PubMed Health⌕ Search

Biomedical subjects

S Na

Publications and source records attributed to S Na.

30 records · Page 2Linked to original sources
PubMed1994

Dominant lethal mutations in the plasma membrane H(+)-ATPase gene of Saccharomyces cerevisiae.

The plasma membrane H(+)-ATPase of Saccharomyces cerevisiae is an essential protein that is required to establish cellular membrane potential and maintain a normal internal pH. An Asp-378 to Asn substitution at the residue phosphorylated during catalysis is dominant lethal when the pma1-D378N mutation is expressed along with a wild-type plasma membrane H(+)-ATPase (PMA1) gene. Several mutations in the first two putative transmembrane domains are also dominant lethal. However, these dominant lethal mutants often appear to be innocuous, because they are frequently lost by gene conversion to the wild-type sequence during the process of introducing the mutant sequence and subsequently removing the wild-type gene. Loss of the mutation by gene conversion does not occur while introducing recessive lethal mutations. Cells carrying the wild-type PMA1 gene on the chromosome and a dominant lethal mutation under the control of a GAL1 promoter on a centromere-containing plasmid exhibit a galactose-dependent lethality. Indirect immunofluorescence staining using anti-Pma1 antibodies shows that induction of dominant lethal PMA1 mutations leads to the accumulation of a number of intensely staining cytoplasmic structures that are not coincident with the nucleus and its immediately surrounding endoplasmic reticulum. These structures also accumulate the endoplasmic reticulum protein Kar2. Expression of the dominant lethal protein also prevents transport of the wild-type ATPase to the plasma membrane.

Asparagine↗
PubMed1994

Mutational analysis of the first extracellular loop region of the H(+)-ATPase from Saccharomyces cerevisiae.

Transmembrane segments 1 and 2 of the yeast plasma membrane H(+)-ATPase are believed to form a helical hairpin structure that is joined by a short extracytoplasmic loop. The hairpin head region (Ala135-Phe144) was probed using site-directed mutagenesis. Scanning alanine mutagenesis produced functional H(+)-ATPase at all positions except Leu138, Asp143, and Phe144. D140A and V142A gave strong hygromycin B resistance and low pH sensitivity suggesting a major kinetic defect in these mutant enzymes. Other amino acid substitutions, such as L138Y, were highly perturbing, while mutations S139E and D140E produced minor effects on phenotype. Small uncharged residues Gly and Ala, which were inserted between Leu138 and Ser139 to examine the importance of loop length on H(+)-ATPase function, were well tolerated, while the insertion of a polar Ser residue was highly perturbing. Other additions were not tolerated by the enzyme. These results suggest that the turn region has limited structural flexibility. The conserved Phe144 residue could be changed to Trp with a minor effect on phenotype. However, neither Tyr, Arg, nor small hydrophobic residues could substitute, suggesting that this region is closely packed and hydrophobic. ATP hydrolysis measurements showed that Vmax was significantly reduced in nearly all mutant enzymes, except D140E; whereas, Km values were nearly normal. Vanadate-sensitivity and pH profiles for ATP hydrolysis were nearly normal for all mutant enzymes except insertion mutant S138+. Mutants with extreme phenotypes (S138+, Tyr138) showed significantly altered medium acidification profiles. These results support the notion that the hairpin head region linking transmembrane segments 1 and 2 forms a tightly packed conformationally sensitive domain that is coupled to the catalytic ATP hydrolysis domain.

Adenosine Triphosphate↗
PubMed1994

Modeling a conformationally sensitive region of the membrane sector of the fungal plasma membrane proton pump.

A molecular model for transmembrane segments 1 and 2 from the fungal proton pumping ATPase has been developed, and this structure is predicted to form a helical hairpin loop structure in the membrane. This region was selected because it is highly conformationally active and is believed to be an important site of action for clinically important therapeutics in related animal cell enzymes. The hairpin loop is predicted to form an asymmetric tightly packed structure that is stabilized by an N-cap between D140 and V142, by hydrogen bonding between residues in the turn region and the helices, and by pi-pi interactions between closely apposed aromatic residues. A short four-residue S-shaped turn is stabilized by hydrogen bonding but is predicted to be conformationally heterogeneous. The principal effect of mutations within the hairpin head region is to destabilize the local close packing of side groups which disrupts the pattern of hydrogen bonding in and around the turn region. Depending on the mutation, this causes either a localized or a more global distortion of the primary structure in the hairpin region. These altered structures may explain the effects of mutations in transmembrane segments 1 and 2 on ATP hydrolysis, sensitivity to vanadate, and electrogenic proton transport. The conformational sensitivity of the hairpin structure around the S-turn may also account for the effects of SCH28080 and possibly ouabain in blocking ATPase function in related animal cell enzymes. Finally, the model of transmembrane segments 1 and 2 serves as a template to position transmembrane segments 3 and 8. This model provides a new view of the H(+)-ATPase that promotes novel structure/function experimentation and could serve as the basis for a more detailed model of the membrane sector of this enzyme.

Amino Acid Sequence↗
PubMed1994

Studies on the plasmid instability in Corynebacteria.

Recombinant plasmid pNAR4, which can be used as a shuttle vector between E. coli and Coryneform bacteria, was constructed by ligating the DNA fragments of pNAT65 and pACYC184. Plasmid instability was found when pNAR4 was transformed to other Corynebacterium strains, segregational instability in C. crenatum B9 and structural instability in C. glutamicum 10147 and C. crenatum T6-13. When pNAT65 was transformed into C. glutamicum 10147, the plasmids in the transformants were found different from pNAT65. The size and main restriction sites in these plasmids were identical to those of pXZ10145. DNA hybridization indicated that in C. glutamicum 10147 a superhelix DNA was showed higher homology with that in pXZ10145. We assume that the existence of this supercoil DNA is related to the phenomena of various plasmid instability. Inter- or/and intra-molecular recombination may be the reason to explain the related phenomena found in our work.

Corynebacterium↗
PubMed1993

Characterization of yeast plasma membrane H(+)-ATPase mutant pma1-A135V and its revertants.

An A135V substitution in the first transmembrane segment of the yeast plasma membrane H(+)-ATPase (PMA1) confers cellular resistance to hygromycin B, exhibits growth sensitivity to low external pH, and results in a defective enzyme that hydrolyzes ATP at 33% of wild type level. The importance of the A135 residue was probed genetically by analysis involving both site-directed mutagenesis and randomly generated second-site intragenic suppressor mutations. No other amino acid at position 135 gave either the wild type phenotype or the normal enzyme activity of A135. Substitutions with the bulkier amino acid residues A135L, A135I, and A135F produced more severe cellular phenotypes than the original A135V mutation. The substitution of the smaller side chain residue Gly was also a mutant, although not as severe as the A135V mutant. The introduction of a bulky Trp or a polar Ser residue produced dominant lethality, while charged amino acids produced recessive lethality. Reduced rates of proton transport measured by acidification of the medium by whole cells correlate closely with the severity of cellular phenotype. Some of the mutant enzymes exhibit an apparent instability in vitro. Thus, the localized structure around A135 is highly constrained. The cellular sensitivity to low external pH of the A135V mutant was used to select intragenic revertants. Most full revertants (low pHR, HygS) restored A135, but second-site mutations in putative transmembrane segments 2 (V146I and V157F) and 4 (L327V) were also observed. Two partial revertants (low pHR, HygR) have secondary mutations at S660C or a double change at F611L-S660F in the putative ATP binding domain. These results provide additional evidence for functional coupling between the cytoplasmic domain catalyzing ATP hydrolysis and transmembrane helices 1 and 2.

Alleles↗
PubMed1993

Determination of nucleotide sequence of Corynebacterium plasmid pXZ10145.

With ABI370A Autosequencer, the total nucleotide sequence of plasmid pXZ10145 from Corynebacterium glutamicum 1014-6T has been determined using the dideoxy chain termination method. The plasmid contain 4887 base pairs (bp). Computer-aided-analysis of the sequence showed the location and number of restriction enzyme cutting sites and revealed eight open reading frames (ORF) on the plasmid. The two sites on the plasmid pXZ10145, where deletion occurred to result in plasmid pNAT65 were confirmed. At these two sites a seven-base pairs sequence "ATCTAGC" was found.

Base Sequence↗
PubMed1992

Genetic probing of the yeast plasma membrane H(+)-ATPase.

The H(+)-ATPase from Saccharomyces cerevisiae has been probed by a random genetic approach that has led to the isolation of primary and secondary site mutations. These H(+)-ATPase (PMA1) mutants help define specific functional, as well as interacting, regions of the H(+)-ATPase. Cellular resistance to hygromycin B has been an important selection tool for the isolation of pmal mutants. One prominent hygromycin B-resistant mutant, pmal-105, was found to have a S368F mutation near the site of phosphorylation (D378) in the catalytic core. This mutation prevents growth in low pH or NH(4+)-containing medium and induces an acid-sensitive Vmax for ATP hydrolysis, as well as a pronounced insensitivity to vanadate. The prominent cellular and biochemical phenotypes of this strain facilitated a detailed revertant analysis to identify protein structure domains that interact directly or indirectly with the localized region defined by the F368 mutation. Partial revertants were isolated which were resistant to low pH or NH4+ but retained hygromycin resistance. Second site mutations were found within the first and second cytoplasmic loop domains, as well as in transmembrane segments 1-3 & 7. All of the revertant enzymes have a stable Vmax but some show changes in the pH optimum for ATP hydrolysis; all display vanadate sensitivities ranging between the insensitive F368 mutant and the fully-sensitive wild type enzyme. Revertant analyses have also been performed on two other pma1 mutants which carry the mutations A135V and G158D in transmembrane segments 1 and 2, respectively. Compensating second site mutations to these mutations were identified in transmembrane segments 1, 2, 4 & 7, as well as within the central catalytic domain. These analyses have helped identify interacting protein structure domains that may participate in coupling ATP hydrolysis to proton transport. Furthermore, they facilitate the construction of structural models to account for these interactions.

Cell Membrane↗
PubMed1991

Characterization of the natural deletion mutant of plasmid pXZ10145 in Corynebacterium glutamicum and construction of a recombinant plasmid.

Plasmid pNAT65, carrying the chloramphenicol resistance marker, was chosen from a number of natural deletion mutants of pXZ10145 when pXZ10145 DNA, originally isolated from Corynebacterium glutamicum 1014-6T, was used to transform the protoplast of Corynebacterium crenatum T6-13. The size of pNAT65 was 2.4 kb, determined by electrophoresis on 0.7% agarose gel. The physical map of plasmid pNAT65 was determined. One recombinant plasmid, pNAR67, was constructed with the DNA fragments of pNAT65 and pBR322 digested respectively with EcoRI. This plasmid was capable of replication in E. coli, expressing ampicillin and tetracycline resistance; but with lower chloramphenicol, the resistance was only about 2 micrograms/ml.

Chromosome Deletion↗
PubMed1990

Animal model of coronary artery spasm-coronary thrombosis-acute myocardial infarction. A study on hemodynamics, EKG, coronary angiography, biochemistry and pathology.

An animal model of coronary artery spasm-coronary thrombosis-acute myocardial infarction (CAS-CATH-AMI) was obtained by injecting ergonovine(0.22 mg/kg) directly into the left coronary artery (LCA) of 17 dogs under general anesthesia. Various parameters of the experimental group were compared with those of the control group consisting of 5 dogs. The following changes were observed: increased average arterial blood pressure (MAP), pulmonary capillary wedge pressure (PCWP), stroke volume (SV, P less than 0.05), PAP (P less than 0.01) transient decreased cardiac output (CO); elevated ST-T in EKG; ventricular arrhythmia in 60% of animals; transient spasm of 50%-75% of LCA in the LCA angiographs; enhancement of platelet aggregation and TXB2 (P less than 0.01) and decline of 6 Keto-PGF1 alpha, SAO2 (P less than 0.05), indicating acute hypoxia and high coagulating pathophysiological changes after CAS. Pathological examination one hour after CAS induction revealed CATH(53%) in addition to CAS induced morphological changes of the CA and myocardium, as well as necrosis of the corresponding sites in its early stage. This study provides a reproducible animal model of CAS-CATH-AMI for research of coronary heart disease and pathomorphological criteria for the diagnosis of CAS. It also shows that CAS may lead to CATH-AMI, therefore, prevention of CATH and AMI is possible.

Animals↗
PubMed1987

Elimination of clonogenic malignant human T cells using monoclonal antibodies in combination with 2'-deoxycoformycin.

2'Deoxycoformycin (dCF) specifically inhibits adenosine deaminase (ADA) and causes selective cytotoxicity of normal and malignant T cells. In clinical trials, dCF caused rapid lysis of malignant T lymphoblasts. Although dCF has been associated with dose-limiting nonhematopoietic toxicities, myelosuppression has not been observed. Since dCF is relatively nontoxic to hematopoietic stem cells, we tested dCF for utility in the ex vivo purging of malignant T lymphoblasts from remission leukemic bone marrow for autologous bone marrow transplantation. We found that T lymphoblast cell lines were sensitive to dCF (plus deoxyadenosine [dAdo]) under conditions that did not ablate human hematopoietic colony-forming cells. Moreover, combined pharmacologic (dCF plus dAdo) and immunologic (anti-T cell monoclonal antibodies [McAb] plus complement) purging resulted in additive reduction in clonogenic T lymphoblasts. These results provide the basis for a clinical trial of bone marrow transplantation using combined pharmacologic/immunologic purging of T lymphoblasts from patients' harvested autologous marrow.

Antibodies, Monoclonal↗