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Results for “variable methylation”

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PubMed2005

Genetic polymorphisms of methylenetetrahydrofolate reductase and colorectal cancer and adenoma.

Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme regulating folate metabolism, which affects DNA methylation and synthesis. Two functional, common polymorphisms (C677T and A1298C) are known in the MTHFR gene. MTHFR activity is lowered in individuals with the 677TT genotype and is somewhat reduced in those with the 1298CC genotype. We reviewed the consistency of reported associations of these polymorphisms with colorectal cancer and adenoma with consideration of the effects of nutritional status. A total of 16 studies have addressed the association between MTHFR C677T polymorphism and colorectal cancer in 10 countries. Decreased risk of colorectal cancer associated with the 677TT genotype has fairly consistently been observed, with few exceptions. This decrease was observable in people with either high or low folate status. Alteration in the thymidylate pool associated with MTHFR activity is postulated as an underlying mechanism. Studies on the A1298C polymorphism are limited, and their results are variable. Almost all of seven studies of colorectal adenoma have found no association between C677T polymorphism and adenoma, but the 677TT genotype seems to be related to increased risk when folate status is poor. Reduced availability of methyl groups for DNA methylation might be more relevant to adenoma formation. Although the underlying mechanisms still remain to be clarified, epidemiological findings regarding MTHFR C677T polymorphism provide strong evidence that adequate folate status confers protection from colorectal cancer.

Adenoma↗

PubMed2004

Formation of molecular complexes by N-methyl-D-aspartate receptor subunit NR2B and ryanodine receptor 2 in neonatal rat myocard.

The N-methyl-d-aspartate (NMDA) receptor is a glutamate gated cation channel prevalent in the postsynaptic membranes of central nervous system neurons. The neurotransmitter receptor complex is thought to represent a tetramer where variable NR2 or NR3 polypeptides form heteromeric assemblies with an obligatory NR1 subunit. Recently, we showed that cardiac myocytes from perinatal rats transiently express the NMDA receptor subunit NR2B, the function of which in heart is unknown. To characterize the cardiac NR2B protein, we determined its subcellular distribution and specific molecular interaction partners. By immunostaining of rat heart tissue slices and acutely dissociated cardiac myocytes, the NR2B antigen was localized at the sarcomeric Z-bands. Using immunoprecipitation of detergent-solubilized NR2B protein and subsequent analysis employing matrix-assisted laser desorption/ionization time of flight mass spectrometry, ryanodine receptor 2 was identified as a molecular interaction partner of the cardiac NR2B polypeptide. Differences in antibody recognition indicate that the cardiac NR2B polypeptide carries a structurally altered C terminus as compared with the NR2B variant prevalent in central nervous system. Based on its localization and protein interaction, the function of cardiac NR2B protein may relate to mechanosensitivity or play a role in the regulation of the contractile apparatus of neonatal heart.

Amino Acid Sequence↗

PubMed2000

Gene conversion of ribosomal DNA in Nicotiana tabacum is associated with undermethylated, decondensed and probably active gene units.

We examined the structure, intranuclear distribution and activity of ribosomal DNA (rDNA) in Nicotiana sylvestris (2n = 2x = 24) and N. tomentosiformis (2n = 2x = 24) and compared these with patterns in N. tabacum (tobacco, 2n = 4x = 48). We also examined a long-established N. tabacum culture, TBY-2. Nicotiana tabacum is an allotetraploid thought to be derived from ancestors of N. sylvestris (S-genome donor) and N. tomentosiformis (T-genome donor). Nicotiana sylvestris has three rDNA loci, one locus each on chromosomes 10, 11, and 12. In root-tip meristematic interphase cells, the site on chromosome 12 remains condensed and inactive, while the sites on chromosomes 10 and 11 show activity at the proximal end of the locus only. Nicotiana tomentosiformis has one major locus on chromosome 3 showing activity and a minor, inactive locus on chromosome 11. In N. tabacum cv. 095-55, there are four rDNA loci on T3, S10, S11/t and S12 (S11/t carries a small T-genome translocation). The locus on S12 remains condensed and inactive in root-tip meristematic cells while the others show activity, including decondensation at interphase and secondary constrictions at metaphase. Nicotiana tabacum DNA digested with methylcytosine-sensitive enzymes revealed a hybridisation pattern for rDNA that resembled that of N. tomentosiformis and not N. sylvestris. The data indicate that active, undermethylated genes are of the N. tomentosiformis type. Since S-genome chromosomes of N. tabacum show rDNA expression, the result indicates rDNA gene conversion of the active rDNA units on these chromosomes. Gene conversion in N. tabacum is consistent with the results of previous work. However, using primers specific for the S-genome rDNA intergenic sequences (IGS) in the polymerase chain reaction (PCR) show that rDNA gene conversion has not gone to completion in N. tabacum. Furthermore, using methylation-insensitive restriction enzymes we demonstrate that about 8% of the rDNA units remain of the N. sylvestris type (from ca. 75% based on the sum of the rDNA copy numbers in the parents). Since the active genes are likely to be of an N. tomentosiformis type, the N. sylvestris type units are presumably contained within inactive loci (i.e. on chromosome S12). Nicotiana sylvestris has approximately three times as much rDNA as the other two species, resulting in much condensed rDNA at interphase. This species also has three classes of IGS, indicating gene conversion has not homogenised repeat length in this species. The results suggest that methylation and/or DNA condensation has reduced or prevented gene conversion from occurring at inactive genes at rDNA loci. Alternatively, active undermethylated units may be vulnerable to gene conversion, perhaps because they are decondensed and located in close proximity within the nucleolus at interphase. In TBY-2, restriction enzymes showed hybridisation patterns that were similar to, but different from, those of N. tabacum. In addition, TBY-2 has elevated rDNA copy number and variable numbers of rDNA loci, all indicating rDNA evolution in culture.

Blotting, Southern↗

PubMed1985

Mapping labeled sites in Escherichia coli ribosomal RNA: distribution of methyl groups and identification of a photoaffinity-labeled RNA region putatively at the peptidyltransferase center.

We have developed a method for the rapid localization of sites of ribosomal RNA labeling to limited regions (approximately 200 bases). The method is based on the formation and polyacrylamide gel electrophoretic separation of hybrids between restriction fragments of rrnB DNA and isotopically labeled rRNA and the subsequent determination of radioactivity across the gel. Using [3H]adenine-labeled rRNA as a control sample, we optimized experimental conditions with respect to a number of variables, including rRNA:DNA stoichiometric ratio, temperature of the annealing step, and levels of nucleases. An important result is that different rRNA X DNA hybrid fragments are obtained in different yields. The method was then applied to analyses of C3H3-labeled rRNA, giving results in good accord with known and proposed sites of rRNA methylation, and of rRNA that has been photoaffinity-labeled with 5-azido-2-nitrobenzoyl-[3H]Phe-tRNAPhe, a probe directed toward the peptidyltransferase center. The latter study showed a single major site of RNA labeling, falling within bases 2445-2668 of 23S rRNA. The extent of labeling was shown to be dependent on light-induced formation of a reactive intermediate and to be decreased in the absence of poly(uridylic acid) or in the presence of puromycin. The location of this major site of labeling is consistent with recent results obtained with an analogous tRNA photoaffinity label [Barta, A., Steiner, G., Brosius, J., Noller, H. F., & Kuechler, E. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 3607-3611] and with related genetic and biochemical studies of antibiotic interaction with ribosomes suggesting that the peptidyltransferase center falls within region V (bases 2043-2625) of 23S rRNA.

Acyltransferases↗

PubMed1998

Overexpression of iron superoxide dismutase in transformed poplar modifies the regulation of photosynthesis at low CO2 partial pressures or following exposure to the prooxidant herbicide methyl viologen.

Chloroplast-targeted overexpression of an Fe superoxide dismutase (SOD) from Arabidopsis thaliana resulted in substantially increased foliar SOD activities. Ascorbate peroxidase, glutathione reductase, and monodehydroascorbate reductase activities were similar in the leaves from all of the lines, but dehydroascorbate reductase activity was increased in the leaves of the FeSOD transformants relative to untransformed controls. Foliar H2O2, ascorbate, and glutathione contents were comparable in all lines of plants. Irradiance-dependent changes in net CO2 assimilation and chlorophyll a fluorescence quenching parameters were similar in all lines both in air (21% O2) and at low (1%) O2. CO2-response curves for photosynthesis showed similar net CO2-exchange characteristics in all lines. In contrast, values of photochemical quenching declined in leaves from untransformed controls at intercellular CO2 (Ci) values below 200 microL L-1 but remained constant with decreasing Ci in leaves of FeSOD transformants. When the O2 concentration was decreased from 21 to 1%, the effect of FeSOD overexpression on photochemical quenching at limiting Ci was abolished. At high light (1000 micromol m-2 s-1) a progressive decrease in the ratio of variable (Fv) to maximal (Fm) fluorescence was observed with decreasing temperature. At 6(o)C the high-light-induced decrease in the Fv/Fm ratio was partially prevented by low O2 but values were comparable in all lines. Methyl viologen caused decreased Fv/Fm ratios, but this was less marked in the FeSOD transformants than in the untransformed controls. These observations suggest that the rate of superoxide dismutation limits flux through the Mehler-peroxidase cycle in certain conditions.

Ascorbate Peroxidases↗

PubMed2006

Experimental design to optimise the analysis of organic volatile compounds in cow slurry by headspace solid-phase microextraction-gas chromatography-mass spectrometry.

The headspace solid-phase microextraction (HS-SPME) of some odorous organic compounds [volatile fatty acids (VFAs), phenols and indoles] from animal wastes was optimised using an experimental design approach. The variables affecting the HS-SPME process studied were as follows: type of fibre, pH of the slurry (3-7), addition of sodium chloride (0-1 mol L(-1)), sample volume (10-30 mL), exposure time (10-30 min), exposure temperature (25-50 degrees C), desorption time (3-10 min) and desorption temperature (230-310 degrees C). As for the sorbent, three different types of fibres were studied: 50/30 microm divinylbenzene/Carboxen/polydimethylsiloxane (DVB/CAR/PDMS), 100 microm PDMS and 85 microm CAR/PDMS. Methylation VFAs was observed when fibres containing PDMS were used. The analytes studied in the optimisation procedure were acetic acid, propanoic acid, butanoic acid, 3-methylbutanoic acid, pentanoic acid, phenol, 4-methylphenol, 4-ethylphenol, indole and skatole (3-methylindole). In order to study the influence of the variables on the responses of the compounds, a Plackett-Burman design was built using the Unscrambler program. The optimisation was carried out using real samples. In order to take into account the matrix effect on the calibration curves, standard solutions were prepared using a purged cow slurry free from the analytes of interest. Repeatability within and among days, accuracy and detection limits were calculated from these calibration curves.

Animals↗

PubMed2003

Spectroscopic and computational studies of a Ni(+)-CO model complex: implications for the acetyl-CoA synthase catalytic mechanism.

The four-coordinate Ni(+) complex [PhTt(t)(Bu)]Ni(I)CO, where PhTt(t)()(Bu) = phenyltris((tert-buthylthio)methyl)borate (a tridentate thioether donor ligand), serves as a possible model for key Ni-CO reaction intermediates in the acetyl-CoA synthase (ACS) catalytic cycle. Resonance Raman, electronic absorption, magnetic circular dichroism (MCD), variable-temperature variable-field MCD, and electron paramagnetic resonance spectroscopies were utilized in conjunction with density functional theory and semiemperical INDO/S-CI calculations to investigate the ground and excited states of [PhTt(t)()(Bu)]Ni(I)CO. These studies reveal extensive Ni(+) --> CO pi-back-bonding interactions, as evidenced by a low C-O stretching frequency (1995 cm(-)(1)), a calculated C-O stretching force constant of 15.5 mdyn/A (as compared to k(CO)(free CO) = 18.7 mdyn/A), and strong Ni(+) --> CO charge-transfer absorption intensities. Calculations reveal that this high degree of pi-back-bonding is due to the fact that the Ni(+) 3d orbitals are in close energetic proximity to the CO pi acceptor orbitals. In the ACS "paramagnetic catalytic cycle", the high degree of pi-back-bonding in the putative Ni(+)-CO intermediate (the NiFeC species) is not expected to preclude methyl transfer from CH(3)-CoFeSP.

Acetate-CoA Ligase↗

PubMed1999

Characterization of cytochrome P-450 2D1 activity in rat brain: high-affinity kinetics for dextromethorphan.

We investigated the enzymatic function, stability, and regional distribution of rat brain cytochrome P-450 (CYP) 2D1 activity. CYP2D1 is the homolog of human CYP2D6, a genetically variable enzyme that activates or inactivates many clinical drugs acting on the central nervous system (e.g., antidepressants, monoamine oxidase inhibitors, serotonin uptake inhibitors, and neuroleptics), drugs of abuse (e.g., amphetamine and codeine), neurotoxins (e.g., 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, 1,2,3, 4-tetrahydroquinoline), and endogenous neurochemicals (e.g., tryptamine). The CYP2D family has been identified in rodent, canine, and primate brain. Conversion of dextromethorphan to dextrorphan by rat brain membranes was assayed by HPLC and was dependent on NADPH, protein concentration, and incubation time. Significant loss of activity was observed in some homogenizing buffers and after freezing of whole tissues or membrane preparations. Dextromethorphan (0.5-640 microM) metabolism was mediated by high- and low-affinity enzyme systems; K(m1) was 2.7 +/- 2.6 and K(m2) was 757 +/- 156 microM (n = 3 rats, mean +/- S.E.). The enzyme activity was significantly (p <.01) and stereoselectively inhibited by CYP2D1 inhibitors quinine and quinidine (not by CYP2C or CYP3A inhibitors), and by anti-CYP2D6 peptide antiserum (not by anti-CYP2C, -CYP2B, or -CYP3A antibodies). The enzymatic activity demonstrated significant brain regional variation (n = 10 regions, p <.001). These data characterize CYP2D1-mediated dextromethorphan metabolism in rat brain and suggest that localized metabolism of other CYP2D1 substrates (drugs, neurotoxins, and possibly endogenous compounds) within the brain will occur. In humans, CYP2D6 is genetically polymorphic; the variable expression of brain CYP2D6 may result in interindividual differences in central drug and neurotoxin metabolism, possibly contributing to interindividual differences in drug effects and neurotoxicity.

Alcohol Oxidoreductases↗

PubMed2006

Proteomic analysis of microglial contribution to mouse strain-dependent dopaminergic neurotoxicity.

Although the pathogenesis of Parkinson's disease (PD) remains unknown, it appears that microglial activation is associated with enhanced neurodegeneration in animal models of PD as well as in PD patients. Experimentally, C57BL/6 and SWR/J mice demonstrate striking differences in the extent of dopaminergic (DAergic) neurodegeneration induced by a parkinsonian toxicant 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The purpose of this study was to determine whether differences in microglial activation between these two strains of mice could provide insight into the variability seen in toxicant induced neuronal death, and subsequently to use a high-throughput proteomic method, combining stable isotope labeling with amino acids in cell culture (SILAC) with liquid chromatography and tandem mass spectrometry, to compare the microglial proteomes of C57BL/6 and SWR/J mice after stimulation with a classical microglial activator, lipopolysaccharide (LPS). We found that DAergic neurotoxicity induced by LPS in a primary neuron-microglia coculture was twofold greater with microglia isolated from the brains of C57BL/6 mice compared with that of SWR/J mice. Upon proteomic analysis we found that, out of over 1,000 proteins identified and quantified, 400 displayed a significant difference in their relative abundance between these two murine strains. Several proteins, which had relatively higher levels in C57BL/6 mice, have previously been implicated in LPS-mediated microglial activation, including those involved in the COX-2 pathway and in prostaglandin E-2 (PGE(2)) production. To validate our proteomic results we confirmed the increased expression level of iNOS in C57BL/6 vs. SWR/J microglia with semiquantitative Western blot. Further analysis of our proteomic discovery data will likely reveal numerous novel proteins involved in inflammation-mediated neurotoxicity in PD.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗

PubMed2001

N-methyl-D-aspartate receptor-mediated increase of neurogenesis in adult rat dentate gyrus following stroke.

Neurogenesis in the adult rat dentate gyrus was studied following focal ischemic insults produced by middle cerebral artery occlusion (MCAO). Animals were subjected to either 30 min of MCAO, which causes damage confined to the striatum, or 2 h of MCAO, which leads to both striatal and cortical infarction. When compared to sham-operated rats, MCAO-rats showed a marked increase of the number of cells double-labelled for 5-bromo-2'-deoxyuridine-5'-monophosphate (BrdU; injected during 4-6 days postischemia) and neuronal-specific antigen (NeuN; a marker of postmitotic neurons) in the ipsilateral dentate granule cell layer and subgranular zone at 5 weeks following the 2 h insult. Only a modest and variable increase of BrdU-labelled cells was found after 30 min of MCAO. The enhanced neurogenesis was not dependent on cell death in the hippocampus, and its magnitude was not correlated to the degree of cortical damage. Systemic administration of the N-methyl-D-aspartate (NMDA) receptor blocker dizocilpine maleate (MK-801) completely suppressed the elevated neurogenesis following 2 h of MCAO. Our findings indicate that stroke leads to increased neurogenesis in the adult rat dentate gyrus through glutamatergic mechanisms acting on NMDA receptors. This modulatory effect may be mediated through changes in the levels of several growth factors, which occur after stroke, and could influence various regulatory steps of neurogenesis.

Animals↗

PubMed2006

Minimal phenotype in a girl with trisomy 15q due to t(X;15)(q22.3;q11.2) translocation.

Few cases of de novo unbalanced X;autosome translocations associated with a normal or mild dysmorphic phenotype have been described. We report a 3-year-old dizygotic female twin with prenatally ascertained increased nuchal translucency. Prenatal chromosome studies revealed nearly complete trisomy 15 due to a de novo unbalanced translocation t(X;15)(q22;q11.2) confirmed postnatally. A mild phenotype was observed with normal birth measurements, minor facial dysmorphic features (hypertelorism, short broad nose, and a relatively long philtrum), and moderate developmental delay at the age of 3 years in comparison to her male fraternal twin. Replication timing utilizing BrdU and acridine-orange staining showed that the der(X) chromosome was late-replicating with variable spreading of inactivation into the translocated 15q segment. The der(X) was determined to be of paternal origin by analyses of polymorphic markers and CGG-repeat at FMR1. Methylation analysis at the SNRPN locus and analysis of microsatellites on 15q revealed paternal isodisomy with double dosage for all markers and the unmethylated SNRPN gene. The Xq breakpoint was mapped within two overlapping BAC clones RP11-575K24 and RP13-483F6 at Xq22.3 and the 15q breakpoint to 15q11.2, within overlapping clones RP11-509A17 and RP11-382A4 that are all significantly enriched for LINE-1 elements (36.6%, 43.0%, 26.6%, 22.0%, respectively). We speculate that the attenuated phenotype may be due to inactivation spreading into 15q, potentially facilitated by the enrichment of LINE-1 elements at the breakpoints. In silico analysis of breakpoint regions revealed the presence of highly identical low-copy repeats (LCRs) at both breakpoints, potentially involved in generating the translocation.

Alleles↗

PubMed1993

Is glycolysis impaired in erythrocytes of diabetic rats?

In erythrocytes from diabetic rats the generation of radioactive metabolites from 3H- or 14C-labeled D-glucose occurs at a lower rate than in cells from control animals, while the total production of L-lactic acid is the same in both cell types. The radioisotopic anomaly is not corrected by agents causing phosphofructokinase activation and is not attributable to interference of metabolites generated from either glycogen or 2,3-diphospho-D-glycerate. It is mimicked by preincubating the erythrocytes at a high concentration of D-glucose and suppressed in diabetic rats treated with insulin in order to lower the blood glucose concentration prior to sacrifice. These findings suggest isotopic dilution of the 3H- or 14C-labeled tracer by endogenous unlabeled D-glucose. The influence of factors such as the concentration of erythrocytes in the incubation medium, the concentration of extracellular D-glucose, and the length of incubation upon radioactive metabolic variables is also compatible with the postulated phenomenon of isotopic dilution.

1-Methyl-3-isobutylxanthine↗

PubMed1998

Cellular and cytoskeleton morphology and strength of adhesion of cells on self-assembled monolayers of organosilanes.

The objective of this study was to explore the potential use of self-assembled monolayers (SAMs) of alkylamine and arylalkyamine as well-defined, homogeneous, tailored in vitro model surfaces for exploring the effect of hydrodynamic flow on morphology and strength of adhesion of human umbilical vein endothelial cells. The cell surface area, shape, f-actin distribution, and adhesion strength of human umbilical vein endothelial cells cultured on self-assembled monolayers of organosilanes were found to be dependent on the chemical composition of the organosilane film and the magnitude of wall shear stress. The direct effects of the differences in chemistry between the two silanes, in modulating cellular response, are probably only secondary to the modulation of cellular functions mediated by differential protein adsorption and conformation on the two silanes. For short seeding times (30 min), prior to application of flow, both substrate chemistry and shear stress modulated the cellular morphology and cytoskeletal organization. For longer seeding times (24 h), prior to application of flow, the chemistry of the underlying surface was the dominant variable in modulating cellular morphology, while the hydrodynamic shear stress modulated the cytoskeleton organization. Cells on N-(2-aminoethyl)-3-aminopropyl trimethoxysilane (EDA) were pleomorphic, while cells on ((((aminoethyl)amino)methyl)phenylethyl)trimethoxysilane (PEDA) expressed a rounded morphology. Application of an incrementally loaded flow regime (0.07-1.25 ml/s) resulted in a time- and shear stress-dependent (10-180 dyn/cm2) detachment of cells, with the cells on EDA depicting higher resistance to wall shear stress. Cellular morphology correlated with the strength of adhesion; cells with rounded morphology on a hydrophobic silane, PEDA, were less tightly bound to the silane, while spread cells on a hydrophilic silane, EDA, were more tightly bound. The higher surface free energy of EDA is speculated to influence the increased cell spreading and strength of adhesion observed in these studies. The presence of the phenyl group in PEDA reduces the surface free energy and may account for the reduced spreading and lower strength of adhesion. The use of well-defined systems, such as monolayer organosilanes, with tunable surface physicochemical properties may serve to deconstruct the complex interaction of cells with extracellular matrix components: surface charge, surface hydrophobicity, and other short- and long-range forces can be individually controlled and correlated with cellular functions. The organosilane monolayers could serve as the building blocks for sequential addition of proteins or cell adhesive/cell repulsive cues to stepwise engineering and construction of more complex systems resembling ECM matrices.

Biocompatible Materials↗

PubMed1982

Decrease in protein content and cell volume of cultured dog kidney epithelial cells during growth.

Protein content per cell and cell volume of the canine kidney epithelial cell line, MDCK, and one of its chemically induced tumorigenic transformants (T1k) were examined during growth in serum-containing and serum-free, hormonally defined media. Both protein content per cell and cell volume (measured with 3-O-methyl-D-glucose) decreased when cell density increased. Significant inverse linear correlations were found between protein content (and cell volume) and the log of the cell number. Equations relating these variables were derived. No difference was detected between cells grown in serum-containing and in serum-free media. Results of 22Na+ uptake experiments differed markedly when expressed as a function of cell number or as a function of protein content or cell volume. However, protein content and cell volume bore a constant relationship to one another, and expression of results as a function of protein content or cell volume yielded qualitatively similar results. These findings show that in MDCK cells, as in other cell lines, protein content and cell volume decrease markedly during growth and that these variations must be taken into account when expressing and interpreting the results of transport measurements.

Animals↗

PubMed2004

The genes for schizophrenia: finally a breakthrough?

A number of susceptibility genes for schizophrenia have recently been identified. They have engendered excitement because replicate studies have attained greater consistency than in the past. In this review, we outline gene mapping methods, and briefly review their strengths and challenges. We also evaluate peer-reviewed genetic association studies that have implicated six selected genes: catechol-O-methyl transferase (COMT), neuregulin 1 (NRG1), dysbindin (DTNBP1), regulator of G-protein signaling 4 (RGS4), and G72 and D-amino-acid oxidase (DAAO). The available supporting evidence is variable. Though credible evidence is available for all of these genes, it is strongest for NRG1 and DTNBP1. Further studies, particularly exhaustive analyses of all polymorphisms at each locus, meta-analyses, and investigations of the likely function of risk alleles (variants) are desirable.

Carrier Proteins↗

PubMed1997

Estrogen as a neuromodulator of MPTP-induced neurotoxicity: effects upon striatal dopamine release.

The effects of estrogen upon MPTP-induced neurotoxicity were examined using in vitro superfusion. In Experiment 1, striatal tissue from ovariectomized rats was infused with MPP+ (10 microM), a combination of MPP+ and 17beta-estradiol (300 nM), the same dose of estradiol preceding MPP+, or no treatment infusion. The effects of these treatments on dopamine release rates during the infusion periods were determined. Infusion of MPP+ resulted in a significant increase in dopamine release as compared to the control. Estradiol added to the MPP+ infusion significantly attenuated this dopamine (DA) release, while estradiol treatment preceding the MPP+ had no effect. In Experiment 2, three different doses of estradiol (0.3, 3, or 300 nM) were infused simultaneously with the MPP+. Doses of estradiol below 300 nM did not attenuate the DA release. In Experiment 3, estradiol alone (300 nM) was infused, to determine dopamine release rate effects of the hormone itself. There was no difference between estradiol treated and non-infused control groups. These results demonstrate that the gonadal steroid hormone estradiol can modulate responses of striatal dopamine neurons to MPP+ by altering the immediate increase in dopamine release which occurs in response to this neurotoxin. These modulating effects of estradiol are dose-dependent, and represent a direct effect upon striatal neurons, most likely involving a non-genomic mechanism of action. These results implicate that hormonal modulation of nigrostriatal dopaminergic neurotoxicity may represent an important variable responsible for the sex differences which are reported in Parkinson's disease.

1-Methyl-4-phenylpyridinium↗

PubMed1989

Neuroprotective effect of MK-801 and U-50488H after contusive spinal cord injury.

One hour before a contusive spinal cord injury either compound MK-801 or compound U-50488H was injected intraperitoneally, and a 14-day-delivery osmotic minipump containing the same drug was placed subcutaneously at the time of surgery. The motor and sensory behavior of the animals was measured over the following 30 days. Both MK-801 and U-50488H treatments had a statistically significant neuroprotective effect. The number of neurons per unit area outside the lesion epicenter was significantly (P less than 0.01) greater in the drug-treated animals (MK-801, 298.9 +/- 74.8 neurons/mm2; U-50488H, 242.7 +/- 16.5 neurons/mm2) than in untreated controls (73.3 +/- 9.3 neurons/mm2). Recovery of sensory and motor behavior was limited but significant differences were observed when drug-treated rats were compared with untreated controls. The effects of the two drugs were not additive for any of the variables studied.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗

PubMed1997

Sources of variability in data from a positive selection lacZ transgenic mouse mutation assay: an interlaboratory study.

Experimental features of a positive selection transgenic mouse mutation assay based on a lambda lacZ transgene are considered in detail, with emphasis on results using germ cells as the target tissue. Sources of variability in the experimental protocol that can affect the statistical nature of the observations are examined, with the goal of identifying sources of excess variation in the observed mutant frequencies. The sources include plate-to-plate (within packages), package-to-package (within animals), and animal-to-animal variability. Data from five laboratories are evaluated in detail. Results suggest only scattered patterns of excess variability below the animal-to-animal level, but, generally, significant excess variability at the animal-to-animal level. Using source of variability analyses to guide the choice of statistical methods, control-vs-treatment comparisons are performed for assessing the male germ cell mutagenicity of ethylnitrosourea (ENU), isopropyl methanesulfonate (iPMS), and methyl methanesulfonate (MMS). Results on male germ cell mutagenesis of ethyl methanesulfonate (EMS) and methylnitrosourea (MNU) are also reported.

Animals↗