80 kDa protein, implying the existence of a potentially important interaction between the effects of cGMP and cAMP. The 65 kDa photolabelled protein appears to be a novel platelet cyclic-nucleotide-binding protein. In contrast, the 49 and 55 kDa photolabelled species are probably the RI and RII regulatory subunits of cAMP-dependent protein kinase, and the 38 kDa protein(s) may be proteolytic fragment(s) of RI and/or RII.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1993

Rapid fixation and embedding method for immunocytochemical studies of tomato spotted wilt tospovirus (TSWV) in plant and insect tissues.

A new rapid fixation and embedding technique using microwave energy was evaluated for immunolabelling and examination of ultrastructure of plant and insect cells. Tissues in gluteraldehyde-paraformaldehyde were fixed for fifteen seconds in a microwave at 100% power, and dehydrated. Microwave energy was then used to polymerize the London Resin White (LR White) acrylic resin during the embedding process. Embedded specimens were then thin sectioned (90 nm) and treated with anti-tomato spotted wilt tospovirus (TSWV) antiserum followed by protein A-gold label, or antisera against a TSWV encoded nonstructural protein followed by goat anti-rabbit gold label. Using this technique, structural and nonstructural proteins of TSWV were readily detected and specifically labelled in cells of the insect vector, the western flower thrips, Frankliniella occidentalis (Pergande), and in infected cells of the plant species, Emilia sonchifolia L.

Animals↗
PubMed1993

Cloning of the capsid protein gene from a blotch isolate of peanut stripe virus.

The 3' terminal 1,367 nucleotides (nts) of a blotch isolate of the potyvirus, peanut stripe virus (PStV), were cloned and sequenced. This region included the 861 nts (287 amino acids) of the PStV capsid protein (CP). The amino acid sequence of the predicted proteinase cleavage site was identified. This region shared limited homology with other potyvirus proteinase cleavage sites. The viral CP gene sequence was followed by 254 nucleotides of 3' nontranslated sequence and a poly-A tail. Based on computer modeling, the 3' nontranslated region could form two lengthy stem-loop structures and two pseudoknot structures. Subclones containing the coding sequences of the CP (amino acids 1-287, 17-287, 17-113 and 106-287) were constructed and expressed in Escherichia coli. These polypeptides were detected using polyclonal antibodies in Western blots.

Amino Acid Sequence↗