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M Solioz

Publications and source records attributed to M Solioz.

At least 55 records · Page 3Linked to original sources
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↗
PubMed1992

Assessment of uncoupling by amiloride analogs.

The amiloride analogs N5-methyl-N5-isobutylamiloride, N5-ethyl-N5-isopropylamiloride, and N5,N5-hexamethyleneamiloride are frequently used to investigate NaH exchange on the premise that they are highly specific inhibitors of the NaH-antiporters. We assessed the relative protonophoric activity of these compounds in reconstituted and native membrane vesicles, using acridine orange fluorescence to measure intravesicular pH. All the compounds tested were found to be potent protonophores at concentrations which are normally used to demonstrate inhibition of NaH exchange. Uncoupling was dependent on both the pH of the assay system and the total amount of lipid present. At the pH optima, which lay in a range from 7.5 to 8.5, these amiloride analogs were more potent uncouplers than the classical protonophore carbonyl cyanide m-chlorophenylhydrazone. Therefore, extreme care must be taken in the interpretation of results obtained using these or similar derivatives of amiloride.

Acridine Orange↗
PubMed1992

Cloning and disruption of a putative NaH-antiporter gene of Enterococcus hirae.

When growing in a sodium-rich environment, wild-type Enterococcus hirae extrudes sodium by two mechanisms, ATP-driven sodium extrusion, and NaH-antiport. Mutant 7683 is unable to grow on sodium-rich media. This is due to two mutations, one inactivating ATP-driven sodium transport and a second rendering NaH-antiport inoperative. 7683 was transformed by electroporation with a gene bank, derived from E. hirae, in an Escherichia coli-E. hirae shuttle vector. Transformants which had regained the ability to grow on sodium-rich media were selected for and the transforming plasmids analyzed. A gene able to restore NaH-antiport activity in 7683 was identified. This gene was named napA. It codes for an extremely hydrophobic protein of 383 amino acids. Hydropathy analysis of this protein indicates that it probably forms 12 transmembraneous helices. In a mutant, possessing only the NaH-antiporter, the napA gene was disrupted by homologous recombination. The resultant strain failed to grow in sodium-rich media, and vesicles isolated from these cells exhibited a defect in sodium proton antiport activity. We conclude that the napA gene codes for a NaH-antiporter. The NapA protein does not exhibit significant homology to any protein in the EMBL genetic data bank.

Amino Acid Sequence↗
PubMed1991

Na/H antiporter mRNA expression in single nephron segments of rat kidney cortex.

Renal cortical tubules consist of polarized epithelial cells where Na/H antiport activity has been demonstrated on the apical and/or basolateral membrane. Apical Na/H antiport activity plays an important role in transcellular bicarbonate (HCO3-) reabsorption, whereas basolateral Na/H antiport activity could be involved in transcellular HCO3- secretion as well as cell volume and pH control. To determine whether this heterogeneity in both localization and function is due to the existence of more than one Na/H antiporter, we studied the tissue distribution of Na/H antiporter mRNA by use of reverse transcription (RT) and polymerase chain reaction (PCR) in isolated nephron segments from rat renal cortex. The primers used were directed against the rat renal cortical Na/H antiporter cDNA which is homologous to the human growth factor-activatable Na/H antiporter. RT/PCR of beta-actin mRNA were performed as positive controls. Na/H antiporter mRNA expression in the proximal tubule was not detectable in S1 and S2 segments from superficial and most midcortical nephrons, which exhibit exclusively luminal Na/H antiport activity. It was expressed in S1 and S2 segments from juxtamedullary nephrons which have also basolateral Na/H antiport activity. Beta-actin mRNA was expressed uniformly in all segments of the proximal tubule. Na/H antiporter mRNA was also expressed in cortical thick ascending limb and cortical collecting duct, segments with basolateral Na/H antiport activity as well as in the glomeruli. In conclusion, at least two different Na/H antiporters exist in the renal cortex, i.e., the proximal tubule. The close correlation between functional localization of basolateral Na/H antiport activity and mRNA expression suggests that the rat kidney Na/H antiporter DNA homologous to the human growth factor activatable Na/H antiporter encodes a basolateral exchanger. The observed expression in a minority of midcortical proximal tubules could reflect a certain heterogeneity in these nephron segments.

Animals↗
PubMed1990

Electrogenic transport by the Enterococcus hirae ATPase.

A transport ATPase from Enterococcus hirae was reconstituted in lipid vesicles and its electrogenic action investigated with the fluorescent dye oxonol VI as membrane potential probe. Reconstitution in bacterial and in soybean phospholipid mixtures led to transport-active vesicle preparations. Inside-out oriented ATPase molecules were activated by the addition of ATP to the extravesicular medium, generating in all experiments an intravesicularly positive potential. The extravesicular pH strongly influenced the initial pumping rate and the duration of the pumping activity. At neutral pH, transient pumping activity was observed, lasting for 1-2 min, while at pH 5.6, pumping was continuous. The transport activity was not dependent on the ionic composition of the buffer on either side of the membrane. These findings can be interpreted as the action of a proton ATPase, regulated by the cytoplasmic proton concentration and electrogenically translocating protons from the cytoplasm to the extracellular space.

Adenosine Triphosphatases↗
PubMed1990

Efficient electrotransformation of Enterococcus hirae with a new Enterococcus-Escherichia coli shuttle vector.

In the present study, an Enterococcus-Escherichia coli shuttle vector, pC3, was constructed that allows efficient transformation by electroporation of Enterococcus hirae ATCC9790. 5 x 10(6) transformants per microgram of plasmid DNA were obtained, using a commercial capacitor discharge device with an improved circuitry and a home-made electrode assembly, delivering pulses of 24 kV/cm across the cell suspension. The transformants were stable without selective pressure and plasmid DNA reisolated from transformed cells displayed no alterations in restriction enzyme analysis. Chromosomal DNA from E coli or E hirae, carried by pC3, was stably maintained in E hirae, making cloning and genetic manipulation in this organism feasible.

DNA, Bacterial↗
PubMed1990

Bacterial genetics by electric shock.

Subjecting bacteria to a high-voltage electric discharge renders the cells permeable to DNA. This powerful method allows the genetic manipulation of bacterial species that cannot easily be transformed by conventional techniques.

Cell Membrane Permeability↗
PubMed1987

Cloning of the K+-ATPase of Streptococcus faecalis. Structural and evolutionary implications of its homology to the KdpB-protein of Escherichia coli.

The K+-ATPase of Streptococcus faecalis consists of a single polypeptide component of relative Mr = 78,000 and serves as an ATP-driven pump for the accumulation of potassium by the bacterial cell. The gene encoding this ATPase was isolated by immunological screening of an S. faecalis genomic library in the Escherichia coli/pUC8 host/vector system. Two independently derived clones express the full-size ATPase polypeptide. Transcription and translation of the cloned DNA apparently proceed by means of the respective S. faecalis signals. DNA sequencing revealed a gene encoding a protein of 583 amino acids and a calculated Mr of 63,070. This protein exhibits in its primary structure regions of homology with the KdpB-subunit of the K+-ATPase of E. coli and, to a lesser extent, with eukaryotic ion-motive ATPases. The hydropathy profiles and secondary structure predictions, respectively, for the S. faecalis ATPase and the E. coli KdpB-protein show striking similarity. Even regions with low homology in the amino acid sequence exhibit structural features that have clearly been conserved in the two proteins. This points to a fundamental role of these domains in the structure and/or function of these transport ATPases.

Adenosine Triphosphatases↗
PubMed1987

Arginine modification with butanedione inhibits the potassium ATPase of Streptococcus faecalis.

The K+-ATPase of Streptococcus faecalis is inhibited by incubation with the arginine-modifying reagent 2,3-butanedione. The inactivation proceeds by pseudo - first order kinetics and a double-logarithmic plot of the pseudo - first order rate constants versus reagent concentrations yields a reaction order of 1.14 with respect to butanedione. Partially inactivated ATPase exhibits a decreased maximal velocity but the same affinity for ATP, as compared to the native enzyme. Butanedione modification is inhibited by adenine nucleotides. These results indicate the involvement of most likely one crucial arginyl residue in adenine nucleotide binding by the ATPase.

Adenosine Triphosphatases↗
PubMed1986

The vanadate-sensitive ATPase of Streptococcus faecalis pumps potassium in a reconstituted system.

The vanadate-sensitive ATPase of Streptococcus faecalis, purified to homogeneity, was reconstituted into soybean phospholipid vesicles in a functional state. Freeze-fracture electron micrographs revealed a relatively uniform population of unilamellar liposomes of 50-100 nm in diameter, with particles protruding from both fracture faces. Transport studies with 42K+ and with a K+-selective electrode showed that the ATP-ase catalyzes electrogenic potassium extrusion in proteoliposomes. The following parameters for potassium transport in the reconstituted system were determined: K+/ATP stoichiometry = 1, Km for potassium = 1.4 mM, Vmax = 0.1 mumol/min/mg. The ATPase could be activated by an electrical membrane potential, vesicle interior positive. This ATPase thus appears to function as a potential regulated, ATP-driven pump that serves in electrogenic potassium accumulation by the bacterial cell.

Adenosine Triphosphatases↗
PubMed1985

Formation of a beta-aspartyl phosphate intermediate by the vanadate-sensitive ATPase of Streptococcus faecalis.

The vanadate-sensitive membrane ATPase of Streptococcus faecalis forms, as part of the reaction cycle, an acylphosphate intermediate. The phosphorylated amino acid residue was identified by reducing the purified reconstituted phosphoenzyme with [3H]borohydride, followed by acid hydrolysis of the protein and quantitative amino acid analysis. Tritiated homoserine was found to be the resulting reaction product, generated through the reduction of a beta-aspartyl phosphate residue. The S. faecalis ATPase thus forms the same phosphorylated intermediate as a number of eukaryotic transport ATPases and appears to be related to these enzymes.

Adenosine Triphosphatases↗
PubMed1983

Dicyclohexylcarbodiimide does not inhibit proton pumping by cytochrome c oxidase of Paracoccus denitrificans.

The effect of dicyclohexylcarbodiimide (DCCD) on the proton pumping two-subunit cytochrome c oxidase from Paracoccus denitrificans was investigated. Purified Paracoccus oxidase was reconstituted into phospholipid vesicles by cholate dialysis. Following incubation with increasing amounts of DCCD, proton ejection was recorded in response to reductant pulses with reduced cytochrome c. Concentrations of DCCD which greatly reduced proton pumping by bovine cytochrome c oxidase used as a control were found to exert only a minor effect on proton translocation by Paracoccus oxidase. Similarly, incubation of the bacterial enzyme with [14C]DCCD failed to reveal the specific covalent interaction previously demonstrated to occur with bovine cytochrome c oxidase, and here also shown for the oxidase of yeast. Thus, Paracoccus oxidase differs in its interaction with DCCD from the functionally analogous eukaryotic enzymes.

Animals↗
PubMed1983

Purification of a putative K+-ATPase from Streptococcus faecalis.

We have purified a novel membrane ATPase from Streptococcus faecalis by the following procedure: extraction of membranes with Triton X-100 followed by fractionation of the extract by successive DEAE-cellulose chromatography, hydroxylapatite chromatography and Cm-Sepharose chromatography. The overall yield was 5%. The purified ATPase appears to consist of a single polypeptide component of Mr = 78,000. The Triton-solubilized purified enzyme has a specific activity of approximately 50 mumol of ATP hydrolyzed per min per mg, is dependent on phospholipids for activity, and is strongly inhibited by vanadate (I50 = 3 microM). Maximal ATPase activity is displayed at pH 7.3. Mg2+-ATP, for which the enzyme has a Km of 60 microM, is the best substrate. The ATPase forms an acylphosphate intermediate that can also be detected in native membranes as the major acylphosphate component. The purified ATPase, when reconstituted into soybean phospholipid vesicles, exhibits coupling, e.g. the ATPase activity can be stimulated at least 8-fold by valinomycin in the presence of potassium. Based on these observations we conclude that the enzyme we have purified is an ion-motive ATPase, most likely a K+-ATPase.

Adenosine Triphosphatases↗
PubMed1982

The cytochrome c oxidase of Paracoccus denitrificans pumps protons in a reconstituted system.

The purified two-subunit cytochrome c oxidase of Paracoccus denitrificans was reconstituted into phospholipid vesicles having a high internal buffering capacity and exhibiting a respiratory control index greater than 6.6. With these proteoliposomes, pH changes of the suspending medium were monitored in response to reductant pulses in the presence of valinomycin and potassium. When reduced cytochrome c was added to allow for a limited number of turnovers (2-12), a net acidification of the extravesicular space could be observed. This apparent proton ejection by the vesicles was abolished by inhibition of the oxidase with azide, by bypassing the oxidase with ferricyanide, or by preventing charge compensation by omitting valinomycin. Addition of uncoupler led to an alkalinization, rather than an acidification, of the extravesicular space in response to reduced cytochrome c. We thus conclude that cytochrome c oxidase of P. denitrificans is a proton pump. Under the conditions described here, an apparent stoichiometry of 0.6 proton ejected/electron was obtained by extrapolation to zero turnovers.

Animals↗