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Biomedical subjects

S Gamble

Publications and source records attributed to S Gamble.

At least 19 recordsLinked to original sources
PubMed1999

The design, construction and calibration of a carefully controlled source for exposure of mammalian cells to extremely low-frequency electromagnetic fields.

Despite some epidemiological evidence for an association between increased risk of cancer and exposure to electromagnetic fields (EMFs), cancer causation by such exposure remains unproven. Furthermore, for reasons such as biological unresponsiveness of the chosen system, poor equipment design and experimental confounders, no reproducible effects on animals or mammalian cells in culture have been demonstrated following exposure to power frequency EMFs at levels normally encountered in residential settings (<10 to 1000 microT). The apparatus described here, designed specifically to perform large, well-controlled cell biology experiments, reduces extraneous variables to the absolute minimum, so that small effects cannot be ascribed to some cause unrelated to the experimental protocol. Our novel apparatus consists of two identical solenoids which, in use, only differ by whether the field-producing current is flowing or not; they do not influence one another in any way. They are supplied with conditioned air from a common tissue culture incubator, are completely screened from environmental a.c. fields with Mumetal shielding and can be operated under normal laboratory conditions. Furthermore, the arrangement is such that the investigator is unaware whether cells have, or have not, been exposed until after the results have been evaluated. We report the design, construction, calibration and potential uses of this source.

Animals↗
PubMed1998

The extravesicular domain of synaptotagmin-1 is released with the latent fibroblast growth factor-1 homodimer in response to heat shock.

The heparin-binding fibroblast growth factor (FGF) prototypes lack a classical signal sequence, yet their presence is required in the extracellular compartment for the activation of cell-surface receptor-dependent signaling. Early studies with FGF-1 demonstrated its presence in bovine brain as a novel high molecular weight complex, and subsequent studies identified a second heparin-binding protein that co-purified with FGF-1. Polypeptide sequence analysis revealed that this heparin-binding protein corresponded to the extravesicular domain of bovine synaptotagmin (Syn)-1, a transmembrane component of synaptic vesicles involved in the regulation of organelle traffic. Since FGF-1 is released in response to heat shock as a mitogenically inactive Cys-30 homodimer, we sought to determine whether this heparin-binding protein was involved in the release of FGF-1. We report that a proteolytic fragment of the extravesicular domain of Syn-1 is associated with FGF-1 in the extracellular compartment of FGF-1-transfected NIH 3T3 cells following temperature stress. By using heparin-Sepharose affinity to discriminate between the monomer and homodimer forms of FGF-1 and resolution by conventional and limited denaturant gel shift immunoblot analysis, it was possible to identify FGF-1 and Syn-1 as potential components of a denaturant- and reducing agent-sensitive extracellular complex. It was also possible to demonstrate that the expression of an antisense-Syn-1 gene represses the release of FGF-1 in response to heat shock. These data indicate that FGF-1 may be able to utilize the cytosolic face of conventional exocytotic vesicles to traffic to the inner surface of the plasma membrane where it may gain access to the extracellular compartment as a complex with Syn-1.

3T3 Cells↗
PubMed1998

Synaptotagmin-1 is required for fibroblast growth factor-1 release.

By using p65 synaptotagmin-1 and fibroblast growth factor (FGF)-1:beta-galactosidase (beta-gal) NIH 3T3 cell co-transfectants, we demonstrate that a proteolytic fragment consisting of the extravesicular domain of synaptotagmin-1 is released into the extracellular compartment in response to temperature stress with similar kinetics and pharmacological properties as FGF-1:beta-gal. Using a deletion mutant that lacks 95 amino acids from the extravesicular domain of synaptotagmin-1, neither synaptotagmin-1 nor FGF-1:beta-gal are able to access the stress-induced release pathway. Furthermore, the p40 extravesicular fragment of synaptotagmin-1 is constitutively released in p40 synaptotagmin-1 NIH 3T3 cell transfectants, and this release is potentiated when the cells are subjected to temperature stress. These data demonstrate that the p40 fragment derived from synaptotagmin-1 is able to utilize the FGF-1 non-classical exocytotic pathway and that the release of FGF-1 is dependent on synaptotagmin-1.

3T3 Cells↗
PubMed1998

Platinum catalysts for the high-yield oxidation of methane to a methanol derivative

Platinum catalysts are reported for the direct, low-temperature, oxidative conversion of methane to a methanol derivative at greater than 70 percent one-pass yield based on methane. The catalysts are platinum complexes derived from the bidiazine ligand family that are stable, active, and selective for the oxidation of a carbon-hydrogen bond of methane to produce methyl esters. Mechanistic studies show that platinum(II) is the most active oxidation state of platinum for reaction with methane, and are consistent with reaction proceeding through carbon-hydrogen bond activation of methane to generate a platinum-methyl intermediate that is oxidized to generate the methyl ester product.

Journal Article↗
PubMed1997

Radiation-induced carcinogenesis: studies using human epithelial cell lines.

It has proved difficult to develop suitable models to study radiation-induced carcinogenesis by using human epithelial cells. However, immortalised human epithelial cell lines have proved useful. Unirradiated cells from the human keratinocyte cell line (HPV-G) and the human embryonic lung cell line (L132) were found to be tumourigenic in T-cell-deficient mice; thus, they are not suitable for transformation studies. Human urothelial cell lines (SV-HUC-1, NT11, BC16) and the human thyroid epithelial cell line (HTori-3) were nontumourigenic. The urothelial cell lines were refractory to radiation-induced carcinogenesis, and only one small tumour was observed in 57 mice that received irradiated cells. Whereas tumours were not produced following irradiation of these urothelial cells, changes in anchorage-independent growth were observed after a single dose of 8 Gy gamma-irradiation but not after 2 or 4 Gy. Irradiation of the human thyroid epithelial cell line (HTori-3) in vitro resulted in tumour formation. Passaging of the cells in vitro before injection did not seem to be critical. Some of the cell lines derived from the primary thyroid tumours exhibited p53 mutations in exons 5, 6, 7, and 8, as detected by single-stranded conformational polymorphism (SSCP) analysis. Thus, the human thyroid epithelial cell line (HTori-3) looks promising as a model for investigating the molecular events in radiation-induced carcinogenesis.

Animals↗
PubMed1997

Incision and drainage of cutaneous abscesses is not associated with bacteremia in afebrile adults.

STUDY OBJECTIVE: To determine the prevalence of bacteremia associated with incision and drainage (I&D) of cutaneous abscesses in afebrile adult emergency department patients. Such information has implications for the ED management of immunocompromised patients, patients with history of endocarditis, and patients with prosthetic appliances such as heart valves and artificial joints. METHODS: We conducted a prospective clinical study in the adult ED of an urban tertiary care teaching hospital. Our subjects were afebrile patients aged 18 to 65 years with localized, nondraining, purulent cutaneous abscesses requiring outpatient surgical management. Before I&D, blood for aerobic and anaerobic blood culture was drawn under sterile conditions. The wound was opened and samples for aerobic wound culture were obtained. Two and 10 minutes after I&D, blood was again drawn, from separate venipunctures. All patients were discharged home with ED follow-up scheduled 48 hours later. RESULTS: From the 50 patients who completed the study, 150 blood samples (50 before and 100 after I&D) and 50 wound samples were obtained. No blood culture was positive, but 30 wound cultures (64%) were positive; the most commonly isolated organism was Staphylococcus aureus. CONCLUSION: I&D of localized cutaneous abscesses in afebrile adults is unlikely to result in transient bacteremia. Larger studies are needed to determine whether routine antibiotic prophylaxis is necessary for afebrile patients undergoing I&D.

Abscess↗
PubMed1995

The cysteine residue responsible for the release of fibroblast growth factor-1 residues in a domain independent of the domain for phosphatidylserine binding.

Fibroblast growth factor (FGF)-1 lacks a classical signal sequence to direct its secretion yet utilizes high affinity cell surface receptors to signal its heparin-dependent angiogenic and neurotrophic activities. We have previously reported that FGF-1 is released in response to temperature stress as a latent homodimer through a pathway that is potentiated by the Golgi inhibitor, brefeldin A (Jackson, A., Tarantini, F., Gamble, S., Friedman, S., and Maciag, T. (1995) J. Biol. Chem. 270, 33-36). In an attempt to further characterize this unconventional secretion mechanism, we sought to define the Cys residue(s) critical for FGF-1 dimer formation and release and to determine whether FGF-1 can associate with known phospholipid components of organelle or plasma membranes, which may be disturbed by brefeldin A. Utilizing FGF-1 Cys mutants, we were able to demonstrate that residue Cys30 is critical for FGF-1 release in response to heat shock. In addition, using solid phase phospholipid binding assays we demonstrate that FGF-1 is able to specifically associate with phosphatidylserine (PS). Heparin inhibits the association between FGF-1 and PS, and synthetic peptide competition assays suggest that the PS-binding domain of FGF-1 lies between residues 114 and 137. These observations indicate that FGF-1 may be able to associate with the PS component of organelle and/or plasma membranes and that the domains responsible for FGF-1 homodimer formation and PS binding are structurally distinct.

3T3 Cells↗
PubMed1995

Identification of the domain within fibroblast growth factor-1 responsible for heparin-dependence.

While the prototype members of the fibroblast growth factor (FGF) family, FGF-1 and FGF-2 are structurally related, the structural differences between these polypeptides predict that they will ultimately exhibit different biological roles. Indeed, a significant difference between these proteins is the dependence of FGF-1 on heparin for the generation of maximal mitogenic activity. In order to gain structural insight into the issue of FGF-1 heparin-dependence, a synthetic gene encoding FGF-2 was constructed with oligonucleotides in a four-cassette format similar to a synthetic gene previously constructed for FGF-1 (Forough et al. 1992, Biochem. Biophys. Acta 1090 293-298). This strategy permitted the molecular shuffling of corresponding cassette(s) between FGF-1 and FGF-2 to yield FGF-1:FGF-2 chimeras. Three amino acid changes (Lys86-->Glu, Tyr120-->His, and Thr121-->Ala) were introduced into the synthetic FGF-2 gene by the cassette format to generate convenient FGF-1 restriction sites, but these alterations did not significantly affect the mitogenic activity or the heparin-binding affinity of the recombinant FGF-2 protein when compared with native FGF-2. Among the various FGF-1:FGF-2 chimeric constructs, one designated FGF-C(1(1/2)1 1), which represents FGF-1 containing FGF-2 amino acid residues 65 to 81, displayed FGF-1-like heparin-binding affinity but it did not require the addition of exogenous heparin to manifest its mitogenic activity. These data suggest that the sequence within residues 65 and 81 from FGF-2 significantly contributes to the heparin-dependent character of FGF-1.

Amino Acid Sequence↗
PubMed1995

The release of fibroblast growth factor-1 from NIH 3T3 cells in response to temperature involves the function of cysteine residues.

Fibroblast growth factor (FGF)-1 is released from NIH 3T3 cells in response to heat shock as a biologically inactive protein that is unable to bind heparin and requires activation by (NH4)2SO4 to generate a biologically active extracellular heparin-binding growth factor (Jackson, A., Friedman, S., Zhan, X., Engleka, K. A., Forough, R., and Maciag, T. (1992) Proc. Natl. Acad. Sci. USA 89, 10691-10695). To further study the mechanism of FGF-1 release in response to heat shock (42 degrees C), we examined the kinetics of FGF-1 release from FGF-1-transfected NIH 3T3 cells and observed that the cells require at least 1 h of exposure to heat shock conditions for the release of FGF-1. Interestingly, agents that interfere with the function of the endoplasmic reticulum-Golgi apparatus, exocytosis, and the multidrug resistance pathway (brefelden A, methylamine, and verapamil, respectively) do not inhibit the release of FGF-1 in response to temperature; rather, they exaggerate the release of FGF-1. Because immunoblot analysis of FGF-1 in the conditioned medium of heat-shocked NIH 3T3 cells revealed the presence of a minor band with an apparent molecular weight of a FGF-1 homodimer and because we have previously shown that FGF-1, but not FGF-2, is able to form a homodimer in response to chemical oxidation by CuCl2 (Engleka, K. A., and Maciag, T. (1992) J. Biol. Chem. 267, 11307-11315), we examined whether reducing agents would substitute for (NH4)2SO4 and activate extracellular FGF-1. Indeed, dithiothreitol and reduced glutathione are able to individually generate a FGF-1 monomer as a heparin-binding protein from the conditioned medium of heat-shocked NIH 3T3 cell transfectants. To confirm that cysteine residues are involved in the release of FGF-1 in response to temperature, we used mutagenesis to prepare a human FGF-1 Cys-free mutant in which Cys30, Cys97, and Cys131 were converted to serine. Analysis of the release of the FGF-1 Cys-free mutant in NIH 3T3 cells transfected with the FGF-1 Cys-free mutant demonstrated that the FGF-1 Cys-free mutant is not released into the conditioned medium in response to temperature. Interestingly, exposure of the NIH 3T3 cell FGF-1 Cys-free transfectants to brefelden A followed by heat shock also demonstrated the absence of the extracellular FGF-1 Cys-free mutant.(ABSTRACT TRUNCATED AT 400 WORDS)

3T3 Cells↗
PubMed1993

Peptide-priming of cytolytic T cell immunity in vivo using beta 2-microglobulin as an adjuvant.

CTL recognize oligopeptides bound to MHC class I molecules. Immunization of animals with antigenic peptides has often failed to stimulate CTL responses. We confirm that immunizations with several peptides, including natural and optimally active antigenic sequences, do not prime cytotoxic immunity in mice. However, immunization with peptides together with human beta 2-microglobulin primes Ag-specific CTL. Priming is observed when animals receive injections either i.v. with ex vivo peptide/beta 2-microglobulin-pulsed cells or s.c. with an admixture of peptide and beta 2-microglobulin. beta 2-Microglobulin promotes the priming of CTL immunity if it is added with peptide, but not if it is added after cells are exposed to peptide. Synthetic peptides and mixtures of peptides from enzymatically cleaved Ag are immunogenic. When a tryptic digest of OVA or the synthetic peptide (OVA258-276) are used as immunogens, the CTL that respond recognize the endogenously processed epitope presented by an OVA-transfected target cell. The peptide + beta 2-microglobulin-primed CTL are CD4-CD8+ and are class I MHC restricted. Using the immunization protocol with beta 2-microglobulin, we have primed CTL responses with peptides from OVA, Sendai virus, and vesicular stomatitis virus. These results may explain previous failures to prime CTL with peptides in vivo and provide a novel approach for developing peptide-based vaccines for viral diseases.

Adjuvants, Immunologic↗
PubMed1993

Characterization of antigen-presenting cells that present exogenous antigens in association with class I MHC molecules.

Exogenous Ag in the extracellular fluids do not gain access to the class I Ag-presenting pathway in most cells. However, there is an APC resident in spleen that can process and present exogenous Ag in association with class I molecules. We characterize the phenotype of this cell. This APC is of low buoyant density, is adherent to Sepharose and glass, and expresses both class II molecules and FcR. This phenotype identifies this APC as a macrophage. Resident, peptone- and thioglycolate-induced peritoneal macrophages also display this Ag-presenting activity. Analysis with CTL clones suggest that this Ag-presenting pathway may be active in only a subset of macrophages. A similar Ag-presenting activity is also present in dendritic cell-enriched populations from spleen although we cannot rule out the possible involvement of contaminating macrophages. In contrast, B and T cells that are resident in spleen and LPS blasts are unable to present exogenous Ag in association with class I molecules. The presentation of exogenous OVA with class I molecules is not inhibited by the inhibitors of thiol proteases, leupeptin, and antipain. The presence of gelonin, a ribosomal inactivating protein, in the extracellular fluids inhibits the ability of these APC to present exogenous OVA. Under identical conditions, gelonin does not inhibit Con A-stimulated T cell proliferation, or LPS-stimulated B cell proliferation and Ag presentation. These results are discussed in relation to the potential pathways through which an Ag in the extracellular fluids is presented with MHC class I molecules.

Animals↗
PubMed1992

Inhibition of class I and class II MHC-restricted antigen presentation by cytotoxic T lymphocytes specific for an exogenous antigen.

Ag in the extracellular fluids can be internalized, processed, and presented in association with class I MHC molecules on specialized APC in normal spleen. We examine the fate of these APC after they present Ag to a CTL. When splenocytes present exogenous OVA to CTL, their ability to subsequently present native Ag in association with both class I and class II molecules is inhibited. CTL do not inhibit the ability of splenocytes to present processing independent peptides with class I or class II molecules. Inhibition of Ag presentation is only observed in the presence of the specific Ag recognized by the CTL. This inhibition is MHC-restricted. In the presence of specific Ag, CTL inhibit the ability of APC to present unrelated Ag. However, bystander APC are not affected by activated CTL. Taken together these results indicate that when APC present exogenous Ag to CTL, they are inhibited or killed. The CTL that mediates this activity has a conventional CD4-CD8+ phenotype and utilizes a TCR-alpha beta. The potential significance of these findings and their possible relationship to phenomena associated with Ts cells are discussed.

Animals↗
PubMed1992

Chemical cross-linking of class I molecules on cells creates receptive peptide binding sites.

Class I heterodimers on the surface of cells are generally unreceptive to binding peptides in the absence of exogenous beta 2-microglobulin. Paraformaldehyde covalently cross-links beta 2-microglobulin to class I heavy chains in situ and stabilizes empty class I heterodimers. Functionally, this cross-linking creates receptive class I peptide binding sites by acting on beta 2-microglobulin-associated molecules. The presentation of preexisting peptide-class I complexes is also enhanced. These findings support a model whereby a structural alteration, the dissociation of beta 2-microglobulin, limits the existence of receptive class I molecules on normal cells and may control the half-life of active class I molecules.

Animals↗
PubMed1991

Dissociation of beta 2-microglobulin leads to the accumulation of a substantial pool of inactive class I MHC heavy chains on the cell surface.

A large pool of free class I heavy chains is detected in situ on the plasma membrane of living cells. These chains are present on cells of different MHC genotypes and appear to exist under physiological conditions in vivo. These molecules arise from the dissociation of previously assembled class I heterodimers at the cell surface. The ratio of intact to dissociated heterodimers is strongly affected by the occupancy of the peptide-binding site of the class I molecule. Upon dissociation of the heterodimer, the class I molecule is functionally inactive. These findings may help to explain why class I molecules on the cell surface are unreceptive to binding peptides yet readily associate with peptides in the presence of exogenous beta 2-microglobulin. These results have implications for understanding the distinct functions of class I versus class II molecules and how the immunological identity of cells is preserved.

Animals↗
PubMed1991

Reassociation with beta 2-microglobulin is necessary for Db class I major histocompatibility complex binding of an exogenous influenza peptide.

A synthetic peptide corresponding to residues 365-380 of the influenza nucleoprotein (NP365-380) has been previously shown to associate with class I major histocompatibility complex-encoded molecules and to stimulate cytotoxic T lymphocytes [Townsend, A. R. M., Rothbard, J., Gotch, F. M., Bahadur, G., Wraith, D. & McMichael, A. J. (1986) Cell 44, 959-968]. We find that intact Db class I heterodimers on the cell surface are unreceptive to binding this antigen. However, NP365-380 readily associates with Db molecules on the plasma membrane in the presence of exogenous beta 2-microglobulin. In addition, there is a second pathway through which this peptide associates with class I molecules that requires energy and de novo protein synthesis. These findings have implications for maintaining the immunological identity of cells and for the use of peptides as vaccines for priming cytolytic T-cell immunity.

Animals↗