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Biomedical subjects

M Metzler

Publications and source records attributed to M Metzler.

At least 19 recordsLinked to original sources
PubMed1999

Life without huntingtin: normal differentiation into functional neurons.

Huntington disease (HD) is a neurodegenerative disorder associated with polyglutamine expansion in a recently identified protein, huntingtin. Huntingtin is widely expressed and plays a crucial role in development, because gene-targeted HD-/- mouse embryos die early in embryogenesis. To analyze the function of normal huntingtin, we have generated HD-/- embryonic stem (ES) cells and used an in vitro model of ES cell differentiation to analyze their ability to develop into neuronal cells. Expression analysis of wild-type ES cells revealed that huntingtin is expressed at all stages during ES cell differentiation with high expression in neurons. Expression levels increased with the maturation of differentiating neurons, demonstrating that expression of huntingtin is developmentally regulated in cell culture and resembles the pattern of expression observed in differentiating neurons in the mouse brain. It is interesting that HD-/- ES cells could differentiate into mature postmitotic neurons that expressed functional voltage- and neurotransmitter-gated ion channels. Moreover, both excitatory and inhibitory spontaneous postsynaptic currents were observed, indicating the establishment of functional synapses in the absence of huntingtin. These results demonstrate that huntingtin is not required for the generation of functional neurons with features characteristic of postmitotic neurons in the developing mouse brain.

Animals↗
PubMed1998

Studies on the genotoxicity of the mammalian lignans enterolactone and enterodiol and their metabolic precursors at various endpoints in vitro.

The mammalian lignans enterolactone (ENL) and enterodiol (END) are formed by intestinal bacteria from the plant lignans matairesinol (MAT) and secoisolariciersinol (SEC), respectively, which are ingested with different types of food. ENL and END are weak estrogens. According to epidemiological and biochemical studies, lignans may act as anticarcinogens, but little is known about their genotoxic potential. We have therefore investigated the effects of ENL, END, MAT and SEC on cell-free microtubule assembly and at the following genetic endpoints in cultured male Chinese hamster V79 cells: disruption of the cytoplasmic microtubule complex, induction of mitotic arrest, induction of micronuclei and their characterization by CREST staining, and mutagenicity at the HPRT gene locus. The lignans were tested at concentrations of 200 microM in the cell-free system and 100 microM in cultured cells, which represents the limit of solubility in each assay. The established aneuploidogen diethylstilbestrol and the clastogen 4-nitroquinoline-N-oxide were used as positive reference compounds. As none of the four lignans had any activity at the endpoints studied, we conclude that ENL, END, MAT and SEC are devoid of aneuploidogenic and clastogenic potential under the experimental conditions used in this study.

4-Butyrolactone↗
PubMed1998

The influence of huntingtin protein size on nuclear localization and cellular toxicity.

Huntington disease is an autosomal dominant neurodegenerative disorder caused by the pathological expansion of a polyglutamine tract. In this study we directly assess the influence of protein size on the formation and subcellular localization of huntingtin aggregates. We have created numerous deletion constructs expressing successively smaller fragments of huntingtin and show that these smaller proteins containing 128 glutamines form both intranuclear and perinuclear aggregates. In contrast, larger NH2-terminal fragments of huntingtin proteins with 128 glutamines form exclusively perinuclear aggregates. These aggregates can form in the absence of endogenous huntingtin. Furthermore, expression of mutant huntingtin results in increased susceptibility to apoptotic stress that is greater with decreasing protein length and increasing polyglutamine size. As both intranuclear and perinuclear aggregates are clearly associated with increased cellular toxicity, this supports an important role for toxic polyglutamine-containing fragments forming aggregates and playing a key role in the pathogenesis of Huntington disease.

Animals↗
PubMed1998

Dose-response studies of the induction of hyperdiploidy and polyploidy by diethylstilbestrol and 17beta-estradiol in cultured human lymphocytes using multicolor fluorescence in situ hybridization.

Diethylstilbestrol (DES) and 17beta-estradiol (E2) are known inducers of aneuploidy and polyploidy in vivo and in vitro. Isolated human lymphocytes were treated with the stilbene estrogen DES (0.05-50 microM) and the steroid estrogen E2 (0.05-75 microM) in culture. Multicolor fluorescence in situ hybridization (FISH) with DNA probes for the centromere and adjacent heterochromatin regions of chromosomes 1, 9, and 16 was used to detect hyperdiploidy, polyploidy, and chromosomal breakage affecting these chromosomes. Using this FISH technique, significant nonlinear increases in hyperdiploidy were observed with both compounds, whereas no induction of chromosomal breakage affecting the pericentric heterochromatin regions of chromosomes 1, 9, and 16 could be detected. DES induced a maximum of approximately 13% hyperdiploid cells at 30 microM, whereas E2 showed its highest induction at 75 microM with 7% hyperdiploid cells. To distinguish hyperdiploidy from polyploidy, a FISH labeling strategy to detect multiple chromosomes simultaneously was established. Using this approach, we could show that most of the cells showing multiple hybridization regions after treatment with both chemicals were most likely the result of polyploidy rather than true hyperdiploidy. These results indicate that the induction of hyperdiploidy/polyploidy with DES and E2 show sublinear dose-response relationships with likely threshold concentrations in human lymphocytes and that FISH with multiple probes targeting different chromosomes can be used to estimate hyperdiploidy and polyploidy frequencies.

Benzimidazoles↗
PubMed1998

A new fingerprint method for sequence analysis of chromosomal translocations at the genomic DNA level.

Chromosomal rearrangements constitute a significant feature of leukemogenesis and malignant transformation in general. Nucleotide patterns in the immediate vicinity of the break point may provide important information about the underlying causalities, eg illegitimate recombination events mediated by topoisomerase II, Alu repeats, or VDJ recombinase. In order to facilitate the determination of those DNA patterns, we developed a new fingerprint approach. In a first step, two DNA fragments were independently amplified by long distance PCR: the genomic region carrying the break point and the normal nonrearranged counterpart. Subsequently, both PCR products were digested with restriction enzymes, end-labelled with a fluorescent dye, and subjected to high resolution polyacrylamide gel electrophoresis. By comparing the restriction patterns of the rearranged and the nonrearranged PCR fragments, the break points could be easily localized within a size range coverable by a single sequencing reaction. Finally, the exact DNA sequence across the break point was directly determined. The 'fingerprint' technique is fast, reliable and enables the assay of multiple samples in parallel.

Base Sequence↗
PubMed1998

Aneuploidogenic and clastogenic potential of the mycotoxins citrinin and patulin.

The aneuploidogenic and clastogenic potentials of the mycotoxins citrinin (CIT) and patulin (PAT) were studied by determining inhibition of microtubule assembly under cell-free conditions and by measuring induction of mitotic arrest and micronuclei in cultured Chinese hamster V79 cells. Both CIT and PAT inhibited cell-free microtubule polymerization in a concentration-dependent manner. PAT, but not CIT, bound covalently to reactive thiol groups of microtubule proteins. At concentrations without gross cytotoxicity, mitotic arrest and CREST-positive micronuclei, i.e. micronuclei containing whole chromosomes/chromatids, were induced by CIT and PAT in V79 cells. The time course of micronucleus induction and positive CREST staining indicate the aneuploidogenic potential of CIT and PAT. CREST-negative micronuclei, i.e. micronuclei containing acentric chromosomal fragments, were induced by PAT but not by CIT, implying a clastogenic potential of PAT. The aneuploidogenic and clastogenic potential of PAT and the aneuploidogenic potential of CIT may well contribute to the putative carcinogenicity of these mycotoxins in long-term animal studies.

Aneuploidy↗
PubMed1998

The estrogenicity of bisphenol A-related diphenylalkanes with various substituents at the central carbon and the hydroxy groups.

The chemical structure of hydroxylated diphenylalkanes or bisphenols consists of two phenolic rings joined together through a bridging carbon. This class of endocrine disruptors that mimic estrogens is widely used in industry, particularly in plastics. Bisphenol F, bisphenol A, fluorine-containing bisphenol A (bisphenol AF), and other diphenylalkanes were found to be estrogenic in a bioassay with MCF7 human breast cancer cells in culture (E-SCREEN assay). Bisphenols promoted cell proliferation and increased the synthesis and secretion of cell type-specific proteins. When ranked by proliferative potency, the longer the alkyl substituent at the bridging carbon, the lower the concentration needed for maximal cell yield; the most active compound contained two propyl chains at the bridging carbon. Bisphenols with two hydroxyl groups in the para position and an angular configuration are suitable for appropriate hydrogen bonding to the acceptor site of the estrogen receptor. Our data suggest that estrogenicity is influenced not only by the length of the substituents at the bridging carbon but also by their nature. Because diphenylalkane derivatives are widespread and their production and use are increasing, potential exposure of humans to estrogenic bisphenols is becoming a significant issue. The hazardous effects of inadvertent exposure to bisphenol-releasing chemicals in professional workers and the general populations therefore deserve investigation.

Benzhydryl Compounds↗
PubMed1997

Interference with microtubules and induction of micronuclei in vitro by various bisphenols.

Bisphenols, in particular bisphenol-A (BP-A), are monomers of various plastics including polycarbonates and epoxy resins which are used in numerous consumer products. The release of BP-A from some of these materials has recently been reported. BP-A is a weak estrogen and structurally related to the aneuploidogenic stilbene estrogen diethylstilbestrol (DES). We have therefore studied BP-A and four other bisphenols for their aneuploidogenic potential by assaying their (i) interference with the cell-free assembly of microtubules (MT); (ii) disruption of the cytoplasmic MT complex in cultured Chinese hamster V79 cells; (iii) disruption of the mitotic spindle and induction of metaphase arrest in V79 cells; and (iv) induction of micronuclei (MN) in V79 cells. At concentrations without gross cytotoxicity, BP-A as well as its alkyl-fluorinated and ring-methylated analog were active at all endpoints tested, whereas the bisphenol without alkyl groups was completely inactive. 4,4'-Dihydroxybenzophenone was inactive against cell-free and cytoplasmic MT but disrupted the mitotic spindle and induced metaphase arrest and MN. The MN caused by the various bisphenols were analyzed for the presence of kinetochores by staining with CREST antibodies. All induced MN were CREST-positive, implying that they contain whole chromosomes/chromatids. The effects on MT and the induction of metaphase arrest and of CREST-positive MN suggest that the environmental estrogen BP-A and some of its analogs are potential aneuploidogens.

Aneuploidy↗
PubMed1997

Induction of micronuclei, DNA strand breaks and HPRT mutations in cultured Chinese hamster V79 cells by the phytoestrogen coumoestrol.

Coumoestrol (COUM), genistein (GEN) and daidzein (DAI) are major phytoestrogens present in numerous plants eaten by humans and food-producing animals. Little is known about the genotoxicity of these natural compounds. The effects of COUM, GEN and DAI were studied in cultured Chinese hamster V79 cells at various endpoints. None of the substances affected the cytoplasmic microtubule complex or the mitotic spindle. However, COUM and GEN but not DAI proved to be strong inducers of DNA strand breaks and micronuclei containing acentric fragments, as shown with antikinetochore antibodies. The clastogenicity of GEN may be due to its non-intercalative inhibitory effect on topoisomerase II, whereas COUM may act through topoisomerase II inhibition and/or DNA intercalation. COUM was also a clear inducer of hypoxanthine guanine phosphoribosyltransferase (HPRT) mutations in V79 cells; GEN was only marginally active and DAI inactive at this endpoint. This is the first report on the clastogenicity and mutagenicity of COUM in mammalian cells.

Animals↗
PubMed1997

HIP1, a human homologue of S. cerevisiae Sla2p, interacts with membrane-associated huntingtin in the brain.

Huntington disease (HD) is associated with the expansion of a polyglutamine tract, greater than 35 repeats, in the HD gene product, huntingtin. Here we describe a novel huntingtin interacting protein, HIP1, which co-localizes with huntingtin and shares sequence homology and biochemical characteristics with Sla2p, a protein essential for function of the cytoskeleton in Saccharomyces cerevisiae. The huntingtin-HIP1 interaction is restricted to the brain and is inversely correlated to the polyglutamine length in huntingtin. This provides the first molecular link between huntingtin and the neuronal cytoskeleton and suggests that, in HD, loss of normal huntingtin-HIP1 interaction may contribute to a defect in membrane-cytoskeletal integrity in the brain.

Amino Acid Sequence↗
PubMed1996

Interaction of p-benzoquinone and p-biphenoquinone with microtubule proteins in vitro.

p-benzoquinone (BQ) and p-biphenoquinone (BPQ) are metabolites of the human myelotoxin and leukemogen benzene, which has been reported to induce aneuploidy in mammalian cells. Because a possible mechanism for the aneuploidogenic effect of benzene may be the disruption of the mitotic spindle by covalent binding of BQ and BPQ to microtubule proteins (MTP), we have studied the reaction of these quinones with MTP and its consequences for microtubule (MT) formation under cell-free conditions. Both BQ and BPQ inhibit the assembly of MTP to MT in a concentration-dependent manner. This interaction is accompanied by a spectral change of the quinones and loss of free sulfhydryl groups of MTP. With 40 microM BQ or BPQ, 50% inhibition of MT assembly was observed and associated with the loss of 1.3 thiol groups per tubulin dimer. Further analysis showed that native MTP form monoadducts, but no diadducts nor disulfide bonds with both BQ and BPQ. The formation of covalent quinone/MTP monoadducts was unequivocally demonstrated by GC/MS analysis of the respective thioanisols liberated by alkaline permethylation. Denatured MTP or glutathione or 2-mercaptoethanol gave rise to the virtually exclusive formation of monoadducts with BQ but led to a high proportion of disulfide bonds with BPQ. Therefore, BQ and BPQ react differently with thiol compounds capable of disulfide bond formation. The fact that both quinones form only monoadducts with native MTP can be explained by the assumption that the thiol groups of native tubulin are not prone to oxidative disulfide bond formation. This proposition was supported by the lack of native MTP to form disulfide bridges upon treatment with hydrogen peroxide/horseradish peroxidase under conditions leading to a complete oxidation of glutathione. The covalent binding of the benzene metabolites BQ and BPQ to critical thiol groups of tubulin inhibits MT formation under cell-free conditions and may also interfere with the formation of a functional spindle apparatus in the mitotic cell, thus leading to the abnormal chromosome segregation and aneuploidy induction reported for benzene.

Animals↗
PubMed1996

Effects of novel 3,4-bisphenylhex(3)enes on cell proliferation in malignant and normal cells.

The growth-inhibitory effect of several newly synthesized alkyl derivatives of 3,4-bisphenylhex(3)ene was studied in four tumor cell lines and three healthy primary cell systems. A marked inhibition of cell proliferation was noted in the neoplastic cells but not in the primary systems. No effect on the cytoplasmic or mitotic microtubule system but an increase in the gross level of 5-methylcytosine in nuclear DNA was observed. It is speculated that the selective growth inhibition of tumor cells is due to DNA-hypermethylation.

5-Methylcytosine↗
PubMed1996

Absence of disease phenotype and intergenerational stability of the CAG repeat in transgenic mice expressing the human Huntington disease transcript.

The mutation underlying Huntington disease (HD) is CAG expansion in the first exon of the HD gene. In order to investigate the role of CAG expansion in the pathogenesis of HD, we have produced transgenic mice containing the full length human HD cDNA with 44 CAG repeats. By 1 year, these mice have no behavioral abnormalities and morphometric analysis at 6 (one animal) and 9 (two animals) months age revealed no changes. Despite high levels of mRNA expression, there was no evidence of the HD gene product in any of these transgenic mice. In vitro transfection studies indicated that the inclusion of 120 bp of the 5' UTR in the cDNA construct and the presence of a frameshift mutation at nucleotide 2349 prevented expression of the HD cDNA. These findings suggest that the pathogenesis of HD is not mediated through DNA-protein interaction and that presence of the RNA transcript with an expanded CAG repeat is insufficient to cause the disease. Rather, translation of the CAG is crucial for the pathogenesis of HD. In contrast to that seen in humans, the CAG repeat in these mice was remarkably stable in 97 meioses. This suggests that genomic sequences may play a critical role in influencing repeat instability.

Animals↗
PubMed1995

Separation and identification of phase I and phase II [14C]antipyrine metabolites in rat and dog urine.

A simple and accurate HPLC procedure was developed to quantify, in a single run, all phase I and phase II [14C]antipyrine metabolites that occur in rat and dog urine. All metabolites were subjected to thermospray-LC-MS and EI-MS in order to establish their structure. The rat metabolizes antipyrine to eight major metabolites, six of which are conjugated; 1.4% of the dose was excreted unchanged, 18.9% in a free form, 30.6% as sulfates and 21.1% as glucuronides. The dog metabolizes antipyrine to four metabolites, all as sulfate (61.0% of the dose) or glucuronide conjugates (16.2% of the dose).

Animals↗
PubMed1995

Complex asparagine-linked oligosaccharides in Mgat1-null embryos.

To investigate the developmental role of complex N-linked oligosaccharides, we previously inactivated the mouse Mgat1 gene which encodes UDP-N-acetylglucosamine: alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I (GlcNAc-TI). Mgat1-null embryos developed morphogenic abnormalities by embryonic day (E) 9.5 and failed to survive beyond E10.5. Prior to E8.5, mutant and wild-type embryos were phenotypically indistinguishable, raising the unexpected possibility that earlier embryonic development may not require complex N-glycans. We have now used in situ RNA hybridization to assess the temporal and spatial pattern of Mgat1 expression in normal embryos, and lectin histochemistry to determine whether Mgat1-null embryos lack complex N-glycans at pre-E9.5 developmental stages. In situ RNA analysis indicated that Mgat1 transcripts normally increase dramatically between E7.0 and E9.5, 1-2 days prior to the death of mutant embryos. However, apparently normal levels of complex N-glycans were observed in E3.5 pre-implantation Mgat1-null embryos prior to declining to undetectable levels by E7.5. Complex N-glycans were not observed in E7.5-E9.5 Mgat1-null embryos with the notable exception of vesicular structures within cells of the visceral extra-embryonic endoderm, perhaps reflecting the ability of these cells to take up and transport maternally derived glycoproteins. Mgat1-null embryos appear to complete pre-implantation development in the presence of maternally derived complex N-glycans, and may die at later stages, post E7.5, when a requirement for embryonically derived complex N-glycans arises.

Animals↗