Biomedical subjects

C W Ward

Publications and source records attributed to C W Ward.

At least 19 recordsLinked to original sources

PubMed1992

Sequence data as the major criterion for potyvirus classification.

Recent knowledge of the structure of the potyvirus particle and its components appears to have resolved what was thought to be an intractable problem of plant virology. This review describes how coat-protein and gene sequence data can be used to provide an hierarchical classification of potyviruses. This classification puts the aphid and non-aphid-transmitted potyviruses into a single family, divides this family into four genera that correspond to the four modes of vector transmission, discriminates distinct potyvirus species from strains, and provides a basis for the formation of subgroups composed of closely related species within a genus.

Capsid↗

PubMed1992

Present status of the sugarcane mosaic subgroup of potyviruses.

Until recently, sugarcane mosaic virus (SCMV) was believed to be a single potyvirus consisting of a large number of strains, differing from each other in certain biological and antigenic properties. The use of affinity-purified polyclonal antibodies directed towards the surface-located, virus-specific amino termini of the coat proteins showed that 17 strains from Australia and the United States represented four distinct potyviruses, namely johnsongrass mosaic virus (JGMV), maize dwarf mosaic virus (MDMV), sorghum mosaic virus (SrMV) and SCMV. Comparisons of strains from each of these four viruses on the basis of reactions on differential sorghum and oat cultivars, cell-free translation of RNAs, morphology and serology of cytoplasmic cylindrical inclusions, amino acid sequence and peptide profiling of coat proteins, 3' non-coding nucleotide sequences, and molecular hybridization with probes corresponding to the 3' non-coding regions, resulted in exactly the same taxonomic assignments as obtained using amino-terminal serology. These results further confirm that the former sugarcane mosaic virus actually consists of four distinct viruses and show that MDMV, SrMV, and SCMV are more closely related to each other than they are to JGMV. Because these four viruses are closely related but distinct, formation of a sugarcane mosaic subgroup in the genus Potyvirus would be appropriate.

Edible Grain↗

PubMed1992

Strains of bean common mosaic virus consist of at least two distinct potyviruses.

Bean common mosaic virus (BCMV) consists of a large number of pathotypes and strains which have largely been identified by their characteristic interactions with a selected number of differential bean cultivars. The relationships among these strains and other potyviruses that infect legumes are complex, with indications that BCMV, blackeye cowpea mosaic virus (BlCMV) and azuki bean mosaic virus (AzMV) may be strains of the one virus. Using high performance liquid chromatographic peptide profiles of coat-protein digests, the NL3 and NY15 strains of BCMV were compared with each other, with the Type and W strains of BlCMV and with the mild mottle strain of peanut stripe virus (PStV). The results suggest that BCMV-NL3 and BCMV-NY15 are distinct potyviruses, not strains of the one virus, and that BCMV-NY15 is a strain of the same potyvirus that includes BlCMV, PStV, AzMV and three potyvirus isolates (74, PM, PN) from soybeans.

Capsid↗

PubMed1992

Serology of potyviruses: current problems and some solutions.

The serological relationships among members of the family Potyviridae are extremely complex and inconsistent. Variable cross-reactivity of polyclonal antisera, unexpected paired relationships between distinct viruses, and lack of cross-reactions between some strains are the major problems associated with the serology of potyviruses. Recent biochemical and immunochemical investigations of coat proteins have established the molecular basis for potyvirus serology and provided explanations for most of the problems with serology of potyviruses. Information from these studies has also formed the basis for the development of several novel approaches to the accurate detection and identification of potyviruses. However, even these novel approaches are not without drawbacks and some of them cannot be applied easily in plant virus laboratories, since they require prior sequence information and facilities for peptide synthesis. These findings suggest that serology is an imperfect criterion for the identification and classification of potyviruses.

Animals↗

PubMed1992

Differentiation of potyviruses and their strains by hybridization with the 3' non-coding region of the viral genome.

Nucleic acid hybridization with the 3' non-coding region of the potyvirus genome as the probe was shown to be a relatively simple means of distinguishing between distinct potyviruses and their strains. Comparisons of the nucleotide sequences of potyvirus genomes (ignoring gaps) showed that the degree of identity between equivalent genes of strains was greater than 96%, while between distinct potyviruses the identity ranged from 42% to 65%, suggesting that any extended sequence could be considered representative of the whole genome and be suitable as a diagnostic probe. The comparisons however, also revealed that some parts of the genome, but not the 3' non-coding region, had local regions of high sequence identity that could lead to cross-hybridization between distinct potyviruses. For this reason, and because its location immediately upstream of the poly(A) tail makes it the most accessible region for the purpose of cloning and sequencing, the 3' non-coding sequence should be most suitable for use as a diagnostic probe. Successful hybridizations (using radiolabeled, polymerase chain reaction-amplified 3' non-coding sequences) have been achieved by probing recombinant clones, purified potyviral RNA, partially purified total RNA from infected plants, and a crude extract of infected plant tissue. The method has been used to support the proposals that watermelon mosaic virus 2 and soybean mosaic virus-N are both strains of the same virus, and to discriminate between several isolates previously believed to be strains of sugarcane mosaic virus. The method should have wide application as a means of differentiating distinct potyviruses from strains.

Blotting, Southern↗

PubMed1992

The isolation, characterization and cloning of a globin-like, host-protective antigen from the excretory-secretory products of Trichostrongylus colubriformis.

An 18-kDa component from the excretory-secretory (ES) products of adults of Trichostrongylus colubriformis was isolated and characterized, and was shown to induce 60-84% protection of guinea pigs from challenge infection following a single intraperitoneal injection. Amino-terminal sequence analysis of gel-purified protein enabled oligonucleotides to be synthesized and used to screen a lambda gt10 cDNA library made from young adult worm mRNA, and to synthesize full-length clones from cDNA using the polymerase chain reaction (PCR). The full-length clones coded for a 20-kDa precursor protein of 173 amino acids which had a strongly hydrophobic leader sequence of 15 residues. The mature protein sequence of 158 amino acid residues was rich in charged amino acids (32%), including 8 oppositely charged pairs of amino acids. The protein sequence contained no half-cystine residues and no potential N-glycosylation sites. Unlike 2 other fully characterized ES components which are expressed only in the parasitic stages, mRNA coding for the 20-kDa component was present in both the parasitic and free-living stages of T. colubriformis. The parasite protein had approximately 20% identity with globins from human and from the larvae of the insect Chironomus thummi thummi. The homology included the invariant distal histidine and phenylalanine, and a number of other residues highly conserved in globins.

Amino Acid Sequence↗

PubMed1992

Altered mechanical responses of malignant hyperthermic skeletal muscle during repetitive stimulation.

This investigation examined the mechanical responses of malignant hyperthermic (MH) and normal porcine skeletal muscle to repetitive stimulation. Twitch and maximal tetanic tensions were not significantly different between muscle types. Tensions produced during stimulation at 20-80 Hz were significantly less in MH muscle than in normal muscle. In addition, MH muscle showed significantly greater force decline (tetanic fade) at the end of contractions evoked by 20-80 Hz stimulation. When stimulated to fatigue, both normal and MH muscle exhibited similar rates of tension decline during the initial minutes. Further stimulation caused additional decline in normal muscle, but a tension plateau in MH muscle. In all cases, normal muscle had greater magnitudes of fatigue than did MH muscle. Results show that there are marked differences between MH and normal muscle in the mechanical responses to repetitive stimulation. Due to its inability to properly regulate intracellular Ca2+ exchange, it is possible that MH muscle might be a useful tool for identifying the mechanisms of muscle fatigue in normal muscle.

Animals↗

PubMed1992

Reduced Ca(2+)-induced Ca2+ release from skeletal muscle sarcoplasmic reticulum at low pH.

The purpose of this investigation was to determine the effects of reduced pH on Ca(2+)-induced Ca2+ release (CICR) from skeletal muscle sarcoplasmic reticulum (SR). Frog semitendinosus fiber bundles (1-3/bundle) were chemically skinned via saponin treatment (50 micrograms/mL, 20 min), which removes the sarcolemma and leaves the SR functional. The SR was first depleted of Ca2+ then loaded for 2 min at pCa (log free Ca2+ concentration) 6.6. CICR was then evoked by exposing the fibers to pCa 5-7 for 5-60 s. CICR was evoked both in the absence of ATP and Mg2+ and in the presence of beta, gamma-methyleneadenosine-5'-triphosphate (AMPPCP, a nonhydrolyzable form of ATP) and Mg2+. Ca2+ remaining in the SR was then assayed via caffeine (25 mM) contracture. In all cases, CICR evoked at pH 6.5 resulted in larger caffeine contractures than that evoked at 7.0, suggesting that more Ca2+ was released during CICR at the higher pH. Accordingly, rate constants for CICR were significantly greater at pH 7.0 than at pH 6.5. These results indicate that reduced pH depresses CICR from skeletal muscle SR.

Adenosine Triphosphate↗

PubMed1992

Coat protein properties suggest that azuki bean mosaic virus, blackeye cowpea mosaic virus, peanut stripe virus, and three isolates from soybean are all strains of the same potyvirus.

The interrelationship of a number of potyviruses infecting legumes has been investigated by comparing molecular properties of their coat proteins. Comparison of the coat proteins by the techniques of amino acid analysis and PAGE was inadequate to distinguish strains from distinct potyviruses. However, high-performance liquid chromatographic peptide profiles of tryptic digests of coat proteins of these legume-infecting potyviruses enabled such assignments to be made. These data indicate that amino acid sequences of coat proteins of azuki bean mosaic virus, the Type and W strains of blackeye cowpea mosaic virus, three isolates (74, PM, PN) of a potyvirus obtained from soybean in Taiwan, and the Blotch and Mild Mottle strains of peanut stripe virus (PStV) may be very similar to the known sequence of PStV Stripe coat protein. In contrast, peptide profiles of coat proteins from soybean mosaic virus, clover yellow vein virus, bean yellow mosaic virus, potato virus Y, and tobacco etch virus were dissimilar to each other and to the profile of PStV Stripe, suggesting that their coat protein sequences were also quite different. Based on observations of the coat protein structure of many potyviruses, the results suggest that the potyvirus isolates with similar coat proteins are strains of the same potyvirus.

Amino Acid Sequence↗

PubMed1991

Coat protein of potyviruses. 7. Amino acid sequence of peanut stripe virus.

The amino acid sequence of the 287-residue coat protein of peanut stripe virus (PStV) was determined from the sequences of overlapping peptide fragments. Results indicated that the amino terminus was blocked by an acetyl group, as has previously been found for the coat protein of Johnsongrass mosaic potyvirus. Comparison of the PStV sequence with coat proteins of 20 distinct potyviruses gave sequence identities of 47-57%, except for zucchini yellow mosaic virus (ZYMV), passionfruit woodiness virus (PWV), and the related strains watermelon mosaic virus 2 (WMV 2) and soybean mosaic virus-N, which showed sequence identities of 70-76%. Several amino acid residues which were common to the core sequences of these coat proteins were at positions previously found to be invariant among potyvirus coat proteins. The degree of these similarities suggests that although PStV, WMV 2, ZYMV, and PWV are distinct potyviruses, they share a common ancestor in their evolutionary development.

Amino Acid Sequence↗

PubMed1991

Correlation between gut hypersensitivity and resistance to Trichostrongylus colubriformis infection of outbred and inbred lines of guinea pigs.

The relationship between gut sensitivity and immunity to challenge infection was examined in outbred and inbred guinea pig lines. Primary infections terminated at 3,6,9 or 13 days and multiple infections of 3 days' duration confirmed the importance of direct gut stimulation and the period of exposure in the induction of immunity and gut hypersensitivity. The studies with the multiple 3-day infections confirmed that the third-stage larvae alone are capable of inducing strong protective immunity and showed that this is accompanied by pronounced gut sensitivity to parasite extracts and secretions. Finally, two inbred guinea pig lines selected for enhanced resistance or susceptibility to T. colubriformis infection displayed corresponding high or low capacities to mount hypersensitivity reactions following a single truncated 3-day primary infection.

Animals↗

PubMed1991

Molecular characterisation of a protective, 11-kDa excretory-secretory protein from the parasitic stages of Trichostrongylus colubriformis.

An 11-kDa protein occurring as a major component of the non-glycosylated fraction of 4th larval stage (L4) and adult Trichostrongylus colubriformis excretory-secretory (ES) fluid has been found to be highly protective in guinea pigs, an alternate host for T. colubriformis. The protein has been purified, characterised and partly sequenced. With a reverse-complement oligonucleotide based on the carboxy-terminal sequence of the protein, recombinant lambda gt11 clones were detected in an L4 cDNA library. The DNA sequence from one clone has a single extended open reading frame coding for a highly charged 11-kDa protein which lacks a leader sequence and contains a potential N-glycosylation site. Expression of the cloned DNA in Escherichia coli was detected with an antibody, raised in rabbits against gel-purified 11-kDa protein.

Amino Acid Sequence↗

PubMed1991

Physicochemical and immunological characterization of recombinant host-protective antigen (VP2) of infectious bursal disease virus.

Small fusions to the N-terminal end of the host-protective antigen (VP2) of infectious bursal disease virus lead to stable expression of VP2 in Escherichia coli and yeast, and reduce the levels of inclusion body formation in E. coli in comparison to VP2 constructs with larger N-terminal fusions. VP2 produced with small N-terminal fusions, like native viral VP2, can be fractionated into a high molecular weight 'multimeric' form and a monomeric form. A virus-neutralizing monoclonal antibody that only recognizes undenatured VP2 preferentially reacts with multimeric forms of recombinant VP2. Both native and recombinant monomeric forms of VP2 are non-immunogenic. The multimeric forms of viral and yeast-derived VP2 are highly immunogenic, while those produced in E. coli are not.

Antibodies, Monoclonal↗

PubMed1991

Effects of the calcium channel agonist, Bay K 8644, on the mechanical output of skeletal muscle fibers.

1. The effects of the calcium agonist, Bay K 8644, on the mechanical output of skeletal muscle were studied in frog semitendinosus fiber bundles and in whole sartorius muscles. 2. Low concentrations of Bay K 8644 (less than or equal to 1 microM) had no significant influence on isometric twitches. Concentrations between 5-20 microM increased peak tension by 28.6 +/- 2.9% while higher concentrations (greater than or equal to 50 microM) initially increased then depressed twitches by 70 +/- 3.5%. 3. 10 microM Bay K 8644 also increased peak tension developed during low frequency stimulation (i.e. less than or equal to 40 Hz), slightly depressed high frequency contractions (i.e. greater than or equal to 80 Hz) but did not reduce maximal tetanic tension which occurred at about 60 Hz. 4. Potentiation of twitches and low frequency tetani and depression of high frequency tetani by Bay K 8644 were partially antagonized by nifedipine (10 microM), low extracellular calcium and D-600 (5 microM). These conditions did not, however, block the depressant actions of greater than or equal to 50 microM Bay K 8644. 5. In skinned fibers, 10 microM Bay K 8644 had no effect on resting or maximal Ca2+ activated tension. Also, 10 microM Bay K 8644 had no effect on caffeine contractures when added to the previous Ca2+ loading solution. 6. These results suggest that Bay K 8644 has both positive and negative inotropic actions on isolated skeletal muscle, which are dependent on drug concentration and muscle activation pattern.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1991

Unexpected sequence diversity in the amino-terminal ends of the coat proteins of strains of sugarcane mosaic virus.

The sequence of the 3'-terminal 1343 nucleotides of the SC strain of the sugarcane mosaic virus (SCMV-SC) genome was compared with the 1376 nucleotides at the 3' terminus of maize dwarf mosaic virus B (MDMV-B). The SCMV-SC sequence includes an open reading frame which codes for the viral coat protein of 313 amino acids (nucleotides 157 to 1116), followed by a 3' non-coding region of 235 nucleotides and a poly(A) tail. The MDMV-B sequence codes for the capsid protein (nucleotides 157 to 1139) of 328 amino acids and has a 3' non-coding region of 236 nucleotides. The coat protein of SCMV-SC has 92% identity with that of MDMV-B except for the region between amino acid residues 27 and 70 of SCMV-SC. This region of SCMV-SC is smaller (44 residues) than the equivalent region in MDMV-B (59 residues) and has only 22% identity with the MDMV-B sequence. Possible mechanisms for the generation of this sequence diversity are discussed. Despite this diversity, the sequence identities of both the major part of the coat proteins and the 3' non-coding regions confirm the proposal, based on previously described serological data, that SCMV-SC and MDMV-B are strains of SCMV.

Amino Acid Sequence↗

PubMed1991

Expression of potyvirus coat protein in Escherichia coli and yeast and its assembly into virus-like particles.

When the full-length coat protein (CP) of the potyvirus, Johnsongrass mosaic virus (JGMV), was expressed in Escherichia coli or yeast, it assembled to form potyvirus-like particles. The particles were heterogeneous in length with a stacked-ring appearance and resembled JGMV particles in their flexuous morphology and width. This cell-free assembly system should permit analysis of the mechanisms of particle assembly and genome encapsidation. Two mutant forms of CP produced by site-directed mutagenesis failed to assemble into virus-like particles.

Amino Acid Sequence↗

PubMed1991

BAY K 8644 and nifedipine alter halothane but not caffeine contractures of malignant hyperthermic muscle fibers.

The purpose of these experiments was to determine if the Ca2+ agonist BAY K 8644 and the Ca2+ antagonist nifedipine alter the mechanical responses of malignant hyperthermia-susceptible (MHS) skeletal muscle to halothane and caffeine. Muscle fiber bundles were dissected from MHS porcine skeletal muscle and exposed to BAY K 8644 (10 microM), nifedipine (1 microM), low-Ca2+ media [Ca2+ replaced by 1 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid], or diltiazem (30 microM) administered alone and with halothane (3%) or caffeine (0.5-0.8 mM). When administered alone, both halothane and BAY K 8644 evoked a significant change in resting tension (i.e., contracture) of 193.7 +/- 61.0 and 51.9 +/- 21.5 mN/cm2, respectively. When administered in combination, BAY K 8644 had no effect on the magnitude of the halothane contracture (195.2 +/- 58.6 mN/cm2) but reduced its onset time from 306.7 +/- 36.3 to 105.9 +/- 8.9 s. Nifedipine, low Ca2+, and diltiazem significantly reduced the halothane contracture (103.1 +/- 30.3, 123.1 +/- 20.6, and 112.6 +/- 16.2 mN/cm2, respectively) but had no effect on its onset time. In addition, low Ca2+ reduced the magnitude of the BAY K 8644 contracture (8.2 +/- 2.1 mN/cm2). BAY K 8644 also increased contractures induced by low caffeine concentrations (0.5-2.0 mM) but did not alter contractures induced by 4.0 and 8.0 mM caffeine, whereas nifedipine, low Ca2+, and diltiazem had no effect on these contractures. These results suggest that extracellular Ca2+ influx may have some influence on halothane but not on caffeine contractures of MHS skeletal muscle.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1991

Effects of BAY K 8644, nifedipine, and low Ca2+ on halothane and caffeine potentiation.

The purpose of this investigation was to examine the effects of the Ca2+ agonist BAY K 8644 and the Ca2+ antagonist nifedipine on halothane- and caffeine-induced twitch potentiation of mammalian skeletal muscle. Muscle fiber bundles were taken from normal Landrace pigs and exposed to BAY K 8644 (10 microM), nifedipine (1 microM), and low Ca2+ media administered alone and in combination with halothane (3%) or with increasing concentrations of caffeine (0.5-8.0 mM). Both BAY K 8644 and halothane potentiated twitches by approximately 80%; when they were administered in combination, twitch potentiation was nearly double that caused by either drug alone. In the presence of nifedipine, halothane increased twitches by less than 30%. Low Ca2+ significantly depressed twitches by approximately 25% but also inhibited halothane's inotropic effect. BAY K 8644 augmented caffeine potentiation but only at low caffeine concentrations (0.5-2.0 mM). Nifedipine and low Ca2+ failed to inhibit caffeine's inotropic effects. These results suggest that halothane potentiates twitches via a mechanism that involves or is influenced by extracellular Ca2+.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗