Biomedical subjects

C W Ward

Publications and source records attributed to C W Ward.

At least 73 records · Page 4Linked to original sources

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↗

PubMed1991

Taxonomy of potyviruses: current problems and some solutions.

There are two major requirements for potyvirus taxonomy; group-specific criteria and criteria which discriminate between distinct viruses and strains. This review discusses the relative merits in potyvirus taxonomy of molecular parameters, such as gene sequence data, nucleic acid hybridization, coat protein sequence data, or high-performance liquid chromatography peptide profiles, and phenotypic characteristics, such as particle morphology, host range, symptomatology, cross-protection, cytoplasmic inclusion morphology, and serology. Coat protein and gene sequence data are the most useful criteria, as they can be used to distinguish viruses from strains and to establish evolutionary relationships between groups of distinct potyviruses. This has led to the revised classification of some viruses and strains and to the clarification of previously conflicting and inconsistent biological properties. It has also provided a sound basis for subgrouping potyviruses. An analysis of the data supports the view that the potyvirus group, including the non-aphid-transmitted viruses, should be elevated to family status, that the vector transmission mode, which correlates with major sequence diversity, should define the four genera, and that distinct potyviruses correspond to species and their variants to strains.

Amino Acid Sequence↗

PubMed1991

Dihydropyridine effects on skeletal muscle fatigue.

1. The purpose of this investigation was to determine if alterations in extracellular calcium (Ca2+) influx by the dihydropyridine derivatives Bay K 8644 and nifedipine affected skeletal muscle fatigue. 2. Tetanic contractions (80 Hz, 100 msec) of frog sartorius muscles were evoked every sec for 3 min. Muscles were fatigued in normal Ringer's solution (NR), in NR containing 1 microM nifedipine of 10 microM Bay K 8644 or in low Ca2+ Ringer's. 3. In each case, the experimental conditions increased the rate and magnitude of fatigue. Rate constants of fatigue obtained during Bay K 8644, nifedipine and low Ca2+ conditions (-.0122 +/- .0016, -.0397 +/- 0022 and 0.0169 +/- .0064 sec-1, respectively) were significantly greater than NR (-.0104 +/- .0006 sec-1, p less than .05). In addition, tetanic forces developed at the end of the stimulation period under the experimental conditions (3.90 +/- 0.81, 1.21 +/- 1.40 and 2.04 +/- 1.10% of initial) were significantly less than NR (7.18 +/- 1.27%, p less than .05). 4. Caffeine contracture forces (10 mM) evoked immediately after stimulation were not significantly different between conditions. 5. These results suggest that alterations in sarcolemmal Ca2+ exchange has some influence on the fatigue process.

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

PubMed1990

Characterization, cloning and host-protective activity of a 30-kilodalton glycoprotein secreted by the parasitic stages of Trichostrongylus colubriformis.

The helminth Trichostrongylus colubriformis is a parasitic nematode infecting the small intestine of sheep. We report the isolation and characterization of a 30-kDa glycoprotein capable of partially protecting guinea-pigs against the parasite. This glycoprotein is secreted by the L4 and adult parasitic stages of the worm. The sequence of three separate cDNA clones predicts the polypeptide to be about 15 kDa, with four N-linked carbohydrate chains and an internal disulphide bond. The clones also indicate the existence of sequence variability in this antigen. Limited sequence homology to a porcine intestinal peptide suggests an influence on host gut physiology.

Amino Acid Sequence↗

PubMed1990

Passive protection against infectious bursal disease virus by viral VP2 expressed in yeast.

Infectious bursal disease virus (IBDV), a pathogen of major economic importance to the world's poultry industries, causes a severe immunodepressive disease in young chickens. Maternal antibodies are able to protect the progeny passively from IBDV infection. The gene encoding the IBDV host-protective antigen (VP2) has been cloned and expressed in yeast resulting in the production of an antigen that very closely resembles native VP2. When injected into specific pathogen free chickens a single dose of microgram quantities of the yeast derived antigen induces high titres of virus neutralizing antibodies that are capable of passively protecting young chickens from infection with IBDV.

Amino Acid Sequence↗