Sensitive, nonradioactive northern blots using alkaline transfer of total RNA and PCR-amplified biotinylated probes.
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Adenosine Triphosphatases↗
Biomedical subjects
T Rausch
Publications and source records attributed to T Rausch.
[Familial incidence of colorectal carcinoma and its significance for a screening program (the example of Upper Basel area)].
Primary prevention of colorectal carcinoma aims at interruption of the adenoma-carcinoma sequence. Colonoscopy is the most reliable screening method, but up to now has only been recommended for persons at high risk. The ongoing 10-year study defines a possible risk group and determines the efficacy of a preventive screening program based on family history. This paper reports on the descriptive statistics of the first five years. All patients with colonic carcinoma diagnosed between 1987 and 1991 living in the upper part of Canton Basel-Land were registered. According to the family history, patients with at least one first degree relative with colonic carcinoma were defined as index patients. Their first degree relatives entered a prospective screening program which includes colonoscopy. 230 colonic carcinomas were diagnosed in the upper part of Basel-Land, representing an incidence of 48/year/100,000 persons. They included 3 (1.3%) hereditary and 227 (98.7%) sporadic carcinomas. 23 (10%) patients had a positive family history. These patients defined 94 relatives at risk. The incidence of colonic carcinoma in the Canton studied does not differ from that reported in Switzerland. Therefore, this part of Basel-Land can be considered representative. The 1.3% hereditary carcinomas are in contrast to the published data of 6%.
Adenomatous Polyposis Coli↗
The hexose transporters at the plasma membrane and the tonoplast of transformed plant cells: kinetic characterization of two distinct carriers.
The plasma membrane hexose transporter and the tonoplast hexose transporter from heterotrophically grown transformed Nicotiana tabacum cells have been studied in vitro using membrane vesicles for trans-zero transport studies. In highly purified phase-partitioned outside-out plasma membrane vesicles (PMV) the hexose transporter showed an apparent Km value of 230 microM (substrate: 3-O-methyl-D-glucose (3-OMG); pHi 7.2/pHo 7.2), which was reduced to 120 microM when a pH gradient was imposed (pHo 5.7/pHi 7.2). However, the Vmax value was not affected indicating that no stable pH gradient was formed. Uptake experiments with 14C-labelled acetate supported this interpretation. Transport was insensitive to N-ethylmaleimide (NEM; up to 1 mM concentration) and p-chloromercuribenzene sulfonate (PCMBS; up to 500 microM), whereas the tonoplast hexose transporter (in mixed inside / out and outside / out vesicles) was inhibited by NEM in a substrate-protectable manner, and PCMBS was also inhibitory. Kinetically two components with apparent Km values of 6 and 20 mM could be distinguished for the tonoplast hexose transporter. Substrate specificities of both transporters were similar except for D-galactose and D-fructose. The results indicate structural differences between the tonoplast and plasma membrane hexose transporters in plants.
3-O-Methylglucose↗
An mRNA from human brain encodes an isoform of the B subunit of the vacuolar H(+)-ATPase.
The B subunit (approximately 60 kDa) of the vacuolar H(+)-ATPase is one of the two major subunits comprising the hydrophilic catalytic complex of the enzyme. Using left and catalytic complex of the enzyme. Using left and right primers which bind two highly conserved sequences of the B subunit, an 836-base pair fragment was amplified from human brain cDNA by the polymerase chain reaction. The amplified fragment was used to probe a Northern blot and to screen a brain cDNA library. A single RNA band, 3.2 kilobases (kb) in length, was detected on Northern blots. A positive cDNA clone containing a 2.5-kb insert was isolated and sequenced. It included a long 3'-untranslated region (greater than 1.2 kb) and was missing a minor portion of the 5'-end of the coding region. The coding region of the brain cDNA sequence was 77% identical at the nucleotide level and 90% identical at the amino acid level to the previously reported sequence for the B subunit of the vacuolar H(+)-ATPase from human kidney (Sudhof, T. C., Fried, V. A., Stone, D. K., Johnston, P. A., and Xie, X.-S. (1989) Proc. Natl. Acad. Sci, U. S. A. 86, 6067-6071). Within the coding region of the brain cDNA, which is 6 amino acid residues shorter at the 3'-end than the kidney sequence, an 11% difference in the GC content was calculated. The 3'-noncoding sequence of the brain cDNA was completely unrelated to that of kidney and was three times longer. We conclude that the B subunit cDNAs from human kidney and brain represent different isoforms. This is the first demonstration of an isoform of a vacuolar H(+)-ATPase subunit.
Aged↗
Structure and function of the promoter of the carrot V-type H(+)-ATPase catalytic subunit gene.
We investigated the 5'-upstream region of the gene encoding the catalytic subunit of the V-type H(+)-ATPase in Daucus carota. A genomic sublibrary was screened with a cDNA probe, and a 4-kilobase genomic clone was obtained covering the first two exons and about 3 kilobases of the 5'-upstream sequence. The intron/exon boundaries matched established consensus sequences. Within 240 base pairs (bp) upstream of the initiation codon three putative TATA boxes were found. Ribonuclease protection and primer extension analysis indicated that the three TATA boxes corresponded to two major and one minor transcription start sites. The flanking sequences of the two more proximal TATA boxes were nearly identical. Additional sequence motifs with putative regulatory function are two CCAAT boxes, an Sp1-binding consensus sequence, and long (TATA)n stretches within 800 bp of the 5'-upstream sequence. Transcriptional fusions to the beta-glucuronidase reporter gene were made for two different promoter constructs, and the resulting plasmids were mobilized into Agrobacterium tumefaciens. The analysis of beta-glucuronidase activities in the transformed carrot calli showed that 240 bp of the upstream sequence, including all three TATA boxes, led to low but detectable beta-glucuronidase expression; however, the larger construct, which included the putative Sp1-binding sequence and the (TATA)n stretches, led to an approximately 6-fold higher beta-glucuronidase expression. Histochemical analysis of beta-glucuronidase activity in the transformed calli showed no preferential expression in any specific cell type, in keeping with the presumed "housekeeping" character of the V-type H(+)-ATPase catalytic subunit gene.
Amino Acid Sequence↗
Cell wall invertase in tobacco crown gall cells : enzyme properties and regulation by auxin.
The cell wall invertase from an Agrobacterium tumefaciens-transformed Nicotiana tabacum cell line (SR1-C58) was purified. The heterogeneously glycosylated enzyme has the following properties: M(r) 63,000, pH optimum at 4.7, K(m sucrose) 0.6 millimolar (at pH 4.7), pl 9.5. Enzyme activity is inhibited by micromolar concentrations of HgCl(2) but is insensitive to H(2)O(2), N-ethylmaleimide and dithiothreitol. Upon transfer of transformed cells from the stationary phase to fresh medium, a cycloheximide- and tunicamycin-sensitive de novo formation of cell wall invertase is demonstrated in the absence or presence of sucrose. While in an auxin mutant (lacking gene 1;SR1-3845) 1 micromolar 1-naphthaleneacetic acid led to a further increased activity, the wild-type transformed cell line (SR1-C58) responded with a decreased activity compared to the control. An analysis of cell wall invertase in and around tumors initiated with Agrobacterium tumefaciens (strain C58) on Nicotiana tabacum stem and Kalanchoë daigremontiana leaves revealed gradients of activity. The results indicate that the auxin-stimulated cell wall invertase is essential for the establishment of the tumor sink.
Journal Article↗
Identification of an Mr 75000 component of the H+/D-glucose cotransporter from Zea mays with monoclonal antibodies directed against the mammalian Na+/D-glucose cotransporter.
Monoclonal antibodies which interact with the mammalian Na+/D-glucose cotransporter and bind to Mr 75,000 and Mr 47,000 polypeptide components of this transporter have been described (Koepsell, H., Korn, K., Raszeja-Specht, A., Bernotat-Danielowski, S. and Ollig, D. (1988) J. Biol. Chem., 263, 18419-18429). The interaction of these antibodies with plasma membranes from Zea mays L. coleoptiles containing an H+/D-glucose cotransporter was studied. Four monoclonal antibodies cross-reacted with Mr 75,000 and Mr 33,000 polypeptides. One of these antibodies, which inhibits Na+/D-glucose cotransport in the kidney and stimulates Na+/D-glucose cotransport in intestine, stimulates electrogenic uptake of 3-O-methyl-D-[14C]glucose in plant membrane vesicles. The data indicate common epitopes in the mammalian Na+/D-glucose cotransporter and the H+/D-glucose cotransporter of plants and suggest that both transporters contain an Mr 75000 polypeptide component.
3-O-Methylglucose↗
The H+ ATPase regulatory subunit of Methanococcus thermolithotrophicus: amplification of an 800 bp fragment by polymerase chain reaction.
An 800 bp fragment of Methanococcus thermolithotrophicus genomic DNA was amplified by the polymerase chain reaction method using primers designed from conserved regions of the V-type H+ ATPase regulatory subunits from the archaebacterium Sulfolobus, and several eukaryotes. Although more than one product was obtained, only one of them had the expected size and was exclusively amplified in the presence of the left and right primers. The DNA and the deduced protein sequences of the putative Methanococcus H+ ATPase subunit revealed homology to the corresponding sequences in Sulfolobus and eukaryotes (about 60% identical residues) and a less evident homology to the eubacterial F1-ATPase alpha-subunit (22% identical residues with E. coli).
Amino Acid Sequence↗
Anion-Sensitive Mg ATP-Dependent Proton Pumping in Microsomal Membranes from Phycomyces blakesleeanus Bgff.
A light microsomal membrane fraction (collected on 28% weight/volume sucrose) from young mycelia of Phycomyces blakesleeanus Bgff. stationary cultures exhibited Mg(2+) ATP-dependent proton pumping as monitored with the dye neutral red or by accumulation of [(14)C]methylamine. The substrate kinetics for ATP (K(m(ATP)) 1.1 millimolar in the presence of 2 millimolar Mg(2+)), the competitive inhibition by ADP (K(i(ADP)) 0.8 millimolar), the anion sensitivity (stimulation by Cl(-), inhibition by NO(3) (-) and SO(4) (2-)), the reversal of acidification by Ca(2+), the inhibition by diethylstilbestrol and dicyclohexylcarbodiimide, as well as the absence of any inhibition by azide, molybdate, and vanadate strongly suggest a tonoplast-type ATPase driven proton pumping. The same membrane fraction showed no pyrophosphate driven H(+)-translocation. Electron microscopic examination of the fungal mycelium demonstrated the presence of vacuoles of different sizes and other nonidentified vesicles but no typical Golgi stacks.
Journal Article↗
Indole-3-Ethanol Oxidase in Phycomyces blakesleeanus Bgff: Characterization of the Enzyme.
Indole-3-ethanol oxidase (IEt oxidase) from Phycomyces blakesleeanus Bgff.(P.b.) is a 56 kD polypeptide as determined by gel filtration. The reaction products are indole-3-acetaldehyde (IAAld) and, possibly, H(2)O(2). Enzyme activity (33-45% ammonium sulfate fraction) shows a broad pH optimum and simple Michaelis-Menten kinetics (K(m) 7 micromolar, Hill coefficient 0.95). Flavin adenine dinucleotide increases enzyme activity particularly under anaerobic conditions. Iodoacetate and HgCl(2) drastically inhibit the enzyme. With IAAld, product inhibition is observed at micromolar concentrations. IAA and some other acidic substituted indoles reduce enzyme activity but only at higher concentrations.
Journal Article↗
Active Glucose Transport and Proton Pumping in Tonoplast Membrane of Zea mays L. Coleoptiles Are Inhibited by Anti-H-ATPase Antibodies.
A tonoplast enriched fraction was obtained from Zea mays L. coleoptiles by isopycnic centrifugation of microsomal membranes in a sucrose step gradient. At the 18/26% interface chloride-stimulated and nitrate-inhibited proton pumping activity coincided with a Mg(2+)-ATP dependent accumulation of 3-O-methyl-d-glucose (OMG) as determined by a membrane filtration technique using (14)C-labeled substrate. OMG transport showed an apparently saturable component with a K(m) of 110 micromolar, and was completely inhibited by 10 micromolar carbonyl cyanide m-chlorophenylhydrazone. Polyclonal antibodies against solubilized native tonoplast H(+)-ATPase and its 62 and 72 kilodalton subunits were assayed for their ability to inhibit proton pumping and OMG accumulation. Antibodies against both the native enzyme and the putative catalytic subunit (72 kilodalton) strongly inhibited proton pumping and OMG transport whereas antibodies against the 62 kilodalton subunit had only a slight effect on both processes.
Journal Article↗
ADP Is a Competitive Inhibitor of ATP-Dependent H Transport in Microsomal Membranes from Zea mays L. Coleoptiles.
An anion-sensitive ATP-dependent H(+) transport in microsomal membranes from Zea mays L. coleoptiles was partially characterized using the pH gradient-dependent decrease of unprotonated neutral red. The following criteria strongly suggest a tonoplast origin of the H(+) transport observed: strict dependence on Cl(-); inhibition by SO(4) (2-) and NO(3) (-); insensitivity against vanadate, molybdate, and azide; reversible inhibition by CaCl(2) (H(+)/Ca(2+) antiport); inhibition by diethylstilbestrol. The substrate kinetics revealed simple Michaelis Menten kinetics for ATP in the presence of 1 millimolar MgCl(2) with a K(m) value of 0.56 millimolar (0.38 millimolar for MgATP). AMP and c-AMP did not influence H(+) transport significantly. However, ADP was a potent competitive inhibitor with a K(i) value of 0.18 millimolar. The same inhibition type was found for membranes prepared from primary roots by the same procedure.
Journal Article↗
Primary Action of Indole-3-acetic Acid in Crown Gall Tumors: Increase of Solute Uptake.
Exogenously added indole-3-acetic acid at a concentration of 100 micromolars stimulates d-glucose uptake (or 3-O-methyl-d-glucose uptake) by 25% in crown gall tumors induced on potato tuber tissue by Agrobacterium tumefaciens strain C 58. The titration of the endogenous IAA with the auxin antagonist 2-naphthaleneacetic acid at 100 micromolars reduces d-glucose uptake by about 80%. The apparent inhibition constant K(i) is 21 micromolars. Other auxin antagonists like 1-naphthoxyacetic acid and 2-(p-chlorophenoxy)-2-methylpropionic acid show similar effects. The uptake of the amino acids leucine, methionine, tryptophan, lysine, and aspartic acid is also inhibited by 2-naphthaleneacetic acid to similar degrees. The auxins 1-naphthaleneacetic acid and 2-naphthoxyacetic acid at concentrations between 10 and 100 micromolars inhibit solute uptake only slightly (inhibition less than 20%). The impact of the results on the postulated role of indole-3-acetic acid as a modifier of the electrochemical proton gradient across the plasmalemma in crown gall tumor tissue is discussed.
Journal Article↗
Nurses' clinical judgments.
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Clinical Competence↗
Molecular evolution of H+-ATPases. I. Methanococcus and Sulfolobus are monophyletic with respect to eukaryotes and Eubacteria.
The classification of methanogenic bacteria as archaebacteria based on 16 s rRNA sequence analysis is currently in dispute. To provide an alternative molecular marker, the polymerase chain reaction technique was used to amplify a 930 bp fragment of Methanococcus thermolithotrophicus genomic DNA corresponding to the catalytic domain of the membrane H+-ATPase. The deduced amino acid sequence was 54-58% identical to the approximately 70 kDa subunits of Sulfolobus acidocaldarius and the eukaryotic vacuolar-type H+-ATPase, and only 29% identical to the beta subunit of the eubacterial-type F0F1-ATPases. Interestingly, a highly conserved aspartate residue in the phosphorylation domain of E1E2-ATPases (P-type) is conserved in the Methanococcus sequence, but is absent from all other known vacuolar and F0F1-ATPases. This suggests that the H+-ATPase of M. thermolithotrophicus, like that of M. voltae, may have a phosphorylated intermediate, despite belonging to the vacuolar-type class of proton pumps. Phylogenetic analysis using Felsenstein's maximum likelihood method and Lake's evolutionary parsimony method confirmed that the H+-ATPases of the two archaebacteria, Methanococcus and Sulfolobus, when compared to eukaryotic vacuolar-type ATPases and eubacterial F0F1-ATPases, form a monophyletic group.
Amino Acid Sequence↗