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Biomedical subjects

J R Stephenson

Publications and source records attributed to J R Stephenson.

At least 73 records · Page 4Linked to original sources
PubMed1986

Development of an enzyme-linked immunosorbent assay for staphylococcal protein A produced in Escherichia coli by pUC8 based plasmids containing the Staphylococcus aureus Cowan I protein A gene.

Staphylococcal protein A, a cell wall component of several strains of Staphylococcus aureus has found many uses as a research tool, as a diagnostic reagent and even as a possible therapeutic agent in cancer. These uses have arisen exclusively out of its almost unique property of binding specifically to the Fc region of many immunoglobulin molecules. As Staphylococcus aureus is pathogenic, it has been desirable for industrial purposes to clone the gene coding for protein A into Escherichia coli and to develop a sensitive assay free from interference by bacterial components. We describe an ELISA assay which is capable of detecting staphylococcal protein A in bacterial lysates at levels as low as 1.0 ng/ml and which is free of interference from lysozyme, lysostaphin, endogenous peroxidases or other bacterial antigens.

Animals↗
PubMed1986

New method for typing Staphylococcus aureus resistant to methicillin based on sulphur-35 methionine labelled proteins: its application in an outbreak.

Sodium dodecyl sulphate polyacrylamide gel electrophoresis of sulphur-35 methionine labelled cellular proteins of Staphylococcus aureus resistant to methicillin was used as a typing method during an outbreak on a cardiothoracic ward. This showed that the outbreak strain was indistinguishable from the epidemic strain of methicillin resistant S aureus prevalent in London. In contrast, 44 epidemiologically separate strains of S aureus gave individually distinct radiolabelled protein profiles. This method permitted rapid confirmation that an epidemic strain was responsible and indicated the need for urgent control measures.

Bacterial Proteins↗
PubMed1986

The purification of alphavirus virions and subviral particles using ultrafiltration and gel exclusion chromatography.

Conventional methods of virus purification using ultracentrifugation frequently result in distorted particles with low levels of biological activity, and are thus unsuitable for preparing samples for high-resolution techniques such as neutron scattering, X-ray scattering in solution, and X-ray crystallography. Moreover, in the event of an instrument failure, ultracentrifugation can also pose a significant hazard when preparing pathogenic viruses or subunits derived from them. By employing exclusively ultrafiltration and gel exclusion chromatography, a method has been developed to prepare highly purified, intact alphavirus particles retaining high levels of biological activity. These procedures have also been adapted to prepare aggregates of viral envelope protein with a defined immunogenic content.

Alphavirus↗
PubMed1986

The insertion of large pieces of foreign genetic material reduces the stability of bacterial plasmids.

The stability of genetically engineered bacterial plasmids under continuous culture fermentation is of crucial importance in the application of microbiology to many processes of potential industrial importance. In order to determine the effect of inserting large pieces of foreign DNA on the stability of bacterial plasmids we have studied the behavior of pAT153 with DNA inserts of various sizes derived from cytomegalovirus. Foreign DNA up to 2 kb in length had no effect on stability, whereas the insertion of an 8-kb fragment resulted in a transient instability. This instability was overcome by the spontaneous appearance of leu+ cells in the culture. Insertion of a 21-kb DNA fragment resulted in a rapid loss of plasmid, which was not prevented by the appearance of leu+ cells. In all cases copy number analyses indicated that plasmid loss was due to segregational instability, probably because the plasmid placed an unacceptable metabolic load on the cell.

Culture Media↗
PubMed1986

Anaerobic bacterial meningitis.

We report a case of anaerobic bacterial meningitis in which a rapid diagnosis was achieved by direct gas-liquid chromatography of cerebrospinal fluid.

Aged↗
PubMed1985

Gastrointestinal colonization and septicaemia with Pseudomonas aeruginosa due to contaminated thymol mouthwash in immunocompromised patients.

An outbreak of septicaemia with Pseudomonas aeruginosa amongst adult men with haematological malignancy involved eight patients on the same ward during a period of 5 weeks. The strains isolated from blood cultures from seven patients were indistinguishable by conventional typing methods. Thymol mouthwash which had been made up and distributed in communal jugs was found to be contaminated with the epidemic strain and was the likely source for this outbreak. A high rate of gastrointestinal colonization with the epidemic strain was found in the patients receiving the contaminated mouthwash. Only those patients with prolonged severe leucopenia developed septicaemia. Communal medications are an unnecessary hazard, particularly in oncology wards.

Adolescent↗
PubMed1985

Rational design of vaccines against enveloped RNA viruses.

The enveloped RNA viruses are responsible for many important infectious diseases both in the UK and worldwide. The most familiar of these would probably be influenza, measles, mumps, rubella, rabies, dengue and yellow fever. Conventional vaccines against all of the most widespread diseases have been available for several years, although with widely varying degrees of safety and efficacy. Although vaccines against diseases such as measles, rubella, and yellow fever have been fairly successful, all vaccines against diseases caused by this group of viruses still have several drawbacks and are in need of improvement for a variety of reasons. During the past decade our knowledge in several diverse areas of the biological sciences has expanded to the extent that it can now be combined and serious attempts made to design and engineer biological molecules with immunogenic potential. First, significant advances have been made in elucidating the mechanisms operating in the immune defence network and in determining the structure of both immunogenic molecules and the components of the immune system with which they interact. Second, the development of recombinant DNA technology has enabled biological molecules to be synthesized under conditions not restricted by the characteristics of their parent organism. Such molecules can then be altered in such a way as to improve their efficiency and their level of production. It is the purpose of this paper to outline the problems associated with the production of vaccines against enveloped RNA viruses and to discuss how recent advances in knowledge and techniques can help to overcome these problems.

Antigen-Presenting Cells↗
PubMed1985

Antigenic relationships between an isolate of the Western Subtype of tick-borne encephalitis virus and an inactivated vaccine derived from it.

High mutation rates resulting from the error prone replicases of RNA viruses could lead to antigenic alterations in viral products and pose significant problems during the manufacture of vaccines against RNA viruses. The production of a vaccine against tick-borne encephalitis virus has been monitored using both polyclonal sera and a library of monoclonal antibodies. Only a few antigenic changes were detected during the alteration of host cell from mouse brain to avian fibroblasts and upon subsequent expansion of the virus population during several rounds of replication. In addition, when the formalin inactivation process was monitored for antigenic change, virtually none was detected.

Animals↗
PubMed1985

Antibody-dependent enhancement of tick-borne encephalitis virus infectivity.

Fourteen mouse monoclonal antibodies raised against tick-borne encephalitis virus (TBEV) and polyclonal antisera raised against six other flaviviruses, Edge Hill (EHV), Japanese encephalitis (JEV), Langat (LGTV), louping ill (LIV), West Nile (WNV) and yellow fever (YFV), were tested for their ability to enhance the replication of TBEV in cells of the mouse macrophage-like line P388 D1, and for their reactivity in ELISA and haemagglutination inhibition (HI) tests. Irrespective of their specificity for either the 51K or 58K polypeptide present in TBEV-infected cells, 13 of the 14 monoclonal antibodies enhanced the replication of TBEV but not of WNV. The remaining monoclonal antibody, which immunoprecipitated the 58K polypeptide of TBEV enhanced WNV but not TBEV, although it reacted strongly with both viruses in ELISA and HI tests. Only polyclonal antisera against viruses within the tick-borne encephalitis virus complex (TBEV, LGTV and LIV) enhanced TBEV replication, although all the polyclonal antisera reacted with TBEV by ELISA; two (against JEV and WNV) also reacted by HI test and all enhanced the replication of WNV. These findings suggest that with TBEV, enhancement may be TBEV complex-specific rather than flavivirus-specific. Data derived from testing both polyclonal and monoclonal antibodies suggest further that not all antibodies that bind to the envelope glycoprotein of TBEV are able to enhance the replication of TBEV, and that enhancement is epitope-specific.

Animals↗
PubMed1985

Molecular cloning of the phenylalanine ammonia lyase gene from Rhodosporidium toruloides in Escherichia coli K-12.

A genomic library of Rhodosporidium toruloides DNA was constructed in bacteriophage lambda 1059. Recombinant phage containing phenylalanine ammonia lyase (PAL) gene sequences were identified by using 32P-labeled cDNA to partially purified PAL mRNA. The PAL gene was subcloned on an 8.5-kilobase PstI DNA restriction fragment into pUC8 to generate the recombinant plasmid pHG2. A restriction map of the PAL gene, together with its flanking regions, was constructed. Northern hybridization analysis of R. toruloides RNA with a restriction fragment encoding part of the PAL gene indicates that PAL mRNA is 2.5 kilobases in length. A single-stranded DNA hybridization probe was constructed and used to quantitate PAL mRNA levels in R. toruloides grown under different physiological conditions. PAL mRNA levels paralleled changes in functional PAL mRNA and antigen. These data are consistent with control of PAL expression being at the level of transcription.

Ammonia-Lyases↗
PubMed1984

Production and purification of murine monoclonal antibodies: aberrant elution from protein A-Sepharose 4B.

A rapid, one-step method for the efficient purification of murine monoclonal antibodies from tissue culture supernatants is described. This process is based on affinity chromatography on protein A-Sepharose columns. It was found that murine monoclonal antibodies raised against tick-borne encephalitis virus frequently eluted at more than one pH value and these pH values did not always correspond to those of antibodies of the same subclass from polyclonal mouse sera. The two populations of antibody molecule eluting at different pH values showed no variation in molecular weight, isoelectric profiles, specific enzyme-linked immunosorbent assay titer, or antibody subclass.

Animals↗
PubMed1984

Philadelphia chromosomal breakpoints are clustered within a limited region, bcr, on chromosome 22.

We have identified and molecularly cloned 46 kb of human DNA from chromosome 22 using a probe specific for the Philadelphia (Ph') translocation breakpoint domain of one chronic myelocytic leukemia (CML) patient. The DNAs of 19 CML patients were examined for rearrangements on chromosome 22 with probes isolated from this cloned region. In 17 patients, chromosomal breakpoints were found within a limited region of up to 5.8 kb, for which we propose the term "breakpoint cluster region" (bcr). The two patients having no rearrangements within bcr lacked the Ph' chromosome. The highly specific presence of a chromosomal breakpoint within bcr in Ph'-positive CML patients strongly suggests the involvement of bcr in this type of leukemia.

Bacterial Proteins↗
PubMed1984

Antigenic variation among members of the tick-borne encephalitis complex.

The antigenic relationships between seven members of the tick-borne encephalitis complex of flaviviruses (group B arboviruses) were examined by raising a library of 16 monoclonal antibodies against one of them and examining their biological and antigenic properties. These clones reacted with only one of two intracellular, virus-specific polypeptides. One polypeptide [mol. wt. 58 X 10(3) (58K)] is related to the major envelope protein E, but the identity of the other is at present unknown, even though it is a major immunogen in experimental infections and vaccinations. Only those clones specific for the 58K polypeptide contain either neutralizing or haemagglutinin-inhibiting activity, but these epitopes are not identical. In general, epitopes on the 51K polypeptide were more heavily conserved than those on the 58K polypeptide, although both conserved and variant epitopes were found on both polypeptides. One epitope was present on the 51K polypeptide which was conserved on all seven isolates studied and another epitope on the same polypeptide was specific for all the western isolates including one isolate of louping-ill virus. Using the monoclonal antibodies raised in the study, it was shown that louping-ill virus was closely related antigenically to isolates of the Western subtype of tick-borne encephalitis virus.

Animals↗
PubMed1983

Chromosomal localization of the human c-fms oncogene.

A molecular probe was prepared with specificity for the human cellular homologue of transforming sequences represented within the McDonough strain of feline sarcoma virus (v-fms). By analysis of a series of mouse-human somatic cell hybrids containing variable complements of human chromosomes it was possible to assign this human oncogene, designated c-fms, to chromosome 5. Regional localization of c-fms to band q34 on chromosome 5 was accomplished by analysis of Chinese hamster-human cell hybrids containing as their only human components, terminal and interstitial deleted forms of chromosome 5. The localization of c-fms to chromosome 5 (q34) is of interest in view of reports of a specific, apparently interstitial, deletion involving approximately two thirds of the q arm of chromosome 5 in acute myelogenous leukemia cells.

Animals↗
PubMed1983

c-sis is translocated from chromosome 22 to chromosome 9 in chronic myelocytic leukemia.

By analysis of a series of somatic cell hybrids derived by fusion of either mouse or Chinese hamster cells with leukocytes from different chronic myelocytic leukemia (CML) patients or from normal donors, we have localized the human oncogene, c-sis, on the q11 to qter segment of chromosome 22 and demonstrated its translocation from chromosome 22 to chromosome 9 (q34) in CML.

Animals↗