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Biomedical subjects

M W Taylor

Publications and source records attributed to M W Taylor.

At least 91 records · Page 5Linked to original sources
PubMed1989

Transfection with bacterial genes as a marker for cells seeded on vascular flow surfaces.

Autologous seeding of vascular grafts has been in use since 1972; however, the fate of seeded cells has never been determined. While short-term retention has been determined by radioactively labeling cells, long-term studies of seeded cells have not been possible due to the lack of an appropriate marker system. We have developed a long-term marker system for endothelial cells by transfecting the cells with bacterial genes that can be detected by fluorescentally-labeled antibodies to these markers. Two bacterial genes, neo and cat both carried by a pSV2 plasmid construct were used to co-transfect cells. Transfects were selected by growth in the presence of G418. Transfected clones were expanded into monolayers that stained positive for cat by fluorescence, and retained the normal cobblestone morphology and factor VIII staining of endothelial cells. By stably transfecting cells with bacterial genes these cells can now be used to seed vascular grafts and follow the long-term fate of seeded cells.

Animals↗
PubMed1989

Interferon gamma-resistant mutants are defective in the induction of indoleamine 2,3-dioxygenase.

Several mutants of the human cell line ME-180 resistant to the cytotoxic effect of interferon gamma (IFN-gamma) were isolated after mutagenesis with nitrosoguanidine. Two of the mutant lines (ME-IR3b and ME-IR6g) examined had significantly lower induction levels of the L-tryptophan (L-Trp) degradative enzyme indoleamine 2,3-dioxygenase activity in response to IFN-gamma. Moreover, culture medium supplemented with low concentrations of L-Trp reversed the cytotoxic effect of IFN-gamma, whereas higher concentrations of L-Trp in the medium were extremely toxic to both parental and mutant cells. These mutants were still protected against herpes simplex virus infection by IFN-gamma and expressed the HLA-DR alpha gene normally in the presence of this lymphokine. Thus, the mutation in these cells is specific to indoleamine 2,3-dioxygenase and not a global effect of an IFN-gamma-induced gene. This genetic evidence indicates that the major pathway of IFN-gamma cytotoxicity in this cell line is mediated primarily by induction of indoleamine 2,3-dioxygenase and deprivation of L-Trp.

Cell Division↗
PubMed1989

In situ amplification of viral signal in infected samples applied directly to nitrocellulose membranes.

A method for the amplification of viral signal present in infected samples is described. Viral suspensions are spotted on nitrocellulose paper, and immediately afterwards an adequate amount of a permissive cell line is added. The nitrocellulose filter is then incubated overnight, fixed and hybridized to labeled viral probes. The technique is extremely fast, reproducible and inexpensive, and may be readily applicable to the clinical diagnosis of many viral diseases.

Biotechnology↗
PubMed1988

Antiproliferative activity of a hybrid protein between interferon-gamma and tumor necrosis factor-beta.

A hybrid protein between interferon-gamma and tumor necrosis factor-beta was made by ligating the respective genes and expressing the fused genes under the control of the trp promoter in Escherichia coli. The antiproliferative activity of the hybrid protein in vitro was greatly increased compared with either interferon-gamma or tumor necrosis factor-beta alone, and both antiviral activity and cytotoxic effect were retained in the hybrid protein. The hybrid protein may have potential clinical application.

Antiviral Agents↗
PubMed1988

Hyperthermia in humans enhances interferon-gamma synthesis and alters the peripheral lymphocyte population.

Induction of hyperthermia (39 degrees C) in human volunteers by immersion in warm water (41-45 degrees C) rapidly alters the cell populations in the peripheral blood. In addition to granulocytosis, there is an alteration of the normal ratios among T-lymphocyte subsets. Following in vitro mitogen stimulation, lymphocytes from hyperthermic individuals produce as much as 10-fold more interferon-gamma (IFN-gamma) than cells withdrawn at basal core temperatures from the same individuals. A temperature threshold of 39 degrees C for this response suggests potential relevance to fever. No change was noted in the activity of the macrophage population. The possible involvement of interleukin-2 (IL-2) in this enhanced production is discussed. No changes were noted in the circulating levels of IFN-gamma.

Adult↗
PubMed1988

A fast and sensitive method for detecting specific viral RNA in mammalian cells.

A quick and sensitive method to quantitate viral RNA synthesis has been developed. Utilizing glutaraldehyde to fix infected cells onto nitrocellulose paper, viral RNA can be probed directly in situ. Viral message can be detected from as few as 10(4) infected cells. This technique can be used to study viral gene expression and can be adapted to screen the activity of antiviral agents such as interferon.

Animals↗
PubMed1988

Analysis of signals controlling expression of the Chinese hamster ovary aprt gene.

The 5' end of the Chinese hamster ovary aprt gene was sequenced and transcription start sites were determined by both S1 nuclease protection and primer extension assays. Deletion mutants covering the same area were constructed, and adenine phosphoribosyltransferase (APRT) or chloramphenicol acetyltransferase (CAT) activity was measured by transient-expression assays. The aprt gene uses a single cluster of transcription start sites and lacks consensus sequences such as TATA and CCAAT, which are general components of eucaryotic promoters. The 5' deletion mutations of the promoter sequences demonstrated that (i) there is no decrease in either APRT activity or transcription extending to position -89 (relative to the main transcription start site); (ii) an additional 29-base-pair (bp) deletion decreases APRT activity and transcription twofold; and (iii) a deletion past the transcription start sites (P5' delta +27) abolishes both APRT activity and transcription, indicating that a 60-bp fragment immediately upstream of the main transcription start site is involved in basic transcription and a 29-bp fragment just upstream of the 60 bp-fragment stimulates transcription twofold. The 3' deletion mutations showed that a deletion of a 61-bp fragment in the 5' leader and coding sequence abolishes the efficient translation of an aprt-CAT gene transcript. In addition, there are two polyadenylation signals at the genomic 3' end, with the proximal one being sufficient for functional polyadenylation.

Animals↗
PubMed1987

Spontaneous and induced interferon production by peripheral blood leucocytes from control population and patients with herpes genitalis.

The spontaneous and PHA induced levels of interferon were measured in peripheral blood leucocyte cultures of twenty-eight individuals diagnosed as positive for herpes genitalis, and in a group of control subjects. As reported by Cunningham and Merigan [1983] for herpes labialis, leucocytes from individuals with herpes genitalis produced low levels of interferon spontaneously; however, similar results were found for individuals within the control population. No statistically significant difference could be found for PHA induced interferon levels, antigen induced interferon levels, or helper/suppressor cell ratios between the herpes genitalis population and control population. Our results indicate that interferon does not play a major role in the latency or recurrence of herpes genitalis.

Antigens, Viral↗
PubMed1987

Mutants of Saccharomyces cerevisiae deficient in adenine phosphoribosyltransferase.

Spontaneous and ethyl methanesulfate induced mutants of Saccharomyces cerevisiae, with partial and complete deficiency of adenine phosphoribosyltransferase (APRT, EC 2.4.2.7), were isolated by selection for resistance to 8-azaadenine. Matings between totally deficient mutants and tester strain resulted in diploid heterozygotes that were sensitive to azaadenine. Upon sporulation and tetrad analysis, azaadenine resistance (and APRT deficiency) segregated as expected for a single Mendelian gene. Hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) activity in the mutants was similar to that in the wild-type cells. There was no detectable activity of adenine aminohydrolase (EC 3.5.4.2) in the wild-type or mutant cells.

Adenine↗
PubMed1987

In vivo hyperthermia enhances plasma antiviral activity and stimulates peripheral lymphocytes for increased synthesis of interferon-gamma.

The effect of in vivo hyperthermia on plasma interferon (IFN) activity and on the induction of IFN-gamma by phytohemagglutinin (PHA) or staphylococcal enterotoxin B (SEB) in isolated leukocyte cultures was investigated. Adult rhesus monkeys (Macaca mulatta) were placed in a climatic chamber maintained at 45 degrees C until their core body temperatures increased 2 degrees C above control levels. Peripheral blood samples were withdrawn both prior to core temperature elevation and at the time of peak body temperature. Plasma IFN-alpha increased slightly from a control value of 12 U/ml to 16 U/ml at the elevated core temperature. However, this alteration of plasma IFN levels appears to be a complex process that includes the loss of certain circulating IFN-alpha subtypes and the influx of acid-labile (Type II) IFN-alpha. Additionally, a non-IFN antiviral factor present in the plasma was elevated 10-fold at the higher body temperature. When mononuclear cells were isolated and cultured at 37 degrees C in the presence of PHA or SEB, those cells isolated from animals at the peak of body temperature showed a 4- to 16-fold increase in IFN-gamma activity relative to cells isolated from the same animal before the temperature increase. Similar results were obtained with cells isolated when fever was induced by the systemic injection of nonviable Escherichia coli. These results demonstrate that increased body temperature results in a circulating lymphocyte pool which is "primed" for the production of elevated levels of IFN-gamma activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

The effect of in vivo hyperthermia on selected lymphokines in man.

We have previously demonstrated that artificially induced hyperthermia in man enhances the subsequent production of interferon gamma (IFN-gamma) in isolated leukocyte cultures. The mechanism(s) responsible for this response may involve changes in the circulating lymphocyte populations and may also reflect activation processes occurring in vivo due to hormonal influences. In order to determine whether hyperthermia was associated with other immunostimulatory effects, we measured lymphocyte activation, natural killer cell activity, interleukin-2 (IL-2) production and endotoxin-induced tumor necrosis factor (TNF) activity in blood samples obtained from normothermic (37 degrees C) and hyperthermic (39 degrees C) individuals. Lymphocyte transformation was significantly depressed in post-hyperthermic cultures compared to pre-hyperthermic control cultures. Pre-hyperthermic autologous human plasma was less effective than fetal calf serum in promoting DNA synthesis, while post-hyperthermic plasma suppressed mitogen-induced activation. Natural killer (NK) cell activity was increased by the elevation of core body temperature. Interleukin-2 (IL-2) production in phytohemagglutinin-stimulated mononuclear cell cultures was also elevated when cells were isolated from hyperthermic individuals relative to a paired normothermic control sample. Lipopolysaccharide-induced tumor necrosis factor (TNF) synthesis in monocyte cultures was unchanged by elevation of the core body temperature. This study indicates that in vivo hyperthermia can produce an immunostimulatory effect, an immunosuppressive effect, or no effect on different parameters of the immune system.

Fever↗
PubMed1986

Nucleotide sequence and deduced amino acid sequence of Escherichia coli adenine phosphoribosyltransferase and comparison with other analogous enzymes.

The Escherichia coli apt gene has been analyzed and its nucleotide (nt) sequence and the deduced amino acid (aa) sequence compared to those of other phosphoribosyltransferases (PRTs). The apt mRNA has a 102-nt leader sequence which may form alternate secondary structures. The RNA transcript may also form several 3' hairpin structures, which, however, do not appear to act as Rho-independent terminators. All PRTs, including E. coli adenine PRT (APRT), have a strongly conserved 13-aa sequence, as well as other regions of aa sequence or structural similarity. E. coli APRT is remarkably similar to the mouse enzyme.

Adenine Phosphoribosyltransferase↗
PubMed1986

Isolation and characterization of mutants at the APRT locus in the L-5178Y TK+/TK- mouse lymphoma cell line.

2,6-Diaminopurine(DAP)-resistant mutants have been isolated from mouse lymphoma 5178Y TK+/TK- heterozygotes. In the presence of 50 microM DAP, two colony types were isolated. Small colonies contained 50% wild-type adenine phosphoribosyl transferase (APRT) activity (partial mutants), whereas large colonies have undetectable levels of APRT (aprt- mutants). aprt- mutants could be isolated following mutagenesis with ICR-191 or EMS from the partial mutants. Southern blot analysis of EcoRI digested wild-type DNA using a 3.1 kb mouse aprt genomic probe indicated sequence polymorphism at one or both EcoRI sites flanking the allele. Southern blot analysis of one of the partial mutants and one ICR-induced aprt- mutant (single step) indicated that both strains were hemizygous at the APRT locus. Such stable hemizygous strains would be useful in short-term mutagen tests.

2-Aminopurine↗
PubMed1985

The synergistic influence of human interferon-gamma and interferon-alpha on suppression of hematopoietic progenitor cells is additive with the enhanced sensitivity of these cells to inhibition by interferons at low oxygen tension in vitro.

The influences of human interferons--natural gamma (2 X 10(7) NIH reference U/mg), recombinant gamma (approximately 5 X 10(6) U/mg), natural alpha (1.4 X 10(8) international reference U/mg), and natural beta (10(6) international reference U/mg)--were evaluated alone or in combination for their effects in vitro on colony formation by low density human bone marrow granulocyte-macrophage (CFU-GM), erythroid (BFU-E), and multipotential (CFU-GEMM) progenitor cells incubated at 5% CO2 in normal incubator (approximately 20%) O2 tension or low (5%) O2 tension. Alone, these interferons demonstrated the same dose response inhibitory curves, as we reported previously, when cells were grown at 20% O2. Recombinant IFN-gamma gave the same dose response curve as natural IFN-gamma. Natural or recombinant interferon synergized with IFN-alpha to suppress colony formation at concentrations that were approximately 2 log units lower than that required by either interferon alone. Equal concentrations of these interferons were not needed for the synergistic effect and were still apparent when one was present at concentrations of 2 log units less than the other. IFN-gamma synergized to a lesser extent with IFN-beta, but IFN-alpha did not synergize with IFN-beta. Cells grown at 5% O2 were more sensitive to inhibition by 2 log units less IFN-gamma or IFN-alpha, and this effect was additive with the synergistic effects of IFN-gamma and IFN-alpha together. These results may have physiological, pathological, and/or clinical relevance.

Bone Marrow Cells↗