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H Weiner

Publications and source records attributed to H Weiner.

At least 73 records · Page 4Linked to original sources
PubMed1996

Amphiphilicity determines binding properties of three mitochondrial presequences to lipid surfaces.

The interactions of three peptides, which correspond to presequences that direct mitochondrial protein import, with model membrane systems were characterized using NMR, fluorescence, and circular dichroism spectroscopies. The positively charged peptides adopted an ordered secondary structure only when the negatively charged phospholipid, cardiolipin, was present in small unilamellar vesicles. Conversely, the peptides adopted an ordered secondary structure in the presence of micelles formed from both formally neutral and negatively charged detergents. The peptides had the same relative affinity for micelles and small unilamellar vesicles containing 20% cardiolipin. Amide proton exchange rates showed that the region of the helical structure which had the greatest hydrophobic moment interacted most readily with micelles. Therefore, it appears that a major determinant of binding to lipid surfaces is the ability of the peptide to attain the correct orientation of hydrophobic and hydrophilic groups. For the three peptides studied, affinity also correlated with the length of the helix, but not with hydrophobic surface area. In each case, the interacting segment of the peptide was toward the C-terminal end of the helix. Previous work has allowed us to postulate that the N-terminus of the presequence is vital for import [Wang, Y., & Weiner, H. (1993) J. Biol. Chem. 268, 4759-4765] and the C-terminal end is essential for membrane interaction [Karslake, C., Piotto, M., Pak, Y. K., Weiner, H., & Gorenstein, D. G. (1990) Biochemistry 29, 9872-9878]. On the basis of the data that are now available, it appears that the interaction with membrane surfaces may depend on the location of an amphiphilic region of the sequence that is near but not necessarily at the C-terminus.

Acetyl-CoA C-Acyltransferase↗
PubMed1996

Similarities and differences in couples' grief reactions following a miscarriage: results from a longitudinal study.

Recent studies have documented grief and depressive reactions in women after a miscarriage. However, the men's reactions to their partner's experience have been neglected. In a controlled follow-up study, 56 couples were studied shortly after the miscarriage, and 6 (N = 47) and 12 months later (N = 45). The participants completed standardized questionnaires for depression, physical complaints, anxiety, and grief. Contrary to commonly held beliefs, men do grieve, but less intensely and enduringly than their partners. The manner in which they experience their grief is similar to that of the women, except that the men cry less and feel less need to talk about it. Unlike the women they do not react with an increased depressive reaction (compared to age- and sex-matched community control groups). Giving up their personal expectations, hopes for, and fantasies about the unborn child is a major source of grieving for both. Some men feel burdened by their wives' grief or depressive reactions. Conflicting reactions may affect the couples' interactions and promote depressive reactions in the women.

Abortion, Spontaneous↗
PubMed1995

Conversion of a nonprocessed mitochondrial precursor protein into one that is processed by the mitochondrial processing peptidase.

Mitochondrial processing peptidase (MPP) cleaves the signal sequence from a variety of mitochondrial precursor proteins. A subset of mitochondrial proteins, including rhodanese and 3-oxoacyl-CoA thiolase, are imported into the matrix space, yet are not processed. Rhodanese signal peptide and translated protein were recognized by MPP, as both were inhibitors of processing. The signal peptide of precursor aldehyde dehydrogenase consists of a helix-linker-helix motif but when the RGP linker is removed, processing no longer occurs (Thornton, K., Wang, Y., Weiner, H., and Gorenstein, D. G. (1993) J. Biol. Chem. 268, 19906-19914). Disruption of the helical signal sequence of rhodanese by the addition of the RGP linker did not allow cleavage to occur. However, addition of a putative cleavage site allowed the protein to be processed. The same cleavage site was added to 3-oxoacyl-CoA thiolase, but this protein was still not processed. Thiolase and linker-deleted aldehyde dehydrogenase signal peptides were poor inhibitors of MPP. It can be concluded that both a processing site and the structure surrounding this site are important for MPP recognition.

Acetyl-CoA C-Acyltransferase↗
PubMed1995

Identification of protein-receptor components required for the import of prealdehyde dehydrogenase into rat liver mitochondria.

Mitochondrial aldehyde dehydrogenase is synthesized as a high-molecular-weight precursor in cytosol and transported into mitochondrial matrix space where it is processed to the mature enzyme. To identify components of the transport machinery on liver mitochondria, anti-idiotypic antibodies against the rabbit anti-prealdehyde dehydrogenase signal peptide antibodies were produced in chicken eggs and rabbit. Both anti-idiotypic antibodies inhibited the import of prealdehyde dehydrogenase (pALDH) into isolated rat liver mitochondria. The rabbit anti-idiotypic antibody could recognize by Western blotting five mitochondrial membrane proteins with apparent molecular weights of 66, 60, 42, 34, and 29 kDa. The anti-idiotypic antibodies were cross-linked to mitochondrial membrane proteins using sulfosuccinimidyl 2-(p-azidosalicylamido)ethyl-1,3'-dithiopropionate which is an iodinatable, heterofunctional, and photoreactive cross-linker. Mitochondrial proteins with apparent molecular weights of 66, 60, and 42 kDa were identified using the chicken antibody. The 66- and 34-kDa proteins were cross-linked to the rabbit antibody as the major components and the 42-kDa protein as a minor one. Antibodies against the 60- and 42-kDa proteins, as well as Fab fragments, inhibited the import of pALDH, suggesting that these proteins are receptor/translocator components for pALDH import.

Aldehyde Dehydrogenase↗
PubMed1995

Proteolysis prevents in vivo chimeric fusion protein import into yeast mitochondria. Cytosolic cleavage and subcellular distribution.

The in vivo import of liver mitochondrial aldehyde dehydrogenase was investigated in yeast by constructing fusion proteins between its leader sequence and beta-galactosidase. Only 7% of the protein was imported. If 21 or 71 amino acids from the mature portion of aldehyde dehydrogenase were included in the construct, 40% was imported. The protein remaining in cytosol was sequenced. When the leader was fused directly to beta-galactosidase, the first 7 residues of the leader were missing. When 21 residues of mature aldehyde dehydrogenase were included, the entire leader plus 6 residues of the mature portion were missing; if 71 residues of mature aldehyde dehydrogenase were included, the first residue found corresponds to the 66th residue of the mature portion. When the leader was fused directly to beta-galactosidase, no processing of the imported protein occurred, and the N-terminal amino acid was blocked, presumably by acetylation. If the 21-amino acid insert was included, processing occurred. A modified leader sequence lacking the three-amino acid linker (RGP) was imported but not processed, just as we found in vitro (Thornton, K., Wang, Y., Weiner, H., and Gorenstein, D.G. (1993) J. Biol. Chem. 268, 19906-19914). The less than 100% import of pre-aldehyde dehydrogenase was due to the action of a post-translational protease attack which prevented import by destroying the leader peptide segment.

Aldehyde Dehydrogenase↗
PubMed1995

Investigation of the active site cysteine residue of rat liver mitochondrial aldehyde dehydrogenase by site-directed mutagenesis.

To determine the active site cysteine residue in aldehyde dehydrogenase, we mutated amino acid residues 49, 162, and 302 of recombinantly expressed rat liver mitochondrial (class 2) aldehyde dehydrogenase. The C49A and C162A mutants were fully active tetrameric enzymes, although the C162A mutant was found to be highly unstable. The C302A mutant was also a tetramer and bound coenzyme, but lacked both dehydrogenase and esterase activities. To test for the role of cysteine 302 as a nucleophile, the residue was mutated to a serine, a poor nucleophile. this C302S mutant was active but was a much poorer catalyst, with a kcat/Km value 7 x 10(5) times lower than that of the recombinant native enzyme. Unlike with native enzyme where deacylation is rate limiting, formation of the serine hemiacetal intermediate appeared to be the rate-limiting step. Cysteine 302 is the only strictly conserved cysteine residue among all the available sequences of the aldehyde dehydrogenase superfamily, supporting the role of this residue as the active site nucleophile of aldehyde dehydrogenase.

Aldehyde Dehydrogenase↗
PubMed1995

Involvement of glutamate 268 in the active site of human liver mitochondrial (class 2) aldehyde dehydrogenase as probed by site-directed mutagenesis.

On the basis of chemical modification studies, it was postulated that glutamate 268 was a component of the active site of liver aldehyde dehydrogenase [Abriola, D. P., Fields, R., MacKerell, A. D., Jr., & Pietruszko, R. (1987) Biochemistry 26, 5679-5684]. To study its role, the residue in human liver mitochondrial (class 2) aldehyde dehydrogenase was mutated to an aspartate, a glutamine, or a lysine, and the enzyme was expressed in Escherichia coli. The mutations did not affect the Km values for NAD or propionaldehyde, but grossly affected the catalytic activity of the enzymes when compared to recombinantly expressed native enzyme; the mutant enzymes had less that 0.4% of the specific activity of the recombinantly expressed native aldehyde dehydrogenase. The mutations also caused a long lag phase to occur prior to the steady state phase of the reaction. The activity of the mutant enzymes could not be restored by the addition of general bases such as sodium acetate, sodium formate, or imidazole. The Kd for NADH was essentially identical for the E268Q mutant and native enzyme. The three mutant forms of the enzyme possessed less than 0.8% of the esterolytic activity of the recombinantly expressed native enzyme. Pre-steady state analysis showed that there was no burst of NADH formation in the dehydrogenase reaction or of p-nitrophenol formation in the esterase reaction. This can be interpreted as implying that glutamate 268 may function as a general base necessary for the initial activation of the essential cysteine residue (302), rather than being involved in only the deacylation or hydride transfer step.(ABSTRACT TRUNCATED AT 250 WORDS)

Aldehyde Dehydrogenase↗
PubMed1995

Site directed mutagenesis to probe for active site components of liver mitochondrial aldehyde dehydrogenase.

Mutational analysis allowed us to rule out an essential role for the histidine residues and for serine 74 in mammalian aldehyde dehydrogenase. The later though, was found to be important in coenzyme interaction. The function of the serine could not be replaced by threonine or by cysteine. The absolute requirement for cysteine 302 and for glutamate 268 was verified using mutational analysis. The fact that these two residues are completed conserved among all aldehyde dehydrogenases is consistent with their being essential in the catalytic process.

Aldehyde Dehydrogenase↗
PubMed1995

Interleukin-1 beta in the dorsal vagal complex inhibits TRH analogue-induced stimulation of gastric contractility.

The effect of murine interleukin-1 beta (mIL-1 beta) microinjected into the dorsal vagal complex (DVC) on thyrotropin-releasing hormone (TRH) analogue (RX-77368)-induced stimulation of gastric contractility was examined in fasted, urethan-anesthetized rats. Gastric corpus contractions were measured with extraluminal force transducers and analyzed by computer. Microinjection of RX-77368 (30 ng) into the right DVC with mIL-1 beta microinjected either into the right (100, 250 pg) or into the left (100, 500 pg) DVC inhibited gastric contractility for 30-120 min postinjection. Peak suppression of gastric contractility (64-78%) occurred at 50-60 min postinjection. Microinjection of mIL-1 beta into the DVC at a lower dose (10 pg) or into sites adjacent to the DVC (100-500 pg) did not suppress the stimulated gastric contractility pattern. Injection of mIL-1 beta (250 pg) or 0.1% bovine serum albumin into the DVC alone did not alter basal gastric contractility. Intracisternal injection of the IL-1 receptor antagonist (250 ng/10 microliters) abolished the inhibitory effect of mIL-1 beta (250 pg) on gastric contractility. These results demonstrate that mIL-1 beta acts in the DVC to inhibit vagally stimulated gastric contractility, and its action is mediated by IL-1 receptors.

Animals↗
PubMed1995

The aldehyde dehydrogenase ALDH2*2 allele exhibits dominance over ALDH2*1 in transduced HeLa cells.

Individuals heterozygous or homozygous for the variant aldehyde dehydrogenase (ALDH2) allele (ALDH2*2), which encodes a protein differing only at residue 487 from the normal protein, have decreased ALDH2 activity in liver extracts and experience cutaneous flushing when they drink alcohol. The mechanisms by which this allele exerts its dominant effect is unknown. To study this effect, the human ALDH2*1 cDNA was cloned and the ALDH2*2 allele was generated by site-directed mutagenesis. These cDNAs were transduced using retroviral vectors into HeLa and CV1 cells, which do not express ALDH2. The normal allele directed synthesis of immunoreactive ALDH2 protein (ALDH2E) with the expected isoelectric point. Extracts of these cells contained increased aldehyde dehydrogenase activity with low Km for the aldehyde substrate. The ALDH2*2 allele directed synthesis of mRNA and immunoreactive protein (ALDH2K), but the protein lacked enzymatic activity. When ALDH2*1-expressing cells were transduced with ALDH2*2 vectors, both mRNAs were expressed and immunoreactive proteins with isoelectric points ranging between those of ALDH2E and ALDH2K were present, indicating that the subunits formed heteromers. ALDH2 activity in these cells was reduced below that of the parental ALDH2*1-expressing cells. Thus, the ALDH2*2 allele is sufficient to cause ALDH2 deficiency in vitro.

Aldehyde Dehydrogenase↗
PubMed1994

Evaluation of electrostatic and hydrophobic effects on the interaction of mitochondrial signal sequences with phospholipid bilayers.

The information that directs a nuclear-coded protein to be imported into mitochondria resides in an N-terminal extension, called a signal sequence. The primary sequences of all known ones differ. The only common feature is their ability to theoretically form an amphiphilic, positively charged, alpha-helix. We previously showed that a short stable helical segment was required for a peptide to be functional in import [Wang, Y., & Weiner, H. (1993) J. Biol. Chem. 268, 4759-4765]. Here we investigate the interaction of three altered signal sequences with phospholipid membranes containing cardiolipin to ascertain the importance of electrostatic and hydrophobic interactions with the membrane. The three already described peptides were derivatives of the signal sequence from aldehyde dehydrogenase, which is composed of three segments, two helices separated by a linker. ANCN had the C-helix replaced by the N-helix of the signal sequence of cytochrome c oxidase subunit IV, ANCC had the C-terminal helix replaced by the C-terminal random coil of cytochrome oxidase subunit IV, and linker deleted had the linker region deleted. ANCC, which functioned poorly as a signal sequence, had a very low affinity for binding to the negatively charged membranes. In contrast, both ANCN and linker deleted showed a relatively high affinity for the membranes and were capable of functioning as a good leader sequence. It appears that linker deleted possessed a stronger hydrophobic effect with membranes while ANCN had a higher electrostatic interaction. On the basic of these studies, a model was proposed to describe the interaction of mitochondrial signal sequences with negatively charged phospholipid membranes involving electrostatic interaction for initial binding and hydrophobic interaction for insertion.(ABSTRACT TRUNCATED AT 250 WORDS)

Aldehyde Dehydrogenase↗
PubMed1994

Involvement of serine 74 in the enzyme-coenzyme interaction of rat liver mitochondrial aldehyde dehydrogenase.

It has been suggested that the active site nucleophile in sheep liver aldehyde dehydrogenase was not a cysteine residue but was a serine located at position 74 [Loomes, K. M., Midwinter, G. G., Blackwell, L. F., & Buckley, P. D. (1990) Biochemistry 29, 2070-2080]. This enzyme form has not yet been cloned and expressed, but since the rat liver mitochondrial enzyme has been and shares 70% sequence homology with other cytosolic aldehyde dehydrogenases, the residue in the rat enzyme was converted into an alanine to test for the necessity of a hydroxyl group at that position. The recombinantly expressed mutant enzyme possessed 10% catalytic activity, but the Km for NAD increased from 10 to 1900 microM while the Kms for various aldehydes were unchanged. Kinetic analysis revealed that the dissociation constant for NAD also increased in the mutant as did k1, the on velocity for NAD binding. The mutant enzyme bound poorly to an AMP-Sepharose column and did not interact as well with NADH, as determined by fluorescence enhancement binding studies, or with ADP-ribose, a competitive inhibitor. Pulse-chase analysis showed that the mutant was as stable as was the recombinantly expressed native enzyme. It was less stable to heat denaturation at 50 degrees C (half-life of 1 min compared to 4). Converting the alanine to a cysteine or a threonine did not restore native-like properties of the enzyme. These mutants had kinetic properties very similar to those of the alanine mutant.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate Ribose↗
PubMed1994

Structure of the signal sequences for two mitochondrial matrix proteins that are not proteolytically processed upon import.

The N-terminal sequences of rhodanese and 3-oxoacyl-CoA thiolase, two mitochondrial matrix proteins that are not proteolytically processed upon import, have been studied by NMR and CD spectroscopy. In aqueous trifluoroethanol, in the presence of micelles, and in the presence of small unilamellar vesicles (SUVs), these peptides form alpha-helical structures beginning near the N-terminus and extending, continuously, for at least three helical turns. This result is consistent with a previous finding that a mutant rat liver mitochondrial aldehyde dehydrogenase signal sequence we designed, which formed a continuous alpha-helix, could successfully direct protein import but was not proteolytically processed (Thornton, K., Wang, Y., Weiner, H., & Gorenstein, D. G. (1993) J. Biol. Chem. 268, 19906-19914). From these three examples, a model is developed which suggests that a mitochondrial signal sequence that has an N-terminal alpha-helix longer than 11 residues can take on the necessary conformation to be imported but cannot adopt the necessary conformation to be processed.

Acetyl-CoA C-Acetyltransferase↗
PubMed1994

Effects of changing glutamate 487 to lysine in rat and human liver mitochondrial aldehyde dehydrogenase. A model to study human (Oriental type) class 2 aldehyde dehydrogenase.

Many Oriental people possess a liver mitochondrial aldehyde dehydrogenase where glutamate at position 487 has been replaced by a lysine, and they have very low levels of mitochondrial aldehyde dehydrogenase activity. To investigate the cause of the lack of activity of this aldehyde dehydrogenase, we mutated residue 487 of rat and human liver mitochondrial aldehyde dehydrogenase to a lysine and expressed the mutant and native enzyme forms in Escherichia coli. Both rat and human recombinant aldehyde dehydrogenases showed the same molecular and kinetic properties as the enzyme isolated from liver mitochondria. The E487K mutants were found to be active but possessed altered kinetic properties when compared to the glutamate enzyme. The Km for NAD+ at pH 7.4 increased more than 150-fold, whereas kcat decreased 2-10-fold with respect to the recombinant native enzymes. Detailed steady-state kinetic analysis showed that the binding of NAD+ to the mutant enzyme was impaired, and it could be calculated that this resulted in a decreased nucleophilicity of the active site cysteine residue. The rate-limiting step for the rat E487K mutant was also different from that of the recombinant rat liver aldehyde dehydrogenase in that no pre-steady-state burst of NADH formation was found with the mutant enzyme. Both the rat native enzyme and the E487K mutant oxidized chloroacetaldehyde twice as fast as acetaldehyde, indicating that the rate-limiting step was not hydride transfer or coenzyme dissociation but depended upon nucleophilic attack. Each enzyme form showed a 2-fold activation upon the addition of Mg2+ ions. Substituting a glutamine for the glutamate did not grossly affect the properties of the enzyme. Glutamate 487 may interact directly with the positive nicotinamide ring of NAD+ for the Ki of NADH was the same in the lysine enzyme as it was in the glutamate form. Because of the altered NAD+ binding properties and kcat of the E487K variant, it is assumed that people possessing this form will not have a functional mitochondrial aldehyde dehydrogenase.

Aldehyde Dehydrogenase↗
PubMed1994

Electroencephalographic coherence in acquired immune deficiency syndrome.

We studied a quantitative electroencephalographic (EEG) measure, coherence, in 28 patients with acquired immune deficiency syndrome (AIDS) and 56 uninfected volunteers. Compared with uninfected subjects, AIDS patients had increased coherence in the 6- to 10-Hz band. The largest increases in coherence were between frontal and occipital regions and between temporal and frontal regions. Coherence within contiguous regions was less affected. Eight of the 28 AIDS patients (29%) had clinically abnormal EEG findings, compared with four of the 56 uninfected control subjects (7%). Among the AIDS patients, 12 had normal neuropsychological performance, nine had mild impairment, and six had moderate impairment. Coherence was increased in each subgroup of AIDS patients, including those with normal neuropsychologic performance and/or normal clinical EEG results. AIDS patients were then classified by quantitative EEG power in frontal head regions as "abnormal" (the upper third of patients) or "normal" (the remainder). Increased coherence was found among both groups. Because the development of abnormal neuropsychological performance or a clinically abnormal EEG examination indicates relatively advanced central nervous system disease, alterations in specific coherence measures may detect subclinical effects of the human immunodeficiency virus on brain function before other changes are evident.

Acquired Immunodeficiency Syndrome↗
PubMed1994

Repeated bereavement, depressed mood, and immune parameters in HIV seropositive and seronegative gay men.

The relationships among bereavement, depressed mood, and immunologic patterns prognostic for the development of acquired immunodeficiency syndrome (AIDS) were determined in a sample of human immunodeficiency virus (HIV) seropositive gay men and a comparison group of HIV seronegative gay men. Immunologic parameters were assessed in 45 men who had recently experienced the deaths of close friends and 45 matched nonbereaved men. No immune differences were found between bereaved and nonbereaved men. Among the HIV seropositive nonbereaved men, higher depressed mood was significantly associated with fewer CD4 (helper/inducer) T lymphocytes, more activated CD8 (suppressor/cytotoxic) T cells, and lower proliferative responses to the mitogen phytohemagglutinin. In summary, HIV seropositive men who reported higher levels of depressed mood not associated with bereavement demonstrated immunologic patterns consistent with HIV activity and progression.

Acquired Immunodeficiency Syndrome↗