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Biomedical subjects

H Weiss

Publications and source records attributed to H Weiss.

At least 181 records · Page 10Linked to original sources
PubMed1992

Altered cell volume regulation in ras oncogene expressing NIH fibroblasts.

Expression of the Ha-ras oncogene has been reported to stimulate the dimethylamiloride sensitive Na+/H+ exchanger and Na+,K+,2Cl- cotransport, both transport systems which are involved in cell volume regulation. The present study has been performed to test for an influence of ras oncogene expression on cell volume regulation in NIH 3T3 fibroblasts expressing the Ha-ras oncogene (+ ras). As controls served NIH 3T3 fibroblasts not expressing the ras oncogene (-ras). In isotonic extracellular fluid, the cell volume of + ras cells (2.70 +/- 0.08 pl) is significantly greater than the cell volume of -ras cells (2.04 +/- 0.10 pl). Both, + ras and -ras cells exhibit a regulatory cell volume increase in hypertonic extracellular fluid and a regulatory cell volume decrease in hypotonic extracellular fluid. The regulatory cell volume decrease is inhibited by 1 mmol/l quinidine and barium, the regulatory cell volume increase is inhibited in -ras and +ras cells by dimethyl-amiloride (100 mumol/l) and, only in +ras cells, by furosemide (100 mumol/l) and bumetanide (10 mumol/l). In conclusion, expression of the ras oncogene leads to a shift of the set point for cell volume regulation to greater cell volumes, which may contribute to the activation of the Na+/H+ exchanger and Na+,K+,2Cl- cotransport.

3T3 Cells↗
PubMed1992

[Morphological and numerical studies of the stress compatibility of patella implants].

Failure of total knee arthroplasty is relatively often caused by problems of the patellofemoral replacement. The purpose of this study was to analyze the distribution of stresses within an anatomical patella and the changes in stress distribution after patellar resurfacing with a Miller-Galante I patellar implant using two- and three dimensional finite element models (FEM). To assess validity, FEM results were compared with morphological findings from contact radiographs and densitographs. Internal orientation of bone trabeculae is in good agreement with the orientation of theoretically calculated principal stresses. Almost unchanged principal tensile stresses after implantation, together with the lack of extreme stress peaks within the cancellous bone ensure stress compatibility of the implant. In the case of a firmly seated implant with good bone ingrowth, increased von Mises stresses are found near the fixation peg/plate junction. Their relevance for improved bone ingrowth near this part of the interface is emphasized. At the same time, material failure at the peg/plate junction can be better understood. An analysis of the early postoperative period assuming nonlinear interface conditions failed to demonstrate an uniform distribution of normal and tangential interface forces.

Aged↗
PubMed1991

Effect of BHT 920 on calcium-activated K+ channels in renal epithelioid MDCK cells.

In Madin Darby canine kidney (MDCK) cells, epinephrine has been shown to increase intracellular calcium, activate calcium-dependent K+ channels and hyperpolarize the cell membrane. The present study has been performed to test for the possible involvement of alpha 2-adrenergic receptors. To this end, the effects of alpha 2-adrenoceptor agonist BHT 920 have been studied on cell membrane potential, ion channel activity and intracellular calcium: Similar to epinephrine, BHT 920 hyperpolarizes the cell membrane, increases intracellular calcium and activates inwardly rectifying K+ channels (single channel slope conductances 30-80 pS). Half-maximal hyperpolarization is achieved at concentrations between 10 and 100 nmol/l. The hyperpolarizing effect of BHT 920 is abolished in the presence of alpha 2-adrenoceptor antagonist yohimbine (100 nmol/l) but not in the presence of alpha 1-adrenoceptor antagonist prazosin (100 nmol/l). At extracellular calcium activity below 100 nmol/l BHT 920 still leads to a transient hyperpolarization of the cell membrane but, in contrast to epinephrine, is unable to significantly increase intracellular calcium or significantly activate the calcium-sensitive K+ channels. The observations indicate that stimulation of alpha 2-receptors participates in the epinephrine-induced increase of intracellular calcium, channel activation and hyperpolarization.

Adrenergic beta-Agonists↗
PubMed1991

Electron microscopic analysis of the peripheral and membrane parts of mitochondrial NADH dehydrogenase (complex I).

Two related forms of the respiratory chain NADH dehydrogenase (NADH:ubiquinone reductase or complex I) are synthesized in the mitochondria of Neurospora crassa. Normally growing cells make a large form that consists of 25 subunits encoded by nuclear DNA and six to seven subunits encoded by mitochondrial DNA. Cells grown in the presence of chloramphenicol, however, make a smaller form comprising only 13 subunits, all encoded by nuclear DNA. When the large enzyme is dissected by chaotropic agents (such as NaBr), all those subunits of the large form that are missing in the small form can be isolated as a distinct, so-called hydrophobic fragment. The small enzyme and the hydrophobic fragment make up, with regard to their redox groups, subunit composition and function, two complementary parts of the large-form NADH dehydrogenase. Averaging of electron microscope images of single particles of the large enzyme was carried out, revealing an unusual L-shaped structure with two domains or "arms" arranged at right angles. The hydrophobic fragment obtained by the NaBr treatment corresponds in size and appearance to one of these arms. A three-dimensional reconstruction from images of negatively stained membrane crystals of the large-form NADH dehydrogenase shows a peripheral domain, protruding from the membrane, with weak unresolved density within the membrane. This peripheral domain was removed by washing the crystals in situ with 2 M-NaBr, exposing a large membrane-buried domain, which was reconstructed in three dimensions. A three-dimensional reconstruction of the small enzyme from negatively stained membrane crystals, also described here, shows only a peripheral domain. These results suggest that the membrane protruding arm of the large form corresponds to the small enzyme, whereas the arm lying within the membrane can be identified as the hydrophobic fragment. The two parts of NADH dehydrogenase that can be defined by the separate genetic origin of (most of) their subunits, their independent assembly, and their distinct contributions to the electron pathway can thus be assigned to the two arms of the L-shaped complex I.

Crystallography↗
PubMed1991

The acyl-carrier protein in Neurospora crassa mitochondria is a subunit of NADH:ubiquinone reductase (complex I).

We determined the primary structure of a 9.6-kDa subunit of the respiratory chain NADH:ubiquinone reductase (complex I) from Neurospora crassa mitochondria and found a close relationship between this subunit and the bacterial or chloroplast acyl-carrier protein. The degree of sequence identity amounts to 80% in a region of 19 residues around the serine to which the phosphopantetheine is bound. The N-terminal presequence of the subunit has the characteristic features of a mitochondrial import sequence. We cultivated the auxotroph pan-2 mutant of N. crassa in the presence of [14C]pantothenate and recovered all radioactivity incorporated into mitochondrial protein in the 9.6-kDa subunit of complex I. We cultivated N. crassa in the presence of chloramphenicol to accumulate the nuclear-encoded peripheral arm of complex I. This pre-assembled arm also contains the 9.6-kDa subunit. These results demonstrate that an acyl-carrier protein with pantothenate as prosthetic group is a constituent part of complex I in N. crassa.

Acyl Carrier Protein↗
PubMed1991

Primary structure of the nuclear-encoded 29.9 kDa subunit of NADH: ubiquinone reductase from Neurospora crassa mitochondria.

We isolated and sequenced cDNA for the 29.9 kDa subunit of mitochondrial NADH: ubiquinone reductase (complex I) from a Neurospora crassa library in the lambda gt11 expression vector. The N-terminus of the mature protein was determined by Edman-degradation. The cDNA contains an open reading frame encoding a preprotein of 273 amino acids. The presequence of the transit protein essential for mitochondrial import is eight residues long. Northern-blot analysis shows, that the level of the corresponding mRNA is increased 3-fold if cells are grown in the presence of chloramphenicol.

Amino Acid Sequence↗
PubMed1991

Primary structure of the nuclear-encoded 18.3 kDa subunit of NADH: ubiquinone reductase (complex I) from Neurospora crassa mitochondria.

The primary structure of the nuclear-encoded 18.3 kDa subunit of the respiratory chain NADH: ubiquinone reductase (complex I) from Neurospora crassa was determined by sequencing cDNA and the N-terminus of the protein. The cDNA contains an open reading frame for a protein of 206 amino acids. The mature protein consists of 173 amino acids and has a molar mass of 18,341 Da. The precursor protein includes a characteristic mitochondrial import sequence with a typical matrix peptidase processing site.

Amino Acid Sequence↗
PubMed1991

The iron-sulfur clusters in the two related forms of mitochondrial NADH: ubiquinone oxidoreductase made by Neurospora crassa.

Two related forms of the respiratory-chain complex, NADH: ubiquinone oxidoreductase (Complex I) are synthesized in the mitochondria of Neurospora crassa. Normally growing cells make a large, piericidin-A-sensitive form, which consists of some 23 different nuclear- and 6-7 mitochondrially encoded subunits. Cells grown in the presence of chloramphenicol make a small, piericidin-A-insensitive form which consists of only approximately 13 nuclear-encoded subunits. The subunits of the small form are either identical or similar to nuclear-encoded subunits of the large form. The iron-sulfur clusters in these two forms of Complex I are characterized by redox potentiometry and EPR spectroscopy. The large form of Complex I contains four EPR-detectable iron-sulfur clusters, N1, N2, N3 and N4, with the spin concentration of the individual clusters equivalent to the flavin concentration, similar to the mammalian counterparts. The small Complex I contains clusters N1, N3 and N4, but it is devoid of cluster N2. A model of the electron-transfer route through the large form of Complex I has been derived from these findings and an evolutionary pathway which leads to the emergence of large Complex I is discussed.

Animals↗
PubMed1991

cDNA and genomic DNA sequence of the 21.3 kDa subunit of NADH:ubiquinone reductase (complex I) from Neurospora crassa.

The primary structure of a nuclear-encoded subunit of the respiratory chain NADH:ubiquinone reductase (complex I) from Neurospora crassa was determined by sequencing cDNA, genomic DNA and the N-terminus of the protein. The sequence correlates to a protein of 200 amino acids and a molecular mass of 21349 Da. The protein is synthesized without a cleavable presequence. It contains two alpha-helices predicted to traverse the bilayer and is a constituent of the membrane part of complex I.

Amino Acid Sequence↗
PubMed1991

Regulation of the proton/electron stoichiometry of mitochondrial ubiquinol:cytochrome c reductase by the membrane potential.

The electron transfer reaction catalysed by mitochondrial ubiquinol:cytochrome c reductase is linked to the outwards translocation of protons with an H+ e- stoichiometry of 1 under non-membrane potential condition. The effect of the electrical membrane potential on the H+/e- stoichiometry was investigated. The enzyme was isolated from Neurospora crassa, reconstituted into phospholipid vesicles and electrical membrane potentials of various values were generated across the membranes by means of the valinomycin-induced potassium-diffusion method. Using lithium ions as counterions for the intravesicular potassium, the induced membrane potential was stable for minutes and was not significantly changed by the protons ejected by the working enzyme. This allowed the assay of steady-state reaction rates at pre-given values of electrical membrane potential. The rate ratio between electron transfer and proton translocation declined from 1 to 0.6 with increase of the membrane potential from 0 to 100 mV. The activity of the quinol/cytochrome c redox reaction followed a parabolic dependence, being activated by low (less than 50 mV) potential and inhibited by high (greater than 100 mV) potential. This apparent non-linear dependence was interpreted in terms of a linear flow/force relationship plus a membrane-potential-dependent slip. Evaluation of the parabolic course by means of a modified linear flow/force relation also indicated a decline of the H+/e- stoichiometry from 1 to 0.5 with increase of the membrane potential from 0 to 120 mV. These observations suggest that the membrane potential controls a change of ubiquinol:cytochrome c reductase between two states that have different reaction routes.

Electron Transport↗
PubMed1991

Relationship between a subunit of NADH dehydrogenase (complex I) and a protein family including subunits of cytochrome reductase and processing protease of mitochondria.

The primary structure of a 40 kDa subunit of the respiratory chain NADH:ubiquinone reductase from Neurospora crassa was determined by sequencing cDNA, genomic DNA and the N-terminus of the mature protein. The gene which is interrupted by 7 introns encodes a preprotein consisting of 375 amino acids with a 26 amino acid long presequence typical for a mitochondrial targeting signal. The sequence of the mature subunit shows conspicuous similarities to the recently [(1989) Nature 339, 147-149] discovered protein family which includes subunits I and II of the ubiquinol:cytochrome c reductase, and the processing proteins, matrix processing peptidase and processing enhancing protein, of mitochondria. The possible role of the subunit is discussed.

Amino Acid Sequence↗
PubMed1991

Effect of trifluoperazine on renal epithelioid Madin-Darby canine kidney cells.

Following exposure to a number of hormones, the cell membrane in Madin-Darby Canine Kidney (MDCK) cells is hyperpolarized by increase of intracellular calcium activity. The present study has been performed to elucidate the possible role of calmodulin in the regulation of intracellular calcium activity and cell membrane potential. To this end trifluoperazine has been added during continuous recording of cell membrane potential or intracellular calcium. Trifluoperazine leads to a transient increase of intracellular calcium as well as a sustained hyperpolarization of the cell membrane by activation of calcium sensitive K+ channels. Half-maximal effects are observed between 1 and 10 mumol/L trifluoperazine. A further calmodulin antagonist, chlorpromazine, (50 mumol/L), similarly hyperpolarizes the cell membrane. The effects of trifluoperazine are virtually abolished in the absence of extracellular calcium. Pretreatment of the cells with either pertussis toxin or phorbol-ester TPA does not interfere with the hyperpolarizing effect of trifluoperazine. In conclusion, calmodulin is apparently involved in the regulation of calcium transfer across the cell membrane but not in the stimulation of K+ channels by intracellular calcium.

Animals↗
PubMed1991

Further analysis of ATP-mediated activation of K+ channels in renal epithelioid Madin Darby canine kidney (MDCK) cells.

ATP activates K+ channels by increasing intracellular calcium activity in Madin Darby canine kidney (MDCK) cells. The present study has been performed to test for the involvement of G-proteins and of protein kinase C in the intracellular transmission of these effects. To this end, the effect of ATP on intracellular calcium and K+ channel activity has been studied in cells pretreated with the phorbol ester 12-O-tetradecanoyl-phorbol 13-acetate (TPA) and/or pertussis toxin. The ATP-induced increase of intracellular calcium is not significantly affected by pretreatment with pertussis toxin, is significantly blunted by pretreatment with TPA and is abolished by pretreatment with both pertussis toxin and the phorbol ester. The ATP activation of K+ channels is similarly blunted by pretreatment with TPA, but is not abolished by pretreatment with both the phorbol ester and pertussis toxin. Furthermore, the ATP-induced hyperpolarization is not abolished in cells pretreated with both pertussis toxin and TPA. In those cells, ATP may activate K+ channels by calcium-dependent mechanisms or lead to localized increases of intracellular calcium sufficient to activate the K+ channels but escaping detection with fura-2 fluorescence.

Adenosine Triphosphate↗