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H Weiner

Publications and source records attributed to H Weiner.

At least 19 recordsLinked to original sources
PubMed1992

Crystallization and preliminary X-ray investigation of bovine liver mitochondrial aldehyde dehydrogenase.

Aldehyde dehydrogenase from bovine liver mitochondria has been crystallized using the sitting drop method of vapor diffusion at 22 degrees C. The crystals formed from solutions containing, 40 mM-sodium citrate, 1 mM-NAD+ and 21% to 24% polyethylene glycol 3400 (pH 5.3 to 5.5). X-ray diffraction data collected from these crystals indicate that the crystals belong to the orthorhombic space group P2(1)2(1)2(1) with cell dimensions of a = 153.7 A, b = 159.37 A and c = 101.45 A. The crystals diffract to at least 2.9 A and a tetramer may comprise the asymmetric unit.

Aldehyde Dehydrogenase↗
PubMed1992

Purification of liver aldehyde dehydrogenase by p-hydroxyacetophenone-sepharose affinity matrix and the coelution of chloramphenicol acetyl transferase from the same matrix with recombinantly expressed aldehyde dehydrogenase.

p-Hydroxyacetophenone was coupled to epoxy-activated Sepharose 6B to generate an affinity chromatographic matrix to purify aldehyde dehydrogenase. Purified beef liver mitochondrial aldehyde dehydrogenase specifically bound to the support and could be eluted with p-hydroxyacetophenone. A post-ammonium sulfate (30-55%) fraction of bovine liver was applied to the affinity gel column and aldehyde dehydrogenase was effectively purified, although not to complete homogeneity, indicating the potential selectivity of the matrix. Both beef liver cytosolic and mitochondrial aldehyde dehydrogenase bound to the column. A post-Cibacron blue Sepharose Cl-6B affinity-fractionated liver mitochondrial aldehyde dehydrogenase was purified to complete homogeneity by p-hydroxyacetophenone-Sepharose, thus eliminating the need for the isoelectric focusing step often employed. p-Hydroxyacetophenone was found to be a competitive inhibitor against propionaldehyde and noncompetitive against NAD. Escherichia coli lysates of recombinantly expressed aldehyde dehydrogenase were purified from E. coli lysates with one major 25-kDa protein contaminant also binding to the column, as detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The 25-kDa contaminant was found to be chloramphenicol acetyl transferase from sequence analysis and binding studies.

Acetophenones↗
PubMed1992

[The role of endogenous opioids in modulation of immunosuppression in fish].

We have previously shown that social confrontation between aggressive fish (e.g., Tilapia) produces a suppression of several immunological parameters--nonspecific cytotoxicity and mitogen-stimulated proliferation in pronephric lymphocytes--in the subordinate fish. By using the opioid antagonist, naltrexone, we now demonstrate indirectly that this immunosuppression is in part mediated by the endogenous opioid system. Evidence is presented that naltrexone-mediated reversal of immunosuppression may be limited to the populations of the cytotoxic and T-cell lineages. The proliferative response to lipopolysaccharide is unaffected by naltrexone. Our data also demonstrate that serum from subordinate (immunosuppressed) fish is immunosuppressive in normal fish: an effect that can be reversed by naltrexone. These results support a link between the neuroendocrine and immune systems in these animals.

Aggression↗
PubMed1991

CRF microinjected into the dorsal vagal complex inhibits TRH analog- and kainic acid-stimulated gastric contractility in rats.

The effect of CRF microinjected into the dorsal vagal complex (DVC) on centrally-stimulated gastric contractility was investigated in fasted, urethane-anesthetized rats. Miniature strain gauge force transducers were acutely implanted on the corpus of the stomach and contractility was analyzed by computer. Microinjection of the stable thyrotropin-releasing hormone (TRH) analog, RX 77368, (26 pmol) into the DVC induced a 12.2-fold stimulation of gastric contractility within 30 min. Corticotropin-releasing factor (CRF) (63-210 pmol) microinjected into the DVC concomitantly with RX 77368 (26 pmol) induced a dose-related inhibition of stimulated gastric contractility. Neither CRF alone (210 pmol) nor vehicle modified basal gastric contractility. Microinjection of kainic acid (141 pmol) into the raphe pallidus nucleus induced a 3.6-fold stimulation of gastric contractility after 45 min. This stimulation was suppressed by bilateral microinjection of CRF (105 pmol/site) into the DVC. These results demonstrate that CRF acts in the DVC to inhibit centrally-stimulated gastric contractility and suggest that TRH and CRF may interact in the DVC to regulate gastric motor function.

Animals↗
PubMed1991

Purification and characterization of catalytically active precursor of rat liver mitochondrial aldehyde dehydrogenase expressed in Escherichia coli.

The cDNA coding for the precursor (p-ALDH) or mature (m-ALDH) rat liver mitochondrial aldehyde dehydrogenase was cloned in an expression vector pT7-7 and expressed in Escherichia coli strain BL21 (DE3)/plysS. The p-ALDH expressed in E. coli was a soluble tetrameric protein. It exhibited virtually the same specific activity and KmS for substrates as m-ALDH. N-terminal sequencing of isolated p-ALDH provided the evidence that the catalytic activity was not derived from a partially processed mature-like enzyme. The assembly states of both p-ALDH and m-ALDH synthesized in a rabbit reticulocyte lysate were also determined. Both of them were monomers and could not bind to a 5'-AMP-Sepharose column, showing that the monomeric form of the enzyme is inactive. The stabilities in vivo and in vitro were compared between p-ALDH and m-ALDH expressed in E. coli. p-ALDH was less stable than was m-ALDH both in vivo and in vitro. Thus, although the conformations of p-ALDH and m-ALDH are similar, the presence of signal peptide is a destabilizing factor to the p-ALDH. p-ALDH expressed in E. coli could bind to and be translocated into rat liver mitochondria, however, with lower efficiency when compared to the import of p-ALDH synthesized in reticulocyte lysate.

Aldehyde Dehydrogenase↗
PubMed1991

Polymorphism of the rat liver mitochondrial aldehyde dehydrogenase cDNA.

In humans, a deficiency in mitochondrial aldehyde dehydrogenase (Class 2 ALDH) activity due to a single base-pair exchange in its structural gene serves as a deterrent to excessive alcohol consumption. Differences in Class 2 ALDH isozyme patterns on isoelectric focusing gels have been observed in the selectively bred, alcohol-preferring (P) and alcohol-nonpreferring (NP) lines of rats. To determine whether the differences are the result of sequence variation in the structural gene, we sequenced the cDNAs for Class 2 ALDH from P and NP rats. A synonymous exchange was seen in the codon for amino acid 473 in both lines, when compared with published sequences. Additionally, when the cDNA from P rats was used as reference, a substitution (G for A) was identified in the cDNA of NP rats which changes amino acid 67 from Gln (CAG codon; ALDH2Q allele) to Arg (CGG codon; ALDH2R allele). The Arg for Gln substitution makes the enzyme more basic and could account for the different electrophoretic mobilities. To determine whether the polymorphism was associated with drinking behavior, we genotyped the ALDH2 locus by amplifying rat genomic DNA encompassing the nucleotide exchange followed by probing with allele-specific oligonucleotides. There are highly significant differences in the frequencies of the two alleles in the P and NP rat lines. The frequency of the ALDH2R allele is 63% in the NP line and only 18% in the P line, whereas the frequency of the ALDH2Q allele is 82% in the P line and 37% in the NP line.

Alcoholism↗
PubMed1991

Effects of protein size on the rate of import of the precursors of aldehyde dehydrogenase and ornithine transcarbamylase into rat liver mitochondria.

It is known that a signal peptide is required for the import of a protein into mitochondrial matrix. It is also known that a signal peptide can be attached to any protein and allow it to be imported. We recently reported that the rate of import of rat liver mitochondrial aldehyde dehydrogenase precursor was slower than that of ornithine transcarbamylase precursor (Wang TTY, Farrés J, and Weiner H. Arch Biochem Biophys 272, 440-449, 1989). It was not known if the difference in the rate of import was related to the fact that the mature portion of aldehyde dehydrogenase is larger (500 amino acids compared with 322 amino acids) or because the signal peptides were different. We further showed that treatment of the mitochondria with alcohols caused an inhibition of the import of the precursor of aldehyde dehydrogenase but not that of ornithine transcarbamylase. In the present study we constructed chimeric proteins that contained the signal peptide from one precursor protein and the mature portion from the other. We found that the rate of import was related to the overall size of the precursor protein. Consistent with this observation was finding that a truncated aldehyde dehydrogenase precursor, which contained 317 amino acids, was imported more rapidly than was the authentic precursor. Consistent with this finding was the fact that butanol caused the inhibition of only the large precursor proteins. Thus, it appears that size of the protein being imported is a major determinant of the rate at which a precursor protein is imported into mitochondria.

Aldehyde Dehydrogenase↗
PubMed1991

Molecular cloning of the mitochondrial aldehyde dehydrogenase gene of Saccharomyces cerevisiae by genetic complementation.

Mutants of Saccharomyces cerevisiae deficient in mitochondrial aldehyde dehydrogenase (ALDH) activity were isolated by chemical mutagenesis with ethyl methanesulfonate. The mutants were selected by their inability to grow on ethanol as the sole carbon source. The ALDH mutants were distinguished from alcohol dehydrogenase mutants by an aldehyde indicator plate test and by immunoscreening. The ALDH gene was isolated from a yeast genomic DNA library on a 5.7-kb insert of a recombinant DNA plasmid by functional complementation of the aldh mutation in S. cerevisiae. An open reading frame which specifies 533 codons was found within the 2.0-kb BamHI-BstEII fragment in the 5.7-kb genomic insert which can encode a protein with a molecular weight of 58,630. The N-terminal portion of the protein contains many positively charged residues which may serve as a signal sequence that targets the protein to the mitochondria. The amino acid sequence of the proposed mature yeast enzyme shows 30% identity to each of the known ALDH sequences from eukaryotes or prokaryotes. The amino acid residues corresponding to mammalian cysteine 302 and glutamates 268 and 487, implicated to be involved at the active site, were conserved. S. cerevisiae ALDH was found to be localized in the mitochondria as a tetrameric enzyme. Thus, that organelle is responsible for acetaldehyde oxidation, as was found in mammalian liver.

Aldehyde Dehydrogenase↗
PubMed1991

[The organism as a body-mind functional unit--consequences for psychosomatic medicine].

Psychosomatic medicine and research has always been more concerned with a broader and more integrated perspective on health, illness, and disease. It has attempted to study and understand persons in terms of the behaviors of interacting systems--the physiology of the organism. In the past 20 years a language has evolved that speaks of the organism's functions in dynamic, time-related terms. Both phenomenologically and mathematically, functions can be described in terms of rhythmus which, with disease, undergo change. In speaking this new language, some long-standing conceptual issues fade away, and others are clarified. At the same time, these new concepts force the investigator into new ways of designing experiments and analyzing data which may extend and modify them.

Animals↗
PubMed1991

Molecular biological studies on liver mitochondrial aldehyde dehydrogenase.

The tools of molecular biology were applied to study different facets of liver aldehyde dehydrogenase. One point investigated was the sequence of the various liver forms of the enzyme and the structure of the genes coding for the enzyme. It was found that an intron existed between the gene sequence coding for amino acids 21 and 22. The presence of the intron could explain why no sequence identity exists between the first 21 residues of the cytosol and mitochondrial isozymes while there is 70% identity between the remaining amino acids. Having the cDNA coding for the mitochondrial enzyme allowed us to produce the precursor form of the enzyme in rabbit reticulocytes and perform in vitro import into isolated mitochondria where it was found that alcohols caused an inhibition of import. The active enzyme was expressed in E. coli and site directed mutagenesis was performed to probe for the active site residues. The role of cysteine and position 302 and glutamate at position 487 was studied.

Alcohols↗
PubMed1991

Stressful experience and cardiorespiratory disorders.

Ever since Selye's time, the belief has persisted that the outcome of stressful experience is disease. The likelihood of this eventuality is increased when the experience is damaging, unavoidable, or uncontrollable. However, in most stressful instances, these conditions do not occur. The experience either is overcome or produces disturbances in physiological functions without structural change. The prevalence of "functional" disorders are far more common in medical practice than is disease. Among the most interesting of these is the hyperventilation syndrome, which may mimic or be confused with ischemic heart disease. Its symptomatology and physiology are complex. The syndrome may produce coronary vasospasm, but it may also complicate ischemic heart disease. It is even believed that chronic hyperventilation may be a risk factor for ischemic heart disease. Stressful experience consisting of various tasks and challenges may also produce myocardial perfusion deficits in ischemic heart disease, presumably secondary to vasospasm. These deficits are in turn considerably more frequent in any one patient than ST segment changes in the electrocardiogram or the incidence of angina pectoris. Vasospasm is in turn related to cardiac arrhythmias, which may occur with ischemic heart disease during a variety of stressful experiences and during outbursts of anger. Finally, the role of stressful experience in inciting ischemic heart disease and its complications remains moot.

Arrhythmias, Cardiac↗
PubMed1990

Bombesin microinjected into the dorsal vagal complex inhibits TRH-stimulated gastric contractility in rats.

The effects of centrally injected bombesin on central and peripheral stimulated gastric contractility were investigated in fasted urethane-anesthetized rats. Miniature strain gauge force transducers were acutely implanted on the corpus of the stomach and gastric contractility was analyzed by computer. Intracisternal injection of the stable thyrotropin-releasing hormone (TRH)-analog RX 77368 (77 pmol) induced a stimulation of gastric contractility for 40 min. Intracisternal injection of bombesin (62-620 pmol) followed 30 min later by that of RX 77368 resulted in a dose-related inhibition of the TRH-analog-induced gastric contractility. Intracisternal injection of bombesin (620 pmol) did not modify gastric contractility stimulated by intravenous carbachol. Stimulation of gastric contractility induced by TRH-analog microinjected into the dorsal vagal complex (DVC) was dose-related suppressed by concomitant injections of bombesin (6.2-620 pmol). Neither bombesin alone (6.2 pmol) nor vehicle modified basal gastric contractility. These results demonstrate that bombesin acts within the brain to inhibit vagally stimulated gastric contractility and that the DVC is a sensitive site for bombesin inhibitory action. These findings suggest a possible interaction between TRH and bombesin in the central vagal regulation of gastric contractility.

Animals↗
PubMed1990

2D NMR and structural model for a mitochondrial signal peptide bound to a micelle.

The 19 amino acid signal peptide of rat liver aldehyde dehydrogenase, possessing a lysine substitution for an arginine and containing 3 extra amino acid residues at the C terminus, was studied by two-dimensional NMR in a dodecylphosphocholine micelle. In this membrane-like environment, the peptide contains two alpha-helical regions, both of which are amphiphilic, separated by a hinge region. The helix located closer to the C terminus is more stable than is the helix located near the N terminus. This suggests that the hydrophobic face of the C-terminal helix is buried within the hydrophobic region of the micelle. On the basis of these results a general model for protein translocation is presented in which the C-terminal amphiphilic helix of the signal region in the preprotein first binds to the mitochondrial membrane and then diffuses to the translocation receptor. The receptor then recognizes the N-terminal helix of the signal region, which is not anchored to the membrane. To explain how this signal peptide was imported into isolated mitochondria in the absence of energy or receptor protein [Pak, Y. K., & Weiner, H. (1990) J. Biol. Chem. 265, 14298-14307], a model for signal peptide translocation across a membrane barrier without the need for auxiliary membrane proteins is proposed. In this model the faces of the two helices fold upon each other, resulting in the mutual shielding of positively charged residues by the complementary hydrophilic face of the other amphiphilic helix.

Aldehyde Dehydrogenase↗
PubMed1990

Import of chemically synthesized signal peptides into rat liver mitochondria.

The signal peptides of pre-aldehyde dehydrogenase (22-mer) and pre-ornithine transcarbamylase (27-mer) were chemically synthesized and their imports into rat liver mitochondria were studied. Both signal peptides were imported rapidly (within 2 min) in the absence of a membrane potential, exogenous ATP, or rabbit reticulocyte lysate. Signal peptides also were imported into mitochondria treated with a low concentration of trypsin which removed the outer membrane proteins. It was concluded that the chemically synthesized signal peptide could be imported differently than the precursor proteins. The imported signal peptide were found to be associated with both outer and inner membranes. Pulse-chase experiments showed that the import was unidirectional and that the signal peptides associated with inner membranes increased during the chase time. The signal peptides inhibited import of precursor proteins to different extents. Association of signal peptides with inner membrane near or at translocator sites might result in inhibition of precursor import.

Aldehyde Dehydrogenase↗
PubMed1990

Sequence of the precursor of bovine liver mitochondrial aldehyde dehydrogenase as determined from its cDNA, its gene, and its functionality.

The partial sequence coding for bovine liver mitochondrial aldehyde dehydrogenase was previously reported (Farres, J., Guan, K.L., and Weiner, H., 1989, Eur. J. Biochem. 180, 67-74); cDNA coding for the N-terminal region has now been obtained by primer extension. The deduced 520 amino acids contained 499 residues corresponding to the mature mitochondrial aldehyde dehydrogenase which was one residue shorter than other mammalian mitochondrial aldehyde dehydrogenases. The N-terminal signal peptide had only 21 amino acids but showed high sequence identity to the signal sequences of human and rat enzymes, which have 17 and 19 amino acids, respectively, and had general characteristic features of typical mitochondrial signal peptides. Although the direct protein sequence of the signal peptide was not available, a mRNA coding for a 57-kDa precursor was proven to exist. The bovine signal peptide was able to direct the import of bovine aldehyde dehydrogenase precursor into isolated liver mitochondria. The complete nuclear gene coding for bovine mitochondrial aldehyde dehydrogenase was found to span at least 25 kb. A restriction map of this gene was constructed. Two potential transcription start sites were identified by primer extension and S1 nuclease protection. Several SpI transcription factor recognition sequences were found in the 5'-region of the gene. TATA box and CAAT box like sequences were found to be exist 18 and 64 bp upstream from the major transcription start site, respectively.

Aldehyde Dehydrogenase↗
PubMed1990

Effects of alcohol on the import of aldehyde dehydrogenase precursor into rat liver mitochondria.

We previously showed that incubation of rat liver mitochondria with alcohols resulted in the inhibition of the import of aldehyde dehydrogenase precursor but not that of ornithine transcarbamylase precursor (Wang TTY, Farrés J, and Weiner H: Arch Biochem Biophys 272:440-449, 1989). The time required for inhibition of import to occur was now measured with ethanol (200 mM) and butanol (100 mM) at 0 degree and 30 degrees C. It required approximately 30 min to achieve 50% inhibition with butanol and 50 min with ethanol. To further substantiate the membrane perturbing effects of alcohols, we also examined the effect of oleic acid on import. We found that incubation of mitochondria with oleic acid (0-100 microM) resulted in inhibition of aldehyde dehydrogenase precursor import in a dose response fashion. In addition to in vitro effects of alcohols on import, we conducted a preliminary study on import of protein into liver mitochondria isolated from rats fed ethanol. We found that the rate of aldehyde dehydrogenase precursor import into liver mitochondria isolated from ethanol fed rats was identical to that from control. The results are consistent with finding that the activity and amount of aldehyde dehydrogenase was the same in mitochondria isolated from the alcohol-fed or control animals.

Alcoholism↗
PubMed1990

Endogenous serotonin produces an inhibitory tone on vagally stimulated gastric function.

The effect of depletion of serotonin stores on vagally stimulated gastric acid secretion and motility was studied in rats. Pretreatment of rats with parachlorophenylalanine (p-CPA) produced a 57% reduction in the intraluminal gastric release of serotonin and a 43-100% potentiation of the gastric acid secretory response elicited by intracisternal injection of the stable TRH analogue RX 77368 in conscious pylorusligated rats or in urethane-anesthetized rats with an acute gastric fistula. p-CPA also enhanced the vagally stimulated gastric acid output produced by intravenous injection of baclofen. In contrast, p-CPA pretreament had no effect on gastric acid secretion stimulated by bethanechol, histamine, or pentagastrin. Selective depletion of central serotonin stores by the neurotoxin 5,7-dihydroxytryptamine (5,7-DHT) given alone or combined with parachloroamphetamine pretreatment did not alter RX 77368-stimulated gastric acid secretion. In addition, gastric contractility stimulated by intracisternal injection of RX 77368 was significantly enhanced by p-CPA but not by 5,7-DHT pretreatment; whereas the contractile response to carbachol was not altered by p-CPA pretreatment. These results suggest that depletion of peripheral but not central serotonergic stores potentiates gastric acid secretion and contractility stimulated by vagally, but not peripherally, acting gastric stimulants. Thus, peripheral serotonin may exert an inhibitory tone on vagally stimulated gastric acid secretion and motility in the rat.

Animals↗