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Biomedical subjects

R T Taylor

Publications and source records attributed to R T Taylor.

At least 19 recordsLinked to original sources
PubMed1992

Hydrodynamic effects on microcapillary motility and chemotaxis assays of Methylosinus trichosporium OB3b.

A study of the random motility and chemotaxis of Methylosinus trichosporium OB3b was conducted by using Palleroni-chamber microcapillary assay procedures. Under the growth conditions employed, this methanotroph was observed qualitatively with a microscope to be either slightly motile or essentially nonmotile. However, the cells did not not respond in the microcapillary assays in the manner expected for nonmotile Brownian particles. As a consequence, several hydrodynamic effects on these Palleroni microcapillary assays were uncovered. In the random-motility microcapillary assay, nondiffusive cell accumulations occurred that were strongly dependent upon cell concentration. An apparent minimal random-motility coefficient (mu) for this bacterial cell of 1.0 x 10(-7) cm2/s was estimated from microcapillary assays. A simple alternative spectrophotometric assay, based upon gravitational settling, was developed and shown to be an improvement over the Palleroni microcapillary motility assay for M. trichosporium OB3b in that it yielded a more-accurate threefold-lower random-motility coefficient. In addition, it provided a calculation of the gravitational-settling velocity. In the chemotaxis microcapillary assay, the apparent chemotactic responses were strongest for the highest test-chemical concentrations in the microcapillaries, were correlated with microcapillary fluid density, and were strongly dependent upon the microcapillary volume. A simple method to establish the maximal concentration of a chemical that can be tested and to quantify any contributions of abiotic convection is described. Investigators should be aware of the potential problems due to density-driven convection when using these commonly employed microcapillary assays for studying cells which have low motilities.

Bacteriological Techniques↗
PubMed1990

Analysis of hydrogen peroxide in freshly prepared coffees.

Coffee has been shown unequivocally to be genotoxic in vitro, but no genotoxicity has been seen in vivo testing. Since the in vitro genotoxicity appears to be dependent on hydrogen peroxide, it is important to know whether hydrogen peroxide is present in prepared coffee and whether it is being formed during the in vitro testing. We have devised a procedure to measure hydrogen peroxide in prepared coffee using disposable reversed-phase columns to decolourize the coffee and retain its catechols. Hydrogen peroxide was assayed in the eluate from the columns by two chromogenic methods: horseradish-peroxidase-mediated oxidation of phenol red and non-enzymatic oxidation of iodide. Three brands of brewed and instant coffee prepared in the manner recommended to the consumer were studied. Although six of the twelve preparations of coffee contained 3-29 microM-hydrogen peroxide, in the other six none could be detected. Sampling, batch, and aging effects may contribute to the variability in the testing, but there is no indication of greater than 100 microM-hydrogen peroxide levels in freshly prepared coffees, as reported in the literature using other methods. Hydrogen peroxide did form slowly in prepared coffee as the beverage became oxygenated, but it formed quickly if the coffee was diluted by addition to an oxygen-containing solution at neutral pH and then incubated at 37 degrees C. These results strongly suggest that adventitious formation of hydrogen peroxide is a confounding factor in the analytical and in vitro genotoxicological testing of coffees.

Beverages↗
PubMed1989

X-ray crystallographic analysis of the structural distortions induced by substitution and annulation of the dodecahedrane nucleus.

Methyl dodecahedranecarboxylate, C22H22-O2, (4), Mr = 318.42, monoclinic, C2/c, a = 12.600 (2), b = 7.992 (4), c = 28.975 (7) A, beta = 101.79 (2) degrees, V = 2856 A3, Z = 8, Dx = 1.48 g cm-3, lambda(Mo K-alpha) = 0.71073 A, mu = 0.84 cm-1, F(000) = 1360, T = 294 K, R = 0.044 for 2109 unique reflections with Fo2 greater than 3 sigma(Fo2). 21-Phenylcyclopropadodecahedrane, C27H24, (5), Mr = 348.49, tetragonal, P4(2)/n, a = 17.528 (3), c = 10.752 (2) A, V = 3303 A3, Z = 8, Dx = 1.40 g cm-3, lambda(Mo K-alpha) = 0.71069 A, mu = 0.85 cm-1, F(000) = 1488, T = 295 K, R = 0.053 for the 1773 unique reflections with Fo2 greater than 1 sigma(Fo2). The molecular geometries of methoxycarbonyldodecahedrane (4) and 21-phenylcyclopropadodecahedrane (5) are compared to those of the parent C20H20 hydrocarbon (1) and its 1,16-dimethyl derivative (2). For both (2) and (4), the framework is modestly distorted by the substituents, the intracavity distance being more extended along the axis that contains the pendant group(s). Remarkably for (5), its cyclopropane ring has all C-C bonds and internal angles essentially equal, and those dodecahedrane bonds in the immediate vicinity of the ring fusion are extensively perturbed.

Molecular Structure↗
PubMed1989

Radioactive-electrophoretic assay of adenosine 5'-triphosphate sulfurylase activity in crude extracts with sulfate or selenate as a substrate.

An assay method for ATP sulfurylase is presented which employs Na2(35)SO4 as a substrate and measures the production of labeled adenosine 5'-phosphosulfate and 3'-phosphoadenosine 5'-phosphosulfate by low-voltage, hanging paper strip electrophoresis. The method is applicable to crude bacterial or mammalian extracts and accurately measures picomole amounts of product(s). Na2(75SeO4 can also be employed as a substrate, if the unstable radioactive product, adenosine 5'-phosphoselenate, is converted to elemental 75Se degrees by inclusion of reduced glutathione in the reaction mixture. The same paper strip electrophoretic technique can then be used to separate 75Se degrees from the radiolabeled substrate. The method also has utility for measuring any direct reduction by crude microbial extracts of radioactive selenate to selenite, independent of ATP sulfurylase.

Adenosine Phosphosulfate↗
PubMed1989

Cytogenetic response to 1,2-dicarbonyls and hydrogen peroxide in Chinese hamster ovary AUXB1 cells and human peripheral lymphocytes.

Mutagenic 1,2-dicarbonyls have been reported to occur in coffee and other beverages and in various foods. We have measured the induction of sister-chromatid exchanges (SCEs) and endoreduplicated cells (ERCs) to determine the genotoxicity of various 1,2-dicarbonyl compounds in Chinese hamster ovary (CHO) AUXB1 cells and human peripheral lymphocytes. The 1,2-dicarbonyls glyoxal, methylglyoxal and kethoxal each induced highly significant increases in both SCEs and ERCs in AUXB1 cells. Glyoxal and kethoxal induced SCEs but not ERCs in human peripheral lymphocytes. In addition, hydrogen peroxide induced highly significant levels of SCEs and ERCs in AUXB1 cells. Bisulfite, which reacts with carbonyl groups to form addition products, significantly reduced the frequency of SCEs and the proportion of ERCs when glyoxal, methylglyoxal, kethoxal and diacetyl were administered to AUXB1 cells. In addition, bisulfite blocked the formation of ERCs, but not SCEs, induced by hydrogen peroxide. These in vitro results suggest that 1,2-dicarbonyls may play an important role in the genotoxicity of some foods and beverages.

Aldehydes↗
PubMed1989

Cytogenetic response to coffee in Chinese hamster ovary AUXB1 cells and human peripheral lymphocytes.

We have investigated the genotoxic effects of three different brands and three types of coffee (freshly brewed regular, instant regular and freshly brewed decaffeinated) in two mammalian systems: the Chinese hamster ovary (CHO) AUXB1 cell line and human peripheral lymphocytes. Sister-chromatid exchanges (SCEs) and endoreduplicated cells (ERCs) were used as the endpoints. Coffee was prepared according to the manufacturer's suggestions, and after cooling, administered to cultured cells at dilutions ranging up to 11% that of full-strength coffee. Each brand and type of coffee induced significant levels of SCEs and ERCs in AUXB1 cells. SCEs, but not ERCs, were induced in human peripheral lymphocytes. Bisulfite, which complexes with carbonyls and reduces their genotoxicity, significantly diminished the number of SCEs and ERCs found after administration of coffee. Catalase and peroxidase, enzymes that destroy hydrogen peroxide activity, had no significant effect upon the SCE and ERC frequencies in AUXB1 cultures treated with freshly brewed regular coffee. These experiments indicate that different brands and types of coffee have sufficient genotoxic activity to increase SCEs and ERCs at levels only a fraction of those normally consumed. 1,2-Dicarbonyls alone and peroxides alone do not appear to be responsible for the majority of SCEs and ERCs that were observed to be induced by dilute coffee.

Aldehydes↗
PubMed1986

Mutagen formation in a model beef supernatant fraction. IV. Properties of the system.

To identify the precursors and elucidate the reaction conditions that yield heterocyclic amine mutagens in cooked meat products and fish, we have used a supernatant 2 (S2) fraction prepared from H2O-homogenized lean round steak. Compounds (MW less than 500) in S2 are the sources of the microsomal-dependent, Salmonella TA 1538 mutagenic activity in open boiled (aqueous), 200 degrees C pressure-heated (aqueous), or 200 to 300 degrees C oven-baked (freeze-dried) homogenates. Combined incorporation-HPLC experiments show that they are also the precursors for frameshift mutagen formation in the outer surfaces of 200 degrees C griddle-fried ground beef. Maximal stimulations of boiled S2 mutagenic activity are given by 10 mM Trp, 2.5 mM creatine phosphate (CP), and synergistically by 10 mM Trp + 2.5 mM CP + 1.0 mM FeSO4 (a mixture abbreviated as S2*). Boiling S2 for 30 hr at the acidic optimum pH of 4.0----600 TA 1538 revertants (no additions) and 1,400 revertants (+CP), while S2*----24,000 revertants/10(8) bacteria/g of dry beef. By the criteria of HPLC, paper electrophoresis, and resistance of the active HPLC fractions to acid-nitrite inactivation, boiled S2 contains 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) and a minor amount of 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ). Boiling S2 with CP doubles the IQ, halves the Trp-P-2, produced traces of MeIQ, and generates an unknown nitrite-resistant mutagen. Boiled S2* contains these same four mutagens, but both the IQ and Trp-P-2 are increased and large amounts of Trp-P-1 also are generated. The identities of IQ, Trp-P-2, and Trp-P-1 were verified by purification and by light-absorption and mass spectra. Their increments in stimulated S2 indicate that Trp (or its degradation products) and CP (or its degradation products) are the beef juice precursors for the indole ring in Trp-P type mutagens and the NH2-imidazole ring in IQ-type mutagens, respectively. Aqueous (pressure) heating or oven-baking S2 for 2 hr at 200 degrees C greatly elevates its TA 1538 activity----45,000 revertants/10(8) bacteria/g of dry beef; dry heating at 300 degrees C----approximately 180,000 revertants/g of dry beef. Along with the increases in total TA 1538 activity at 200 to 300 degrees C, the number of mutagens formed from the less than 500 MW S2 precursors also multiplies.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1985

The effects of indomethacin and interleukin-2 on the proliferation of lymphocytes from patients with lung cancer.

The effects of the addition of indomethacin and interleukin-2 (IL-2) to phytohemagglutinin (PHA)-stimulated lymphocytes from patients with untreated squamous-cell carcinoma of the lung and with chronic obstructive pulmonary disease and from normal individuals were studied. In 13 of 21 patients with lung carcinoma, the response of lymphocytes to PHA stimulation was significantly augmented by indomethacin. In these 13 patients, the nonaugmented PHA response was also significantly depressed, with only 1 patient falling in the normal range. In the other 8 patients, the average PHA reactivity was normal, as was the degree of augmentation by indomethacin. In additional studies, IL-2 was added to PHA-stimulated lymphocytes with and without the addition of indomethacin. In the normal group, IL-2 further increased PHA reactivity by an average of 46.0%. The addition of indomethacin to these cultures increased the PHA reactivity by only 8.8%, which is similar to the indomethacin effect for the PHA-stimulated cells without the addition of IL-2. In the cancer group, the PHA reactivity of 13 of 19 patients was significantly increased by the addition of IL-2. When indomethacin was added simultaneously with IL-2, the proliferative response for 12 of 19 patients was increased significantly more than for the normal controls. Thus, this study indicates that the PHA reactivity of lymphocytes from many lung carcinoma patients is augmented to a greater extent than for normal individuals by inhibiting prostaglandin synthesis. Also, the addition of IL-2 to these PHA-stimulated cultures did not mimic the addition of indomethacin, but instead the effects were usually additive.(ABSTRACT TRUNCATED AT 250 WORDS)

Cells, Cultured↗
PubMed1985

Induced reversion of a Chinese hamster ovary triple auxotroph. Validation of the system with several mutagens.

A Chinese hamster ovary triple auxotroph (CHO AUXB1) requires glycine, adenosine, and thymidine (GAT) for growth and survival due to a defect in the structural gene for folylpolyglutamate synthetase (FPGS). This auxotroph and others like it contain less than 3% of the parental amounts of FPGS activity. In order to develop a reverse mutation assay with CHO AUXB1, we determined the optimal conditions for measuring reversion and characterized some of the revertants. We also obtained quantitative mutagenicity data for several direct-acting mutagens for comparison to the parental CHO-S/HGPRT locus. Induced revertants appear in the culture immediately following 20-22 h exposures in +GAT complete medium, indicative of dominant genetic changes. They are maximally expressed after 2 population doublings and can be conveniently selected after 44-48 h of expression growth by plating 1 X 10(6) cells/100-mm dish into -GAT-deficient medium and incubating 12-13 days. Plating reconstruction experiments show that the cloning efficiencies of revertants in -GAT medium are not influenced by the presence of up to 1 X 10(6) CHO AUXB1 cells. Dose-dependent increases above the spontaneous revertant frequency (average = 5 X 10(7)) are induced with cis-Pt(NH3)2Cl2 (14-fold) (but not trans-Pt(NH3)2Cl2), PtCl4(10-fold), Pt(SO4)2 (14-fold), K2CrO4 (8-fold), EMS (10-fold), 4-NQO (53-fold), ICR-191 (60-fold), and ICR-170 (30-fold). All of the revertants that have been isolated are stable to repeated subculturing in -GAT medium; 40 out of 42 that have been analyzed are characterized by an increased 72-h growth incorporation of labeled folate and their extracts contain 5-94% as much FPGS as the original, parental CHO-S line. Spontaneous and induced reversion to the GAT+ phenotype primarily reflects mutations involving the FPGS gene locus. But the re-acquisition by most of the revertants of much less than normal amounts of FPGS activity suggests that they arise from compensatory second-site mutations within this gene. Comparison of the mutagenicity patterns of the foregoing compounds as a function of the applied concentration and the relative percent survival reveals some interesting similarities, as well as differences, between the CHO AUXB1/FPGS and CHO-S/HGPRT loci. In particular, the FPGS locus is rather insensitive to EMS (or other simple alkylating agents). However, it seems to be quite susceptible to reversion by other chemicals that are known to react selectively with guanine bases in DNA. CHO AUXBI is a useful supplemental mammalian assay system for assessing quantitatively the generally weak mutagenic activities of metal compounds.

Animals↗
PubMed1982

Acute toxicity of methanol in the folate-deficient acatalasemic mouse.

Formate acidosis is the chief measurable biochemical characteristic of acute methanol toxicity in man. Its marked elevation in the blood stream of primates has been proposed to account for their much greater susceptibility versus rodents to methanol poisoning. Therefore, a study was undertaken to assess whether folic acid deficient (FAD) mice which accumulate formate are much more sensitive to the lethal effects of this alcohol than folic acid sufficient (FAS) mice. Moreover, because some formate is oxidized by catalase-H2O2 in rodents, but not in primates, we also compared the urinary excretion and blood plasma accumulation of formate and the methanol sensitivity of acatalasemic mice. Methanol-dosed C57BL/6Csb (acatalasemic) mice exhibit slightly lower LD50S than CSa (normal catalase) mice, irrespective of their folate state. CSb-FAD mice excreted much more formate and developed higher plasma formate concentrations (11-17 mM) than identically dosed CSa-FAD animals (6 mM). However, in no instance did a folate deficiency produce a large reciprocal decrease in the oral or i.p. LD50 that would be expected from a huge increase (greater than 10-fold) in the 24-h blood plasma formate level. A low methionine (0.2%) intake did not decrease the oral methanol LD50 of CSb-FAD mice, although excess dietary methionine (1.8%) did lower it from 7.1 to 6.4 g/kg. Methanol treated (4 g/kg) Csb-FAD mice excreted 30.8-48.2% of the oral dose as urinary formate, depending on the level of dietary methionine. Csb-FAS and -FAD mice which were given 2 g/kg sodium formate orally (LD50 = 4.7 and 3.7 g/kg) cleared this dose from the blood within 24 h and excreted 58% and 76% of it, respectively, in the urine. Our results indicate that the plasma formate concentration does not correlate well with methanol lethality in Csb-FAS vs. -FAD mice. In addition, urinary excretion, not oxidation, is the primary means by which mice, and probably rats, eliminate high levels of blood formate. Since the Csb-FAD mouse attains high plasma formate levels and low blood pH-values similar to those which have been reported for methanol poisoned monkeys, it appears to be of value as an inexpensive small animal model for further studies of lethal methanol toxicity and the contribution of formate to this process.

Acatalasia↗
PubMed1981

Description of a disposable individual-mouse urine collection apparatus.

An inexpensive, individual mouse urine collection apparatus was constructed from a 250 ml conical polypropylene centrifuge tube and wire cloth. The construction did not require special tools, and was completed in 1 hour. This apparatus allowed the ad libitum feeding and watering of individual mice during the urine collection period. Volumes of urine collected ranged from 0.5-2.0 ml per 24 hours per 25 g mouse.

Animals↗
PubMed1980

Chromosomal assignment of the gene for folylpolyglutamate synthetase to human chromosome 9.

An auxotrophic mutant, GAT-, derived from the Chinese hamster cell line CHO-K1 and exhibiting multiple growth requirements for glycine, adenine, and thymidine, has been shown to be deficient in one of the folate-dependent enzymes, folylpolyglutamate synthetase (FPGS). This mutant was fused with normal human lymphocytes and human lymphoblasts and 41 GAT+ primary hybrid clones were isolated in medium lacking glycine, adenine, and thymidine. In addition, 71 secondary clones were isolated after growth of four primary hybrid clones in enriched medium, which allows losing the complementing human chromosome without losing cell viability. Analysis of human isozyme markers in the 112 primary and secondary clones established a syntenic relationship between the GAT marker and AK1, an isozyme marker for human chromosome 9. Enzyme studies of the parental and hybrid cells not only showed restored enzyme activities of FPGS in the hybrids, but also demonstrated that several enzyme characteristics of FPGS in the hybrids are consistent with those of the human enzyme. Thus, it is concluded that the human gene coding for the enzyme FPGS which complements the auxotrophic mutant GAT- in the CHO-K1 cells can be assigned to human chromosome 9. This assignment provides an additional selective marker for mammalian cell genetic analysis in which human chromosome 9 can be selectively and stably retained in the cell hybrids.

Animals↗