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S A Jeffs

Publications and source records attributed to S A Jeffs.

18 recordsLinked to original sources
PubMed2006

Comparative analysis of HIV-1 recombinant envelope glycoproteins from different culture systems.

The productivity of stable Chinese hamster ovary cell lines secreting HIV-1 monomeric (IIIB gp120) and oligomeric (UG21 gp140) recombinant envelope glycoproteins was compared in serum-containing (S+), serum-free (S-) and protein-free (P-) culture media. UG21 gp140 expression was greatest in S+ medium, while IIIBgp120 production was lower than gp140 in all three media but highest in S-. UG21 gp140 production was highest in standard 850-cm2 roller bottle cultures in S+ media, peaking after 14 days of incubation, while expression levels in the three media were 0.5 (S+), 0.4 (S-) and 0.2 (P-) mg/l, from which 90, 80 and 12% of gp140, respectively, could be purified by immunoaffinity chromatography. Purified UG21 gp140 from S+ and S- media possessed biological functionality as evidenced by CD4 and monoclonal antibody (Mab) binding. In contrast, UG21 gp140 from P- medium appears to be misfolded and non-functional. Despite the possession of a different N-linked glycan profile, UG21 gp140 from S- media shows very similar CD4 and Mab binding characteristics to S+ UG21 gp140. The relevance of these findings to HIV vaccine development is discussed.

Animals↗
PubMed2004

Expression and characterisation of recombinant oligomeric envelope glycoproteins derived from primary isolates of HIV-1.

The production, purification and characterisation of recombinant gp140 oligomeric envelope glycoproteins derived from six primary isolates of HIV-1 (covering clades A, B, C, D, F and O) are described. Using a Chinese hamster ovary cell expression system, expression levels of between 0.1 and 1 mg/l cell-conditioned culture media were obtained, and purified to >95% by affinity chromatography. A, B, D, F and O clade gp 140s were found to be multimeric, bind to a panel of defined env-specific monoclonal antibodies and interact with CD4 and CXCR4, demonstrating correct folding. Their immunogenicity was confirmed by the generation of high-titre anti-gp140 antibodies in rabbits. The C clade gp140 was incorrectly folded and poorly antigenic. Despite the presence of an unmodified gp120/41 cleavage site, only the B clade gp140 showed significant processing to gp120 and gp41. Each gp140 has a specific pattern of oligomerisation, and varies in its resistance to reducing agents and salt concentration. The binding of gp140 to soluble and cell-surface CD4 and CXCR4 is related to the degree of oligomerisation. The C1 and C5 regions, CD4 binding domain and the epitope defined by the 2G12 monoclonal antibody were well exposed, but the C-terminal region of the extracellular domain of gp41 appears to be occluded by oligomerisation. These reagents have potential as immunogens for use in vaccine development.

Animals↗
PubMed2002

Truncated gp120 envelope glycoprotein of human immunodeficiency virus 1 elicits a broadly reactive neutralizing immune response.

Removal of the V1-V3 loops from IIIB gp120 results in a protein, PR12, with altered immunogenicity compared to the full-length protein. Polyclonal immune sera raised in rats using PR12 as immunogen recognizes envelope glycoproteins of clades A, B, C, E, F and G and can neutralize chimeric human immunodeficiency virus type 1 (HIV-1) HXB2 viruses expressing envelopes from primary HIV-1 clades B, C, E and F. These data suggest that the immune response to PR12 is directed toward conserved epitopes expressed by viral glycoproteins of diverse genotypes. Five monoclonal antibodies (mAb) derived from PR12-immunized rats were unable to neutralize virus infectivity; hence the epitopes responsible for the induction of this cross-clade neutralizing activity remain to be elucidated. However, PR12 immune sera were able to compete with the human neutralizing mAb 2G12 for gp120 binding, implying that this epitope may be immunogenic when expressed in the context of this truncated protein.

Animals↗
PubMed2002

Comparison of morphine alone with morphine plus clonidine for postoperative patient-controlled analgesia.

BACKGROUND: Clonidine is an alpha 2 adrenergic agonist with analgesic properties. This study aimed to see if the addition of clonidine to morphine when given by patient-controlled analgesia (PCA) would improve analgesia beyond the first 12 h after surgery. METHODS: Sixty patients undergoing lower abdominal surgery were recruited into a randomized double blind study. At the end of surgery Group C received an infusion of clonidine 4 micrograms kg-1 over 20 min, PCA clonidine 20 micrograms and morphine 1 mg bolus. Group M received an infusion of saline and then PCA morphine 1 mg bolus. Pain, sedation and nausea and vomiting were assessed after 12, 24 and 36 h, and satisfaction with analgesia was assessed at 36 h. RESULTS: Pain scores were significantly lower in Group C between 0 and 12 h, but thereafter there was no difference. Morphine consumption was the same for both groups until 24-36 h. Nausea and vomiting was significantly reduced in Group C between 0 and 24 h. Patients in Group C were significantly happier with their pain relief (four-point scale).

Abdomen↗
PubMed2001

Characterization of human monoclonal antibodies selected with a hypervariable loop-deleted recombinant HIV-1(IIIB) gp120.

Recombinant gp120 of the HIV-1(IIIB) isolate (BH10 clone) has been mutated to form the PR12 protein with the first 74 C-terminal amino acids and the V1, V2 and V3 hypervariable loops deleted. A variety of studies have shown that the CD4 binding domain (CD4bd) is very well exposed in PR12 in contrast to rgp120(LAI). Using PR12 for selection of human monoclonal antibodies (MAbs) from HIV-infected individuals, five MAbs were generated with specificities to the epitopes overlapping the CD4bd (1570A,1570C,1570D,1595 and 1599). The three MAbs, 1570A, C and D, generated from one HIV-infected individual, represent one MAb as determined by sequence analysis of the V(H)3 region. Since the epitopes overlapping the CD4bd exhibit variability among HIV-1 clades, the specificity of anti-CD4bd MAbs were distinguished by differing patterns of binding to recombinant envelope proteins derived from clade A, B, C, D and E viruses. The PR12-selected MAbs were also compared with a panel of gp120-selected anti-CD4bd MAbs and showed a different range of specificities. MAb 1599 is clade B specific, MAb 1595 reacts with the A, B and D clades, while MAb 1570 recognises the most conserved epitope, as it binds to all proteins. The results show that the exposure of different epitopes in the CD4bd of the PR12 protein allows this protein to serve as an immunogen and to induce anti-CD4bd antibodies.

Antibodies, Monoclonal↗
PubMed1996

Antigenicity of truncated forms of the human immunodeficiency virus type 1 envelope glycoprotein.

Chinese hamster ovary (CHO) cell lines secreting a series of truncated forms of human immunodeficiency virus type 1 (HIV-1) IIIB (clone BH10) gp12O were assembled. Using purified glycoproteins, we demonstrated the functional and structural integrity of these truncates by their reactivity with both sCD4 and anti-gp 120 monoclonal antibodies (MAbs). Deletion of the Vl, V2 and V3 regions had minimal effects on CD4 binding, but deletion of the NH2 terminus affected the folding of the truncated molecule. Deletion of either V1/V2 or V1/V2/V3 regions led to enhanced recognition by some, but not all, MAbs mapping to the CD4 binding site. In contrast, deletion of the V1/V2 regions had no effect on the ability of V3-specific MAbs to bind to the truncate. These results support the use of truncated forms of gp12O as components of potential HIV vaccines.

Animals↗
PubMed1994

Partial characterization and kinetics of expression of Sm15, a Schistosoma mansoni tegumental antigen.

Differential antibody screening of an adult Schistosoma mansoni cDNA expression library constructed in lambda gt11 identified a partial cDNA clone, A70. This cDNA encodes a fusion protein recognized by antibodies raised against highly irradiated schistosomula and adult worm tegumental membranes but not by anti-egg antibodies. Anti-tegumental membrane antisera affinity-purified on the A70 cDNA fusion protein were used for Western blotting analysis and indirect immunofluorescence, resulting in the identification of a 15-kDa protein (Sm15) in the tegument of adult worms. This is one of the principal tegumental antigens recognized by antibodies from mice protectively vaccinated with adult worm tegumental membranes. Sm15 is much smaller than the protein encoded by its gene, suggesting that it results from a highly processed precursor. It was found that Sm15 behaves as an integral membrane protein upon partitioning in Triton X-114 and that it is present in worms of 2 weeks or older but not in schistosomula or miracidia. The affinity-purified antibodies also revealed the presence of a 23-kDa antigen in whole-worm homogenates that is apparently coexpressed with Sm15. The 23-kDa antigen was not found associated with membranes and is probably a soluble protein. A further series of Western blots were undertaken using antibodies affinity-purified from serum raised against schistosomula. In this case, the 23- and 15-kDa products were not recognized, but rather soluble proteins ranging from 45- to 150-kDa were detected in almost all larval stages investigated. The results suggest that the precursor is differentially processed during maturation.

Animals↗
PubMed1993

Structure of the gene encoding a putative Schistosoma mansoni tegumental antigen precursor.

In order to obtain the complete gene encoding the putative precursor of a 15-kDa Schistosoma mansoni tegumental antigen (Sm15), two cDNAs (A70 and A184) and two fragments of independent genomic clones were subcloned and sequenced. The collated sequence contains 4700 nucleotides and represents the full length open reading frame of the gene, encoding a protein of 1032 amino acids with a calculated molecular mass of 116,900. Thus, the gene encodes a much longer protein than that identified in the tegumental membranes, suggesting that it encodes a precursor that is subsequently highly processed. A 964-bp region composed of 5 closely related repeats was found to be present within the translated frame. The predicted protein is highly acidic and there is no indication of hydrophobic domains that may represent transmembrane regions or indicate attachment of a GPI anchor. The coding region has no homologies in the currently available data bases. In the 5' non-transcribed area a copy of the SM alpha repeat family is present. The coding region is preceded by putative CCAAT and TATA boxes that may be involved in the control of expression.

Amino Acid Sequence↗
PubMed1991

Structure of Sm25, an antigenic integral membrane glycoprotein of adult Schistosoma mansoni.

Sm25 is the principal antigen recognised by antibodies from mice protectively vaccinated with isolated tegumental membranes of adult Schistosoma mansoni. The full-length amino acid sequence of this protein has been deduced from the sequence of two cDNAs, one isolated by screening a cDNA library and the other, including the 5' end of the gene, amplified directly from adult worm RNA using the polymerase chain reaction. The predicted sequence represents a nascent polypeptide of Mr 21,500. Following cleavage of a predicted signal sequence, the Mr of the resulting polypeptide is 17,600. The polypeptide contains 2 potential sites for N-linked glycosylation and a hydrophobic domain at the C-terminus that could facilitate membrane association. Analysis of the mature gene product confirmed that Sm25 is an N-glycosylated integral membrane protein and that the Mr of the deglycosylated polypeptide is between 15,000 and 20,000.

Amino Acid Sequence↗
PubMed1991

Molecular cloning and characterisation of the 22-kilodalton adult Schistosoma mansoni antigen recognised by antibodies from mice protectively vaccinated with isolated tegumental surface membranes.

A cDNA clone from an adult Schistosoma mansoni lambda gt11 expression library (A12) encoding an antigenic polypeptide of 22 kDa is described. A12 is 797 bp long and has one open reading frame encoding a protein of 190 amino acids which does not contain a signal sequence or membrane anchor motif and has no homologies with any sequences on the currently available data bases. Its product (sm22.6) is recognised by antibodies from mice protectively vaccinated with purified adult S. mansoni tegumental membranes and by serum from S. mansoni-infected Brazilians. It is present in all post-snail life cycle stages except the egg, is not sex-specific, and is found in 9 species of Schistosoma, but not in a range of other helminths. Data are presented which suggest that sm22.6 is a soluble, peripheral membrane protein.

Amidohydrolases↗
PubMed1988

Echinococcus granulosus (Cestoda): uptake of L-amino acids by secondary hydatid cysts.

The uptake of cycloleucine, L-proline, L-alanine and L-threonine by secondary hydatid cysts of Echinococcus granulosus (U.K. horse strain 3-8 mm in diameter, derived from Balb/c mice infected 300-400 days previously) occurs by passive diffusion into the cyst wall (laminated layer plus germinal layer) and by mediated mechanisms into the fluid-filled interior. The maximal concentrations of these compounds are achieved after incubation for 2 h in vitro and approach those in vivo. Kt and Vmax values describing the uptake of these compounds are given. The flux rates for these compounds are extremely slow compared to those obtained with the protoscolex. A rationale for standardizing the experimental method for uptake studies with hydatid cysts is described.

Absorption↗
PubMed1987

Echinococcus granulosus: specificity of amino acid transport systems in protoscoleces.

Protoscoleces of Echinococcus granulosus absorb the L-amino acids proline, methionine, leucine, alanine, serine, phenylalanine, lysine and glutamic acid by a combination of mediated transport and diffusion. All eight amino acids were accumulated against a concentration gradient. Comparison of Kt and Vmax values suggests that a low affinity for a particular compound is compensated for by a relatively larger number of transport sites for that compound. Four systems serve for the transport of the eight substrates studied: 2 for neutral (EgN1, EgN2) and 1 each for acidic (EgA) and basic (EgB) amino acids. All eight amino acids are incorporated into protein to varying degrees and substantial portions of absorbed L-alanine and L-methionine are metabolized into other compounds.

Absorption↗
PubMed1986

Echinococcus granulosus: absorption of cycloleucine and alpha-aminoisobutyric acid by protoscoleces.

Protoscoleces of Echinococcus granulosus absorb the amino acids cycloleucine and alpha-aminoisobutyric acid (AIB) by a combination of mediated uptake and diffusion. After correcting for the latter, values for Kt and Vmax of 0.124 mM and 0.947 nmoles/mg protein/2 min for cycloleucine were calculated; corresponding values for AIB were 0.039 mM and 0.139 nmoles/mg protein/2 min. Both amino acids were accumulated against a concentration gradient and a comparison of Kt and Ki values determined in mutual inhibition experiments suggested that both cycloleucine and AIB share a common uptake locus (loci). Cycloleucine uptake was pH-dependent and could be inhibited by a variety of other amino acids. Neither D- nor L-proline inhibited cycloleucine absorption but D-methionine, D-alanine, D-leucine, D-valine and D-serine were much more effective inhibitors than their L-counterparts.

Absorption↗
PubMed1985

Hymenolepis diminuta (Cestoda): uptake of cycloleucine by metacestodes.

Cycloleucine uptake by metacestodes of H. diminuta of various ages was investigated. Absorption occurs by active mediated transport, mean Kt = 0.28 mM. Vmax values are age-related, and can be correlated to developmental changes. Cycloleucine uptake in the metacestode is very similar to that in the adult worm and the implications of this are discussed.

Amino Acids↗