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PubMed1993

Development and optimization of reactivation techniques for carbamate-inhibited brain and plasma cholinesterases in birds and mammals.

Two biochemical assays were developed which promote and measure the induced reactivation of carbamate-inhibited cholinesterases in avian and mammalian brain and plasma samples. The effects of inhibitor concentration, temperature, and the extent of dilution on the achievement of a steady state equilibrium and the subsequent level and rate of recovery of brain cholinesterase activity were investigated. A similar procedure for reactivation of carbamate-inhibited plasma cholinesterase activity involved the removal of excess carbamate from a small sample volume (< 400 microliters). Both methods begin by measuring cholinesterase activity immediately following dilution and involve an incubation period during which conditions for spontaneous reactivation of the inhibited enzymes are maximized. Both assays are suitable for large-scale, rapid use and appear able to restore inhibited cholinesterase activity to levels closely approximating that of control values for each species tested. These methods will not only maximize the usefulness of cholinesterases in monitoring carbamate pesticide exposure but should prove to be extremely useful tools in the forensic assessment of carbamate exposure in human and wildlife pesticide incidents.

Aldicarb↗
PubMed1995

Nucleotide sequence analysis of the M genomic segment of El Moro Canyon hantavirus: antigenic distinction from four corners hantavirus.

El Moro Canyon hantavirus (ELMC, previously known as HMV-1) is associated with the western harvest mouse Reithrodontomys megalotis. The interpretation that ELMC is a novel hantavirus was based upon comparisons of the nucleotide sequence of the S genomic segment with those of other hantaviruses. We now show that the ELMC M genome, like the S genome, is genetically similar to but distinct from that of Four Corners hantavirus (FC). The ELMC M genome is 3801 nt in length and encodes a glycoprotein precursor of 1139 amino acids. The G1 and G2 genes are 71 and 73.1% identical to those of FC and the corresponding glycoproteins are 73.5 and 82.3% identical. A portion of the G1 glycoprotein of ELMC that is homologous to an important linear epitope of FC differs from the FC epitope by 10 of 31 residues. That domain and flanking sequences were expressed in Escherichia coli. G1 antibodies from 3 of 19 FC-infected patients showed cross-reactivity with ELMC by Western blot assay. The portion of the ELMC G1 antigen recognized by those antibodies is the region of homology to the FC epitope. These data support our previous interpretation that ELMC is distinct from FC and other hantaviruses.

Amino Acid Sequence↗
PubMed1996

Sin Nombre virus mRNA synthesis.

Sin Nombre (SN) virus is the major etiologic agent of hantavirus pulmonary syndrome, a severe respiratory disease with high mortality. Like other hantaviruses, SN virus causes an inapparent chronic infection of the natural rodent reservoir and tends to grow slowly and produce little cytopathic effect even in highly susceptible Vero E6 tissue culture cells. An electrochemiluminescent quantitative PCR approach was developed to allow examination of SN virus RNA transcription in synchronously infected cells. Although virion particles contain equimolar ratios of the three negative-strand genome RNA segments (S, M, and L), rates and levels of accumulation of the corresponding N, GPC, and L viral mRNAs varied. The smallest mRNA (N) was detectable earliest and plateaued at the highest level, where as the largest mRNA (L) appeared latest and at the lowest plateau level. In addition, all three mRNAs were found to share a common 5' capped primer initiation mechanism, but appeared to have different mRNA termination mechanisms. The N mRNA 3' terminus mapped to position 1435 on the S segment, in close proximity to a CCC-rich suspected transcription termination motif. The GPC mRNA 3' terminus contained a poly(A) tall and mapped to a U8 transcription termination-polyadenylation motif reminiscent of those seen in other negative-strand RNA viruses. Finally, the L mRNA 3' terminus appeared identical to the L segment antigenome 3' terminus.

Animals↗
PubMed1995

Identifying chromosomal fragile sites from individuals: a multinomial statistical model.

The inability to identify fragile sites from data for single individuals remains the major obstacle to determining whether these chromosomal loci are predisposed to cancer-causing and evolutionary rearrangements. We describe a novel statistical model that is amenable to data from single individuals and that establishes site-specific chromosomal breakage as nonrandom with respect to the distribution of total breakage. Our method tests incrementally smaller subsets of the data for homogeneity under a multinomial model that assigns equal probabilities to a maximal set of nonfragile sites and unrestricted probabilities to the remaining fragile sites with significantly higher numbers of breaks. We show how standardized Pearson's chi-square (X2) and likelihood-ratio (G2) statistics can be appropriately used to measure goodness-of-fit for sparse contingency (individual-based) data in this model. A sample application of this approach indicates extensive variation in fragile sites among individuals and marked differences in fragile-site inferences from pooled as opposed to per-individual data.

Animals↗
PubMed1977

Visualization of centriole-bodies using silver stain.

Duplicate staining of human metaphase chromosomes, first with Giemsa followed by silver, revealed the presence of two small silver-stained bodies not seen in the Giemsa stained metaphases. Similar bodies were subsequently found in the metaphases of several animal groups. The size, structure, spatial relationships to the nucleus, behavior throughout the cell cycle, and apparent universal presence of these bodies suggest that they are either centrioles or associated centriolar structures. These centriole-bodies can be seen throughout the cell cycle, even in hypotonically spread C-metaphase chromosome plates. The silver stain procedure allows enough resolution to distinguish parent and daughter centriole-bodies at interphase, thus permitting visualization of the replication, maturation and separation stages of these bodies with the light microscope.

Animals↗
PubMed1989

Altitudinal and seasonal effects on aerobic metabolism of deer mice.

I compared the maximal aerobic metabolic rates (VO2max), field metabolic rates (FMR), aerobic reserves (VO2max-FMR), and basal metabolic rates (BMR) of wild and recently captured deer mice from low (440 m) and high (3800 m) altitudes. To separate the effects of the thermal environment from other altitudinal effects, I examined mice from different altitudes, but similar thermal environments (i.e., summer mice from high altitude and winter mice from low altitude). When the thermal environment was similar, VO2max, FMR, and aerobic reserve of low and high altitude mice did not differ, but BMR was significantly higher at high altitude. Thus, in the absence of thermal differences, altitude had only minor effects on the aerobic metabolism of wild or recently captured deer mice. At low altitude, there was significant seasonal variation in VO2max, FMR, and aerobic reserve, but not BMR. BMR was correlated with VO2max, but not with FMR. The significant positive correlation of BMR with VO2max indicates a cost of high VO2max, because higher BMR increases food requirements and energy use during periods of thermoneutral conditions.

Aerobiosis↗
PubMed1992

A comparison of methods for sampling the deer tick, Ixodes dammini, in a Lyme disease endemic area.

The purpose of this study was to compare the trapping and examining of mice, drag sampling, and CO2-baited traps for their ability to detect the presence and abundance of immature deer ticks, Ixodes dammini, in a Lyme disease endemic area in southern New York State. Eight study sites were sampled 14 times between 28 May and 31 August by setting 49 live-traps, four CO2-baited traps, and drag sampling 500 m2. A total of 1540 nymphs and 3079 larvae was collected during the study. Drag sampling collected the most nymphs (705), while more larvae were recovered from CO2-baited traps (1105). Comparisons among the methods showed a significant difference in the numbers of both larval and nymphal ticks collected (P less than 0.01). There was a positive correlation between the numbers of nymphs collected by drag sampling and CO2-baited tick traps (rs = 0.83, P less than 0.05), and between the numbers of larvae collected by drag sampling and mouse trapping (rs = 0.75, P less than 0.05). These results suggest that drag sampling would be the single most reliable method for quantitatively sampling immature I. dammini populations in a Lyme disease endemic area.

Analysis of Variance↗
PubMed1995

Ultrastructural characteristics of Sin Nombre virus, causative agent of hantavirus pulmonary syndrome.

A previously unrecognized disease, hantavirus pulmonary syndrome, was described following an outbreak of severe, often lethal, pulmonary illness in the southwestern United States in May-June, 1993. We have now studied the morphologic features of the causative agent, Sin Nomber virus (SNV), by thin section electron microscopy and immunoelectron microscopy of infected Vero E6 cells. SNV virions were roughly spherical and had a mean diameter of 112 nm. They had a rather dense envelope and closely apposed fine surface projections, 7 nm in length. Filamentous nucleocapsids were present within virions. Viral inclusion bodies were present in the cytoplasm of infected cells; these appeared granular or filamentous, depending on the plane of section. All of these characteristics were similar to published descriptions of other hantaviruses; however, unlike all other hantaviruses and virtually all other member viruses of the family Bunyaviridae which bud upon smooth intracytoplasmic membranes, SNV budding occurred almost entirely upon the plasma membrane of infected cells. Virus budding was associated with the formation of long 28 nm diameter tubular projections. Occasional elongated 47 nm diameter virus-like particles were seen to bud upon intracytoplasmic membranes. As shown by immunoelectron microscopy, viral antigens were localized over virions, inclusions, and tubular projections associated with virion morphogenesis.

Animals↗
PubMed1993

Seasonal acclimation of prairie deer mice.

Prairie deer mice responded to long nights by reducing their metabolic rates, core temperatures, thermal conductances and incremental metabolic responses to cold stimulus, while increasing their capacities for nonshivering thermogenesis. Some winter animals spontaneously entered daily torpor in the mornings and thereby further reduced their metabolic rates and core temperatures. Provision of exogenous melatonin (by subdermal implants) mimiced short photoperiod effects on metabolic rates and core temperatures of wild-caught, laboratory maintained animals. Provision of supplemental dietary tryptophan to laboratory animals conditioned to natural light cycles mimiced metabolic effects of long nights in summer animals, and further reduced metabolic rates of winter mice, but did not affect their core temperature levels. Newly caught, laboratory maintained deer mice responded to natural seasonal clues of short-photoperiod and increased dietary tryptophan by reducing their resting energy requirements through both lower metabolic and lower core temperature levels. Short photoperiod and seasonal change also promoted gonadal involution, and resulted in more socially tolerant huddling by mice with reduced core temperature. Reduced 24-hour LH excretion rates were also observed in winter animals which were exposed to seasonal light cycles at warm (25 degrees C) room temperatures. We propose that seasonal acclimatization involves pineal effects on sex hormone-influenced social behaviors and on resting metabolism. These effects serve to conserve resting energy expenditure and promote hypothermic insulation by wild prairie deer mice.

Acclimatization↗
PubMed1987

Respective roles of the microsomal ethanol oxidizing system and catalase in ethanol metabolism by deermice lacking alcohol dehydrogenase.

To evaluate the roles of MEOS (microsomal ethanol oxidizing system) and catalase in non-alcohol dehydrogenase (ADH) ethanol metabolism, MEOS and catalase activities in vitro and ethanol oxidation rates in hepatocytes from ADH-negative deermice were measured after treatment with catalase inhibitors and/or a stimulator of H2O2 generation. Inhibition of ethanol peroxidation by 3-amino-1,2,4-triazole (aminotriazole) was found to be greater than 85% up to 3 h and 80% at 6 h in liver homogenates. Urate (1 mM) which stimulates H2O2 production in living systems, increased ethanol oxidation fourfold in control but not in cells from aminotriazole-treated animals, documenting effective inhibition of catalase-mediated ethanol peroxidation by aminotriazole. While aminotriazole slightly depressed (15%) basal ethanol oxidation in hepatocytes, in vitro experiments showed a similar decrease in MEOS activity after aminotriazole pretreatment. Azide (0.1 mM), a potent inhibitor of catalase in vitro, also did not affect ethanol oxidation in control cells. By contrast, 1-butanol, a competitive inhibitor of MEOS, but neither a substrate nor an inhibitor of catalase, decreased ethanol oxidation rates in a dose-dependent manner. These results show that, in deermice lacking ADH, catalase plays little if any role in hepatic ethanol oxidation, and that MEOS mediates non-ADH metabolism.

1-Butanol↗
PubMed1987

NAD+-dependent retinol dehydrogenase in liver microsomes.

A microsomal NAD+-dependent retinol dehydrogenase is being described with optimal activity at physiological pH. The enzyme was present in liver microsomes of rats and also in a strain of deermice which lacks the cytosolic retinol dehydrogenase. Unlike the latter enzyme, the microsomal retinol dehydrogenase was not inhibited by either ethanol or 4-methylpyrazole; its activity was insensitive to CO and not oxygen dependent, in contradistinction with that of the microsomal cytochrome P-450 and NADPH-dependent retinol oxidase. Chronic ethanol consumption resulted in an increased activity of the microsomal retinol dehydrogenase which may contribute to hepatic retinol depletion, especially in view of the insensitivity of the enzyme to ethanol inhibition.

Alcohol Oxidoreductases↗
PubMed1989

The microsomal ethanol oxidizing system mediates metabolic tolerance to ethanol in deermice lacking alcohol dehydrogenase.

Metabolic tolerance to ethanol has been attributed to enhanced mitochondrial reoxidation of reducing equivalents produced in the alcohol dehydrogenase (ADH) pathway or to non-ADH mechanisms. To resolve this issue, deermice lacking low Km hepatic ADH were fed for 2 weeks a liquid diet containing ethanol or isocaloric carbohydrate and hepatocytes were isolated. Ethanol (50 mM) oxidation increased (9.8 vs 4.5 nmol/min/10(6) cells in controls). To differentiate which of two non-ADH pathways (the microsomal ethanol oxidizing system (MEOS) or catalase) was responsible for the induction, four approaches were used. First, MEOS was assayed in hepatic microsomes and found to be increased (24.4 vs 6.8 nmol/min/mg protein in controls). Second, hepatocyte ethanol metabolism was measured after addition of the catalase inhibitor azide (0.1 mM) and found to be unchanged. By contrast, the competitive MEOS inhibitor, 1-butanol, depressed metabolism in a concentration-dependent manner. A third approach relied on measurement of isotope effects known to be different for MEOS and catalase. From the isotope effect values, MEOS was calculated to contribute 85% or more of total ethanol oxidation by cells from both ethanol-fed and control animals. A fourth approach involved in vivo pretreatment with pyrazole (300 mg/kg/day for 2 days), which reduced peroxidation by catalase to 13% of control values in liver homogenates while inducing MEOS activity to 152% of controls. Hepatocytes from pyrazole-treated deermice showed a 47% increase in ethanol metabolism, paralleling the MEOS induction and contrasting with the catalase suppression. These results indicate that since metabolic tolerance occurs in the absence of ADH, it is not necessarily ADH mediated, and further, that MEOS rather than catalase accounts for basal ethanol metabolism and its increase after chronic ethanol treatment.

1-Butanol↗
PubMed1989

Retinoic acid synthesis by cytosol from the alcohol dehydrogenase negative deermouse.

Cytosolic alcohol dehydrogenase in the deermouse is coded by a single genetic locus and a strain of the deermouse which is alcohol dehydrogenase negative exists. These two strains of the deermouse were used to extend insight into the role of cytosolic alcohol dehydrogenases in the conversion of retinol into retinoic acid. Retinoic acid synthesis from physiological concentrations of retinol (7.5 microM) with cytosol from the alcohol dehydrogenase negative deermouse was 13% (liver), 14% (kidney), 60% (testes), 78% (lung), and 100% (small intestinal mucosa) of that observed with cytosol from the positive deermouse. The rates in the negative strain ranged from 0.3 to 0.7 nmol/h/mg protein: sufficient to fulfill cellular needs for retinoic acid. Ten millimolar 4-methylpyrazole inhibited retinoic acid synthesis 92, 94, 26, and 30% in kidney, liver, lung, and testes of the positive deermouse, respectively, but only 50, 30, 0, and 0% in the same tissues from the negative deermouse. Ethanol (300 mM) did not inhibit retinoic acid synthesis in kidney cytosol from the negative strain. Therefore multiple cytosolic dehydrogenases, including alcohol dehydrogenases, contribute to retinol metabolism in vitro. The only enzyme(s) likely to be physiologically significant to retinoic acid synthesis in vivo, however, is the class of dehydrogenase, distinct from ethanol dehydrogenase, that is common to both the positive and the negative deermouse. This conclusion is supported by the data described above, the kinetics of retinoic acid synthesis and retinal reduction in kidney cytosol from the negative deermouse, and the very existence of the alcohol dehydrogenase negative deermouse. This work also shows that microsomes inhibit the cytosolic conversion of retinol into retinoic acid and that the synthesis of retinal, a retinoid that has no known function outside of the eye, does not reflect the ability or capacity of a sample to synthesize retinoic acid.

Alcohol Dehydrogenase↗