scholarly journals Glucose represses transcription of Saccharomyces cerevisiae nuclear genes that encode mitochondrial components.

1984 ◽  
Vol 4 (5) ◽  
pp. 939-946 ◽  
Author(s):  
E Szekely ◽  
D L Montgomery
Keyword(s):  
Blot Hybridization ◽  
Beta Subunit ◽  
Alpha Subunit ◽  
Northern Blot Hybridization ◽  
Rich Medium ◽  
Genes Encoding ◽  
Atpase Subunits ◽  
Steady State Levels ◽  
Alpha Beta ◽  
Kinetics Of

By Northern blot hybridization analysis, we demonstrated that the steady-state levels of mRNAs specifying the alpha subunit of ATPase, the beta subunit of ATPase, and the ATP/ADP translocator are all reduced in cells grown in glucose-rich medium. The extent to which glucose represses the levels of alpha, beta, and translocator mRNAs varies from strain to strain, from 2.5- to 7-fold. Furthermore, by hybridization experiments with an excess of DNA, we showed that glucose represses the rates of synthesis of these mRNAs. The kinetics of repression and depression of transcription were also studied. Finally, a mutant was characterized which appears to be defective in depression of transcription of the genes encoding the alpha and beta ATPase subunits as well as the ATP/ADP translocator.

Glucose represses transcription of Saccharomyces cerevisiae nuclear genes that encode mitochondrial components

1984 ◽  
Vol 4 (5) ◽  
pp. 939-946
Author(s):  
E Szekely ◽  
D L Montgomery
Keyword(s):  
Blot Hybridization ◽  
Beta Subunit ◽  
Alpha Subunit ◽  
Northern Blot Hybridization ◽  
Rich Medium ◽  
Genes Encoding ◽  
Atpase Subunits ◽  
Steady State Levels ◽  
Alpha Beta ◽  
Kinetics Of

By Northern blot hybridization analysis, we demonstrated that the steady-state levels of mRNAs specifying the alpha subunit of ATPase, the beta subunit of ATPase, and the ATP/ADP translocator are all reduced in cells grown in glucose-rich medium. The extent to which glucose represses the levels of alpha, beta, and translocator mRNAs varies from strain to strain, from 2.5- to 7-fold. Furthermore, by hybridization experiments with an excess of DNA, we showed that glucose represses the rates of synthesis of these mRNAs. The kinetics of repression and depression of transcription were also studied. Finally, a mutant was characterized which appears to be defective in depression of transcription of the genes encoding the alpha and beta ATPase subunits as well as the ATP/ADP translocator.

Mercury weakens membrane anchoring of Na-K-ATPase

1992 ◽  
Vol 262 (5) ◽  
pp. F837-F842 ◽  
Author(s):  
E. Imesch ◽  
M. Moosmayer ◽  
B. M. Anner
Keyword(s):  
Atpase Activity ◽  
Model Compound ◽  
Beta Subunit ◽  
Alpha Subunit ◽  
Structural Effects ◽  
Subunit Interaction ◽  
Alpha Beta ◽  
Membrane Anchoring ◽  
Atpase Inhibition

The presence of circulating inhibitors able to decrease the renal Na-K-adenosinetriphosphatase (ATPase) activity (natriuretic hormones) was postulated some 30 years ago. In the present work, the natriuretic inhibitor HgCl2 was selected as a model compound for the structural characterization of a possible natriuretic pathway for Na-K-ATPase modification. The structural effects of Na-K-ATPase inhibition by HgCl2 were assessed by trypsinolysis of the blocked enzyme in comparison with untreated preparations. The results show that inactivation of Na-K-ATPase by HgCl2 leads to the release of the alpha-subunit from the membrane preferentially in the E2 conformation but also in the E1 conformation. Apparently, HgCl2 weakens the membrane anchoring of the alpha-subunit, presumably by loosening the alpha-beta-subunit interaction. By this mechanism, the sensitivity of the Na-K-ATPase to extracellular drugs, hormones, and antibodies, as well as to intracellular proteases and other regulatory factors, could be altered.

scholarly journals Molecular characterization and expression of the stratification-related cytokeratins 4 and 15.

1988 ◽  
Vol 106 (4) ◽  
pp. 1249-1261 ◽  
Author(s):  
R E Leube ◽  
B L Bader ◽  
F X Bosch ◽  
R Zimbelmann ◽  
T Achtstaetter ◽  
...  
Keyword(s):  
Basal Cell ◽  
Blot Hybridization ◽  
Northern Blot Hybridization ◽  
Type I ◽  
Specific Expression ◽  
Genes Encoding ◽  
Cell Layers ◽  
Hybridization In Situ ◽  
Stratified Epithelia

A number of human cytokeratins are expressed during the development of stratified epithelia from one-layered polar epithelia and continue to be expressed in several adult epithelial tissues. For studies of the regulation of the synthesis of stratification-related cytokeratins in internal tissues, we have prepared cDNA and genomic clones encoding cytokeratin 4, as a representative of the basic (type II) cytokeratin subfamily and cytokeratin 15, as representative of the acidic (type I) subfamily, and determined their nucleotide sequences. The specific expression of mRNAs encoding these two polypeptides in certain stratified tissues and cultured cell lines is demonstrated by Northern blot hybridization. Hybridization in situ with antisense riboprobes and/or synthetic oligonucleotides shows the presence of cytokeratin 15 mRNA in all layers of esophagus, whereas cytokeratin 4 mRNA tends to be suprabasally enriched, although to degrees varying in different regions. We conclude that the expression of the genes encoding these stratification-related cytokeratins starts already in the basal cell layer and does not depend on vertical differentiation and detachment from the basal lamina. Our results also show that simple epithelial and stratification-related cytokeratins can be coexpressed in basal cell layers of certain stratified epithelia such as esophagus. Implications of these findings for epithelial differentiation and the formation of squamous cell carcinomas are discussed.

scholarly journals Expression of rat endopeptidase-24.18 in COS-1 cells: membrane topology and activity

1994 ◽  
Vol 300 (1) ◽  
pp. 37-43 ◽  
Author(s):  
P E Milhiet ◽  
D Corbeil ◽  
V Simon ◽  
A J Kenny ◽  
P Crine ◽  
...  
Keyword(s):  
Cell Surface ◽  
Culture Media ◽  
Enzymic Activity ◽  
Beta Subunit ◽  
Membrane Topology ◽  
Site Directed Mutagenesis ◽  
Alpha Subunit ◽  
Cell Extracts ◽  
Membrane Bound ◽  
Alpha Beta

Endopeptidase-24.18 (E-24.18; EC 3.4.24.18) is a metallopeptidase of the astacin family and is highly expressed in kidney brush-border membranes of rodents. Rat E-24.18 consists of two disulphide-linked alpha/beta dimers [(alpha/beta)2]. In order to investigate the mechanisms of assembly and the importance of each subunit in the enzymic process, the cloned cDNAs for the rat alpha and beta subunits were transiently expressed either alone or together in COS-1 cells. Immunoblotting of cell extracts and spent culture media showed that, when expressed alone, the alpha subunit is secreted, whereas the beta subunit is membrane-bound. In alpha/beta-transfected cells, the alpha subunit remained membrane-bound, but could be released from the cell surface after papain treatment or after incubation with 10 mM dithiothreitol. Furthermore, mutants of the alpha subunit in which the putative C-terminal anchor domain was deleted could still form cell-associated alpha/beta dimers. These results are consistent with a topological model of E-24.18 in which the beta subunit is anchored in the plasma membrane and the alpha subunit is retained at the cell surface through disulphide bridge(s) with the beta subunit. Both the alpha and beta recombinant subunits expressed in COS-1 cells showed little azocasein-degrading activity. However, activity of either individual subunits of alpha/beta dimers was increased after mild trypsin digestion, suggesting that in COS-1 cells the enzymes are synthesized as zymogens. Finally, inactivation of the alpha subunit by site-directed mutagenesis of Glu-157, which is believed to play a role in catalysis, showed that both subunits participate in the enzymic activity of the heterodimer.

scholarly journals Binding Interaction of a Ring-hydroxylating Dioxygenase with Fluoranthene in Pseudomonas aeruginosa DN1

2021 ◽  
Author(s):  
Shu-Wen Xue ◽  
Yue-Xin Tian ◽  
Jin-Cheng Pan ◽  
Ya-Ni Liu ◽  
Yanling Ma
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Amino Acid ◽  
Gene Knockout ◽  
Amino Acid Sequences ◽  
Beta Subunit ◽  
Alpha Subunit ◽  
Binding Interaction ◽  
Mononuclear Iron ◽  
Genes Encoding ◽  
Substrate Conversion

Abstract Pseudomonas aeruginosa DN1 can efficiently utilize fluoranthene as its sole carbon source, and the first step in the biodegradation process is catalyzed by a ring-hydroxylating dioxygenase (RHD). To better understand the binding interaction of RHD with fluoranthene in the strain DN1, the genes encoding alpha subunit (RS30940) and beta subunit (RS05115) of the RHD were functionally characterized using gene knockout approach and homology modeling combined with molecular docking. The results showed that the mutants lacking the characteristic alpha subunit and/or beta subunit failed to degrade fluoranthene effectively. Based on the translated protein sequence and Ramachandran plot, 96.5 % of the primary amino-acid sequences of the alpha subunit in the modeled structure of the RHD were in the permitted region, 2.3 % in the allowed region, but 1.2 % in the disallowed area. The active center of the alpha subunit constituted a triangle structure of the mononuclear iron atom and the two oxygen atoms coupled with a catalytic ternary of His217-His222-Asp372 for the dihydroxylation reaction with fluoranthene. Amino acid residues adjacent to fluoranthene were nonpolar groups, and the C7-C8 positions on the fluoranthene ring were estimated to be the best oxidation sites. The distance of C7-O and C8-O was 3.77 Å and 3.04Å respectively, and both of them were parallel. The results demonstrated that the dihydroxylation reaction was initiated at C7-C8 positions of the fluoranthene ring by RHD in P. aeruginosa DN1, indicating that the binding interaction may be useful for predicting substrate conversion of RHDs.

Synthesis and translocation of Na(+)-K(+)-ATPase alpha- and beta-subunits to plasma membrane in MDCK cells

1992 ◽  
Vol 262 (2) ◽  
pp. C470-C483 ◽  
Author(s):  
A. K. Mircheff ◽  
J. W. Bowen ◽  
S. C. Yiu ◽  
A. A. McDonough
Keyword(s):  
Plasma Membrane ◽  
Mdck Cells ◽  
Subcellular Fractionation ◽  
Beta Subunit ◽  
Alpha Subunit ◽  
Beta Subunits ◽  
Madin Darby Canine Kidney ◽  
Intracellular Membranes ◽  
Alpha Beta ◽  
Darby Canine Kidney

Synthesis and translocation of Na(+)-K(+)-ATPase alpha-catalytic and beta-glycoprotein subunits from intracellular membranes to the plasma membrane were studied in Madin-Darby canine kidney cells (MDCK-T) by combining the methods of pulse-chase labeling, subcellular fractionation on sorbitol gradients, and immunoprecipitation. Immunoprecipitation from homogenates revealed that radioactive methionine incorporated into beta-subunit was equal to that incorporated into alpha-subunit after 15 min of labeling. Because the ratio of total methionines in alpha- vs. beta-subunit is approximately 5:1, these results suggest that beta-subunit is synthesized in molar excess over alpha-subunit. Half of the newly synthesized beta-subunit, likely unassembled units, were degraded by 60 min after labeling, while alpha-subunits were stable through 120 min after synthesis, suggesting alpha may be limiting for alpha beta-assembly. By 120 min the ratio of counts incorporated into alpha vs. beta approached 5, which is predicted by a 1:1 ratio of alpha to beta. The sorbitol gradient resolved two major membrane samples: a mixture of endoplasmic reticulum and Golgi populations and a plasma membrane-enriched sample. Immature beta (beta i) could not be detected in the plasma membrane-enriched samples at levels greater than could be attributed to cross-contamination by intracellular membranes. Mature beta (beta m) became detectable after 30 min, and conversion of beta i to beta m was 90% complete at 120 min. A peak of labeled alpha-subunit appeared in the plasma membrane-enriched sample at 60 min, coincident with the appearance of labeled beta m-subunit in this sample, suggesting movement as alpha beta-heterodimers.

LH subunit mRNA concentrations during LH surge in ovariectomized estradiol-replaced rats

1988 ◽  
Vol 254 (1) ◽  
pp. E99-E103
Author(s):  
D. J. Haisenleder ◽  
A. L. Barkan ◽  
S. Papavasiliou ◽  
S. M. Zmeili ◽  
C. Dee ◽  
...  
Keyword(s):  
Blot Hybridization ◽  
Beta Subunit ◽  
Alpha Subunit ◽  
Dot Blot ◽  
Ether Anesthesia ◽  
Lh Surge ◽  
Subunit Mrna ◽  
Dot Blot Hybridization ◽  
Ovx Rats ◽  
Lh Secretion

In cycling rats, pituitary concentrations of luteinizing hormone (LH) beta-subunit mRNA increase two- to threefold before the afternoon proestrus LH surge without a corresponding increase in alpha-subunit mRNA. Estradiol (E2) treatment is known to allow expression of daily LH surges in ovariectomized (OVX) rats, and the timing, magnitude, and duration of LH secretion is similar to the LH surge on proestrus. The present study was conducted to examine whether the regulation of LH subunit mRNAs during the LH surge in OVX-E2-treated rats is similar to that present on proestrus. Female Holtzman rats were OVX and Silastic implants containing E2 were inserted subcutaneously under ether anesthesia. Some animals received bromocriptine (0.6 mg sc, twice/day beginning 1 h before surgery). On the 2nd day after surgery, groups of animals (n = 4-10/group) were decapitated at intervals between 1000 and 2100. LH and prolactin (PRL) levels were measured in trunk blood. LH subunit mRNA concentrations in the pituitaries were measured by dot-blot hybridization assay. In OVX-E2 rats the LH surge occurred at 1830 and was accompanied by a selective twofold increase in alpha-subunit mRNA (from 266 +/- 18 to 459 +/- 61 pg cDNA bound/100 micrograms pituitary DNA) and maximum values were present at 1730. LH beta-subunit mRNA (m = 29 +/- 1 pg cDNA bound/100 micrograms pituitary DNA) was unchanged throughout the day. Bromocriptine treatment resulted in the suppression of serum PRL (m = 23 +/- 2 ng/ml) and the LH surge was delayed by 1-1.5 h and somewhat blunted.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Characterization of Bacteroides fragilis Hemolysins and Regulation and Synergistic Interactions of HlyA and HlyB

2006 ◽  
Vol 74 (4) ◽  
pp. 2304-2316 ◽  
Author(s):  
Kirstin P. Robertson ◽  
C. Jeffrey Smith ◽  
Andrea M. Gough ◽  
Edson R. Rocha
Keyword(s):  
Hemolytic Activity ◽  
Blot Hybridization ◽  
Bacteroides Fragilis ◽  
Blood Agar ◽  
Northern Blot Hybridization ◽  
Rich Medium ◽  
Independent Manner ◽  
Hemolytic Assay ◽  
Growth Deficiency

ABSTRACT This study describes the presence of 10 hemolysin orthologs in the genome of the opportunistic human anaerobic pathogen Bacteroides fragilis, which is currently classified as a nonhemolytic bacterium. The hemolysins were designated HlyA through HlyI plus HlyIII. All cloned hemolysin genes were able to confer hemolytic activity to a nonhemolytic Escherichia coli strain on blood agar plates. Interestingly, HlyH was found to be present in the genome of the B. fragilis NCTC9343 strain but absent in strains 638R, YCH46, and Bacteroides thetaiotaomicron VPI-5482. The hemolysins HlyA, HlyB, and HlyIII were selected for further characterization. HlyA, HlyB, and HlyIII were cytolytic to erythrocytes on liquid hemolytic assay. When hlyA and hlyB were expressed together in a nonhemolytic E. coli strain, the strain showed enhanced hemolytic activity on blood agar plates. Further analysis revealed that HlyA and HlyB have synergistic hemolytic activity as detected by the liquid hemolytic assay. In addition, the two-component hemolysins HlyA and HlyB form a protein-protein complex in vivo as determined by bacterial two-hybrid system assay. The hlyB and hlyA genes are organized in an operon that is coordinately regulated by iron and oxygen. Northern blot hybridization analysis revealed that hlyBA were expressed as a bicistronic mRNA induced approximately 2.5-fold under low-iron conditions and repressed in iron-rich medium. The normal iron-regulated expression of hlyBA mRNA was lost in the furA mutant strain. In contrast, the hlyA gene was also expressed as a single mRNA in iron-rich medium, but its expression was reduced approximately threefold under low-iron conditions in a Fur-independent manner. This suggests that hlyA alone is regulated by an unidentified iron-dependent regulator. Moreover, the expression levels of hlyBA and hlyA were reduced about threefold following oxygen exposure and treatment with hydrogen peroxide. Taken together, these results suggest that iron and oxidative stress have an effect on the control of hlyBA and hlyA transcriptional levels. A hlyBA mutant was constructed, and its hemolytic activity was greatly diminished compared to those of the hlyIII mutant and parent strains. In addition, the hlyBA mutant had a significant modification in colony morphology and growth deficiency compared to the parent strain. The implications of these findings for the pathophysiology of B. fragilis in extraintestinal infections and competition in ecological systems for this organism are discussed.

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