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M Solioz

Publications and source records attributed to M Solioz.

At least 19 recordsLinked to original sources
PubMed1997

CopY is a copper-inducible repressor of the Enterococcus hirae copper ATPases.

The cop operon of Enterococcus hirae effects copper homeostasis in this organism. It encodes a repressor, CopY, an activator, CopZ, and two P-type copper ATPases, CopA and CopB. Expression of all four genes is regulated by the ambient copper. In this regulation, CopY apparently acts as a copper-inducible repressor. By DNase I footprinting, it was shown that purified CopY protected two discrete sites in the region encompassing nucleotides -71 to -11 relative to the translational start site and containing hyphenated inverted repeats. Transcription is initiated between these repeats at nucleotide -42, in a domain that remained accessible to DNase I in the DNA-repressor complex. Chemical cross-linking revealed that CopY exists as a dimer in solution. In DNA band-shift assays, it was apparent that the CopY-DNA interaction occurred in two discrete steps. Half-maximal binding of repressor to the two operator sites was observed at 2 x 10(-9) M and 5 x 10(-9) M CopY, respectively. Copper ions released CopY from the promoter/operator with an apparent half-binding constant for Cu(I) of 20 microM. The site-directed mutations A-61T and A-30T essentially abolished the binding of CopY to the respective binding sites, and the double mutation A-61T/A-30T inactivated both binding sites. Thus, CopY is a copper-inducible repressor of the cop operon of E. hirae, exhibiting highly specific DNA-protein interactions with two sites on the cop promoter/operator and playing a key role in copper homeostasis in E. hirae.

Adenosine Triphosphatases↗
PubMed1997

The pyrethroids permethrin and cyhalothrin are potent inhibitors of the mitochondrial complex I.

The synthetic pyrethroid derivatives permethrin and cyhalothrin are widely used insecticides that are considered to be relatively nontoxic to higher animals. However, a variety of toxic effects on mammals have been reported. We investigated the effect of these drugs on energy coupling by mitochondria and on the activity of the individual respiratory complexes. Using isolated rat liver mitochondria, a concentration-dependent inhibition of glutamate and succinate sustained state 3 respiration was found for both compounds in the micromolar range. The effect of pyrethroids on the activities of the complexes I to V were assessed individually in submitochondrial particles (complex I) and in freeze-thawed mitochondria (complexes II-V). Complex I (EC 1.6.5.3) was found to be the most sensitive link within the electron transport chain. Half-maximal inhibition was observed at 0.73 microM permethrin and 0.57 microM cyhalothrin, respectively, and exhibited sigmoidal inhibition kinetics. Complexes II, III, IV and V (EC 1.3.5.1, 1.10.2.2, 1.9.3.1, 3.6.1.34) were not significantly inhibited by up to 50 microM of these drugs. Thus, our results reveal a model of action of synthetic pyrethroid insecticides not previously reported.

Animals↗
PubMed1996

Phosphoenzyme formation by purified, reconstituted copper ATPase of Enterococcus hirae.

The Enterococcus hirae CopB ATPase serves in the secretion of excess copper from cells and belongs to the recently discovered, new class of heavy metal transport ATPases. We here report the affinity purification of CopB to near homogeneity and its reconstitution into phospholipid vesicles. In these proteoliposomes, the ATPase formed an acylphosphate reaction intermediate with the gamma-phosphate of ATP. ATPase activity and phosphoenzyme formation were inhibited by vanadate with an I(50) of 0.1 mM. Our results suggest that heavy metal and non-heavy metal ATPases operate by the same underlying mechanism.

Adenosine Triphosphatases↗
PubMed1996

Application of mRNA differential display to liver cirrhosis: reduced fetuin expression in biliary cirrhosis in the rat.

We here show the application of mRNA differential display to investigate changes in gene expression in rat liver cirrhosis and address problems inherent in the technique when applied to this complex disease model. A number of differentially expressed mRNA species could be identified and two were analyzed in more detail here. One was found to derive from a new gene while the other corresponded to fetuin, a 41 kDa N-glycoprotein that specifically inhibits tyrosine kinase activity of the insulin receptor when phosphorylated. Fetuin expression was reduced by 45% in liver cirrhosis induced by bile duct ligation, but not in cirrhosis induced by carbon tetrachloride/Phenobarbital, as compared to controls. Our results raise the possibility that fetuin plays a regulatory role in the proliferation of parenchymal liver cells.

Animals↗
PubMed1996

Fixed versus titrated interferon-alpha 2B in chronic hepatitis C. A randomized controlled multicenter trial. The Swiss Association for the Study of the Liver.

BACKGROUND/AIM: Interferon has become the mainstay of treatment of chronic hepatitis C; however, duration of treatment and dose remain unresolved questions. The present study aimed to compare standard dose interferon with a titrated dose regimen carried out for 1 year. METHODS: This was a randomized, controlled multicenter trial. Patients with chronic hepatitis C were randomly allocated to a control group (n = 30), to a fixed dose group (n = 31) where interferon-alpha 2b 3 MU thrice weekly was given for 1 year or a titrated group (n = 34) where interferon was titrated starting at 5 MU thrice weekly to the lowest dose keeping the patient in remission as assessed by a normal ALT value. Liver biopsies were obtained before and at the end of treatment; in addition, galactose elimination capacity was measured as a measure of cytosolic function. RESULTS: In the control, fixed and titrated groups a complete response was achieved in 2/29, 10/28 and 15/31, respectively (p < 0.001 in favor of treatment, p = n.s. for the two treatments). The corresponding figure for sustained response was 1/29, 5/28 and 6/ 31 (p = n.s). In the titrated group, a complete (sustained) response was achieved with 5 MU in 2 (2), with 4 MU in 1 (0), with 3 MU in 4 (0), with 2 MU in 3 (0) and with 1 MU in 5 (4). Liver biopsy score and galactose elimination capacity improved significantly in responders but not in treatment failures. CONCLUSIONS: Both fixed and titrated dosing of interferon given for 1 year induced virus clearance in only a minority of treated patients. However, in a small number of patients, a complete and sustained response can be achieved with low doses of interferon. Dose titration could be an interesting approach to decreasing the cost and side effects in the treatment of chronic hepatitis C.

Adult↗
PubMed1996

CPx-type ATPases: a class of P-type ATPases that pump heavy metals.

ATP-driven heavy metal pumps represent a newly defined class of proteins that translocate toxic and essential metals across biological membranes. These transporters form a separate evolutionary branch of the ion-transporting P-type ATPases. We propose to call these enzymes CPx-type ATPases, based on the common novel feature of a conserved intramembranous cysteine-proline-cysteine or cysteine-proline-histidine motif.

Adenosine Triphosphatases↗
PubMed1995

[Does serum HCV-RNA-positive hepatitis C differ from serum HCV-RNA-negative hepatitis C?].

Viral RNA is detectable in the serum of the majority but not all patients with chronic hepatitis C. Whether the viremic form differs from the non-viremic form of the disease is unknown. We therefore compared histology (modified Knodell score) and liver function (conventional liver function tests, galactose elimination capacity and aminopyrin breath test) of viremic (n = 45) and non-viremic (n = 37) patients with chronic hepatitis C. Neither the total histologic score, nor any of the individual histologic parameters assessed differed significantly in serum HCV RNA positive and negative patients. Compared to non-viremic subjects, patients with detectable HCV RNA in serum had slightly higher transaminases (p = ns), lower serum albumin (p < 0.05) and decreased galactose elimination capacity (p < 0.05). This trend towards more severe functional hepatic impairment in serum HCV RNA positive patients persisted when cirrhotics and non-cirrhotics were analyzed separately; it fell just short of reaching statistical significance, however, most probably due to the small number of subjects per patient group. The median age of serum HCV RNA positive cirrhotics was 17 years, and that of serum HCV RNA negative cirrhotics 22 years higher than that of the respective non-cirrhotics. Biochemical (transaminases) and histological (intralobular and piece-meal necrosis) markers of disease activity, as well as metabolic functional reserve (aminopyrin breath test, galactose elimination capacity) and histologic severity of fibrosis correlated only loosely (Rs = 0.24-0.56), albeit significantly (p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1995

Copper and silver transport by CopB-ATPase in membrane vesicles of Enterococcus hirae.

The P-type ATPase, CopB, of Enterococcus hirae is required for the copper resistance displayed by this organism and thus was postulated to be a copper pump. Using 64Cu+ and 110mAg+, we here show ATP-driven copper and silver accumulation catalyzed by CopB in native inside-out membrane vesicles of E. hirae. CopB ATPase exhibited an apparent Km for Cu+ and Ag+ of 1 microM and for ATP of 10 microM. Transport was maximal at pH 6 and had an apparent Vmax of 0.07 nmol.min-1.mg-1 for both copper and silver transport. Vanadate displayed a biphasic effect on transport: maximal inhibition was observed at 40 microM vanadate for copper transport and 60 microM for silver transport, respectively. At higher vanadate concentrations, these inhibitions were reversed. The CopB ATPase of E. hirae is thus a pump for the extrusion of monovalent copper and silver ions, with copper probably being the natural substrate.

Adenosine Triphosphatases↗
PubMed1995

Two trans-acting metalloregulatory proteins controlling expression of the copper-ATPases of Enterococcus hirae.

Enterococcus hirae possesses two P-type ATPases, CopA and CopB, that are involved in copper homeostasis. These enzymes are induced by extracellular copper concentrations that are either too low or too high for optimal growth. To identify the regulatory proteins involved in induction, the DNA upstream of copA was cloned and sequenced. Following a putative promoter region, it contains two genes, copY and copZ, that encode proteins of 145 and 69 amino acids, respectively. Both proteins contain metal binding motifs and exhibit significant sequence similarity to known regulatory proteins. Gene disruption of copY by reverse genetics caused constitutive overexpression of CopA and CopB, generating a copper-dependent phenotype. In contrast, disruption of copZ suppressed the expression of the two copper-ATPases, rendering the cells copper-sensitive. Both null mutations could be complemented in trans with plasmids bearing copY or copZ. Thus, copY and copZ encode trans-acting metalloregulatory proteins that are required for induction of the cop operon by copper. In this mechanism, CopY apparently acts as a metal-fist type repressor and CopZ as an activator.

Adenosine Triphosphatases↗
PubMed1995

Identification of a gene (arpU) controlling muramidase-2 export in Enterococcus hirae.

Muramidase-2 of Enterococcus hirae is a 74-kDa peptidoglycan hydrolase that plays a role in cell wall growth and division. To study its regulation, we isolated a mutant defective in muramidase-2 release under certain growth conditions. This mutant had cell walls which apparently lacked 74-kDa muramidase-2 but which accumulated two proteolytic fragments of 32 and 43 kDa, which exhibited muramidase-2 activity in the membrane fraction. By complementation cloning, we identified a 2.6-kb fragment of the E. hirae chromosome containing a gene cluster coding for proteins of 58 to 137 amino acids. One of these genes (arpU), which encoded a 15.9-kDa protein, was shown to complement the defect of the A9 mutant in trans. We propose that this gene may be involved in the regulation of muramidase-2 export.

Amino Acid Sequence↗
PubMed1994

Induction of the putative copper ATPases, CopA and CopB, of Enterococcus hirae by Ag+ and Cu2+, and Ag+ extrusion by CopB.

The two P-type ATPases CopA and CopB are effecting regulation of cellular copper activity in Enterococcus hirae. With antibodies against these ATPases, we showed on Western blots the simultaneous induction of CopA and CopB by copper or silver ions. Copper contents of wild type and mutant cells lacking either CopA, CopB or both enzymes were measured by atomic absorption. Strains disrupted in copB showed clearly enhanced copper contents. Mutants lacking CopB also lost the ability of energy dependent efflux of silver ions. Our results demonstrate that CopA and CopB are under the same genetic control and support the proposal that CopB is a copper and silver exporting ATPase.

Adenosine Triphosphatases↗
PubMed1994

Copper pumping ATPases: common concepts in bacteria and man.

Recently, four genes encoding putative copper pumping ATPases have been cloned from widely different sources: two genes from Enterococcus hirae that are involved in copper metabolism and two human genes that are defective in the copper-related Wilson and Menkes disease. The predicted gene products are P-type ATPases. They exhibit extensive sequence similarity and appear to be members of a new class of ATP driven copper pumps involved in the regulation of cellular copper.

Adenosine Triphosphatases↗
PubMed1994

Operon of vacuolar-type Na(+)-ATPase of Enterococcus hirae.

The Gram-positive bacteria Enterococcus hirae expel sodium by two systems: a Na+/H(+)-antiporter and a vacuolar-type Na(+)-ATPase. We isolated a mutant, NalkA, defective in the Na(+)-ATPase. NalkA grew normally at neutral pH but was unable to grow in the presence of > 100 mM sodium at pH 9.5. By functional complementation at high pH, we cloned pES1, a plasmid from an E. hirae gene bank containing a 5.2-kilobase pair region of genomic DNA. The genomic DNA in pES1 contains five complete open reading frames, ntpM, -N, -O, -P, and -Q, encoding proteins of 75, 16, 23, 38, and 11 kDa. A sixth incomplete open reading frame, ntp 'L, precedes ntpM. The 3'-end of the cloned DNA overlaps with a previously published sequence encoding the ntpA and ntpB subunits of the E. hirae Na(+)-ATPase (Takase, K., Yamato, I., and Kakinuma, Y. 1993) J. Biol. Chem. 268, 11610-11616). The insert of pES1 therefore represents the upstream region of the ntp operon that encodes the E. hirae Na(+)-ATPase. Complementation analysis with various deletions derived from pES1 suggest that the original mutation is in the ntpM gene. Of the new genes described here, three exhibited significant sequence similarity to known proteins; ntpM shares 24% identical amino acid residues with the "116-kDa" subunits of eukaryotic vacuolar ATPases, ntpN exhibits 28% sequence identity with the 16-kDa proteolipid of human vacuolar ATPase, and ntpO has sequence homology to the 31-kDa subunit of the bovine kidney vacuolar ATPase. No known proteins with sequence similarity to ntp'L, -P, or -Q could be identified. Disruption of either ntpM, -N, or -O in wild-type cells by cassette mutagenesis resulted in mutants unable to effect ATP-driven sodium extrusion. NtpM, -N, and -O therefore represent three new gene products involved in sodium extrusion by the vacuolar-type Na(+)-ATPase of E. hirae, and three more gene products, NtpL, -P, and -Q, may also be constituents of this enzyme. The ntp operon thus contains at least eight genes.

Adenosine Triphosphatases↗
PubMed1994

Technical report. Video imaging of ethidium bromide-stained DNA gels with surface UV illumination.

We describe here the use of surface UV illumination to record ethidium bromide-stained DNA gels with a video camera. This mode of illumination allows the use of a standard video camera equipped with a red filter and results in a high signal strength. The assembly of a low-cost video system on this basis is described. It uses the public domain software called Image on a Macintosh computer and PostScript laser printer or a thermal printer to generate hard copies. The setup is sensitive enough to detect 500 pg of DNA on an ethidium bromide-stained DNA gel. The UV illumination method described here can also greatly improve the sensitivity of existing video recording equipment.

DNA↗
PubMed1993

Functional expression of the Enterococcus hirae NaH-antiporter in Escherichia coli.

We recently described the cloning of napA, the putative structural gene for the NaH-antiporter of Enterococcus hirae (Waser, M., Bienz-Hess, D., Davies, K., and Solioz, M. (1992) J. Biol. Chem. 267, 5396-5400). To analyze the gene product of napA, we expressed it in Escherichia coli. When placed under the control of a T7 promoter, napA could be transcribed and labeled specifically with [35S]methionine. The resultant gene product exhibited an apparent M(r) of 3,4 x 10(4) when subjected to sodium dodecyl sulfate-gel electrophoresis. The function of NapA was tested by expressing it from its own promoter in the E. coli mutant EP432. This mutant lacks both of the endemic NaH-antiporters, NhaA and NhaB; its growth is thus very sensitive to Na+ and Li+ and membranes derived from this strain do not exhibit NaH-antiport activity. When complemented with napA, EP432 gained tolerance to Na+ or Li+. Membranes prepared from the complemented mutant exhibited NaH-antiport activity. The properties of this activity were determined by acridine fluorescence measurements on vesicles energized with lactate. The NaH-antiporter expressed by napA exhibited a Km of 1 mM for Na+ and 0.1 mM for Li+ at pH 7.5. At pH 8.5, the relative rate of NaH-antiport activity was 50%, with little change in the Km, and approached zero at pH 9. These results demonstrate that napA is the structural gene for the NaH-antiporter of E. hirae. NapA exhibits properties different from those of the two E. coli NaH-antiporters encoded by nhaA and nhaB, yet functionally complements a defect in these genes.

Amino Acid Sequence↗
PubMed1993

Primary structure of two P-type ATPases involved in copper homeostasis in Enterococcus hirae.

We cloned an operon, copAB, from Enterococcus hirae encoding two P-type ATPases of 727 and 745 amino acids, respectively. Both enzymes display heavy metal ion binding motifs in their polar N-terminal region. With an antibody against CopB, we showed on Western blots that expression of the operon is induced by either low or high ambient copper concentrations. Disruption of the copA gene renders the cells dependent, whereas copper disruption of copB results in a copper-sensitive phenotype. CopA exhibits 35% sequence similarity to CopB and 43% similarity to the ATPase encoded by the recently cloned human Mc1 gene, a gene responsible for the Menkes inborn error of copper metabolism. Our results imply that CopA and CopB are heavy metal ion ATPases that regulate the cytoplasmic copper activity, with CopA serving in the uptake and CopB in the extrusion of copper.

Adenosine Triphosphatases↗
PubMed1993

Low-dose interferon in chronic hepatitis non-A/non-B: effects on quantitative liver function and structure in a randomized, controlled multicenter trial.

In this randomized, controlled multicenter trial we evaluated the effects of recombinant interferon-alpha 2b on galactose elimination capacity and histological activity index in 88 patients with chronic active hepatitis non-A/non-B. Forty-five patients were randomly assigned to treatment with interferon at 1.5 x 10(6) U three times per for 1 year; 43 patients were assigned to no treatment. A complete response (normalization of alanine aminotransferase) was observed, respectively, in 47% and 5% of the two groups (P < 0.006); 47% of these patients suffered a relapse. Thus 22% of patients had a sustained response. Histological activity decreased significantly in responders (P < 0.04) while the biopsy score did not change significantly in nonresponders. In contrast, galactose elimination capacity--a surrogate marker for survival in chronic active hepatitis--was not affected by response to treatment. None of the parameters evaluated, including hepatitis C virus RNA, was able to predict response or relapse. We conclude that low-dose interferon treatment for 1 year is as effective as the recommended treatment schedule.

Adolescent↗