Multiple sclerosis and paramyxovirus.
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Antibodies, Viral↗
Biomedical subjects
W C Russell
Publications and source records attributed to W C Russell.
Recognition mechanisms in the synthesis of animal virus DNA.
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Animals↗
Characterization of the adenovirus proteinase: development and use of a specific peptide assay.
An assay system has been developed for the adenovirus endoproteinase which utilizes the synthetic peptide MSGGAFSW, derived from the cleavage site of the adenovirus type 2 (Ad2) protein pVI. MSGGAFSW was shown to be cleaved at the G-A bond when incubated with a source of Ad2 proteinase. Digestions were readily monitored by either fast protein liquid chromatography or thin layer electrophoresis, enabling the rapid production of quantitative data. Comparison of the peptide assay with a previously described [35S]methionine assay system showed it to be faster, cleaner and less prone to extreme conditions of pH and ionic strength. The effect on adenovirus proteinase activity of a number of inhibitors was assessed using both the [35S]methionine and peptide assays. Identical inhibitor profiles were obtained and these suggested that the adenovirus enzyme is a cysteine proteinase. The 23K gene product, thought to be the proteinase, contains cysteine and histidine residues at positions 122 and 54, respectively, that could constitute part of the active site of a cysteine proteinase. These amino acids and their surrounding residues are conserved in all serotypes examined and appear to bear some resemblance to those in the putative active sites of the 3C proteinases in picornaviruses.
Adenoviruses, Human↗
Characterization of the adenovirus proteinase: substrate specificity.
Peptides were synthesized based on the cleavage sites in the adenovirus type 2 proteins pVI and pVII. The synthetic peptides were incubated with disrupted, purified adenovirus as a source of proteinase and specific cleavages were monitored by fast protein liquid chromatography and amino acid analysis. Using this approach it was established that all the peptides cleaved were of the form M(L)XGX decreases G or M(L)XGG decreases X. Thus we have shown that the adenoviral proteinase recognizes a specific secondary structure formed by a sequence of at least five amino acids, the main determinants of specificity being two and four residues to the N-terminal side of the bond cleaved. We were able to examine the relevant structural features of the peptide substrates by utilizing the CHEM-X molecular modelling package. Using our consensus sequence we were able to predict the cleavage sites in the viral proteins pVIII, pre-terminal protein (pTP), 11K and IIIa. Octapeptides containing the predicted sites in pVIII and the pTP were synthesized and shown to be cleaved by the proteinase.
Adenoviruses, Human↗
Adenovirus subviral particles and cores can support limited DNA replication.
Adenovirus type 2 cores can function effectively as templates in an in vitro replication system. Viral DNA replication assays using cores as templates do not differ in their requirements to the well characterized assays using DNA-complex templates, i.e. there is a dependence on terminal protein precursor (pTP), DNA polymerase and DNA binding protein and the assay is greatly stimulated by certain host transcription factors. The products of initiation and limited elongation are easily distinguishable and, in the system described, there is specific proteolysis of the pTP adducts as a function of the adenovirus-coded protease, present in the nuclear extracts from infected cells, or the core templates. Substitution of Mn2+ ions for Mg2+ ions in the replication assay has a dramatic effect on the nature of the replication events, in most cases resulting in the stimulation of initiation without elongation. Similar results can be achieved by utilizing subviral particles as templates, obtained by dialysis of purified adenovirus in a hypotonic buffer at pH 6.4. Restriction enzyme analysis of the replicated products confirmed that DNA synthesis proceeds from the adenovirus termini using both the core and subviral templates. By adding an ATP-regenerating system elongation can be further stimulated, particularly in the case of the subviral templates. Quantification of nucleotide incorporation into the appropriate restriction fragments indicates that for the subviral templates replication can proceed for at least 2000 to 3000 bases from either terminus. These results suggest that the adenovirus genome is packaged in the virion in a conformation readily available for at least the initial replication events. Such a conformation might also be appropriate for early transcription.
Adenoviruses, Human↗
Phosphorylation of adenovirus DNA-binding protein.
Evidence is presented here which indicates that the adenovirus DNA-binding protein (DBP) is phosphorylated at a tyrosine residue early in infection. This was suggested by the discovery that a proportion of the label in 32P-labelled DBP was resistant to alkali, and was substantiated by acid hydrolysis of DBP immunoprecipitates and by immunoblotting with a monoclonal antibody against phosphotyrosine. Treatment of [35S]methionine-labelled DBPs with chymotrypsin produced fragments of apparent Mr 45K and 39K whereas digestion of 32P-labelled DBP resulted in fragments of 45K and 26K. Consideration of the distribution of 32P label and its alkali stability in these fragments suggested that chymotrypsin cleaved populations of DBP at different sites depending on their phosphorylation states. The conservation, in all of the seven adenovirus serotypes sequenced, of a tyrosine residue (at amino acid 195 in adenovirus type 2) together with its surrounding residues, suggests that phosphorylation/dephosphorylation at this tyrosine residue may be important in various functions ascribed to the DBP.
Adenoviruses, Human↗
The urease of Ureaplasma urealyticum.
The urease from Ureaplasma urealyticum (serotype 8) has been purified by immuno-affinity column chromatography. Two active nickel-containing forms of the enzyme were demonstrated by non-denaturing electrophoretic analysis and a single active peak of apparent molecular mass 190 kDa was shown by FPLC. Total inactivation and denaturation of the enzyme to give three subunit polypeptides (one of 72 kDa containing nickel, one of 14 kDa and one of 11 kDa) was achieved by treatment with SDS and boiling. Densitometry suggested that the active enzyme contains equimolar ratios of the three subunits and hence is a hexamer. The enzyme displayed a pH optimum of 6.9 and pI values were determined. Storage of the purified enzyme at -70 degrees C followed by thawing to 20 degrees C caused a partial breakdown to inactive subunits. Anti-urease monoclonal antibodies bound both to the active enzyme and to the inactive 72 kDa subunit, and the antibodies cross-reacted with ureases from all of the other human serotypes. Competition assays with the antibodies revealed four distinct epitopes of the enzyme, all distinct from its active site.
Antibodies, Monoclonal↗
Serotype 8- and serocluster-specific surface-expressed antigens of Ureaplasma urealyticum.
The polypeptides of all 14 serotypes of Ureaplasma urealyticum were analyzed by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining. The electrophoretic patterns did not allow ready discrimination of individual serotypes or seroclusters. The analysis of the antigens of serotype 8 was reported previously (B. L. Precious, D. Thirkell, and W. C. Russell, J. Gen. Microbiol. 133:2659-2676, 1987). In this study, three of the surface-expressed membrane antigens of 16, 17, and 96 kilodaltons were further investigated, and monoclonal antibodies were raised against these three polypeptides. The major 96-kilodalton polypeptide was serotype 8 specific, and the 16-kilodalton polypeptide was present only in the larger serocluster. We describe monoclonal antibody probes that unequivocally differentiate serotype 8 from the other serotypes and that separate the two seroclusters of the organism.
Animals↗
Influence of age and testosterone levels on masculine development in rams.
Fifty wethers and 51 spring-born rams were divided into five groups and slaughtered at different seasons of the year at average ages of 271, 361, 459, 557 or 652 d to determine the age and season at which differences in secondary sex characteristics could be detected. Serum testosterone concentrations and testes weights were low in January when the rams were 271 d of age and again in April at 361 d of age. By July, at 459 d of age, testosterone concentrations and testes weights had peaked and then decreased the following November at 557 d and February at 652 d. In contrast with plasma testosterone concentrations and testes weights, buckiness scores, splenius to semimembranosus or semitendinosus muscle ratios, splenius muscle weights and neck and shoulder percentages were not seasonal. All of these measures increased significantly up to July and continued to increase slowly, but not significantly, thereafter. Muscle color and texture scores and rib eye color scores tended to increase in a linear manner for both rams and wethers as age increased. Subcutaneous fat from rams was yellower and softer than that from wethers over all age groups. Ram fat firmness did not change (P greater than .05) with age, and the only significant change in ram fat color was between the groups at 271 and 361 d of age. Overall, season of year coupled with higher levels of serum testosterone was related to initial development of secondary sex characteristics in ram lambs.
Adipose Tissue↗
Influence of gender, breed and age on maturity characteristics of sheep.
Fifty-one ewes, 50 wethers and 51 rams of Finn x whiteface or Suffolk x whiteface breeding were slaughtered at an average age of 271, 361, 459, 557 or 652 d to study changes in maturity characteristics with changes in age. When metacarpal epiphyseal cartilage (growth plates) ossifies with age, break joints on the distal end of the metacarpals fuse and the end of the bone then appears as a spool joint rather than as a break joint. This fusion occurs earlier chronologically in ewes than in rams or wethers. Most Finn x whiteface-cross ewes, which normally reach reproductive maturity earlier than Suffolk x whiteface-cross ewes, exhibited ossified metacarpal growth plates by 459 d of age; break joints of Suffolk x whiteface crosses were not fused until 557 d of age. Both breed groups of rams exhibited fused joints by 557 d of age, but only 3 of 10 wethers exhibited spool joints at 652 d of age. Microscopic examination showed that even in these three wethers the epiphyseal growth plate could still be detected. Yearling 2-yr-old classifications of sheep from teeth were not gender-dependent within the discrete age classes of this study. About half the sheep in each gender group were classified as yearlings at 459 d of age and about half in each group were classified as 2-yr-olds by 652 d of age. Stepwise multiple regression equations to estimate age of sheep in days were developed. Overall maturity, lean color and texture and rib bone maturity were the best carcass measures of maturity. Maturity scores for characteristics of the break joint did not enter any of the equations studied. When eye lens weight was included in equations with the best measures of carcass maturity, accuracy of estimates for age of sheep was improved.
Age Determination by Teeth↗
Complications and inappropriate use of minitracheostomy.
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Equipment Safety↗
Detection of Z DNA binding proteins in tissue culture cells.
A gel electrophoresis DNA binding assay to detect Z DNA binding proteins has been developed utilising [32P] labelled poly [d(G-C)] which was converted to the Z form by incubation in 100 microM Co(NH3)6Cl3. The parameters of the assay were established using a Z DNA antibody as a model system and then applied to extracts of Hela and BHK21 cells. Using an anti-Z DNA antibody conditions were established which allowed resolution of antibody-DNA complexes and free DNA in the presence of 100 microM Co(NH3)6Cl3. The inclusion of unlabelled complementary homopolymers eliminated non-specific binding to the labelled Z-DNA probe. Competition experiments demonstrated that the assay was highly specific for double stranded non-B DNA. Application of the technique to extracts of mammalian cells demonstrated that human and hamster cells contain Z-DNA binding proteins; further characterisation by a blotting technique indicated that a 56,000 molecular weight cell protein preferentially binds Z-DNA.
Antibodies↗
An antigenic analysis of the adenovirus type 2 fibre polypeptide.
Twenty-seven monoclonal antisera were generated against the SDS-denatured fibre of adenovirus type 2. The antisera were characterized using radioimmune assay, fluorescent antibody tests, immune precipitation, Western blotting, haemagglutination and neutralization, and formed six groups as follows: A, type-specific neutralizing antisera which exhibited haemagglutination inhibition (Hi+); B, type-specific non-neutralizing antisera which did not exhibit haemagglutination inhibition (Hi-); C, subgroup-specific neutralizing antisera Hi+; D, a subgroup-specific neutralizing antiserum Hi-; E, subgroup-specific non-neutralizing antisera Hi-; F, a subgroup-specific neutralizing antiserum Hi- which did not react in Western blotting tests. The C-terminal 201 amino acids of the fibre were expressed in Escherichia coli and a total of six antisera from groups A, B and C recognized five epitopes carried on this region which in several models is thought to form the knob of the fibre. At least eight epitopes were expressed by the entire native fibre. The five epitopes of the C-terminal end of the fibre formed three antigenic sites. Two sites each consisted of a neutralizing type-specific and a neutralizing group-specific epitope which overlapped in position. The remaining site consisted of a type-specific, non-neutralizing epitope.
Adenoviridae↗
Isolation and characterization of monoclonal antibodies to simian virus 5 and their use in revealing antigenic differences between human, canine and simian isolates.
Hybridomas secreting monoclonal antibodies to simian virus 5 (SV5) were obtained following immunization of mice with purified preparations of a human isolate (LN) of SV5. Immune precipitation studies showed that these monoclonal antibodies had specificities for the haemagglutinin-neuraminidase (HN), fusion (F), nucleo-, matrix and phospho- (P) proteins of SV5. By use of a radioimmune competition assay the monoclonal antibodies to the HN protein were assigned to four groups, members of which recognized different antigenic sites on the protein. All the anti-HN antibodies and the anti-F antibody neutralized virus infectivity. The 54 monoclonal antibodies obtained were used to determine whether there were antigenic differences between five human, two canine and one simian isolate of SV5. Although most of the monoclonal antibodies reacted with all isolates, a few did reveal antigenic differences in the HN, F and P proteins. Furthermore, analysis by SDS-PAGE showed that while the electrophoretic mobilities of most of the virus polypeptides of these isolates were similar some differences could be detected. In particular the P protein showed the most marked mobility differences between the human, canine and simian isolates. Slight differences in the mobility of the F1 glycoprotein could also be visualized.
Animals↗
Preliminary characterization of the urease and a 96 kDa surface-expressed polypeptide of Ureaplasma urealyticum.
Analysis by SDS-PAGE of Ureaplasma urealyticum (predominantly serotype 8), propagated in a growth medium containing 10% (v/v) foetal calf serum, revealed a complex series of polypeptides apparently free of medium contaminants. Serological analysis using an immobilized antibody reagent, and immunoblotting using a polyclonal serum, showed the presence of two major and several minor antigens. One major antigen, a putative surface component of apparent molecular mass 96 kDa was shown, with a monoclonal antibody, to be serotype-specific. Growth of the organism was partially suppressed in the presence of the antibody. The second major antigen had an apparent molecular mass of 76 kDa and was presumed to be an internal component since it failed to label with the Bolton and Hunter reagent, in contrast to the 96 kDa antigen. Another monoclonal antibody was characterized which detected the canonical urease enzyme of the organism serotype 8 and of the two other human serotypes tested. Purification of this urease antigen by affinity chromatography and electrophoretic analysis of polypeptides after denaturation revealed a single polypeptide of molecular mass 76 kDa, putatively related to the above major antigen. Enzymic activity could be recovered after purification and demonstrated by in situ techniques only when electrophoretic analysis was done under non-denaturing conditions suggesting that the functional enzyme is a multimeric complex.
Antigens, Bacterial↗
On the trail of paramyxoviruses in Paget's disease of bone.
The ultrastructural discovery of microcylindric inclusions in the nuclei and cytoplasm of osteoclasts in tissue affected by Paget's disease of bone has created a new approach. The morphologic similarity of the inclusions to viral structures has stimulated further studies involving immunocytologic techniques and in situ hybridization. Polyclonal antibodies reveal the presence of paramyxovirus antigens and measles virus and respiratory syncytial virus in pagetic osteoclasts. Monoclonal and monospecific polyclonal antibodies demonstrate paramyxovirus antigens of measles virus, simian virus (SV5), and human parainfluenza virus (PF3). In situ hybridization carried out with a 3H-labeled DNA probe, specific for the measles nucleocapsid protein, detects measles virus nucleotide sequences in the nuclei and cytoplasm of pagetic osteoclasts, confirming ultrastructural and immunocytologic findings. Surprisingly, the tritiated probe also hybridizes with a large proportion of mononucleated cells: osteoblasts, osteocytes, fibroblasts, and lymphocytes. This suggests a very wide host cell range for measles virus genomic information which, however, would appear to undergo translation only in osteoclasts. The cause-effect relation between the viral information contained by diseased bone cells and Paget's disease of bone remains to be established.
Antibodies, Monoclonal↗
In situ hybridization with biotinylated DNA probes: a rapid diagnostic test for adenovirus upper respiratory infections.
Adenovirus DNA was detected in cells from nasopharyngeal secretions of children with acute respiratory infections by in situ hybridization with biotinylated probes. The technique was easy to perform, giving rapid results which were well correlated with those of immunofluorescence assays of the same samples. Adenoviruses of subgroups B, C and E were detected equally well by probes prepared either from purified adenovirus type 5 or from a plasmid (A1) carrying a cloned insertion of BamH1 fragments C and D of adenovirus type 2 in pAT 153. The use of stable non-radioactive probes makes in situ hybridization a feasible assay for use in clinical laboratories with moderate resources.
Adenoviridae Infections↗
A neutralising monoclonal antibody against a paramyxovirus reacts with a brain antigen.
A monoclonal antibody against simian virus (SV5) HN glycoprotein which specifically neutralises the virus cross-reacts in immunofluorescence with an antigen in the cytoplasm of Purkinje cells of adult rat brain. After fixation in acetic acid-ethanol a cross-reaction is also seen with a myelin component. Cross-reaction of this antibody with human brain was also observed on section using immunofluorescence and in tissue extracts using ELISA and radioimmune assays. Monoclonal antibodies against other viruses, against another epitope on the same virus polypeptide and against another SV5 polypeptide of the same isotype were all negative in these tests.
Animals↗