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S Wain-Hobson

Publications and source records attributed to S Wain-Hobson.

118 records · Page 7Linked to original sources
PubMed1977

The determination of molecular-motion parameters from proton-relaxation-enhancement measurements in a number of Gd(III) - antibody-fragment complexes. A comparative study.

Longitudinal and transverse proton relaxation rates for water in the hydration spheres of Gd(III) bound to the non-immune rabbit IgG fragments Fc (C-terminal half of heavy-chain dimer), pFc' (C-terminal quarter of heavy-chain dimer) and Fab (N-terminal half of heavy and light chain) have been measured at a number of frequencies and temperatures using pulsed nuclear magnetic resonance spectrometry. For the fragments Fc and pFc', a full computer analysis showed that the results could be fitted by parameters of similar magnitude to those found previously for IgG. In contrast to the results for the other complexes the Fab -Gd(III) complex showed no slow exchange contribution to the relaxation rates. Under these circumstances it was found possible to obtain an accurate value for the hydration number (q) from measurements of the longitudinal and transverse relaxation rates at a chosen frequency such that the product of the nuclear Larmor frequency (omega1) and the correlation time for the dipolar relaxation processes (tauc) was approximately unity. Water-proton relaxation rates were also determined for the complex of Gd(III) with the Fv fragment of the mouse myeloma protein MOPC 315. A computer analysis of the results revealed a slow exchange contribution to the rates and this gave errors in the variable parameters similar to those observed previously for IgG, Fc and pFc'. The conclusions drawn from the different systems are discussed in terms of the present state of application of the proton relaxation enhancement technique in biology.

Animals↗
PubMed1977

Structure of an antibody combining site by magnetic resonance.

The structure of the combining site of the DNP binding IgA mouse myeloma protein MOPC 315 has been determined by a combination of high resolution nuclear magnetic resonance, electron spin resonance, model building and chemical modifications. This approach yields that general dimensions of the site, its polarity and asymmetry features, the assignment of the DNP-contact residues and their three-dimensional coordinates.

Animals↗
PubMed1976

Difficulties in determining accurate molecular motion parameters from proton relaxation enhancement measurements as illustrated by the immunoglobulin G-Gd(III) system.

Longitudinal and traverse proton magnetic relaxation rates for water in the hydration sphere of Gd(III) bound to non-immune rabbit IgG (immunoglobulin G) have been determined over a wide range of frequencies (4-84 MHz) at constant temperature (19 degrees C) using pulsed nuclear magnetic resonance spectrometry. The rates have also been determined at temperatures between 0 and 40 degrees C for two frequencies (61 and 84 MHz). The rates were fitted to existing theory using a computer least-squares procedure. Further computer analysis was then carried out to determine the sensitivity of the best-fit error to variation in the variable parameters in the theoretical expressions used. These include the water co-ordination number (q) for which it was found large variations could occur (between approximately 2 and 8) for only small changes in the error of best-fit. It was concluded that a slow exchange contribution to the relaxation rates was important in deciding which parameters are poorly determined. A rotational correlation time (tau r) was obtained which suggested there might be considerable internal motion of the Fc region (C-terminal half of heavy-chain dimer) of the IgG molecule. However the possibility of large errors in this value prevented unequivocal conclusions being drawn.

Animals↗
PubMed1976

Interactions of the lanthanide- and hapten-binding sites in the Fv fragment from the myeloma protein MOPC 315.

1. The interactions of lanthanide metals and dinitrophenyl spin-label haptens with the Fv fragment of the mouse myeloma protein MOPC 315 were investigated by the techniques of fluorescence, e.s.r. (electron spin resonance) and high-resolution n.m.r. (nuclear magnetic resonance). 2. The protein fluorescence of Fv fragment at 340nm is quenched by the haptens (fluorescence enhancement, epsilon=0.15) and enhanced by Gd(III) (epsilon=1.14) and other lanthanides. The binding of the haptens studied here is insensitive to pH in the range 5.5-7.0 (dissociation constant KH=0.3-1.0 muM) and shows 1:1 stoicheiometry. The binding of Gd(III) also shows 1:1 stoicheiometry, but is pH-dependent; the binding constant (KM) varies from 10 muM at pH7.0 to 700 muM at pH4.8. La(III) binding is less sensitive to pH. The pH-dependences of the metal-binding constants imply that a group in the protein with pKa greater than or equal to 6.2 is involved in the binding, and probably also other groups with lower pKa values. 3. The apparent binding of the haptens is weakened about 20-fold by Gd(III), and vice versa. An equilibrium scheme involving a ternary complex with an interaction between the two binding sites is derived in Appendix I to explain the experimental results at two pH values. 4. Time-dependent fluorescence changes are observed in the presence of Gd(III) at pH5.5. A two-state kinetic scheme involving a 'slow' conformational change in the Fv fragment is derived in Appendix II to explain this time-dependence. This scheme is consistent with the antagonistic equilibrium behaviour. 5. The e.s.r. changes in the spin-label haptens on binding to Fv fragment and on the subsequent addition of lanthanides are consistent with the binding scheme for haptens and lanthanides proposed from the fluorescence studies. A difference between the limiting quenching of the e.s.r. signal from the bound haptens in the presence of saturating concentrations of Gd(III) and La(III) is attributed to dipolar interactions between bound Gd(III) and the nitroxide moiety of the bound hapten. The residual quenching with Gd(III) allows an estimate of 1.2nm to be made for the distance between the two paramagnetic centres. 6. The 270 MHz proton difference spectrum of the Fv fragment resulting from the addition of La(III) suggests that any metal-induced conformational changes are small and involve relatively few amino acid residues on the Fv fragment...

Animals↗
PubMedDate not supplied

Residual expression of reporter genes in constructs mimicking HIV genome organization.

Plasmids were constructed whereby the expression of a reporter gene, either the cDNA corresponding to the secreted form of human alkaline phosphatase (SEAP) or the herpes simplex virus type 1 (HSV1) thymidine kinase (tk) gene, was rendered dependent upon the expression of the human immunodeficiency virus type 1 (HIV1) tat and rev proteins. The SEAP or tk genes were placed between HIV1 splice donor and acceptor sites. One SEAP construct carried a series of alternating splice donor and acceptor sites. In all cases, the rev response element mapped within an intron. Despite such mimicry of the HIV1 genome, residual expression of the reporter gene in the absence of tat and rev was observed. These results, as well as non-specific T-cell recruitment, suggest limits to the specificity of using HIV-activated toxic gene expression to kill HIV-infected cells.

Acquired Immunodeficiency Syndrome↗
PubMedDate not supplied

Stochastic events in the amplification of HTLV-I integration sites by linker-mediated PCR.

Human T-cell leukaemia virus type I (HTLV-I) proviral integration sites from an asymptomatic carrier and from the MT4 cell line were analysed by linker-mediated PCR (LMPCR) and inverse PCR (IPCR). LMPCR was more sensitive, allowing detection of a greater number of integrated proviruses. Reconstruction experiments using a cloned integrated HTLV-1 provirus indicated that > 100 copies were necessary to be detected frequently by LMPCR. To circumvent this problem, the LMPCR analysis was performed approximately 20 times per sample. Thus, for the MT4 cell line, the seven major integration sites were accompanied by approximately 20 clones of lesser frequency. For an asymptomatic HTLV-I carrier, nine integration sites were identified in a single amplfication, while a further 9 followed from 14 additional reactions. These findings show that there is a stochastic element to sampling HTLV-I integration sites by LMPCR, which tends to underestimate the actual number of HTLV-I bearing clones. Accordingly, those detected in at least two reactions represent the most abundant clones.

Base Sequence↗
PubMedDate not supplied

Molecular cloning of lymphadenopathy-associated virus.

Lymphadenopathy-associated virus (LAV) is a human retrovirus first isolated from a homosexual patient with lymphadenopathy syndrome, frequently a prodrome or a benign form of acquired immune deficiency syndrome (AIDS). Other LAV isolates have subsequently been recovered from patients with AIDS or pre-AIDS and all available data are consistent with the virus being the causative agent of AIDS. The virus is propagated on activated T lymphocytes and has a tropism for the T-cell subset OKT4 (ref. 6), in which it induces a cytopathic effect. The major core protein of LAV is antigenically unrelated to other known retroviral antigens. LAV-like viruses have more recently been independently isolated from patients with AIDS and pre-AIDS. These viruses, called human T-cell leukaemia/lymphoma virus type III (HTLV-III) and AIDS-associated retrovirus (ARV), seem to have many characteristics in common with LAV and probably represent independent isolates of the LAV prototype. We have sought to characterize LAV by the molecular cloning of its genome. A cloned LAV complementary DNA was used to screen a library of recombinant phages constructed from the genomic DNA of LAV-infected T lymphocytes. Two families of clones were characterized which differ in a restriction site. The viral genome is longer than any other human retroviral genome (9.1-9.2 kilobases).

Acquired Immunodeficiency Syndrome↗
PubMedDate not supplied

A novel type of human papillomavirus associated with genital neoplasias.

The role of human papillomaviruses (HPVs) in the development of genital neoplasias has been well documented. The genomes of two HPV types, HPV16 and HPV18, have been found to be associated with about 70% of invasive carcinomas of the uterine cervix. As, under non-stringent hybridization conditions, HPV DNA sequences have been detected in about 90% of cervical carcinomas, it seems likely that additional HPV types are associated with these tumours. Here we report the molecular cloning and characterization of a novel HPV type, tentatively named HPV33, whose sequences have been detected in 4-8% of biopsies of genital intra-epithelial neoplasias and cervical invasive carcinomas, usually as free monomeric or oligomeric molecules. However, in one specimen of vulvar Bowen's disease, HPV33 DNA sequences were integrated in the host-cell genome. Thus, HPV33 probably represents an additional type of potentially oncogenic genital HPV.

Carcinoma↗
PubMedDate not supplied

AIDS virus-specific cytotoxic T lymphocytes in lung disorders.

Human immunodeficiency virus (HIV) is implicated in the development of AIDS (acquired immune deficiency syndrome). HIV infection leads to the generation of HIV-specific thymus-derived (T) lymphocytes in humans and apes. We describe an experimental system permitting the quantitative and systematic analysis of HIV-specific cytotoxic T lymphocytes (CTL). Functional, HIV-specific CTL are obtained by broncho-alveolar lavage (BAL) from the lungs of seropositive patients with lymphocytic alveolitis. These alveolar CTL: (1) recognize and kill HIV-infected alveolar macrophages in vitro under autologous, but not heterologous, conditions; (2) correspond to standard CTL as they express the CD3 and CD8 surface markers, but not the CD4 marker; and (3) are restricted by class I HLA transplantation antigens in their cytotoxic activities. We propose the hypothesis that interactions between HIV-specific CTL and infected macrophages induce major inflammatory reactions in seropositive patients.

Cytotoxicity, Immunologic↗