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J G Lewis

Publications and source records attributed to J G Lewis.

At least 37 records · Page 2Linked to original sources
PubMed1996

Production and characterization of monoclonal antibodies to dehydroepiandrosterone sulfate: application to direct enzyme-linked immunosorbent assays of dehydroepiandrosterone sulfate and androsterone/epiandrosterone sulfates in plasma.

Mice were immunized with 5-androstene-3 beta-ol-7,17-dione-7-CMO:bovine serum albumin (DHEA-7-O-CMO-BSA) or 5-androstene-3 beta-ol-17-one hemisuccinate-bovine serum albumin (DHEA-3HS-BSA) conjugates and monoclonal antibodies were produced, characterized, and selected for maximum DHEAS binding. Of these hybridomas, four clones from DHEA-3HS-BSA-immunized mice had acceptable criteria for the development of a competitive enzyme-linked immunosorbent assay (ELISA) for DHEAS in plasma. One hybridoma supernatant from DHEA-7-O-CMO-BSA-immunized mice showed 360% cross-reactivity to both androsterone sulfate and epiandrosterone sulfate. This allows the possibility of the direct determination of androsterone sulfate and epiandrosterone sulfate in plasma after correction for the DHEAS contribution. Both ELISAs employ a DHEA-3HS-thyroglobulin conjugate adsorbed to the wells of a standard 96-well microtiter plate. DHEAS in the standards or diluted plasma sample competes with immobilized DHEA-3HS-thyroglobulin for antibody-binding sites. Antibody is detected with anti-mouse-lg peroxidase by further washing, adding o-phenylenediamine substrate, and reading the absorbance at 492 nm. The ELISAs are simple, reproducible, and reliable and, to our knowledge, they are the first tests employing monoclonal antibodies to DHEAS.

Adult↗
PubMed1995

Site and mechanism of antisense inhibition by C-5 propyne oligonucleotides.

Antisense gene inhibition occurs when an oligonucleotide (ON) has sufficient binding affinity such that it hybridizes its reverse complementary target RNA and prevents translation either by causing inactivation of the RNA (possibly by RNase H) or by interfering with a cellular process such as stalling a ribosome. The mechanisms underlying these processes were explored. Cellular antisense inhibition was evaluated in a microinjection assay using ON modifications which precluded or allowed in vitro RNase H cleavage of ON/RNA hybrids. RNase H-independent inhibition of protein synthesis could be achieved by targeting either the 5'-untranslated region or the 5'-splice junction of SV40 large T antigen using 2'-O-allyl phosphodiester ONs which contained C-5 propynylpyrimidines (C-5 propyne). Inhibition at both sites was 20-fold less active than inhibition using RNase H-competent C-5 propyne 2'-deoxy phosphorothioate ONs. In vitro analysis of association and dissociation of the two classes of ONs with complementary RNA showed that the C-5 propyne 2'-O-allyl phosphodiester ON bound to RNA as well as the C-5 propyne 2'-deoxy phosphorothioate ON. In vitro translation assays suggested that the two classes of ONs should yield equivalent antisense effects in the absence of RNase H. Next, ON/T antigen RNA hybrids were injected into the nuclei and cytoplasm of cells. Injection of C-5 propyne 2'-O-allyl phosphodiester ON/RNA hybrids resulted in expression of T antigen, implying that the ONs dissociated from the RNA in cells which likely accounted for their low potency. In contrast, when C-5 propyne 2'-deoxy phosphorothioate ON/T antigen RNA complexes were injected into the nucleus, the duplexes were stable enough to completely block T antigen translation, presumably by RNA inactivation. Thus, a dramatic finding is that C-5 propyne 2'-deoxy phosphorothioate ONs, once hybridized to RNA, are completely effective at preventing mRNA translation. The implication is that further increases in complex stability coupled with effective RNase H cleavage will not result in enhanced potency. We predict that the development of more effective ONs will only come from modifications which increase the rate of ON/RNA complex formation within the nucleus.

Alkynes↗
PubMed1995

Effects of oxidized LDL on mononuclear phagocytes: inhibition of induction of four inflammatory cytokine gene RNAs, release of NO, and cytolysis of tumor cells.

A critical step in development of atherosclerosis is the interaction of oxidized low-density lipoprotein (LDL) with mononuclear phagocytes. Oxidized LDL, as well as acetyl-LDL, is rapidly taken up into macrophages via a family of scavenger receptors. We report that macrophages treated with oxidized LDL have markedly lower levels of mRNA specific for the genes MCP-1, TNF-alpha, IL-1 alpha, and KC as measured by Northern blot analyses of lipopolysaccharide (LPS)-stimulated macrophages. By contrast, acetyl-LDL does not inhibit these genes at the doses at which oxidized-LDL is effective. Similar effects are observed whether the LDL is oxidized in the presence of Cu2+ or of Fe2+. Such inhibition also occurs when maleylated bovine serum albumin (BSA), which also clears by one or more scavenger receptors on macrophages, is used as the stimulant. Fe2+ or Cu2+ oxidized LDL inhibits release of nitric oxide when triggered by LPS and direct cytolysis of tumor cells when triggered by maleylated BSA or LPS. Taken together, the data presented indicate that oxidized LDL inhibits induction of several important gene RNAs as well as functional markers that characterize the development of inflammatory and fully activated macrophages.

Animals↗
PubMed1995

Selective sensitivity of macrophages to cytotoxicity by inhibitors of macromolecular synthesis: induction of apoptosis.

Initial studies designed to measure the effect of inhibiting RNA synthesis by dactinomycin on macrophage functions revealed that the cells were uniformly killed at concentrations that have been routinely used to inhibit RNA synthesis in other cell types. We, thus, determined the dose curve for the cytotoxicity of dactinomycin for macrophages and two other cell types, L929 cells and splenic lymphocytes. Macrophages were extremely sensitive to the cytotoxicity of dactinomycin compared to the other cell types. Submicromolar concentrations that induced 100% cytotoxicity in macrophages caused little death in L929 cells or lymphocytes. Concentrations of dactinomycin that inhibited RNA synthesis by 40% in macrophages induced almost complete cell death but inhibition of over 80% of RNA synthesis in L929 cells or lymphocytes induced no measurable cytotoxicity. Macrophages did take up more dactinomycin than other cells but the amount was not sufficient to account for the large differences in cytotoxicity. We next tested the effects of doxorubicin and cycloheximide and found that macrophages were also extremely sensitive to killing by these compounds, and there was a very close association between the amount of inhibition of protein synthesis and the amount of toxicity. The morphology of macrophages exposed to these agents was consistent with death by apoptosis. This was further supported by assays measuring membrane integrity and DNA fragmentation. These data demonstrate that inhibition of macromolecular synthesis in macrophages, by different mechanisms, causes macrophages to undergo apoptosis. They further suggest that, in contrast to other cell types that require protein synthesis for apoptosis, macrophages require the synthesis of certain proteins to avoid apoptosis.

Animals↗
PubMed1995

Induction of heat, freezing and salt tolerance by heat and salt shock in Saccharomyces cerevisiae.

Stress tolerance of Saccharomyces cerevisiae was examined after exposure to heat and salt shock in the presence or absence of the protein synthesis inhibitor cycloheximide. Cells heat-shocked (37 degrees C for 45 min) in the absence of cycloheximide demonstrated increased tolerance of heat, freezing and salt stress. For cells heat-shocked in the presence of cycloheximide, heat and salt tolerance could still be induced, although at lower levels, while induction of freezing tolerance was completely inhibited. These results indicated that while heat shock proteins (hsps) may contribute to induced heat and salt tolerance they are not essential, although induction of freezing tolerance appears to require protein synthesis. Exposure of cells to salt shock (300 mM NaCl for 45 min) induced stress protein synthesis and the accumulation of glycerol, responses analogous to induction of hsp synthesis and trehalose accumulation in cells exposed to heat shock. Cells salt-shocked in the absence of cycloheximide showed a similar pattern of induced stress tolerance as with heat, with increased tolerance of heat, salt and freezing. Cells salt-shocked in the presence of cycloheximide continued to show induced heat and salt tolerance, but freezing tolerance could not be induced. These results lend support to the hypothesis that hsp synthesis is not essential for induced tolerance of some forms of stress and that accumulated solutes such as trehalose or glycerol may contribute to induced stress tolerance.

Cycloheximide↗
PubMed1994

Cryoprotection of yeast by alcohols during rapid freezing.

We have investigated the cryoprotective effect of alcohols on Saccharomyces cerevisiae and other yeast under various freezing conditions. For S. cerevisiae, at a cooling rate of 3 degrees C min-1 methanol and ethanol acted as cryosensitizers. However, at a cooling rate of 200 degrees C min-1, both methanol and ethanol proved superior to all other cryoprotectants tested, including glycerol, dimethyl sulfoxide, lactose, trehalose, polyethylene glycol, and polyvinyl pyrrolidone. Propan-2-ol also demonstrated a small but measurable effect although propan-1-ol and butan-1-ol demonstrated no cryoprotective effect. A minimum cooling rate of 25 degrees C min-1 to elicit the cryoprotective effect of ethanol was necessary; below this rate it acted as a cryosensitizer. At cooling rates up to 650 degrees C min-1 substantial cryoprotective effect was still evident. Although the effect of ethanol was variable for other yeast genera tested, ethanol acted positively for all strains of S. cerevisiae. We hypothesize that the cryoprotective effect of alcohols during rapid cooling is a result of their ability to induce increased membrane permeability, allowing rapid water equilibration during extracellular freezing and avoidance of intracellular ice crystal formation.

Alcohols↗
PubMed1994

Effects of benzene metabolites on receptor-mediated phagocytosis and cytoskeletal integrity in mouse peritoneal macrophages.

Exposure to benzene can induce a number of hematotoxicities and decrease host resistance to microorganisms and tumors. Several studies have shown that metabolism of benzene to reactive intermediates is required for myelotoxicity. Since receptor-mediated phagocytosis by macrophages is an important host defense, we have examined the effects of benzene metabolites on receptor-mediated phagocytosis in cultured murine peritoneal macrophages. 1,4-Benzoquinone (BQ) was the most potent of the metabolites examined. Ten-minute exposures to a 12.5 microM concentration inhibited Fc and complement receptor-mediated phagocytosis by > or = 90%. Macrophage viability was largely unaffected by BQ treatment. Exposure to 50 and 100 microM 1,2,4-benzenetriol (BT) inhibited Fc receptor-mediated phagocytosis by 70 and 95%, respectively. Hydroquinone (HQ) elicited a major decrease (50%) only at 100 microM. The comparative inhibitory potencies of BT and HQ correlate with previously published data on their relative facility for autooxidation to quinones at physiological pH. Catechol had no effect at the concentrations employed. Macrophages treated with BQ and BT failed to recover their Fc receptor-mediated phagocytic capacity when incubated overnight in the absence of the xenobiotics. Only small differences in the inhibition of Fc receptor-mediated phagocytosis were observed between macrophages exposed to BQ at 4 versus 37 degrees C. BQ also had little effect on the Fc receptor binding of target cells. Fluorescent digital imaging microscopy demonstrated that BQ treatment markedly decreased the filamentous actin content of macrophages. However, BQ bound in low amounts to purified actin and did not affect its assembly. Our findings suggest that a mechanism for inhibition of Fc receptor-mediated phagocytosis by BQ is disruption of filamentous actin via an effect(s) other than the direct alkylation of actin by BQ.

Actins↗
PubMed1994

Re-examining steroid hormone metabolites as ovulation markers using monoclonal antibodies.

Plasma progesterone and plasma estradiol levels are commonly used to monitor ovulation in women although for the adequate documentation of ovulation the expense and discomfort of multiple venipuncture sampling may be required. Accurate and definitive information on ovulation in women can be obtained by the simple measurement of metabolites of progesterone and estradiol in early morning urine samples. These analyses have been made possible by the use of our own highly specific monoclonal antibodies to both pregnanediol-3-glucuronide (P-3-G) and estrone-3-glucuronide (E-3-G) and the development of simple, direct, automated enzyme-linked immunosorbent assays. Sequential sampling and the generation of ovulation profiles allows detection of ovulation and identification of the infertile/fertile phases of the cycle for either planned pregnancies or natural family planning. Aberrations in ovulation are easily detected as is documentation of the transition to menopause. The use of sequential, spaced, early morning urine samples for P-3-G and E-3-G allows accurate assessment of ovulatory function rather than relying on the usual single plasma sampling. The data presented also show that the direct determination of plasma pregnanediol-3-glucuronide in plasma is as informative as plasma progesterone measurement.

Adult↗
PubMed1994

Molecular evolution and epidemiology of dengue-3 viruses.

The nucleic acid sequences of the pre-membrane/membrane and envelope protein genes of 23 geographically and temporally distinct dengue (DEN)-3 viruses were determined. This was accomplished by reverse transcriptase-PCR amplification of the structural genes followed by automated DNA sequence analysis. Comparison of nucleic acid sequences revealed that similarity among the viruses was greater than 90%. The similarity among deduced amino acids was between 95% and 100%, and in many cases identical amino acid substitutions occurred among viruses from similar geographical regions. Alignment of nucleic acid sequences followed by parsimony analysis allowed the generation of phylogenetic trees, demonstrating that geographically independent evolution of DEN-3 viruses had occurred. The DEN-3 viruses were separated into four genetically distinct subtypes. Subtype I consists of viruses from Indonesia, Malaysia, the Philippines and the South Pacific islands; subtype II consists of viruses from Thailand; subtype III consists of viruses from Sri Lanka, India, Africa and Samoa; subtype IV consists of viruses from Puerto Rico and the 1965 Tahiti virus. Phylogenetic analysis has also contributed to our understanding of the molecular epidemiology and worldwide distribution of DEN-3 viruses.

Amino Acid Sequence↗
PubMed1994

Glucocorticoid responsive ACTH secreting bronchial carcinoid tumours contain high concentrations of glucocorticoid receptors.

Cushing's syndrome due to a bronchial ACTH secreting carcinoid tumour may be difficult to distinguish from a pituitary microadenoma (corticotrophinoma) causing Cushing's disease, since in both disorders ACTH secretion may be responsive to glucocorticoids. Why some bronchial carcinoid tumours are responsive is unknown but it could be because of co-secretion of corticotrophin releasing factor (CRF) and/or expression of glucocorticoid receptors. We report two patients with glucocorticoid responsive ACTH secreting bronchial carcinoid tumours, neither of whom produced or responded to CRF. Significant glucocorticoid receptor binding capacity (92 and 102 pmol/g protein), compared with control lung tissue, was found in extracts from both tumours. These findings suggest that corticotrophinoma-like responses to glucocorticoids observed in some ACTH secreting bronchial carcinoids result from expression of glucocorticoid receptors and are not necessarily related to the production of CRF.

ACTH Syndrome, Ectopic↗
PubMed1994

Ouabain is not detectable in human plasma.

An enzyme-linked immunosorbent assay is described for the measurement of ouabain in human plasma. This assay is specific for ouabain, strophanthidin, and ouabagenin, with other steroids, including digoxin and vasopressor hormones, exhibiting negligible cross-reactivity. Assay sensitivity was 0.06 nmol/L if 1 mL plasma was extracted and less than 0.005 nmol/L when 20 mL plasma was analyzed. Extracted plasma samples showed ouabainlike immunoreactivity that diluted in parallel with the ouabain standard curve. Repeated extraction and assay of single plasma samples, however, did not produce consistent results in the assay. Increased specificity was obtained by high-performance liquid chromatography of sample extracts before assay. When high-performance liquid chromatographic profiles of plasma spiked with ouabain standard or following bolus intravenous injections of ouabain into normal human volunteers were compared with profiles of unspiked plasma, there was no support for the immunoreactive material in the latter samples being ouabain. We propose that if ouabain is present in the human circulation, its concentration is less than 0.005 nmol/L.

Animals↗
PubMed1993

Antisense gene inhibition by oligonucleotides containing C-5 propyne pyrimidines.

Phosphorothioate oligodeoxynucleotides containing the C-5 propyne analogs of uridine and cytidine bind RNA with high affinity and are potent antisense inhibitors of gene expression. In a cellular assay, gene-specific antisense inhibition occurred at nanomolar concentrations of oligonucleotide, was dose-dependent and exquisitely sensitive to sequence mismatches, and was correlated with the melting temperature and length of oligonucleotide. Activity was independent of RNA target site and cell type but was detectable only when the oligonucleotides were microinjected or delivered with cell-permeabilizing agents. These oligonucleotides may have important applications in therapy and in studies of gene function.

Alkynes↗
PubMed1993

Selection and partial characterization of dengue 2 virus mutants that induce fusion at elevated pH.

Two types of dengue (DEN) 2 virus mutants were selected either by repeated exposure to acidic pH (acid mutant, AM), or by the addition of ammonium chloride to Aedes albopictus C6/36 cells prior to and during viral infection (fusion mutant, FM). Both mutants grew more slowly than the parent strain and induced smaller plaques in Vero cells. The 50% fusion from within index for both mutants occurred at least 0.65 pH units higher than with the wild-type DEN virus. A single amino acid substitution (Asn-153 to Asp) was found in the envelope (E)-glycoprotein of the AM virus. Three amino acid substitutions were detected on the E-glycoprotein of the FM virus: Ile-6 to Met, Asn-134 to Ser, and Asn-153 to Tyr. No mutations were found in the precursor to the membrane protein, prM. The DEN virus E-glycoprotein has two potential glycosylation sites: Asn-67 and Asn-153. The loss of the potential glycosylation site at Asn-153 or the change in the chemical characteristics resultant from the amino acid substitutions in both mutants implicates these regions of the E-glycoprotein in virus-mediated membrane fusion.

Acids↗
PubMed1993

The need for consistent nomenclature and assessment of growth phases in diauxic cultures of Saccharomyces cerevisiae.

The need for consistent nomenclature and accurate assessment of late growth phases in diauxic yeast cultures is highlighted by the substantial variation of stress tolerance in Saccharomyces cerevisiae after the exhaustion of the initial fermentable carbon source. At present, a wide variety of assessment methods and confused terminology exists in the literature, leading to difficulties in the interpretation and comparison of published results. A method based on the depletion of ethanol accumulated during the respiro-fermentative growth phase is suggested as suitable for assessing subsequent growth phases and reporting results. Consistent application of nomenclature for growth phases is recommended to assist the interpretation of published experimental results. It is suggested that the phases of growth in diauxic batch culture should be referred to using the terms (1) initial lag phase, (2) respiro-fermentative phase, (3) diauxic lag phase, (4) respiratory phase, (5) stationary phase, and (6) death phase.

Fermentation↗
PubMed1993

Role of growth phase and ethanol in freeze-thaw stress resistance of Saccharomyces cerevisiae.

The freeze-thaw tolerance of Saccharomyces cerevisiae was examined throughout growth in aerobic batch culture. Minimum tolerance to rapid freezing (immersion in liquid nitrogen; cooling rate, approximately 200 degrees C min-1) was associated with respirofermentative (exponential) growth on glucose. However, maximum tolerance occurred not during the stationary phase but during active respiratory growth on ethanol accumulated during respirofermentative growth on glucose. The peak in tolerance occurred several hours after entry into the respiratory growth phase and did not correspond to a transient accumulation of trehalose which occurred at the point of glucose exhaustion. Substitution of ethanol with other carbon sources which permit high levels of respiration (acetate and galactose) also induced high freeze-thaw tolerance, and the peak did not occur in cells shifted directly from fermentative growth to starvation conditions or in two respiratorily incompetent mutants. These results imply a direct link with respiration, rather than exhaustion of glucose. The role of ethanol as a cryoprotectant per se was also investigated, and under conditions of rapid freezing (cooling rate, approximately 200 degrees C min-1), ethanol demonstrated a significant cryoprotective effect. Under the same freezing conditions, glycerol had little effect at high concentrations and acted as a cryosensitizer at low concentrations. Conversely, under slow-freezing conditions (step freezing at -20, -70, and then -196 degrees C; initial cooling rate, approximately 3 degrees C min-1), glycerol acted as a cryoprotectant while ethanol lost this ability. Ethanol may thus have two effects on the cryotolerance of baker's yeast, as a respirable carbon source and as a cryoprotectant under rapid-freezing conditions.

Cryoprotective Agents↗
PubMed1992

Clinical, biochemical, and neuropsychiatric evaluation of a patient with a contiguous gene syndrome due to a microdeletion Xp11.3 including the Norrie disease locus and monoamine oxidase (MAOA and MAOB) genes.

Norrie disease is a rare X-linked recessive disorder characterized by blindness from infancy. The gene for Norrie disease has been localized to Xp11.3. More recently, the genes for monoamine oxidase (MAOA, MAOB) have been mapped to the same region. This study evaluates the clinical, biochemical, and neuropsychiatric data in an affected male and 2 obligate heterozygote females from a single family with a submicroscopic deletion involving Norrie disease and MAO genes. The propositus was a profoundly retarded, blind male; he also had neurologic abnormalities including myoclonus and stereotopy-habit disorder. Both obligate carrier females had a normal IQ. The propositus' mother met diagnostic criteria for "chronic hypomania and schizotypal features." The propositus' MAO activity was undetectable and the female heterozygotes had reduced levels comparable to patients receiving MAO inhibiting antidepressants. MAO substrate and metabolite abnormalities were found in the propositus' plasma and CSF. This study indicates that subtle biochemical and possibly neuropsychiatric abnormalities may be detected in some heterozygotes with the microdeletion in Xp11.3 due to loss of the gene product for the MAO genes; this deletion can also explain some of the complex phenotype of this contiguous gene syndrome in the propositus.

Adolescent↗
PubMed1992

Production of a monoclonal antibody to cortisol: application to a direct enzyme-linked immunosorbent assay of plasma.

Cortisol mouse monoclonal antibodies were produced and characterized. Of the four clones studied, supernatant from one clone (A2), compared with other cortisol monoclonal antibodies, showed minimal cross-reactivity to other C21 steroids and was suitable for the direct determination of cortisol in plasma by enzyme-linked immunosorbent assay using a standard 96-well microtiter plate. The enzyme-linked immunosorbent assay uses the immobilized antigen approach, in which cortisol in plasma samples or standards competes with immobilized steroid for antibody-binding sites. After washing, the cortisol antibody bound to the wells of the microtiter plate is detected with antimouse immunoglobulin conjugated to horseradish peroxidase. Following further washing, o-phenylenediamine substrate is added. The enzyme-linked immunosorbent assay is robust and semiautomated. The mean +/- SD recovery from plasma was 97% +/- 6%. Precision studies on three different plasma pools showed mean coefficients of variation of 7.6% and 8.6% for within- and between-assay variation, respectively. The satisfactory performance criteria allow its use in the routine laboratory.

Animals↗