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PubMed2001

Oxyanion binding alters conformation and quaternary structure of the c-terminal domain of the transcriptional regulator mode. Implications for molybdate-dependent regulation, signaling, storage, and transport.

The molybdate-dependent transcriptional regulator ModE of Escherichia coli functions as a sensor of intracellular molybdate concentration and a regulator for the transcription of several operons that control the uptake and utilization of molybdenum. We present two high-resolution crystal structures of the C-terminal oxyanion-binding domain in complex with molybdate and tungstate. The ligands bind between subunits at the dimerization interface, and analysis reveals that oxyanion selectivity is determined primarily by size. The relevance of the structures is indicated by fluorescence measurements, which show that the oxyanion binding properties of the C-terminal domain of ModE are similar to those of the full-length protein. Comparisons with the apoprotein structure have identified structural rearrangements that occur on binding oxyanion. This molybdate-dependent conformational switch promotes a change in shape and alterations to the surface of the protein and may provide the signal for recruitment of other proteins to construct the machinery for transcription. Sequence and structure-based comparisons lead to a classification of molybdate-binding proteins.

Amino Acid Sequence↗
PubMed2001

Tungstate Uptake by a highly specific ABC transporter in Eubacterium acidaminophilum.

The Gram-positive anaerobe Eubacterium acidaminophilum contains at least two tungsten-dependent enzymes: viologen-dependent formate dehydrogenase and aldehyde dehydrogenase. (185)W-Labeled tungstate was taken up by this organism with a maximum rate of 0.53 pmol min(-)1 mg(-)1 of protein at 36 degrees C. The uptake was not affected by equimolar amounts of molybdate. The genes tupABC coding for an ABC transporter specific for tungstate were cloned in the downstream region of genes encoding a tungsten-containing formate dehydrogenase. The substrate-binding protein, TupA, of this putative transporter was overexpressed in Escherichia coli, and its binding properties toward oxyanions were determined by a native polyacrylamide gel retardation assay. Only tungstate induced a shift of TupA mobility, suggesting that only this anion was specifically bound by TupA. If molybdate and sulfate were added in high molar excess (>1000-fold), they were also slightly bound by TupA. The K(d) value for tungstate was determined to be 0.5 microm. The genes encoding the tungstate-specific ABC transporter exhibited highest similarities to putative transporters from Methanobacterium thermoautotrophicum, Haloferax volcanii, Vibrio cholerae, and Campylobacter jejuni. These five transporters represent a separate phylogenetic group of oxyanion ABC transporters as evident from analysis of the deduced amino acid sequences of the binding proteins. Downstream of the tupABC genes, the genes moeA, moeA-1, moaA, and a truncated moaC have been identified by sequence comparison of the deduced amino acid sequences. They should participate in the biosynthesis of the pterin cofactor that is present in molybdenum- and tungsten-containing enzymes except nitrogenase.

ATP-Binding Cassette Transporters↗
PubMed2004

Oxidative stress contributes to methotrexate-induced small intestinal toxicity in rats.

BACKGROUND: Gastrointestinal toxicity is one of the most serious side effects in the methotrexate (MTX) treatment. However, the mechanism of the toxicity has not been completely clarified, which may be the reason why symptomatic therapy is carried out. On the other hand, the oxidative stress is known to play an important role in various diseases and drug-induced side effects. In this study the focus was on the oxidative stress in order to clarify the mechanism of MTX-induced small intestinal damage, especially neutrophil infiltration. METHODS: MTX (20 mg/kg body wt) was administered to rats intravenously. Mucosal homogenates were prepared from the small intestine and used for assay of biochemical parameters, by which induction of oxidative stress and neutrophil infiltration were evaluated. N-acetylcysteine (NAC; 80 mg/kg body wt), an antioxidant or sodium tungstate (tungsten; 0.7 g/kg body wt), an inhibitor of xanthine dehydrogenase (XD)/xanthine oxidase (XO) known as an important source of reactive oxygen species (ROS) was given to rats with MTX to investigate the contribution of ROS to neutrophil infiltration. RESULTS: The MTX treatment of rats induced the oxidative stress in the small intestine. The ROS production was seen preceding an increase of myeloperoxidase activity, which suggested neutrophil infiltration. Both treatments of NAC and tungsten prevented the MTX-induced ROS production and neutrophil infiltration. CONCLUSIONS: These results suggest that oxidative stress plays an important role in the MTX-induced small intestinal damage, especially neutrophil infiltration. Thus, the modulation of oxidative stress would be useful in reducing intestinal damage in MTX treatment.

Acetylcysteine↗
PubMed2002

Role of abscissic acid in water stress-induced antioxidant defense in leaves of maize seedlings.

Roles of abscissic acid (ABA) in water stress-induced oxidative stress were investigated in leaves of maize (Zea mays L.) seedlings exposed to water stress induced by polyethylene glycol (PEG 6000). Treatment with PEG at -0.7 MPa for 12 and 24 h led to a reduction in leaf relative water content (RWC) by 7.8 and 14.1%, respectively. Duration of the osmotic treatments is considered as mild and moderate water stress. The mild water stress caused significant increases in the generation of superoxide radical (O2-) and hydrogen peroxide (H2O2), the activities of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR) and the contents of ascorbate (ASC), reduced glutathione (GSH). The moderate water stress failed to further enhance the capacity of antioxidant defense systems, as compared to the mild water stress. The contents of catalytic Fe, which is critical for H2O2-dependent hydroxyl radical (OH) production, and the oxidized forms of ascorbate and glutathione pools, dehydroascorbate (DHA) and oxidized glutathione (GSSG), markedly increased, a significant oxidative damage to lipids and proteins took place under the moderate water stress. Pretreatment with ABA caused an obvious reduction in the content of catalytic Fe and significant increases in the activities of antioxidant enzymes and the contents of non-enzymatic antioxidants, and then significantly reduced the contents of DHA and GSSG and the degrees of oxidative damage in leaves exposed to the moderate water stress. Pretreatment with an ABA biosynthesis inhibitor, tungstate, significantly suppressed the accumulation of ABA induced by water stress, reduced the enhancement in the capacity of antioxidant defense systems, and resulted in an increase in catalytic Fe, DHA and GSSG, and oxidative damage in the water-stressed leaves. These effects were completely prevented by addition of ABA, which raised the internal ABA content. Our data indicate that ABA plays an important role in water stress-induced antioxidant defense against oxidative stress.

Abscisic Acid↗
PubMed1992

Use of fluidizing bed aerosol generators to establish a dust mixture of two substances at a fixed ratio for inhalation toxicity studies.

A method was developed to use two fluidizing bed generators to deliver a mixture of 1 mg cobalt + 15 mg tungsten carbide/m3 to an inhalation exposure chamber with the output from the cobalt generator split to provide the same cobalt concentration to a cobalt-only chamber. To provide a more uniform delivery of material and to minimize the amount of starting dust needed, a subsystem that produced timed bursts of compressed air was used to prevent the accumulation of dust along the aerosol transport tubes. The addition of an electrostatic precipitator placed in the exhaust lines reduced the amount of dust delivered to the high-efficiency particulate air filters, thereby reducing the number of filter changes.

Aerosols↗
PubMed2005

Rapid and simple procedure for homogenizing leaf tissues suitable for mini-midi-scale DNA extraction in rice.

We describe a rapid and simple procedure for homogenizing leaf samples suitable for mini/midi-scale DNA preparation in rice. The methods used tungsten carbide beads and general vortexer for homogenizing leaf samples. In general, two samples can be ground completely within 11.3+/-1.5 sec at one time. Up to 20 samples can be ground at a time using a vortexer attachment. The yields of the DNA ranged from 2.2 to 7.6 microg from 25-150 mg of young fresh leaf tissue. The quality and quantity of DNA was compatible for most of PCR work and RFLP analysis.

DNA, Plant↗
PubMed1986

Reverse transcriptase activity (RTA) in lymphocyte cultures of AIDS patients treated with HPA-23.

HPA-23 was used in a parallel, multiple dose study in patients with Centers for Disease Control (CDC)-defined AIDS. Sixteen patients were divided into four dosage groups, receiving 0.25, 0.5, 1.0, or 2.0 mg/kg HPA-23 respectively, by rapid IV infusion five days/week for eight weeks. Blood was collected before, at weeks 1, 3, and 7 of treatment, and two weeks post-therapy. Patient peripheral blood lymphocytes (PBL) were cultivated in the presence of fresh PBL from a healthy donor for 30 days. Media were changed and reverse transcriptase activity (RTA) was tested every four to five days. The results showed a significant decrease in RTA in patients treated with a dose of 0.5 or 1 mg/kg, but only a slight decrease in patients who received the lowest dose. In the group treated with the 2 mg/kg dose, two patients had toxic reactions and were discontinued; the other two showed a slight decrease in RTA. In 40% of treated patients, RTA did not increase again two weeks after the end of treatment. No significant immunologic and clinical changes were noticed during the observation period. In vitro experiments of Con A stimulated PBL in presence and absence of HPA-23 showed an increase in proliferation in the presence of the drug.

Acquired Immunodeficiency Syndrome↗
PubMed1997

In vitro genotoxic effects of hard metal particles assessed by alkaline single cell gel and elution assays.

Hard metals (WC-Co) are made of a mixture of cobalt metal (Co, 5-10%) and tungsten carbide particles (WC, >80%). Excessive inhalation of WC-Co is associated with the occurrence of different lung diseases including an excess of lung cancers. The elective toxicity of hard metal is based on a physico-chemical interaction between cobalt metal and tungsten carbide particles to produce activated oxygen species. The aim of the present study was to assess the genotoxic activity of hard metal particles as compared with Co and WC alone. In human peripheral lymphocytes incubated with Co or WC-Co, a dose- and time-dependent increased production of DNA single strand breaks (ssb) was evidenced by alkaline single cell gel electrophoresis (SCGE) and modified alkaline elution (AE) assays. Addition of 1 M formate, a hydroxyl radical scavenger, had a protective effect against the production of ssb by both WC-Co or Co alone. On the basis of an equivalent cobalt-content, WC-Co produced significantly more ssb than Co. WC alone did not produce DNA ssb detectable by the AE assay, but results obtained with the SCGE assay may suggest that it either allows some uncoiling of the chromatin loops or induces the formation of slowly migrating fragments. Overall, this in vitro study is the first demonstration of the clastogenic property of cobalt metal-containing dusts. The results are consistent with the implication of an increased production of hydroxyl radicals when Co is mixed with WC particles. The SCGE results also suggest that WC may modify the structure of the chromatin, leading to an increased DNA sensitivity to clastogenic effects. Both mechanisms are not mutually exclusive and may concurrently contribute to the greater clastogenic activity of WC-Co dust. This property of WC-Co particles may account for the excess of lung cancers observed in hard metal workers.

Cobalt↗
PubMed1995

Quantitative and qualitative assessment of enamel surface following five composite removal methods after bracket debonding.

The search for an ideal enamel surface finishing method following bracket debonding has met with equivocal response. In this study, four other methods were assessed to determine their effectiveness against the slow speed tungsten carbide bur which is regarded as the established method of removing residual composite. Orthodontic brackets were bonded on 50 premolar teeth extracted for orthodontic purpose. After debonding with a standardized technique, the teeth were randomly divided into five equal groups. Samples in each group were subjected to different finishing procedures: Group A--Ormco band removing plier. Group B--Komet slow speed tungsten carbide bur. Group C--High speed ultrafine diamond bur. Group D--Jet high speed tungsten carbide bur. Group E--High speed white stone finishing bur. For consistency, the finishing procedures were limited to 15 seconds with each particular method. The composite remnants which then remained on the enamel surface were graded by four independent raters using the Composite Remnant Index (CRI). Subsequent to this, the enamel surface was examined in a Scanning Electron Miscroscope at x203 magnification. The photomicrographs were then graded by the same four independent raters using the Surface Roughness Index (SRI). Statistical analysis with the Friedman test indicated that there was no significant difference in inter-examiner variability in both the CRI and SRI assessment, but there existed significant difference among groups (P < 0.01) for both the CRI and SRI. It was found that no single absolute method was considered to be ideal for composite removal.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of Variance↗
PubMed2004

Enamel surface roughness following debonding using two resin grinding methods.

The purpose of this study was to assess quantitatively the roughness of the enamel surface following debonding using two resin removal methods. The enamel surface of 30 premolar crowns was covered with black tape with a 3 mm window on the middle buccal third to standardize the area of analysis. The initial enamel surfaces were subjected to profilometry, registering four roughness parameters (Ra, Rq, Rt and Rz). The brackets were bonded to the plaster-embedded enamel surfaces with a chemically cured, no-mix adhesive, and debonded after 1 week. Resin removal in half of the specimens was performed with an eight-bladed carbide bur, and in the other half with an ultra-fine diamond bur, both attached to a high speed hand piece; a second profilometric measurement was made after resin removal. Finishing of all surfaces was achieved with Soflex discs and a third registration of roughness followed. The duration of each resin removal protocol was also recorded. The results were analysed with two-way ANOVA and the Newman-Keuls test with the two resin grinding modes and the three intervals serving as discriminating variables (n = 15). For the duration results, a one-way ANOVA was used. Significant differences (P < 0.05) were observed with respect to enamel roughness parameters between the two resin grinding methods used, while there was no consistent roughness-reducing effect of finishing with Soflex discs. Resin removal with a diamond bur was achieved in approximately half the time compared with the eight-bladed bur. The increase in most roughness variables induced by the debonding procedures was not reversed at the end of the finishing stage, regardless of the resin removal protocol used, suggesting an irreversible effect on enamel texture.

Analysis of Variance↗
PubMed1999

Isolation of acetate auxotrophs of the methane-producing archaeon Methanococcus maripaludis by random insertional mutagenesis.

To learn more about autotrophic growth of methanococci, we isolated nine conditional mutants of Methanococcus maripaludis after transformation of the wild type with a random library in pMEB.2, a suicide plasmid bearing the puromycin-resistance cassette pac. These mutants grew poorly in mineral medium and required acetate or complex organic supplements such as yeast extract for normal growth. One mutant, JJ104, was a leaky acetate auxotroph. A plasmid, pWDK104, was recovered from this mutant by electroporation of a plasmid preparation into Escherichia coli. Transformation of wild-type M. maripaludis with pWDK104 produced JJ104-1, a mutant with the same phenotype as JJ104, thus establishing that insertion of pWDK104 into the genome was responsible for the phenotype. pWDK104 contained portions of the methanococcal genes encoding an ABC transporter closely related to MJ1367-MJ1368 of M. jannaschii. Because high levels of molybdate, tungstate, and selenite restored growth to wild-type levels, this transporter may be specific for these oxyanions. A second acetate auxotroph, JJ117, had an absolute growth requirement for either acetate or cobalamin, and wild-type growth was observed only in the presence of both. Cobinamide, 5', 6'-dimethylbenzimidazole, and 2-aminopropanol did not replace cobalamin. This phenotype was correlated with tandem insertions in the genome but not single insertions and appeared to have resulted from an indirect effect on cobamide metabolism. Plasmids rescued from other mutants contained portions of ORFs denoted in M. jannaschii as endoglucanase (MJ0555), transketolase (MJ0681), thiamine biosynthetic protein thiI (MJ0931), and several hypothetical proteins (MJ1031, MJ0835, and MJ0835.1).

ATP-Binding Cassette Transporters↗
PubMed2006

Vanadate but not tungstate prevents the fructose-induced increase in GLUT5 expression and fructose uptake by neonatal rat intestine.

Intermediary signals, precociously enhancing GLUT5 transcription in response to perfusion of its substrate, fructose, in the small intestine of neonatal rats, are not known. Because glucose-6-phosphatase (G6Pase), glucose-6-phosphate translocase (G6PT), and fructose-1,6-bisphosphatase (FBPase) expression increases parallel to or precedes that of GLUT5, we investigated the link between these gluconeogenic genes and GLUT5 by using vanadate or tungstate, potent inhibitors of gluconeogenesis. Small intestinal perfusions of 20-d-old rats were performed with fructose alone, fructose + vanadate or tungstate, glucose alone, and glucose + vanadate or tungstate. As expected, fructose, but not glucose nor glucose + inhibitor perfusion, increased GLUT5 mRNA abundance and fructose transport. Fructose perfusion dramatically increased G6Pase mRNA abundance but had no effect on G6Pase activity. In sharp contrast, fructose perfusion did not increase FBPase gene expression but stimulated FBPase activity. Both vanadate and tungstate significantly inhibited G6Pase activity but did not prevent the fructose-induced increases in G6Pase and G6PT gene expression. Perfusion with fructose + vanadate prevented the fructose-induced increases in fructose transport and GLUT5 mRNA abundance, whereas perfusion with fructose + tungstate did not. Interestingly, vanadate, but not tungstate, inhibited the fructose-induced increase in FBPase activity. Thus, vanadate inhibition of fructose-induced increases in FBPase activity paralleled exactly vanadate inhibition of fructose-induced increases in GLUT5 mRNA abundance and activity. Fructose-induced changes in FBPase activity may regulate changes in GLUT5 expression and activity in the small intestine of neonatal rats. The marked increases in intestinal G6Pase and GLUT5 mRNA abundance may be a parallel response to different factors released during fructose perfusion.

Animals↗
PubMed2002

Independent induction of two blue light-dependent monovalent anion transport systems in the plasma membrane of Monoraphidium braunii.

In the plasma membrane of the green alga Monoraphidium braunii there are at least two monovalent anion transport systems. One of them is specific for bicarbonate. This transport system is activated by blue light and its induction is triggered by a decrease in the external CO2 concentration. The second transport system is responsible for nitrate uptake at least. This transport system is also activated by blue light and its induction occurs when there is no ammonium in the external medium. Both transport systems are synthesized independently. Hence, when M. braunii cells grow with nitrate as the only nitrogen source under high CO2, they have a nitrate transport system but lack a bicarbonate transporter. Conversely, cells grown with ammonium under low CO2, have a bicarbonate transport system but lack a nitrate transporter. Both transport systems are induced in cells irradiated with white light in the absence of a carbon source, suggesting that there may be precursors in the plasma membrane that only need the synthesis and assembly of some component(s) to become fully active. The induction of nitrate and nitrite reductases, however, only takes place when a carbon source is supplied to the cells.

Anion Transport Proteins↗
PubMed2003

In vitro genotoxic effects of different combinations of cobalt and metallic carbide particles.

Occupational exposure to hard metal dust, consisting of tungsten carbide (WC) and metallic cobalt particles (Co), is associated with an increased risk of lung cancer, while no increased risk was observed in workers exposed to Co alone. In vitro, in human peripheral blood mononucleated cells (PBMC), we previously demonstrated that WC-Co is more genotoxic than Co and WC alone. A possible mechanism underlying this higher genotoxicity is a specific physicochemical interaction between Co and WC particles leading to the enhanced short-term formation of active oxygen species. The aim of this study was to evaluate the in vitro genotoxicity of other combinations of Co with metal carbide particles in comparison with WC-Co. The ability of Cr(3)C(2), Mo(2)C and NbC and of their powder mixtures with Co to induce DNA strand breaks and alkali-labile sites was assessed by the alkaline Comet assay and their potential to induce chromosome(/genome) mutations by the cytokinesis-block micronucleus test on human PBMC from two donors. PBMC were treated in vitro for 15 min, 24 h after the onset of PHA stimulation. In the micronucleus test, while the metal carbides alone did not increase the micronucleus frequency, Co alone and the four tested carbide-Co mixtures induced a statistically significant concentration-dependent increase in micronucleated binucleates. In addition to WC, NbC and Cr(3)C(2) particles were able to interact with Co, producing a higher mutagenic effect than the individual metal particles. Mo(2)C particles did not display interactive mutagenicity with Co in the micronucleus test, possibly related to their small specific surface area, compactness and/or spherical shape. With the Comet assay, applied directly at the end of the treatment, less clear results, due to inter-experimental and inter-donor variation, were obtained. These data indicate that particular interaction of a metal carbide with Co leading to enhanced mutagenicity is not specific for WC.

Adult↗
PubMed1998

Lung cancer risk in hard-metal workers.

An industry-wide mortality study on the association between lung cancer and occupational exposure to cobalt and tungsten carbide was carried out in the French hard-metal industry. This case-control study was nested in the historical cohort of workers ever employed in this industry's 10 facilities, most of which are located in eastern France. Workers were followed up from 1968 to 1991. Occupational exposure was assessed using a job-exposure matrix that provided semiquantitative scores for 320 job periods. These scores were significantly correlated with the levels of cobalt measured in 744 historical air samples. In this cohort, which comprised 5,777 males and 1,682 females, the death rate from lung cancer was significant (63 deaths, standardized mortality ratio=1.30, 95% confidence interval (CI) 1.00-1.66) when compared with national death rates. Sixty-one cases and 180 controls were included in the study. When the exposures during the last 10 years were ignored, a twofold lung cancer risk was observed among workers simultaneously exposed to cobalt and tungsten carbide (odds ratio (OR)=1.93, 95% CI 1.03-3.62) adjusted for other cobalt exposure (OR=2.21, 95% CI 0.99-4.90). The odds ratios increased with cumulative exposure (first quartile, OR=1.00; second quartile, OR=2.64; third quartile, OR=2.59; fourth quartile, OR=4.13) and, to a lesser degree, with duration of exposure (one decade, OR=1.00; two decades, OR=1.61; three decades, OR=2.77; four decades, OR=2.03). Adjustments for smoking and for exposures to known or suspected carcinogens did not change the results, yet the odds ratio for smoking (3.38) was lower than expected, suggesting the possibility of some misclassification. Occupational risk was highest among smokers. This study supports the hypothesis that workers who manufacture hard metals have an increased mortality from lung cancer due to simultaneous exposure to cobalt and tungsten carbide.

Adult↗