Biomedical subjects

M Metzler

Publications and source records attributed to M Metzler.

At least 37 records · Page 2Linked to original sources

PubMed2000

Huntingtin is required for normal hematopoiesis.

Huntington's disease (HD) is a neurodegenerative disease associated with polyglutamine expansion in huntingtin, a widely expressed protein. The function of huntingtin is unknown although huntingtin plays a fundamental role in development since gene targeted HD (-) (/-)mouse embryos die shortly after gastrulation. Expression of huntingtin is detected in spleen and thymus but its role in hematopoiesis has not been examined. To determine the function of huntingtin and to provide insight into potential pathologic mechanisms in HD, we analyzed the role of huntingtin in hematopoietic development. Expression of huntingtin was analyzed in a variety of hematopoietic cell types, and in vitro hematopoiesis was assessed using an HD ( +/-)and several HD( -) (/-)embryonic stem (ES) cell lines. Although wild-type, HD ( +/-)and HD( -) (/-)ES cell lines formed primary embryoid bodies (EBs) with similar efficiency, the numbers of hematopoietic progenitors detected at various stages of the in vitro differentiation were reduced in HD ( +/-)and HD( -/-)() ()ES cell lines examined. Expression analyses of hematopoietic markers within the EBs revealed that primitive and definitive hematopoiesis occurs in the absence of huntingtin. However, further analysis using a suspension culture in the presence of hematopoietic cytokines demonstrated a highly significant gene dosage-dependent decrease in proliferation and/or survival of HD ( +/-)and HD( -) (/-)cells. Enrichment for the CD34(+)cells within the EB confirmed that the impairment is intrinsic to the hematopoietic cells. These obser- vations suggest that huntingtin expression is required for the generation and expansion of hematopoietic cells and provides an alternative system in which to assess the function of huntingtin.

Animals↗

PubMed2000

HIP12 is a non-proapoptotic member of a gene family including HIP1, an interacting protein with huntingtin.

Huntingtin-interacting protein I (HIP1) is a membrane-associated protein that interacts with huntingtin, the protein altered in Huntington disease. HIP1 shows homology to Sla2p, a protein essential for the assembly and function of the cytoskeleton and endocytosis in Saccharomyces cerevisiae. We have determined that the HIP1 gene comprises 32 exons spanning approximately 215 kb of genomic DNA and gives rise to two alternate splice forms termed HIP1-1 and HIP1-2. Additionally, we have identified a novel protein termed HIP12 with significant sequence and biochemical similarities to HIP1 and high sequence similarity to Sla2p. HIP12 differs from HIP1 in its pattern of expression both at the mRNA and protein level. However, HIP1 and HIP12 are both found within the brain and show a similar subcellular distribution pattern. In contrast to HIP1, which is toxic in cell culture, HIP12 does not confer toxicity in the same assay systems. Interestingly, HIP12 does not interact with huntingtin but can interact with HIP1. suggesting a potential interaction in vivo that may influence the function of each respective protein.

Alternative Splicing↗

PubMed2000

Oxidative metabolites of the mammalian lignans enterodiol and enterolactone in rat bile and urine.

Recent studies have shown that the mammalian lignans enterodiol (END) and enterolactone (ENL) are biotransformed in vitro by hepatic microsomes from rats and humans to various metabolites carrying one additional hydroxy group either at the aromatic or at the aliphatic moiety. To clarify whether these metabolites are also formed in vivo, each lignan was administered intraduodenally at a dose of 10 mg/kg of bw to bile duct-catheterized female Wistar rats and the 6 h bile analyzed by HPLC and GC-MS. With END-dosed rats, three products of aromatic and two of aliphatic monohydroxylation were found, whereas six aromatic and five aliphatic monohydroxylated biliary metabolites were detected after administration of ENL. The metabolites hydroxylated at the aromatic rings were unequivocally identified by comparison with synthetic reference compounds. The structures of the in vivo metabolites arising from aliphatic hydroxylation could not be completely elucidated; they were identical with some of the formerly reported microsomal products according to GC retention times and mass spectra. Significant amounts of most of the metabolites of the mammalian lignans identified in bile were also found in the urine of female rats after oral administration of 10 mg/kg of bw END or ENL and in the urine of female and male Wistar rats after they had been fed a diet containing 5% flaxseed. Thus, the mammalian lignans END and ENL give rise to several hydroxylated metabolites in vivo, which may contribute to the biological effects of these important food constituents.

4-Butyrolactone↗

PubMed2000

Oxidative in vitro metabolism of the soy phytoestrogens daidzein and genistein.

The oxidative metabolism of the major soy isoflavones daidzein and genistein was investigated using liver microsomes from Aroclor-treated male Wistar rats. Both daidzein and genistein were extensively metabolized and are therefore excellent substrates for cytochrome P450 enzymes. The identity of the metabolites was elucidated using high-performance liquid chromatography (HPLC) with diode array detection, gas chromatography-mass spectrometry (GC/MS), and HPLC/atmospheric pressure ionization electrospray mass spectrometry (API-ES MS) as well as reference substances. Daidzein was converted to nine metabolites, comprising four monohydroxylated, four dihydroxylated, and one trihydroxylated metabolite. Genistein was metabolized to four monohydroxylated and two dihydroxylated products. With both isoflavones the additional hydroxy groups are exclusively introduced into the ortho positions of existing phenolic hydroxy groups. The major metabolites of daidzein were identified as 6,7,4'-trihydroxyisoflavone, 6,7,3',4'-tetrahydroxyisoflavone, 7,8, 4'-trihydroxyisoflavone, and 5,6,7,4'-tetrahydroxyisoflavone. The main microsomal metabolites of genistein were 5,6,7, 4'-tetrahydroxyisoflavone and 5,7,8,4'-tetrahydroxyisoflavone. Furthermore, the GC/MS and HPLC/API-ES MS analysis support the conclusion that one monohydroxylated metabolite of daidzein and genistein is hydroxylated at the aliphatic position C-2 of the C-ring. The UV-vis, GC/MS, and HPLC/MS data of all detected metabolites as well as the derived chemical structure of the metabolites are presented. Most metabolites are reported in this paper for the first time. On the basis of these findings it is suggested that hydroxylation reactions may also play an important role in the in vivo metabolism of the soy isoflavones daidzein and genistein.

Animals↗

PubMed2000

Electrophilic properties of patulin. Adduct structures and reaction pathways with 4-bromothiophenol and other model nucleophiles.

The mycotoxin patulin (PAT) is believed to exert its cytotoxic and chromosome-damaging effects by forming covalent adducts with essential cellular thiols. Since the chemical structures of such adducts are unknown to date, we have studied the reaction of PAT and its O-acetylated derivative with the monofunctional thiol model compound 4-bromothiophenol (BTP), which was chosen due to analytical advantages. By means of analytical and preparative high-performance liquid chromatography, 16 adducts of PAT and 3 adducts of acetyl-PAT were isolated and their chemical structures elucidated by (1)H and (13)C NMR, IR, and UV spectroscopy. Time course studies and analysis of daughter product formation from isolated intermediate adducts led to a detailed scheme for the reaction of PAT with BTP. The structures of adducts of PAT formed with other model nucleophiles, e. g., the aliphatic thiol 2-mercaptoethanol and the aromatic amine 4-bromoaniline, were also elucidated and found to corroborate the reaction scheme. In addition, one further reaction pathway was observed with 2-mercaptoethanol, which appears to be independent from those found for BTP. Our study with model nucleophiles provides insights into the electrophilic reactivity of PAT and proved to be useful for the structure elucidation of PAT adducts with biological nucleophiles of toxicological relevance, as will be reported by Fliege and Metzler [(2000) Chem. Res. Toxicol. 13, 373-381].

Aniline Compounds↗

PubMed2000

Electrophilic properties of patulin. N-acetylcysteine and glutathione adducts.

In our studies on the electrophilic properties of the mycotoxin patulin (PAT), we have now investigated the nonenzymatic reaction of PAT with the thiol-containing tripeptide glutathione and its metabolic degradation product N-acetyl-L-cysteine (NAC). Adduct formation in aqueous phosphate buffer (pH 7.4) was studied by analytical HPLC/DAD, and most of the products were isolated by preparative HPLC. Structure elucidation was carried out mainly by means of high-resolution NMR experiments and comparison of the data with those previously obtained for PAT adducts formed with simple model nucleophiles such as 4-bromothiophenol and 2-mercaptoethanol [Fliege, R., and Metzler, M. (2000) Chem. Res. Toxicol. 13, 363-372]. The assigned structures were confirmed by UV spectroscopy, formation of daughter products from isolated adducts, and partly FAB-MS. The reaction pathways of PAT with NAC were qualitatively the same as those previously observed for the aliphatic thiol model compound 2-mercaptoethanol. Due to the chiral nature of NAC and the new chiral center generated during the reaction with PAT, two diastereomers of each adduct were formed and observed in HPLC analysis. The major products formed in the reaction of PAT with GSH were of the same structural type as obtained with NAC. In addition, three cyclic adducts were formed with GSH, arising from the nucleophilic activity of the alpha-amino groups of the glutamic acid and the cysteine residue. In contrast, free cysteine yielded a markedly different adduct pattern, possibly due to the preferred formation of mixed thiol/amine-type adducts involving the alpha-amino group.

Acetylcysteine↗

PubMed2000

Nuclear transport of oligonucleotides in HepG2-cells mediated by protamine sulfate and negatively charged liposomes.

PURPOSE: The aim of this study was to characterize the intracellular fate and nuclear uptake kinetics of oligonucleotides (ON) that were complexed with protamine sulfate (PS) and negatively charged liposomes at different ratios of ON to PS. METHODS: Double-fluorescence labelling of ON and liposomal lipid was applied to simultaneously monitor the interaction as well as the individual fate of active agent and carrier upon intracellular delivery using confocal laser scanning microscopy (CLSM). A DNA-analogue of a 68-mer intramolecular double-stranded RNA:DNA-hybridoligonucleotide (chimeraplasts) with unmodified phosphate backbone was employed. This construct was condensed with PS and coated with a liposomal formulation (AVE-3 = artificial viral envelope). RESULTS: PS-ON complexes and AVE -3-coated complexes with a defined composition were very effective in nuclear transport of ON for a ON:PS charge ratio of 1:3. Nucleus:cytosol fluorescence ratios peaked at about 10 hrs and started to decrease again at 21 hrs. CONCLUSIONS: AVE associates with PS-condensed ON, and this complex is able to be taken up by cells and to deliver ON to the nucleus. PS-ON complexes are released from the liposomal formulation, mainly as an extranuclear enzymatic degradation of the liposomal phospholipids. The results of the kinetic analysis can be used to optimize transfection protocols with ON in HepG2 cells.

Anions↗

PubMed2000

Quantitative DNA fragment analysis for detecting low amounts of hepatitis B virus deletion mutants in highly viremic carriers.

Many variants of hepatitis B virus (HBV) with deletions in the viral genome have been identified. Some of these variants are indicator or even effector of a more severe course of hepatitis. These deletion mutants contribute a variable and sometimes very low proportion to the viral population. For early detection of small amounts of deletion mutants among a large number of wild-type genomes, we applied a new screening method designated quantitative fragment analysis (QFA). By QFA the whole viral genome can be scanned for the presence of deletions or insertions of >/=3 nucleotides representing more than 2% of the viral population. Using QFA we showed that an often described deletion of 8 nucleotides is packaged in viral capsids and not a polymerase chain reaction (PCR) artifact. QFA was applied to study the emergence of deletion mutants in a group of 18 pediatric patients who had been infected from a common source while being under multidrug cancer chemotherapy. All patients had developed a highly viremic asymptomatic HBV carrier state. In 3 of these patients 3 different kinds of HBV deletion mutants were found by QFA: 8 bp deletions within the core promoter, core gene deletions from 8 to 86 bp, and large deletions of up to 1,989 bp spanning the precore/core and the preS/S reading frames. PCR primers that specifically amplify deletion variants enabled the detection of additional patients harboring the investigated variant.

Adolescent↗

PubMed2000

Neutral wrist splinting in carpal tunnel syndrome: a comparison of night-only versus full-time wear instructions.

OBJECTIVE: To compare the effects of night-only to full-time splint wear instructions on symptoms, function, and impairment in carpal tunnel syndrome (CTS). DESIGN: Randomized clinical trial with 6-week follow-up. SETTING: Veterans Administration Medical Center, outpatient clinic. SUBJECTS: Outpatients with untreated CTS were consecutively recruited from our electrodiagnostics lab. Twenty-one patients (30 hands) were enrolled, and 17 patients (24 hands) completed the study. INTERVENTIONS: Thermoplastic, custom-molded, neutral wrist splints with subjects receiving either full-time or night-only wear instructions. OUTCOME MEASURES: Symptoms and functional deficits were measured by Levine's self-administered questionnaire, and physiologic impairment was measured by median nerve sensory and motor distal latency. COMPLIANCE AND CROSSOVER: Almost all (92%) of the combined sample reported frequent splint use, but their adherence to specific wearing instructions was limited. A majority (73%) of the full-time group reported splint wear less than one half of waking hours, and some (23%) of the night-only group reported occasional daytime wear. Despite this tendency for treatment crossover, the two treatment groups differed in daytime wear as intended (chi2 analysis, p = .004). RESULTS: The combined sample improved in three of four outcome measures: sensory distal latency (mean = .28msec, standard deviation [SD] = .37, p = .004), symptom severity (mean = .64, SD = .46, p = .0001), and functional deficits (mean = .49, SD = .51, p = .0001). Severity of CTS was a factor only in sensory distal latency improvement (more improvement in severe CTS). Subjects receiving full-time wear instructions showed superior distal latency improvement, both motor (.35 vs -.07msec, p = .04) and sensory (.46 vs . 13msec, p = .05) when compared with subjects receiving night-only wear instructions. CONCLUSIONS: This study provides added scientific evidence to support the efficacy of neutral wrist splints in CTS and suggests that physiologic improvement is best with full-time splint wear instructions.

Adult↗

PubMed1999

The mycotoxin patulin induces intra- and intermolecular protein crosslinks in vitro involving cysteine, lysine, and histidine side chains, and alpha-amino groups.

As previous studies have indicated a multiple electrophilic reactivity of patulin (PAT) towards simple thiol nucleophiles, we have methodically investigated the ability of PAT to covalently crosslink proteins in vitro. By means of sodium dodecylsulphate polyacrylamide gel electrophoresis, the formation of PAT-induced intermolecular protein-protein crosslinks was clearly demonstrated for bovine serum albumin containing one thiol group per molecule, but also for the thiol-free hen egg lysozyme. Characterization of the crosslink sites was carried out by (1) modulation of the thiol groups with N-ethylimaleimide and 2-iminothiolane; (2) comparison with various known crosslinking agents, i.e. phenylenedimaleimide, glutardialdehyde, and dimethylsuberimidate, and (3) fluorescence incorporation studies using dansyl-labeled amino acids and a fluorescent glutathione derivative. The thiol group of cysteine was preferred for PAT-mediated crosslink reactions, but the side chains of lysine and histidine, and alpha-amino groups also exhibited reactivity. PAT can act both as a homobifunctional as well as a heterobifunctional crosslinking agent. The initial formation of a monoadduct with a thiol group appears to activate PAT for the subsequent reaction with an amino group, but also leads to rapid loss of further electrophilic properties when no second nucleophile for crosslink completion is available. Studies using microtubule proteins as a protein with experimentally controllable quarternary structure and a proposed cellular target for PAT toxicity emphasized the influence of specific sterical conditions on crosslink formation at low protein concentrations. Non-polymerized microtubule proteins, i.e. tubulin alpha,beta-dimers, formed a defined product with PAT consisting of an intramolecularly crosslinked beta-tubulin, whereas guanosine triphosphate- or paclitaxel-induced polymerization to microtubule-like quarternary structures prior to treatment with PAT gave rise to intermolecular crosslink formation between alpha- and beta-tubulin. In contrast, denaturated tubulin yielded none of those two new protein species, but only unspecific intramolecular crosslinks and highly crosslinked aggregates. Thus, in addition to the amino acid composition, the tertiary and quarternary superstructures of proteins appear to markedly influence their reactivity towards PAT. Under appropriate conditions, the generation of protein crosslinks could easily be observed at concentrations of PAT equal to or even below the concentration of the protein. The relevance of these novel reaction pathways of PAT demonstrated in vitro for its in vivo mechanisms of toxicity remains to be investigated.

Animals↗

PubMed1999

The phytoestrogens coumoestrol and genistein induce structural chromosomal aberrations in cultured human peripheral blood lymphocytes.

The clastogenic potential of the phytoestrogens coumoestrol (COUM), genistein (GEN) and daidzein (DAI) has been studied in human peripheral blood lymphocytes in vitro. After exposure of the cultured lymphocytes to 50 to 75 microM COUM or 25 microM GEN for 6 h, a clear induction of structural chromosomal aberrations was observed by cytogenetic analysis. The major alterations were chromatid breaks, gaps and interchanges. In contrast, DAI did not induce chromosome aberrations even at 100 microM. These results, together with previously published reports on the induction of micronuclei and DNA strand breaks in cultured Chinese hamster V79 cells by COUM and GEN, but not DAI, suggest that some but not all phytoestrogens have the potential for genetic toxicity.

Adult↗

PubMed1999

Novel metabolites of the mammalian lignans enterolactone and enterodiol in human urine.

Enterolactone (ENL) and enterodiol (END) are found in high concentrations in human body fluids after ingestion of flaxseed and whole-grain products. Although much interest is presently focused on these mammalian lignans because of their putative beneficial health effects, little is known about their metabolic fate in humans. We have now identified nine novel metabolites of ENL and END in the urine of female and male humans ingesting flaxseed for five days. The chemical structures of six ENL metabolites and of three END metabolites were elucidated by GC/MS analysis and comparison with authentic reference compounds obtained by chemical synthesis. The six identified metabolites of ENL were the products of monohydroxylation at the para-position and at both ortho-positions of the parent hydroxy group of either aromatic ring. Likewise, the three END metabolites were formed through aromatic monohydroxylation at the para- and ortho-positions. The biological significance of these metabolites remains to be established.

4-Butyrolactone↗

PubMed1999

Oxidative metabolism of the mammalian lignans enterolactone and enterodiol by rat, pig, and human liver microsomes.

Hepatic microsomes from aroclor-treated male Wistar rats biotransform enterolactone to 12 metabolites, six of which carry an additional hydroxy group at the aromatic and six at the aliphatic moiety according to HPLC/MS and GC/MS analysis. The aromatic hydroxylation products were identified with the help of synthesized reference compounds as enterolactone monohydroxylated in the para position and in both ortho positions of the original phenolic hydroxy group of either aromatic ring. The synthesis of the reference compounds and their spectroscopic characterization is described. Enterodiol is metabolized by hepatic microsomes from aroclor-treated male rats to three aromatic and four aliphatic monohydroxylated metabolites. Aromatic hydroxylation occurs in the para position and the two ortho positions of the original phenolic hydroxy group. Most of the metabolites of enterolactone and enterodiol were also formed with microsomes from uninduced rat, pig, and human liver, suggesting that oxidative metabolism is a common feature in the disposition of these lignans in the mammalian organism.

4-Butyrolactone↗

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↗

PubMed1999

Subtype-specific enhancement of NMDA receptor currents by mutant huntingtin.

Evidence suggests that NMDA receptor-mediated neurotoxicity plays a role in the selective neurodegeneration underlying Huntington's disease (HD). The gene mutation that causes HD encodes an expanded polyglutamine tract of >35 in huntingtin, a protein of unknown function. Both huntingtin and NMDA receptors interact with cytoskeletal proteins, and, for NMDA receptors, such interactions regulate surface expression and channel activity. To determine whether mutant huntingtin alters NMDA receptor expression or function, we coexpressed mutant or normal huntingtin, containing 138 or 15 glutamine repeats, respectively, with NMDA receptors in a cell line and then assessed receptor channel function by patch-clamp recording and surface expression by western blot analysis. It is interesting that receptors composed of NR1 and NR2B subunits exhibited significantly larger currents when coexpressed with mutant compared with normal huntingtin. Moreover, this effect was selective for NR1/NR2B, as NR1/NR2A showed similar currents when coexpressed with mutant versus normal huntingtin. However, ion channel properties and total surface expression of the NR1 subunit were unchanged in cells cotransfected with NR1/NR2B and mutant huntingtin. Our results suggest that mutant huntingtin may increase numbers of functional NR1/NR2B-type receptors at the cell surface. Because NR1/NR2B is the predominant NMDA receptor subtype expressed in medium spiny neostriatal neurons, our findings may help explain the selective vulnerability of these neurons in HD.

Cell Line↗

PubMed1999

Multicenter clinical trial of recombinant human insulin-like growth factor I in patients with acute renal failure.

BACKGROUND: Patients with acute renal failure (ARF) have high morbidity and mortality rates, particularly if they have serious comorbid conditions. Several studies indicate that in rats with ARF caused by ischemia or certain nephrotoxins, insulin-like growth factor-I (IGF-I) enhances the recovery of renal function and suppresses protein catabolism. METHODS: Our objective was to determine whether injections of recombinant human IGF-I (rhIGF-I) would enhance the recovery of renal function and is safe in patients with ARF. The study was designed as a randomized, double-blind, placebo-controlled trial in intensive care units in 20 teaching hospitals. Seventy-two patients with ARF were randomized to receive rhIGF-I (35 patients) or placebo (37 patients). The most common causes of ARF in the rhIGF-I and placebo groups were, respectively, sepsis (37 and 35% of patients) and hypotension or hemodynamic shock (42 and 27% of patients). At baseline, the mean (+/- SD) APACHE II scores in the rhIGF-I and placebo-treated groups were 24 +/- 5 and 25 +/- 8, respectively. In the rhIGF-I and placebo groups, the mean (median) urine volume and urinary iothalamate clearances (glomerular filtration rate) were 1116 +/- 1037 (887) and 1402 +/- 1183 (1430) ml/24 hr and 6.4 +/- 5.9 (4.3) and 8.7 +/- 7.2 (4.4) ml/min and did not differ between the two groups. Patients were injected subcutaneously every 12 hours with rhIGF-I, 100 microgram/kg desirable body weight, or placebo for up to 14 days. Injections were started within six days of the onset of ARF. The primary end-point was a change in glomerular filtration rate from baseline. Other end points included changes from baseline in urine volume, creatinine clearance and serum urea, creatinine, albumin and transferrin, frequency of hemodialysis or ultrafiltration, and mortality rate. RESULTS: During the treatment period, which averaged 10.7 +/- 4.1 and 10.6 +/- 4.5 days in the rhIGF-I and placebo groups, there were no differences in the changes from baseline values of the glomerular filtration rate, creatinine clearance, daily urine volume, or serum urea nitrogen, creatinine, albumin or transferrin. In patients who did not receive renal replacement therapy, there was also no significant difference in serum creatinine and urea between the two groups. Twenty patients in the rhIGF-I group and 17 placebo-treated patients underwent dialysis or ultrafiltration. Twelve rhIGF-I-treated patients and 12 placebo-treated patients died during the 28 days after the onset of treatment. CONCLUSIONS: rhIGF-I does not accelerate the recovery of renal function in ARF patients with substantial comorbidity.

Acute Kidney Injury↗

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↗