Biomedical subjects

C W Ward

Publications and source records attributed to C W Ward.

At least 55 records · Page 3Linked to original sources

PubMed1995

Mutagenic structure/function analysis of the cytoplasmic cysteines of the insulin receptor.

Native human insulin receptor (hIR) has been reported to contain only one free thiol group proposed to lie near the ATP-binding. domain of its beta-subunit [Finn, Ridge and Hofmann (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 419-423]. The present study investigated the role of the six cytoplasmic cysteines of the beta-subunit of the hIR using a mutagenic approach in which insulin receptors, mutated at each cytoplasmic cysteine (to alanine) in turn, were transfected into Chinese hamster ovary (CHO) cells. Cell lines expressing hIR mutation at high level were obtained which, by both flow-cytometric analysis towards an hIR-specific monoclonal antibody (83-7) and insulin-binding analysis, were similar to the well-characterized CHOT cell line which overexpresses native hIR. The ED50 and Kd values of the mutant receptors were the same as those of the wild-type hIR. Each of the mutant receptors signalled insulin action to stimulate receptor autophosphorylation and kinase activity as well as glucose utilization to levels appropriate for the receptor level expressed. In contrast, insulin-stimulated thymidine uptake and glucose-transport responses of two of the six mutant cell lines, those expressing Cys981Ala and Cys1245Ala, were impaired compared with that of the native hIR-expressing cell line, CHOT. The beta-subunits of each of the hIR cytoplasmic cysteine mutant cell lines could be alkylated specifically with N-[3H]ethylmaleimide. The kinase activity of each receptor was inhibited by N-ethylmaleimide and stimulated by iodoacetamide, indicating that none of the cytoplasmic cysteines alone contributes the single free thiol group to the hIR structure. We conclude that the cytoplasmic cysteines of the hIR have a predominantly passive role in hIR activity although Cys-981 and Cys-1245 do affect mitogenic and glucose-transport responses of the receptor. Our findings indicate that the stoicheiometry of a single free thiol group/mol of insulin-binding activity noted in previous studies is either spread fractionally over a number of the cytoplasmic cysteines or is one of the four cysteines in the ectodomain of the hIR beta-subunit. Alternatively, the mutagenesis performed in the present study may enable differential exposure of a second titratable cysteine in wild-type and mutant receptors.

Amino Acid Sequence↗

PubMed1995

Insulin and epidermal growth factor receptors contain the cysteine repeat motif found in the tumor necrosis factor receptor.

The insulin receptor (INSR) and epidermal growth factor receptor (EGFR) are representatives of two structurally related subfamilies of tyrosine kinase receptors. Using the Wisconsin GCG sequence analysis programs, we have demonstrated that the cysteine-rich regions of INSR and EGFR conform to the structural motif found in the tumor necrosis factor receptor (TNFR) family. The study also revealed that these regions were not composed of simple repeats of eight cysteine residues as previously proposed and that the second Cys-rich region of EGFR contained one fewer TNFR repeat than the first. The sequence alignments identified two cysteine residues in INSR that could be responsible for the additional disulfide bonds known to be involved in dimer formation. The published data on the alignments for the fibronectin type III repeat region of the INSR together with previous cysteine mutagenesis studies indicated that there were two disulfide bonds linking the alpha and beta chains of the INSR, but only one alpha-beta linkage in the insulin-like growth factor 1 receptor (IG1R). Database searches and sequence alignments showed that the TNFR motif is also found in the cysteine-rich repeats of laminins and the noncatalytic domains of furin-like proteases. If the starting position of the repeat is altered the characteristic laminin repeat of eight cysteine residues can be shown to consist of a TNFR-like motif fused to the last half of an EGF-like repeat. The overlapping regions of these two motifs are known to have identical disulfide bonding patterns and similar protein folds.

Amino Acid Sequence↗

PubMed1995

Purification and properties of insulin receptor ectodomain from large-scale mammalian cell culture.

Ectodomain of the exon 11+ form of the human insulin receptor (hIR) was expressed in the mammalian cell secretion vector pEE6.HCMV-GS, containing the glutamine synthetase gene. Following transfection of the hIR ectodomain gene into Chinese hamster ovary (CHO-K1) cells, clones were isolated by selecting for glutamine synthetase expression with methionine sulphoximine. The expression levels of ectodomain were subsequently increased by gene amplification. Production was scaled up using a 40-liter airlift fermenter in which the transfected CHO-K1 cells were cultured on microcarrier beads, initially in medium containing 10% fetal calf serum (FCS). By continuous perfusion of serum-free medium into the bioreactor, cell viability was maintained during reduction of FCS, which enabled soluble hIR ectodomain to be harvested for at least 22 days. Harvests were concentrated 20-fold by anion-exchange chromatography. Optimal recovery of ectodomain from early harvests containing large quantities of serum proteins was achieved by insulin-affinity chromatography, whereas in later harvests purification was achieved by multistep chromatography. Analysis of the purified hIR ectodomain showed that it had a molecular weight by sedimentation equilibrium analysis of 269,500. Amino-terminal amino acid sequence analysis showed that the ectodomain was correctly processed to alpha and beta chains and that glycosylation characteristics were similar to those of native hIR. The integrity of the ectodomain was demonstrated by the recognition of conformation-dependent anti-hIR antibodies and by its binding of insulin (Kd approximately 2 x 10(-9) M). These results demonstrate the successful production and purification of hIR ectodomain by processes amenable to scale-up and in a form appropriate for structure/function studies of the ligand-binding domain of the receptor.

Animals↗

PubMed1995

Evaluation of complete genome sequences and sequences of individual gene products for the classification of hepatitis C viruses.

Comparisons of genome and polyprotein sequences of hepatitis C virus (HCV) isolates world-wide has led to the identification of nine major genotypes and many subtypes. This classification is based on either complete genome/polyprotein sequences or sequence data from the 5' noncoding region, core, E1, NS3 or NS5B genes. The relative merit of different gene segments as taxonomic markers and the validity of the resulting assignments is not clear at this stage. To resolve the taxonomy of HCV genotypes and subtypes, we have compared the complete genome and polyprotein sequences of 19 HCV isolates available in the databases as well as sequences of individual genes and gene products of these isolates. Based on the correlation between sequence relationships and taxonomic assignments of other RNA viruses, we show that the nine major genotypes of HCV represent nine distinct virus species and their subtypes subspecies. Our sequence comparison of the 5' noncoding regions and the individual gene products suggests that E2, NS2, NS5B, E1, NS4A, NS4B and NS5A (in that order) are the most appropriate regions for the discrimination between species, subspecies and strains of HCV. The 5' noncoding, core and NS3 regions are less effective in distinguishing between species, subspecies and strains. Based on a comparison of the polymerase sequence identities of HCVs, pestiviruses and flaviviruses as well as the recent information on the size and morphology of HCV virions, we propose that HCVs, pestiviruses and flaviviruses should be classified into three separate families, named Hepciviridae, Pestiviridae and Flaviviridae, respectively rather than three genera of the Flaviviridae as currently classified. We also propose "Hepcivirus" as the genus name for HCVs.

Base Sequence↗

PubMed1994

Cysteine-524 is not the only residue involved in the formation of disulphide-bonded dimers of the insulin receptor.

The human insulin receptor (hIR) is a member of the transmembrane tyrosine kinase receptor family. It is a disulphide-linked homodimer which can be reduced to two insulin-binding monomers by mild reduction of class-I disulphide bonds. The number of disulphide bonds between the alpha- and beta-chains within the monomer or between the monomers in the dimer is not known, although one dimer bond involving hIR Cys-524 has recently been identified [Schaffer and Ljungqvist (1992) Biochem. Biophys. Res. Commun. 189, 650-653]. In the present report hIR Cys-524 was converted into alanine by site-directed mutagenesis and expressed at high levels in Chinese hamster ovary (CHO) cells. The mutant receptor was processed normally and shown to bind insulin normally, with ED50 and KD values not different from those of the wild-type hIR. It was still a disulphide-linked dimer as judged by SDS/PAGE, indicating that there are alpha-alpha-chain disulphide bonds additional to the Cys-524 linkage in the insulin receptor dimer. Insulin-stimulated receptor autophosphorylation and kinase activity of the mutated receptor were both impaired compared with that of the wild-type receptor by 49% and 53% respectively. CHO cells overexpressing the mutant receptor, however, did not show a reduced capacity to stimulate glucose utilization, indicative that the level of receptor expression was sufficient to saturate downstream insulin action. These findings indicate that alpha-alpha disulphides additional to that provided by Cys-524 hold the receptor dimer together and that mutagenesis of Cys-524 reduces the ability of the receptor to signal insulin action subsequent to hormone binding.

Alanine↗

PubMed1994

Nucleotide sequence of the 3'-terminal region of the genome confirms that pea mosaic virus is a strain of bean yellow mosaic potyvirus.

The 1,035 nucleotides at the 3'end of the I strain of pea mosaic potyvirus (PMV-I) genomic RNA, encoding the coat protein, have been cloned and sequenced. A comparison of the derived coat protein sequence with those of the bean yellow mosaic virus (BYMV) strains, CS, S, D and GDD, indicates that PMV-I is a strain of BYMV. Sequence comparisons and hybridisation studies using the 3'-noncoding region support this classification. The nucleotide and protein sequence data also suggest that PMV-I and BYMV-CS form one subset of BYMV strains while the other three strains form another.

Amino Acid Sequence↗

PubMed1994

Cowpea aphid borne mosaic virus-Morocco and South African Passiflora virus are strains of the same potyvirus.

High performance liquid chromatography (HPLC) profiles of tryptic peptides and partial amino acid sequence analysis have been employed to establish the taxonomic status of the Moroccan isolate of cowpea aphid-borne mosaic virus (CABMV). Some previous reports have suggested CABMV to be very closely related to blackeye cowpea mosaic virus (B1CMV) while other reports have concluded that this relationship is distant. In this report a tryptic digest of the coat protein of CABMV-Morocco was compared with those of the coat proteins of B1CMV-Type, B1CMV-W, the mild mottle strain of peanut stripe virus (PStV-MM) and the NY15 strain of bean common mosaic virus (BCMV-NY15), all of which are now recognised as strains of BCMV. The comparisons also included the NL-3 strain of bean necrosis mosaic virus (BNMV-NL3), which had previously been classified as a strain of BCMV. The HPLC peptide profiles indicated that CABMV-Morocco was distinct from BCMV and BNMV. Amino acid sequence analysis of peptides accounting for more than half of the coat protein confirmed that CABMV-Morocco was not a strain of BNMV or BCMV but was a distinct member of the BCMV subset of viruses that previously has been shown to include BCMV, BNMV, soybean mosaic virus, zucchini yellow mosaic virus, passionfruit woodiness virus and South African Passiflora virus (SAPV). Comparison of the partial sequence data with these and other published sequences revealed that the coat protein of CABMV-Morocco is very similar to that of SAPV suggesting that they are strains of the same virus. Since CABMV was described over 25 years earlier than SAPV, the name CABMV should take precedence and SAPV should be renamed CABMV-SAP, the South African Passiflora strain of CABMV.

Amino Acid Sequence↗

PubMed1993

Sequence diversity in the surface-exposed amino-terminal region of the coat proteins of seven strains of sugarcane mosaic virus correlates with their host range.

The N-terminal region of the coat proteins of five strains (Isis, Brisbane, Sabi, Bundaberg, and BC) of sugarcane mosaic virus (SCMV) isolated from four different plant species (sugarcane, sabi grass, wild sorghum, and blue couch grass) have been compared with the previously published data for SCMV-SC and SCMV-MDB, isolated from sugarcane and maize, respectively. The region, beginning at residue 11 and ending 16 residues beyond the second trypsin cleavage site of the coat protein, varied in size from 68 amino acid residues (Bundaberg) to 115 residues (BC) and contained repeat sequence motifs. Comparisons of the sequence identity and the nature of the repeats in the seven sequences showed that there were five different sequence patterns. These could be grouped further into three subsets which appeared to correlate with the host range of the strains. SCMV-Brisbane, SC, and Isis, isolated from sugarcane, showed almost identical sequence patterns and formed one subset. The other four strains had different sequence patterns and could be grouped further into a Sabi and Bundaberg subset (isolated from sabi grass), and a BC and MDB subset.

Amino Acid Sequence↗

PubMed1993

Watermelon mosaic virus-Morocco is a distinct potyvirus.

The relationship of the Morocco isolate of watermelon mosaic virus (WMV) to WMV2, soybean mosaic virus (a virus closely related to WMV2) and the W strain of papaya ringspot virus (PRSV-W), formerly WMV1, was examined by comparing tryptic peptide profiles using high performance liquid chromatography. The profiles indicated that the coat protein sequence of WMV-Morocco differed substantially from those of the other potyviruses. This conclusion was supported by sequence data from five tryptic peptides from the coat protein of WMV-Morocco, which showed only 61-68% identity to equivalent sequences in PRSV-W, WMV2 and zucchini yellow mosaic, another potyvirus infecting cucurbits. Based on the above data, and on known correlations between coat protein sequence similarities and potyvirus relationship, it is concluded that WMV-Morocco should be regarded as a distinct potyvirus.

Amino Acid Sequence↗

PubMed1993

Characterization of the mRNA encoding a proline-rich 37-kilodalton glycoprotein from the excretory-secretory products of Trichostrongylus colubriformis.

A glycoprotein, with apparent molecular weight in SDS-polyacrylamide gels of 37 kDa, has been isolated from the excretory-secretory (ES) products of the adult stage of Trichostrongylus colubriformis, a parasitic nematode. This protein is the major ES product recognized in immunoblots by lymph from a naturally infected sheep. A synthetic oligonucleotide, based on peptide sequence data from a digest of the purified protein was used to successfully screen a cDNA library. A cDNA clone was isolated which encoded a presumptive protein precursor of 220 amino acids that contained a 63 amino acid region of which more than 35% of the residues were proline, three peptide sequences determined from the natural component, and three potential N-glycosylation sites, consistent with the protein being isolated from the lectin-bound fraction of the adult ES products. The presumptive, processed, amino terminus encoded by the cDNA clone was preceded by a signal-like, hydrophobic-rich region of 16 amino acids.

Amino Acid Sequence↗

PubMed1993

High level production of hybrid potyvirus-like particles carrying repetitive copies of foreign antigens in Escherichia coli.

Synthesis in E. coli of native coat protein of Johnsongrass mosaic virus, and hybrid protein molecules containing foreign antigens, resulted in the intracellular formation of potyvirus-like particles (PVLPs). The foreign antigens used were an octapeptide epitope from Plasmodium falciparum and a decapeptide hormone (luteinizing hormone releasing hormone) at the N- or at both N- and C-terminal regions of the coat protein molecule, and a full length protein antigen (Sj26-glutathione S-transferase of 26 kD from Schistosoma japonicum) replacing the N-terminal 62 amino acids of the coat protein. Electron microscopy of ultrathin sections of E. coli revealed that PVLPs resulting from coat protein molecules containing peptide fusions appeared in vast arrays of parallel strands within the cytoplasm sometimes extending the length of the cell and at times the cells were strung together, with "threads" of PVLPs appearing to connect individual bacterial cells. PVLPs resulting from the fusion of the 26 kD antigen Sj26 to coat protein were shorter and wider. The physical form of the high molecular weight PVLPs enabled purification by simple size exclusion column chromatography. The Sj26-PVLPs administered to mice without adjuvant elicited antibody responses comparable to monomeric Sj26 administered with Freund's Complete Adjuvant.

Animals↗

PubMed1993

Site-directed mutagenesis of a potyvirus coat protein and its assembly in Escherichia coli.

Multiple copies of the Johnsongrass mosaic virus coat protein synthesized in Escherichia coli can readily assemble to form potyvirus-like particles. This E. coli expression system has been used to identify some of the key amino acid residues, within the core region of the coat protein, required for assembly. The two charged residues R194 and D238 previously proposed theoretically to be involved as a pair in the construction of a salt bridge crucial for the assembly process were targeted for site-directed mutagenesis. The results from our experiments suggest that the two residues are required for the assembly process but are not necessarily involved as a pair in a common salt bridge.

Capsid↗

PubMed1993

Fatigue-induced alterations in Ca2+ and caffeine sensitivities of skinned muscle fibers.

The purpose of this investigation was to examine the Ca2+ and caffeine sensitivities of skinned skeletal muscle fibers after fatigue. Single frog semitendinosus fibers were chemically skinned in either a rested state or after tetanic contractions (80 Hz, 100 ms) evoked at 2 s-1 for 5 min. This protocol reduced tetanic force to 1.8 +/- 0.2% of control. Maximal Ca(2+)-activated force (F0, 20 degrees C) was not significantly different between rested and fatigued fibers. However, the concentration of Ca2+ required to evoke 50% of F0 was significantly lower in the fatigued fibers (1.80 +/- 0.18 vs. 1.33 +/- 0.16 microM; P < 0.05), an effect that persisted as the skinned fiber was allowed to incubate in the relaxing solution for > 90 min. The addition of caffeine (25 mM) after Ca2+ loading of the sarcoplasmic reticulum (SR) for periods of 5-30 s (0.25 microM free [Ca2+]) evoked smaller contractures in fatigued fibers than in rested fibers. However, when the loading period was prolonged (60-240 s), force developed after caffeine application was not significantly different between conditions. This suggests that the rate, but not the maximal capacity of Ca2+ loading by the SR, is reduced by fatigue. After Ca2+ loading (120 s), the minimal caffeine concentration required to evoke a contracture in fatigued fibers (5.7 +/- 0.3 mM) was significantly greater than that of control fibers (3.1 +/- 0.4 mM), an effect that persisted with prolonged incubation of the skinned fibers. In addition, the rate of force increase in response to 8 mM caffeine was reduced in fatigued fibers by 41%.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Effects of alpha 1-receptor blockade on the cardiovascular and thermoregulatory responses to severe exercise in the heat.

Up to an internal temperature of 38 degrees C, nonacral skin blood flow increases with mild exercise in a warm environment. While this appears to be due predominately to an active vasodilator system and not to altered vasoconstrictor activity, it is uncertain if more intense exercise affects this vasodilator response. Consequently, six fit (VO2pk = 62.8 +/- 2.2 ml.kg-1.min-1, mean +/- SEM) college aged men performed 30 min bouts of cycle ergometry exercise at 70% VO2pk in a hot environment (35 degrees C, 55% rh) while under the influence of either an alpha 1-receptor blocker (prazosin, PRAZ) or a placebo (CTL). Resting rectal temperature (Tre) was lower (p = 0.002) and heart rate (HR) higher (p = 0.018) during PRAZ. Neither resting nor exercise forearm blood flow (FBF, plethysmography) and forearm vascular conductance (FVC) differed between PRAZ and CTL. Exercise Tre and mean skin temperature (Tsk) did not differ significantly between treatments, rising to 38.7 and 36.4 degrees C, respectively, by the 30th min of exercise. Exercise HR was 4 beats.min-1 higher (p = 0.016) with PRAZ. Mean arterial pressure (MAP) did not differ significantly between drug treatments, As FBF, FVC, Tre, Tsk and MAP were similar between treatments while only HR was significantly affected by PRAZ, it appears that alpha 1-receptor blockade does not alter the thermoregulatory responses to severe exercise in the heat. However, the elevated HR in the presence of a stable MAP suggests that central venous return may have been attenuated during PRAZ.

Blood Pressure↗

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↗