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Biomedical subjects

S Gillam

Publications and source records attributed to S Gillam.

At least 19 recordsLinked to original sources
PubMed1992

Characterization of the specificity and genetic restriction of human CD4+ cytotoxic T cell clones reactive to capsid antigen of rubella virus.

Using 11 overlapping synthetic peptides covering more than 95% of the amino acid sequence of capsid protein of rubella virus, 7 CD4+ T cell clones (R10, R11, R18, A2, A10, A11, and A12) isolated from 2 rubella seropositive donors reacted strongly to rubella capsid peptides C6 (residues 119-152), C9 (residues 205-233), or C11 (residues 255-280), respectively, in both proliferation and cytotoxicity assay. Truncated peptides C6E (residues 125-139), C9B (residues 205-216), and C11E (residues 260-272) were shown to be involved directly to the T cell determinants of C6, C9, and C11, respectively. Genetic restriction of these T cell clones was analyzed by using human cell lines with various HLA-DR phenotypes as targets and/or antigen-presenting cells in cytotoxicity assay and/or proliferation assays. The results indicated that the recognition of peptide C6 by T cell clones (R11 and R18) was associated with DRw9 molecule, while the HLA restriction element of the responses of other T cell clones (A2 and A11, A10, and A12) that reacted with peptide C9 or C11 was DR4 molecule. However, there may be a cross-recognition by the T cell clone (A12) between DR1 and DR4 subtypes.

Amino Acid Sequence↗
PubMed1992

Localization of the virus neutralizing and hemagglutinin epitopes of E1 glycoprotein of rubella virus.

Current serological assays using whole rubella virus (RV) as a target antigen for detecting RV-specific antibodies fail to define specific RV proteins and antigenic determinants such as hemagglutinin (HA) and virus-neutralizing (VN) epitopes of rubella virus. A panel of E1 deletion mutants and a subset of E1-specific monoclonal antibodies (MAb) were used for the initial analysis of HA and VN epitopes of E1 glycoprotein. A peptide region (E1(193) to E1(269)) was found to contain HA and VN epitopes. Using both overlapping synthetic peptides and truncated fusion proteins within this region, the HA epitope defined by MAb 3D9F mapped to amino acid residues E1(214) to E1(240), while two VN epitopes defined by MAb 21B9H and MAb 16A10E mapped to amino acid residues E1(214) to E1(233) and E1(219) to E1(233), respectively. The epitopes defined in this study are recognized by antibody whether or not the epitopes are glycosylated.

Amino Acid Sequence↗
PubMed1992

The influence of N-linked glycosylation on the antigenicity and immunogenicity of rubella virus E1 glycoprotein.

Rubella virus E1 glycoprotein contains three functional N-linked glycosylation sites. The role of N-linked glycosylation on the antigenicity and immunogenicity of E1 glycoprotein was studied using vaccinia recombinants expressing E1 glycosylation mutants. Expressed E1 glycosylation mutant proteins were recognized by a panel of E1-specific monoclonal antibodies in radioimmunoprecipitation, immunofluorescence, and immunoblotting, indicating that carbohydrate side chains on E1 are not involved in the constitution of epitopes recognized by these monoclonal antibodies. This observation was further supported by the fact that removal of oligosaccharides on E1 by glycosidase digestion did not significantly change the antigenicity of E1. All the glycosylation mutants were capable of eliciting anti-RV E1 antibodies. The single glycosylation mutants (G1, G2, and G3), but not the double mutant (G23) or the triple mutant (G123), were found to be capable of inducing virus neutralizing antibodies. Among the single glycosylation mutants, only G2 and G3 were active in producing hemagglutination inhibition antibodies in mice. Our findings suggest that although carbohydrate on E1 is not directly involved in the antigenic structures of E1, it is important in maintaining proper protein folding and stable conformation for expression of immunological epitopes on E1.

Animals↗
PubMed1992

Evaluating the drug dependency unit.

An evaluation of the St Mary's Drug Dependency Unit is described. Unit staff were interviewed and a retrospective analysis of 156 records was performed. The advent of HIV infection has led to an emphasis on public health priorities in the treatment of drug misuse. However, DDU staff retain a highly individualistic approach to their work. Staff aiming at abstinence within treatment may compromise important advice on harm minimization. The mean delay between first contact and entering treatment was 10 days; 42% of clients failed to complete the initial assessment. The need to extend access to the service is not easy to reconcile with the increasingly specialised support role for such units implied in recent strategy documents. Little is known of the effectiveness of different services for drug misusers. In this study, only 6% of clients were known to have achieved abstinence at one year. The efficiency of the DDU could be improved by closer definition of the unit's goals and target population, regular progress reviews, shortening the assessment procedure and the computerisation of data collection.

Adult↗
PubMed1992

Cellular and humoral immune responses to rubella virus structural proteins E1, E2, and C.

Better understanding of cell-mediated immune responses to rubella virus would provide the basis for the development of safe and effective vaccines against rubella and would aid in analysis of the pathophysiology of congenital rubella syndrome. We have expressed individual rubella virus structural proteins, E1, E2 and C, via vaccinia virus recombinants. Using the expressed recombinant proteins as antigens, we were able to demonstrate antigen-specific lymphocyte proliferative responses in control individuals and individuals with congenital rubella syndrome. Among the two human groups studied, E1 glycoprotein proved to be a better immunogen than E2 or C. For the control individuals, significant differences in proliferative responses to the structural proteins E1, E2, and C were observed. These differences were not significant in individuals with congenital rubella syndrome. In parallel to the lymphoproliferative responses, immunoglobulin G responses were also found directed mainly to the E1 glycoprotein. These results suggest that E1 may be the most important rubella virus antigen to study in determining the domains required for constructing subunit vaccines against rubella.

Adult↗
PubMed1992

Identification of T-cell epitopes on E2 protein of rubella virus, as recognized by human T-cell lines and clones.

T-cell epitopes on the E2 protein of rubella virus were studied by using 15 overlapping synthetic peptides covering the E2 protein sequence. The most frequently recognized epitopes on E2 were E2-4 (residues 54 to 74), with 5 of 10 tested T-cell lines responding to it. Two CD4+ cytotoxic T-cell cloned isolated from one T-cell line responded strongly in proliferation assays with peptide E2-4 and were cytotoxic to target cells presenting the E2-4 determinant. Truncated peptides contained within the E2-4 peptide sequence were used to define the T-cell determinants. Results indicated that amino acid residues 54 to 65 were directly involved. Human cell lines with different HLA phenotypes were tested for the capacity to present the antigenic determinants. The results suggested that recognition of peptide E2-4 by T-cell clones was associated with HLA DR7.

Amino Acid Sequence↗
PubMed1992

Induction of an organ-specific autoimmune disease, lymphocytic hypophysitis, in hamsters by recombinant rubella virus glycoprotein and prevention of disease by neonatal thymectomy.

Glycosylated, membrane-associated E1 (58-kDa) and E2 (47- to 49-kDa) rubella virus proteins and unglycosylated nucleoprotein C (33 kDa), from separately expressed vaccinia virus recombinants, were injected into golden Syrian hamsters. Rubella virus E1 and E2 glycoproteins consistently induced an organ-specific autoimmune disease, autoimmune lymphocytic hypophysitis, which was evidenced by the induction of autoantibodies against pituitary cells and by lymphocytic infiltration of the pituitary. Neonatal thymectomy prevented the disease. In contrast, rubella virus nucleoprotein C did not induce either autoantibodies against pituitary cells or lymphocytic infiltration of the pituitary. This finding raises the possibility that virus-specific protein itself can induce an organ-specific autoimmune disease in certain circumstances.

Animals↗
PubMed1992

Analysis of T- and B-cell epitopes of capsid protein of rubella virus by using synthetic peptides.

A nested set of 11 overlapping synthetic peptides covering the entire sequence of rubella virus capsid protein was synthesized, purified, and tested against human rubella virus-specific T-cell lines and rubella virus-seropositive sera. T-cell lines derived from four donors responded strongly to four synthetic peptides containing residues 96 to 123, 119 to 152, 205 to 233, and 255 to 280. Only one peptide (residues 255 to 280) was recognized by all four T-cell lines. Two human immunodominant linear B-cell epitopes were mapped to residues 1 to 30 and 96 to 123 by using peptide-specific enzyme-linked immunosorbent assay. All 11 synthetic peptides were highly immunogenic and induced strong antibody responses in rabbits against the respective immunized peptides. Seven of the 11 rabbit antipeptide antisera (anti-1-30, -74-100, -96-123, -119-152, -205-233, -231-257, and -255-280) specifically recognized the capsid protein on immunoblots. Identification of these T- and B-cell epitopes represents the first step toward rational design of synthetic vaccines against rubella.

Amino Acid Sequence↗
PubMed1992

Role of N-linked oligosaccharides in processing and intracellular transport of E2 glycoprotein of rubella virus.

The role of N-linked glycosylation in processing and intracellular transport of rubella virus glycoprotein E2 has been studied by expressing glycosylation mutants of E2 in COS cells. A panel of E2 glycosylation mutants were generated by oligonucleotide-directed mutagenesis. Each of the three potential N-linked glycosylation sites was eliminated separately as well as in combination with the other two sites. Expression of the E2 mutant proteins in COS cells indicated that in rubella virus M33 strain, all three sites are used for the addition of N-linked oligosaccharides. Removal of any of the glycosylation sites resulted in slower glycan processing, lower stability, and aberrant disulfide bonding of the mutant proteins, with the severity of defect depending on the number of deleted carbohydrate sites. The mutant proteins were transported to the endoplasmic reticulum and Golgi complex but were not detected on the cell surface. However, the secretion of the anchor-free form of E2 into the medium was not completely blocked by the removal of any one of its glycosylation sites. This effect was dependent on the position of the deleted glycosylation site.

Animals↗
PubMed1991

The influence of capsid protein cleavage on the processing of E2 and E1 glycoproteins of rubella virus.

The structural polyprotein of rubella virus is cotranslationally processed by host cell signal peptidase. Oligonucleotide-directed mutagenesis was used to alter the cleavage site between capsid and E2 proteins and to examine the importance of this cleavage for the transport and processing of E2 and E1 glycoproteins. The in vitro and in vivo expression of the cleavage site mutant revealed that the E2 polypeptide can cross the endoplasmic reticulum membrane without the cleavage of its signal peptide, while the transport of E2 beyond the endoplasmic reticulum requires the cleavage of E2 from capsid. We have shown that capsid protein does not appear to undergo further proteolytic processing after it is cleaved from E2 by signal peptidase. Some of the requirements for the cleavage by signal peptidase between capsid and E2 were examined by the in vitro analysis of wild-type and mutant cDNAs.

Animals↗
PubMed1991

Analysis of rubella virus E1 glycosylation mutants expressed in COS cells.

cDNA clones encoding the envelope glycoprotein E1 of rubella virus (RV) were altered by site-directed mutagenesis at consensus sites for addition of N-linked glycans. The resulting plasmids were introduced into COS cells and the mutant E1 proteins were analyzed by indirect immunofluorescence, radioimmunoprecipitation, and immunoblotting. We found that RV E1 contains three N-linked oligosaccharides, each approximately 2 kDa in size. Although lack of glycosylation did not appear to affect targeting of E1 to the Golgi region, mutants lacking N-linked glycans at Asn 177 and Asn 209 failed to bind anti-E1 antibodies under nonreducing conditions. Our results suggest that glycosylation may be important for expression of important immunologic epitopes on RV E1.

Animals↗
PubMed1991

pH-dependent solubility shift of rubella virus capsid protein.

The mechanism of capsid uncoating in rubella virus and other togaviridae is not well understood. This study presents data which suggest that rubella virus capsid undergoes a structural change from having hydrophilic to hydrophobic properties, between pH 5 and 5.5. Such a conformational change would allow capsid uncoating to occur within the lysosome, allowing RNA penetration to occur upon fusion of the viral envelope with the limiting membrane of the lysosome.

Animals↗
PubMed1979

RNA synthesis and RNA polymerase activities in germ cells of developing rainbow trout testis.

Spermatogenesis is a complex developmental process which sequentially generates several different germ cell types. These cell types from rainbow trout (Salmo gairdnerii) testis were separated by sedimentation in serum albumin gradients and characterized on the basis of their physical properties, chronological appearance, and protein synthesis. The rate of RNA synthesis, the types of RNA made, and the RNA polymerase activities present were determined for each cell type. The rate of RNA synthesis decreased from a high level in spermatogonia and spermatocytes to a low level in early spermatids and was absent in late spermatids and mature spermatozoa. Newly synthesized RNA in spermatogonia and spermatocytes consisted of a variety of molecular weight species, including 18 S and 28 S ribosomal RNAs. The synthesis of high molecular weight RNAs, especially ribosomal RNAs, decreased drastically in early spermatids, leading to the synthesis of only small molecular weight RNAs. RNA polymerase I and II were present in all cell types but the activities of both showed large decreases between spermatocytes and middle spermatids. Both RNA polymerase activities were almost absent from spermatozoa. The activities of RNA polymerase I and II from unfractionated testis cells at different stages of hormone-induced spermatogenesis were quantitated by fractionation of the solubilized extract on DEAE-cellulose. Both polymerases showed major decreases in activity which began near the chronological mid-point of development. For polymerase I the decrease in activity was over 400 fold, for polymerase II over 200 fold. The number of RNA polymerase II molecules per testis cell, quantitated by the binding of [3H]amanitin to cell extracts, also decreased markedly during spermatogenesis. The reduction in polymerase II activity was accompanied by a parallel 200-fold decrease in[3H]amanitin binding. The reduction in polymerase activity appears, therefore, to be due to an actual reduction in the cellular content of RNA polymerase II molecules. These results suggest that transcription in maturing testes is regulated, at least in part, by the concentrations of the RNA polymerases.

Amanitins↗
PubMed1979

Defined transversion mutations at a specific position in DNA using synthetic oligodeoxyribonucleotides as mutagens.

The oligodeoxyribonucleotides, pCCCAGCCTCAA, which is complementary to nucleotides 5274--4284 of bacteriophage phi X174 viral DNA , and pCCCAGCCTAAA, which corresponds to the same sequence with a C leads to A change at the ninth nucleotide, were synthesized enzymatically. The second of these oligonucleotides was used as a primer for E. coli DNA polymerase I, from which the 5'-exonculease has been removed by proteolysis (Klenow enzyme), on wild-type phi X174 viral DNA template. After ligation, this yielded closed circular heteroduplex DNA with a G, A mismatch at nucleotide 5276. Transfection of E. coli spheroplasts with the heteroduplex DNA produced phage mutated at this nucleotide (G leads to T in the viral DNA) with high efficiency (13%). The mutant DNA, which corresponds to the gene B mutant am16, was reverted (T leads to G) by the wild type oligonucleotide with an efficiency of 19%. The nucleotide changes were established by sequence determination of the mutated viral DNA using the enzymatic terminator method. The production of specific transversion mutations, together with a previous demonstration of specific transition mutations (1), established that short enzymatically synthesized oligodeoxyribonucleotides can be used to induce any class of single nucleotide replacement with high efficiency and thus provide a powerful tool for specific genetic manipulations in circular genomes like that of phi X174.

Bacteriophage phi X 174↗
PubMed1979

Site-specific mutagenesis using synthetic oligodeoxyribonucleotide primers: I. Optimum conditions and minimum ologodeoxyribonucleotide length.

A synthetic oligodeoxyribonucleotide mismatched at a single nucleotide to a specific complementary site on wild-type circular phi X174 DNA can be used to produce a defined point mutation after in vitro incorporation into closed circular duplex DNA by elongation with DNA polymerase and ligation followed by transfection of Escherichia coli (Hutchison et al., 1978; Gillam et al., 1979). The present study is an investigation of the optimum conditions required for the oligodeoxyribonucleotide-primed reaction for production of transition and transversion mutations in phi X174 DNA, using the large (Klenow) fragment of E. coli DNA polymerase I. Under optimum conditions up to 39% of the progeny of transfection are the desired mutant and significant mutation is observed using a heptadeoxyribonucleotide.

Bacteriophage phi X 174↗