PubMed Health⌕ Search

Biomedical subjects

L Zimniak

Publications and source records attributed to L Zimniak.

14 recordsLinked to original sources
PubMed2000

Cl(-) channels in basolateral TAL membranes XV. Molecular heterogeneity between cortical and medullary channels.

We have isolated two new and highly homologous cDNAs, mmClC-Ka from mouse outer medulla and mcClC-Ka from mouse cortex. In both cases, mRNA was obtained from the indicated region and subjected to RT-PCR using primers from the nucleotide sequence of rbClC-Ka, which encodes basolateral Cl(-) channels (termed rbClC-Ka) in rabbit MTAL. The predicted protein products of mmClC-Ka and mcClC-Ka, mmClC-Ka and mcClC-Ka, respectively, were 85% homologous and had predicted molecular weights of 75 kDa. The predicted protein sequences for mmClC-Ka and rbClC-Ka had three cytosolic sites-threonine 185, threonine 187 and serine 270-which were absent in mcClC-Ka. These three moieties represent potential sites for phosphorylation of mmClC-Ka and rbClC-Ka, but not of mcClC-Ka, and may account for the failure of (ATP + PKA) to increase the open time probability P(o) in basolateral CTAL Cl(-) channels. We prepared antisense oligonucleotides specific for nonhomologous regions of these two cDNAs, mmAntisense for mmClC-Ka and mcAntisense for mcClC-Ka. Using anti-rbClC-Ka, a polyclonal antibody to rbClC-Ka, we found that, when transfected into cultured mouse MTAL and CTAL cells, mmAntisense suppressed the appearance of the 75 kDa band by 50% in vesicles from MTAL but not CTAL cells, while transfection of MTAL and CTAL cells with mcAntisense suppressed appearance of the 75 kDa band in vesicles from CTAL but not MTAL cells. mmAntisense transfection also prolonged the half-time (T(1/2), sec) for (36)Cl(-) efflux in cultured MTAL cells from 82.4 +/- 6.8 sec (sem) to 187.8 +/- 9.5 sec (n = 5; P = 0.0001) while mcAntisense transfection had no such effect. Conversely, in cultured CTAL cells, mcAntisense transfection prolonged the T(1/2) for (36)Cl(-) efflux from 80.9 +/- 6.3 sec to 191.8 +/- 6.5 sec (n = 5; P = 0.00005), while mmAntisense had no such effect. We conclude that mmClC-Ka and mcClC-Ka may encode the basolateral Cl(-) channels mediating net Cl(-) absorption in mouse MTAL and CTAL, respectively.

Amino Acid Sequence↗
PubMed1997

Medullary thick limb basolateral Cl- channels.

Chloride transport by the medullary thick ascending limb is a major determinant of urinary concentrating and diluting power and also of tubuloglomerular feedback. In this review, we discuss the electrophysiologic, regulatory, and molecular features of a Cl- channel present in basolateral membranes from the medullary thick ascending limb. Functional and immunohistochemical data support the view that rbCIC-Ka, a CIC Cl- channel cloned from rabbit kidney medulla, may be the Cl- channel which mediates basolateral Cl- efflux.

Animals↗
PubMed1997

Increased resistance to oxidative stress in transfected cultured cells overexpressing glutathione S-transferase mGSTA4-4.

Peroxidation of unsaturated fatty acids in membrane phospholipids is one of the multiple cytotoxic effects of oxidative stress. Lipid peroxidation is significant because a single initiating event triggers a chain reaction, thus amplifying the initial stimulus. Many oxidative stress-related pathologies have been linked to lipid peroxidation. Mouse glutathione S-transferase mGSTA4-4 exhibits high glutathione conjugating activity with toxic products of lipid peroxidation, e.g., 4-hydroxynon-2-enal. In addition, mGSTA4-4 has glutathione peroxidase activity toward phospholipid hydroperoxides. On the basis of these catalytic properties, we have previously proposed that the enzyme may be physiologically important in alleviating the cytotoxic effects of lipid peroxidation. We have now experimentally confirmed this hypothesis by transfecting HepG2 cells with mGSTA4 cDNA, and demonstrating a protective effect of expressed mGSTA4-4 protein on cells exposed during plating to H2O2, organic hydroperoxides, and phosphatidylcholine hydroperoxide. As compared to cells transfected with insert-free vector, a larger proportion of mGSTA4-transfected cells was able to attach to the culture dish, and continued to divide in the presence of the above compounds. In addition to alleviating the cytotoxic effects of oxidative stress, mGSTA4-4 may interfere with the subtoxic but cytostatic signals generated by a low-level pro-oxidant state.

Animals↗
PubMed1997

Cl- channels in basolateral renal medullary membranes. XII. Anti-rbClC-Ka antibody blocks MTAL Cl- channels.

Cl- channels in the medullary thick ascending limb (MTAL) studied by either patch-clamp technique or reconstitution into lipid bilayers are activated by increases in intracellular Cl- concentrations. rbClC-Ka, a ClC Cl- channel, may represent this channel. We therefore evaluated the role of rbClC-Ka in transcellular MTAL Cl- transport in two separate ways. First, an antibody was raised against a fusion protein containing a 153-amino acid fragment of rbClC-Ka. Immunostaining of rabbit kidney sections with the antibody was localized to basolateral regions of MTAL and cortical thick ascending limb (CTAL) segments and also to the cytoplasm of intercalated cells in the cortical collecting duct. Second, Cl- uptake and efflux were measured in suspensions of mouse MTAL segments. Cl- uptake was bumetanide sensitive and was stimulated by treatment with a combination of vasopressin + forskolin + dibutyryl adenosine 3',5-cyclic monophosphate (DBcAMP). Cl- efflux was also increased significantly by vasopressin + forskolin + DBcAMP from 114 +/- 20 to 196 +/- 36 nmol.mg protein-1.45 s-1 (P = 0.003). Cl- efflux was inhibited by the Cl- channel blocker diphenylamine-2-carboxylate (154 +/- 26 vs. 70 +/- 21 nmol.mg protein-1.45 s-1, P = 0.003). An anti-rbClC-Ka antibody, which inhibits the activity of MTAL Cl- channels in lipid bilayers, reduced Cl- efflux from intact MTAL segments (154 +/- 28 vs. 53 +/- 14 nmol.mg protein-1.45 s-1, P = 0.02). These results support the view that rbClC-Ka is the basolateral membrane Cl- channel that mediates vasopressin-stimulated net Cl- transport in the MTAL segment.

Animals↗
PubMed1996

Cl- channels in basolateral renal medullary vesicles XI. rbClC-Ka cDNA encodes basolateral MTAL Cl- channels.

The present experiments examined whether rbClC-Ka, a CIC family Cl-channel cDNA from rabbit outer medulla, encodes a basolateral membrane Cl- channel mediating net medullary thick ascending limb (MTAL) Cl- absorption. MTAL cells contain a Cl- channel having certain properties that make it a plausible candidate for the basolateral Cl- channel in that segment. Especially pertinent among properties is the fact that cytosolic Cl- increases in the range 2-25 mM activated these Cl- channels. Cultured mouse MTAL cells were grown in the presence of an antisense oligonucleotide specific for rbCIC-Ka or a random oligonucleotide with no complementarity to rbCIC-Ka. The abundance of Cl- channels was assessed by the frequency of incorporation of Cl- channels from membrane vesicles prepared from these cells into lipid bilayers and by Western blot analysis using an antiserum to the COOH terminus of the rbClC-ka protein. With the use of vesicles from untreated cells or cells treated with the random oligonucleotide, Cl- channels were incorporated into bilayers in 17% and 16% of trials, respectively. However, when vesicles were prepared from cells pretreated with antisense oligonucleotide, there was a virtual abolition of Cl- channel incorporation into bilayers but no effect on the frequency of K+ channel incorporation. In parallel with the reduction in Cl- channel incorporation, the abundance of rbClC-Ka protein was reduced approximately 50% on Western blots. Finally, exposure of Cl- channels in lipid bilayers to the rbClC-Ka antiserum resulted in a block in channel activity. These results support the contention that the basolateral Cl- channel mediating net Cl- absorption in the MTAL is encoded by rbClC-Ka.

Animals↗
PubMed1995

Cl- channels in basolateral renal medullary vesicles. X. Cloning of a Cl- channel from rabbit outer medulla.

These experiments were intended to identify candidate cDNAs which might encode basolateral membrane Cl- channels of the mTAL using a homology-based cloning strategy. We prepared a cDNA library using a 1.8 to 3.2 kb mRNA fraction from rabbit outer medulla that induces a Cl- conductance in cellular membranes of Xenopus laevis oocytes. The cDNA library was screened with two 32P-oligonucleotide probes corresponding to highly conserved sequences in other Cl- channels. We isolated two cDNAs: rbClC-Ka and rbClC-Kb. The protein sequences deduced from these two cDNAs had 99% homology. Using RT-PCR technology, cultured mouse mTAL cells were found to contain mRNA corresponding to those two cDNAs. Expression of the mRNAs corresponding to these two cDNAs was kidney-specific and was greater in rabbit renal medulla than rabbit renal cortex. Finally, by using RT-PCR technology in combination with microdissected glomeruli or tubule segments, we found mRNA for rbClC-Ka in glomeruli, proximal convoluted tubules, mTAL and cortical collecting tubules.

Amino Acid Sequence↗
PubMed1992

Unusual resistance of peptidyl transferase to protein extraction procedures.

Peptidyl transferase, the ribosomal activity responsible for catalysis of peptide bond formation, is resistant to vigorous procedures that are conventionally employed to remove proteins from protein-nucleic acid complexes. When the "fragment reaction" was used as a model assay for peptide bond formation, Escherichia coli ribosomes or 50S subunits retained 20 to 40 percent activity after extensive treatment with proteinase K and SDS, but lost activity after extraction with phenol or exposure to EDTA. Ribosomes from the thermophilic eubacterium Thermus aquaticus remained more than 80 percent active after treatment with proteinase K and SDS, which was followed by vigorous extraction with phenol. This activity is attributable to peptidyl transferase, as judged by specific inhibition by the peptidyl transferase-specific antibiotics chloramphenicol and carbomycin. In contrast, activity is abolished by treatment with ribonuclease T1. These findings support the possibility that 23S ribosomal RNA participates in the peptidyl transferase function.

Electrophoresis, Polyacrylamide Gel↗
PubMed1992

The pgpA and pgpB genes of Escherichia coli are not essential: evidence for a third phosphatidylglycerophosphate phosphatase.

To further define the genes and gene products responsible for the in vivo conversion of phosphatidylglycerophosphate to phosphatidylglycerol in Escherichia coli, we disrupted two genes (pgpA and pgpB) which had previously been shown to encode gene products which carried out this reaction in vitro (T. Icho and C. R. H. Raetz, J. Bacteriol. 153:722-730, 1983). Strains with either gene or both genes disrupted had the same properties as the original mutants isolated with mutations in these genes, i.e., reduced in vitro phospholipid phosphatase activities, normal growth properties, and an increase in the level of phosphatidylglycerophosphate (1.6% versus less than 0.1% in wild-type strains). These results demonstrate that these genes are not required for either normal cell growth or the biosynthesis of phosphatidylglycerol in vivo. In addition, the total phosphatidylglycerophosphate phosphatase activity in the doubly disrupted mutant was reduced by only 50%, which indicates that there is at least one other gene that encodes such an activity and thus accounts for the lack of a dramatic effect on the biosynthesis of anionic phospholipids in these mutant strains. The phosphatidic acid and lysophosphatidic acid phosphatase activities of the pgpB gene product were also significantly reduced in gene-interrupted mutants, but the detection of residual phosphatase activities in these mutants indicated that additional genes encoding such phosphatases exist. The lack of a significant phenotype resulting from disruption of the pgpA and pgpB genes indicates that these genes may be required only for nonessential cell function and leaves the biosynthesis of phosphatidylglycerophosphate as the only step in E. coli phospholipid biosynthesis for which a gene locus has not been identified.

Escherichia coli↗
PubMed1992

Cl- channels in basolateral renal medullary membranes, VI. Cl- conductance expression in Xenopus oocytes.

Cl- channels from basolateral membranes of the mammalian thick ascending limb of Henle differ significantly, in certain of their functional characteristics, from Cl- channels in apical membranes of secretory epithelia such as trachea or small intestine and from certain Cl- channels in the central nervous system. Yet there is no sequence information available about basolateral thick ascending limb Cl- channels. As an initial step in the isolation of a Cl- channel from the thick ascending limb of Henle's loop, we attempted a functional expression of a Cl- conductance in oocytes from Xenopus laevis. Oocytes injected with mRNA isolated from the outer medulla of rabbit kidney had a membrane conductance, measured using a two-electrode voltage clamp, which was sixfold greater than in water-injected oocytes (9.05 +/- 1.56 vs. 1.43 +/- 0.15 microS, respectively). Ion substitution experiments showed the conductance in the RNA-injected oocytes to be Cl- selective. In addition, the Cl(-)-channel blocker diphenylamine-2-carboxylate (1 mM) almost completely inhibited (79 +/- 6%) the increased conductance seen in the RNA-injected oocytes. Fractionation of the mRNA by sucrose gradient centrifugation revealed that peak Cl- channel activity was expressed using an mRNA fraction of 1.8-3.2 kb in size. These results demonstrate that a membrane Cl- conductance can be expressed in X. laevis oocytes injected with size-selected fractions of mRNA from rabbit outer renal medulla.

Animals↗
PubMed1989

Identification of RegA protein from Pseudomonas aeruginosa using anti-RegA antibody.

The product of Pseudomonas aeruginosa regA gene acts as a positive regulator of exotoxin A expression. The protein, RegA, was overproduced in E. coli transformed with an expression vector containing the regA gene. The overproduced RegA accumulated in E. coli in the form of inclusion bodies. The latter were isolated and served as a source of antigen for raising polyclonal antibodies. The antibodies reacted specifically with a P. aeruginosa protein whose molecular weight corresponded to that predicted for RegA from its known DNA sequence, and whose response to modulating factors matched that expected for RegA. The immunodetectable RegA was localized in the membrane fraction of P. aeruginosa strain PA103.

ADP Ribose Transferases↗
PubMed1988

The cDNA sequence of the 69-kDa subunit of the carrot vacuolar H+-ATPase. Homology to the beta-chain of F0F1-ATPases.

Vacuolar ATPases constitute a novel class of N-ethylmaleimide- and nitrate-sensitive proton pumps associated with the endomembrane system of eukaryotic cells. They resemble F0F1-ATPases in that they are large multimeric proteins, 400-500 kDa, composed of three to nine different subunits. Previous studies have indicated that the active site is located on the approximately 70-kDa subunit. Using antibodies to the approximately 70-kDa subunit of corn to screen a carrot root lambda gt11 cDNA library, we have isolated cDNA clones of the carrot 69-kDa subunit. The complete primary structure of the 69-kDa subunit was then determined from the nucleotide sequence of its cDNA. The 69-kDa subunit consists of 623 amino acids (Mr 68,835), with no obvious membrane-spanning regions. The carrot cDNA sequence was over 70% homologous with exons of a Neurospora 69-kDa genomic clone. The protein sequence of the carrot 69-kDa subunit also exhibited 34.3% identity to four representative F0F1-ATPase beta-chains over a 275-amino-acid core stretch of similar sequence. Alignment studies revealed several regions which were highly homologous to beta-chains, including sequences previously implicated in catalytic function. This provides definitive evidence that the vacuolar ATPase is closely related to the F0F1-type ATPases. A major functional difference between the 69-kDa and beta-subunits is the location of 3 critical cysteine residues: two in the putative catalytic region (Cys-248 and Cys-256) and one in the proposed Mg2+-binding site (Cys-279). These cysteines (and two others) probably account for the sensitivity of the vacuolar H+-ATPase to the sulfhydryl reagent, N-ethylmaleimide. It is proposed that the two ATPases may have arisen from a common ancestor by the insertion or deletion of a large stretch of nonhomologous sequence near the amino-terminal end of the subunit.

Amino Acid Sequence↗