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H Weiner

Publications and source records attributed to H Weiner.

At least 19 recordsLinked to original sources
PubMed2007

Cortical abnormalities in epilepsy revealed by local EEG synchrony.

Abnormally strong functional linkage between cortical areas has been postulated to play a role in the pathogenesis of partial epilepsy. We explore the possibility that such linkages may be manifest in the interictal EEG apart from epileptiform disturbances or visually evident focal abnormalities. We analyzed samples of interictal intracranial EEG (ICEEG) recorded from subdural grids in nine patients with medically intractable partial epilepsy, measuring interelectrode synchrony using the mean phase coherence algorithm. This analysis revealed areas of elevated local synchrony, or "hypersynchrony" which had persistent spatiotemporal characteristics that were unique to each patient. Measuring local synchrony in a subdural grid results in a map of the cortical surface that provides information not visually apparent on either EEG or structural imaging. We explore the relationship of hypersynchronous areas to the clinical evidence of seizure localization in each case, and speculate that local hypersynchrony may be a marker of epileptogenic cortex, and may prove to be a valuable aid to clinical ICEEG interpretation.

Adolescent↗
PubMed2007

Number of nitrate groups determines reactivity and potency of organic nitrates: a proof of concept study in ALDH-2-/- mice.

BACKGROUND AND PURPOSE: Mitochondrial aldehyde dehydrogenase (ALDH-2) has been shown to provide a pathway for bioactivation of organic nitrates and to be prone to desensitization in response to highly potent, but not to less potent, nitrates. We therefore sought to support the hypothesis that bioactivation by ALDH-2 critically depends on the number of nitrate groups within the nitrovasodilator. EXPERIMENTAL APPROACH: Nitrates with one (PEMN), two (PEDN; GDN), three (PETriN; glyceryl trinitrate, GTN) and four (pentaerithrityl tetranitrate, PETN) nitrate groups were investigated. Vasodilatory potency was measured in isometric tension studies using isolated aortic segments of wild type (WT) and ALDH-2-/- mice. Activity of the cGMP-dependent kinase-I (reflected by levels of phosphorylated VAsodilator Stimulated Phosphoprotein, P-VASP) was quantified by Western blot analysis, mitochondrial dehydrogenase activity by HPLC. Following incubation of isolated mitochondria with PETN, PETriN-chromophore and PEDN, metabolites were quantified using chemiluminescence nitrogen detection and mass spectrometry. KEY RESULTS: Compared to WT, vasorelaxation in response to PETN, PETriN and GTN was attenuated about 10fold in ALDH-2-/- mice, identical to WT vessels preincubated with inhibitors of ALDH-2. Reduced vasodilator potency correlated with reduced P-VASP formation and diminished biotransformation of the tetranitrate- and trinitrate-compounds. None of these findings were observed for PEDN, GDN and PEMN. CONCLUSIONS AND IMPLICATIONS: Our results support the crucial role of ALDH-2 in bioactivating highly reactive nitrates like GTN, PETN and PETriN. ALDH-2-mediated relaxation by organic nitrates therefore depends mainly on the number of nitrate groups. Less potent nitrates like PEDN, GDN and PEMN are apparently biotransformed by other pathways.

Aldehyde Dehydrogenase↗
PubMed2005

Bacterial signal peptide recognizes HeLa cell mitochondrial import receptors and functions as a mitochondrial leader sequence.

Phage display was used to identify new components of the mammalian mitochondrial receptor complex using Tom20 as a binding partner. Two peptides were identified. One had partial identity (SMLTVMA) with a bacterial signal peptide from Toho-1, a periplasmic protein. The other had partial identity with a mitochondrial inner membrane glutamate carrier. The bacterial signal peptide could carry a protein into mitochondria both in vivo and in vitro. The first six residues of the sequence, SMLTVM, were necessary for import but the two adjacent arginine residues in the 30-amino-acid leader were not critical for import. The signal peptides of Escherichia coli beta-lactamase and Bacillsus subtilis lipase could not carry proteins into mitochondria. Presumably, the Toho-1 leader can adopt a structure compatible for recognition by the import apparatus.

Amino Acid Sequence↗
PubMed2005

Stable transformation of CHO Cells and human NARP cybrids confers oligomycin resistance (oli(r)) following transfer of a mitochondrial DNA-encoded oli(r) ATPase6 gene to the nuclear genome: a model system for mtDNA gene therapy.

Point and deletion mutations and a general depletion of mammalian mitochondrial DNA (mtDNA) give rise to a wide variety of medical syndromes that are refractory to treatment, possibly including aging itself. While gene therapy directed at correcting such deficits in the mitochondrial genome may offer some therapeutic benefits, there are inherent problems associated with a direct approach. These problems are primarily due to the high mitochondrial genome copy number in each cell and the mitochondrial genome being "protected" inside the double-membrane mitochondrial organelle. In an alternative approach there is evidence that genes normally present in the mitochondrial genome can be incorporated into the nuclear genome. To extend such studies, we modified the Chinese Hamster Ovary (CHO) mtDNA-located ATPase6 gene (possessing a mutation which confers oligomycin resistance- oli(r)) by altering the mtDNA code to the universal code (U-code) to permit the correct translation of its mRNA in the cytoplasm. The U-code construct was inserted into the nuclear genome (nucDNA) of a wild type CHO cell. The expressed transgene products enabled the transformed CHO cell lines to grow in up to 1000 ng mL(-1) oligomycin, while untransformed sensitive CHO cells were eliminated in 1 ng mL(-1) oligomycin. This approach, termed allotopic expression, provides a model that may make possible the transfer of all 13 mtDNA mammalian protein-encoding genes to the nucDNA, for treatments of mtDNA disorders. The CHO mtATPase6 protein is 85% identical to both the mouse and human mtATPase6 protein; these proteins are highly conserved in the region of the oligomycin resistance mutation. They are also well conserved in the regions of the oligomycin resistance mutation of the mouse, and in the region of a mutation found in Leigh's syndrome (T8993G), also called NARP (neurogenic weakness, ataxia, retinitis pigmentosum). It is likely that the CHO oli(r) mtATPase6 Ucode construct could impart oligomycin-resistance in human and mouse cells, as well as function in place of the mutant ATPase subunit in a NARP cell line. Preliminary experiments on human cybrids homoplasmic for the NARP mutation (kindly supplied by D.C. Wallace), transformed with our construct, display an increased oligomycin resistance that supports these suppositions.

Animals↗
PubMed2005

A randomized blinded trial of combination therapy with cyclophosphamide in patients-with active multiple sclerosis on interferon beta.

OBJECTIVE: To evaluate the efficacy and safety of combination therapy with pulse cyclophosphamide given with methylprednisolone (MP) and interferon beta (IFNbeta)-Ia in multiple sclerosis (MS) patients with active disease during IFNbeta monotherapy. METHODS: This was a randomized, single-blind, parallel-group, multicenter trial in MS patients with a history of active disease during IFNbeta treatment. Patients were randomized to either cyclophosphamide 800 mg/m2 plus methylprednisolone 1 g IV (CY/MP) or methylprednisolone once a month for six months and then followed for an additional 18 months. All patients received three days of methylprednisolone 1 g IV at screening and 30 mcg IFNbeta-Ia IM weekly for the entire 24 months. The primary endpoint was change from baseline in the mean number of gadolinium-enhancing (Gd+) lesions. Secondary clinical endpoints included time to treatment failure. RESULTS: Fifty-nine patients were randomized to treatment: 30 to CY/MP and 29 to MP Change from baseline in the number of Gd+ lesions was significantly different between treatment groups at three (P =0.01), six (P =0.04) and 12 months (P =0.02), with fewer lesions in the CY/MP group. The cumulative rate of treatment failure was significantly lower in the CY/MP group compared with the MP group (rate ratio =0.30; 95% confidence interval, 0.12-0.75; P =0.011). CY/MP treatment was well tolerated. CONCLUSION: Combination therapy with CY/MP and IFNbeta-Ia decreased the number of Gd+ lesions and slowed clinical activity in patients with previously active disease on IFNbeta alone.

Adjuvants, Immunologic↗
PubMed2001

Protective autoimmunity is a physiological response to CNS trauma.

Primary damage caused by injury to the CNS is often followed by delayed degeneration of initially spared neurons. Studies in our laboratory have shown that active or passive immunization with CNS myelin-associated self-antigens can reduce this secondary loss. Here we show, using four experimental paradigms in rodents, that CNS trauma spontaneously evokes a beneficial T cell-dependent immune response, which reduces neuronal loss. (1) Survival of retinal ganglion cells in rats was significantly higher when optic nerve injury was preceded by an unrelated CNS (spinal cord) injury. (2) Locomotor activity of rat hindlimbs (measured in an open field using a locomotor rating scale) after contusive injury of the spinal cord (T8) was significantly better (by three to four score grades) after passive transfer of myelin basic protein (MBP)-activated splenocytes derived from spinally injured rats than in untreated injured control rats or rats similarly treated with splenocytes from naive animals or with splenocytes from spinally injured rats activated ex vivo with ovalbumin or without any ex vivo activation. (3) Neuronal survival after optic nerve injury was 40% lower in adult rats devoid of mature T cells (caused by thymectomy at birth) than in normal rats. (4) Retinal ganglion cell survival after optic nerve injury was higher (119 +/- 3.7%) in transgenic mice overexpressing a T cell receptor (TcR) for MBP and lower (85 +/- 1.3%) in mice overexpressing a T cell receptor for the non-self antigen ovalbumin than in matched wild types. Taken together, the results imply that CNS injury evokes a T cell-dependent neuroprotective response.

Animals↗
PubMed2001

Subunit communication in tetrameric class 2 human liver aldehyde dehydrogenase as the basis for half-of-the-site reactivity and the dominance of the oriental subunit in a heterotetramer.

Data has been published showing that in heterotetrameric liver mitochondrial aldehyde dehydrogenase composed of the active (E487) and the inactive Oriental-variant (K487) subunit, the Oriental variant was dominant and caused the inactivation of the E487 subunit. The published structures of the enzyme showed that the glutamate at position 487 is salt bonded to an arginine (475) in a different subunit. Arg475 was mutated to a glutamine to test for its importance in causing the Oriental variant to be an enzyme with a high Km for NAD and a low specific activity. Unexpectedly, the R475Q mutant exhibited positive cooperativity in NAD binding with a Hill coefficient of 2. Individual heterotetramers composed of subunits of E487 and K487 were produced by making changes to two residues on the surface of the enzyme and then co-expressing both cDNAs in E. coli. The E(3)K form had essentially 50% the activity of the E(4) homotetrameric form while EK(3) had essentially the same properties as did the homotetrameric K(4) Oriental variant. This showed that in a dimer pair composed of one K- and one E- subunit the K-subunit became dominant and caused the inactivation of its E-partner. Further, pre-steady state burst data and steady state kinetic data make it appear that there was one functioning active subunit in each of the dimer pairs that made up the tetrameric enzyme. Thus, the half-of-the-site reactivity is a result of having one functioning and one non-functioning subunit in each dimer pair. The actual structural basis for this is still not understood, but could be related to the E487-R475 inter-dimer salt bond.

Aldehyde Dehydrogenase↗
PubMed2001

Interaction of human aldehyde dehydrogenase with aromatic substrates and ligands.

The substrate benzaldehyde (but not propionaldehyde) could elute aldehyde dehydrogenase from a p-hydroxyacetophenone-affinity column, and inhibit the esterase activity (K(i)=47 microM), indicating that this simple aromatic aldehyde binds to the free enzyme and possibly in the substrate-binding site. Thus, the kinetic mechanism for aldehyde dehydrogenase might be dependent upon which aldehyde is used in the reaction. Chloramphenicol which also elutes the enzyme from the affinity column, shows a discriminatory effect by inhibiting the ALDH1 oxidation of benzaldehyde and activating that of propionaldehyde while showing no effect when assayed with hexanal or cyclohexane-carboxaldehyde. Chloramphenicol is an uncompetitive inhibitor against NAD when benzaldehyde is the substrate. We propose that this drug might interact with both the benzaldehyde and NAD binding sites.

Aldehyde Dehydrogenase↗
PubMed2001

Role of the C-terminal tail on the quaternary structure of aldehyde dehydrogenases.

To assess the importance of the C-terminal tail in the structure of aldehyde dehydrogenases (ALDH), mutants of tetrameric ALDH1 were generated by adding a tail of 5 amino acids (ALDH1-5aa) or the tail from the class 3 enzyme. A mutant of dimeric ALDH3 was made, where 17 amino acids from the C-terminus were deleted to generate ALDH3DeltaTail. The expression and solubility of the ALDH1 mutants was slightly lower than the wild type. Expression of ALDH3DeltaTail mutant was similar to wild type, but the solubility was only about 30%. The activity of ALDH1-5aa mutant was 30%, while ALDH1-H3Tail mutant was 60% active, compared to the wild type. The activity of the class 3 mutant was similar to the activity of the parent ALDH3 enzyme. Analysis of stability against temperature demonstrated that ALDH1-5aa was more stable than ALDH1 wild type, while the ALDH1-H3Tail mutant was considerably less stable than ALDH1, showing a stability similar to ALDH3. However, native gel and size exclusion analysis, showed no changes in the oligomerization state of these mutants. ALDH3DeltaTail mutant was more stable than wild type; the stability against temperature was similar to ALDH1. The ALDH3DeltaTail mutant showed an elution similar to that of ALDH1 from the size exclusion column, indicating that it was possibly a tetramer. These results show that the tail in ALDH3, is involved in the determination of the quaternary structure of ALDH3, but has no effect on the ALDH1 enzyme; the absence of the C-terminal tail is not the only factor participating in holding the dimers together. Thus, the interaction between single residues, or interactions with the N-terminal region might be more important for maintaining stable tetramers.

Aldehyde Dehydrogenase↗
PubMed2001

Making an Oriental equivalent of the yeast cytosolic aldehyde dehydrogenase as well as making one with positive cooperativity in coenzyme binding by mutations of glutamate 492 and arginine 480.

Yeast has at least three partially characterized aldehyde dehydrogenases. Previous studies by gene disrupted in our laboratory revealed that the Saccharomyces cerevisiae cytosol ALDH1 played an important role in ethanol metabolism as did the class 2 mitochondrial enzyme. To date, few mutagenesis studies have been performed with the yeast enzymes. An important human variant of ALDH is one found in Asian People. In it, the glutamate at position 487 is replaced by a lysine. This glutamate interacts with an arginine (475) that is located in the subunit that makes up the dimer pair in the tetrameric enzyme. Sequence alignment shows that these two residues are located at positions 492 and 480, respectively, in the yeast class 1 enzyme which shares just 45% sequence identity with the human enzymes. Mutating glutamate 492 to lysine produced an enzyme with altered kinetic properties when compared to the wild-type glutamate-enzyme. The K(m) for NADP of E492K increased to nearly 3600 microM compare to 40 microM for wild-type enzyme. The specific activity decreased more than 10-fold with respect to the recombinant wild-type yeast enzyme. Moreover, substituting a glutamine for a glutamate was not detrimental in that the E492Q had wild-type-like K(m) for NADP and V(max). These properties were similar to the changes found with the human class 2 E487K mutant form. Further, mutating arginine 480 to glutamine produced an enzyme that exhibited positive cooperativity in NADP binding. The K(m) for NADP increased 11-fold with a Hill coefficient of 1.6. The NADP-dependent activity of R480Q mutant was 60% of wild-type enzyme. Again, these results are very similar to what we recently showed to occur with the human enzyme [Biochemistry 39 (2000) 5295-5302]. These findings show that the even though the glutamate and arginine residues are not conserved, similar changes occur in both the human and the yeast enzyme when either is mutated.

Aldehyde Dehydrogenase↗
PubMed2001

Repeat endoscopic third ventriculostomy: is it worth trying?

OBJECT: The goal of this study was to evaluate the safety, efficacy, and indications for repeat endoscopic third ventriculostomies (ETV). METHODS AND RESULTS: We reviewed the records of 20 patients who had undergone repeat ETV from 1987 to 1999. Their ages ranged from 8 months to 53 years (mean 17 years). The primary etiologies of hydrocephalus were: primary aqueductal stenosis (9 cases), tumor (5), Chiari malformation (2), prior infection (2), prior intraventricular hemorrhage (1), and blocked foramen of Monro (1 patient). The interval between the first and second ETVs ranged from 8 days to almost 6 years (mean 12.8 months). The intraoperative findings at repeat surgery were: occlusion of the primary orifice by scar (10 cases), virginal floor of the third ventricle (5 cases), pinhole ventriculostomy (3 cases), incompletely penetrated membrane (1 case), and blood clot occluding the orifice (1 case). The follow-up period ranged from 3 to 47 months (median 20 months). Repeat ETV was successful in 13 patients (65%). These patients did not require further shunting or other procedures during follow-up. Seven patients (35%) required placement of a shunt after repeat ETV. Several complications were observed in 1 patient (5%), including seizures, elevated ICP, bilateral pulmonary edema, and cardiac arrhythmia. This patient ultimately recovered fully; the ETV was successful, and the patient did not require a shunt. CONCLUSIONS: Based on the experience of this group of patients, repeat ETV is as effective and as safe as a primary ETV procedure, and should be attempted in selected patients.

Adolescent↗
PubMed2001

Mitochondrial aldehyde dehydrogenase activity is required for male fertility in maize.

Some plant cytoplasms express novel mitochondrial genes that cause male sterility. Nuclear genes that disrupt the accumulation of the corresponding mitochondrial gene products can restore fertility to such plants. The Texas (T) cytoplasm mitochondrial genome of maize expresses a novel protein, URF13, which is necessary for T cytoplasm-induced male sterility. Working in concert, functional alleles of two nuclear genes, rf1 and rf2, can restore fertility to T cytoplasm plants. Rf1 alleles, but not Rf2 alleles, reduce the accumulation of URF13. Hence, Rf2 differs from typical nuclear restorers in that it does not alter the accumulation of the mitochondrial protein necessary for T cytoplasm-induced male sterility. This study established that the rf2 gene encodes a soluble protein that accumulates in the mitochondrial matrix. Three independent lines of evidence establish that the RF2 protein is an aldehyde dehydrogenase (ALDH). The finding that T cytoplasm plants that are homozygous for the rf2-R213 allele are male sterile but accumulate normal amounts of RF2 protein that lacks normal mitochondrial (mt) ALDH activity provides strong evidence that rf2-encoded mtALDH activity is required to restore male fertility to T cytoplasm maize. Detailed genetic analyses have established that the rf2 gene also is required for anther development in normal cytoplasm maize. Hence, it appears that the rf2 gene was recruited recently to function as a nuclear restorer. ALDHs typically have very broad substrate specificities. Indeed, the RF2 protein is capable of oxidizing at least three aldehydes. Hence, the specific metabolic pathway(s) within which the rf2-encoded mtALDH acts remains to be discovered.

Aldehyde Dehydrogenase↗
PubMed2001

The N-terminal portion of mature aldehyde dehydrogenase affects protein folding and assembly.

Human liver cytosolic (ALDH1) and mitochondrial (ALDH2) aldehyde dehydrogenases are both encoded in the nucleus and synthesized in the cytosol. ALDH1 must fold in the cytosol, but ALDH2 is first synthesized as a precursor and must remain unfolded during import into mitochondria. The two mature forms share high identity (68%) at the protein sequence level except for the first 21 residues (14%); their tertiary structures were found to be essentially identical. ALDH1 folded faster in vitro than ALDH2 and could assemble to tetramers while ALDH2 remained as monomers. Import assay was used as a tool to study the folding status of ALDH1 and ALDH2. pALDH1 was made by fusing the presequence of precursor ALDH2 to the N-terminal end of ALDH1. Its import was reduced about 10-fold compared to the precursor ALDH2. The exchange of the N-terminal 21 residues from the mature portion altered import, folding, and assembly of precursor ALDH1 and precursor ALDH2. More of chimeric ALDH1 precursor was imported into mitochondria compared to its parent precursor ALDH1. The import of chimeric ALDH2 precursor, the counterpart of chimeric ALDH1 precursor, was reduced compared to its parent precursor ALDH2. Mature ALDH1 proved to be more stable against urea denaturation than ALDH2. Urea unfolding improved the import of precursor ALDH1 and the chimeric precursors but not precursor ALDH2, consistent with ALDH1 and the chimeric ALDHs being more stable than ALDH2. The N-terminal segment of the mature protein, and not the presequence, makes a major contribution to the folding, assembly, and stability of the precursor and may play a role in folding and hence the translocation of the precursor into mitochondria.

Aldehyde Dehydrogenase↗
PubMed2001

The Sonic Hedgehog-Gli pathway regulates dorsal brain growth and tumorigenesis.

The mechanisms that regulate the growth of the brain remain unclear. We show that Sonic hedgehog (Shh) is expressed in a layer-specific manner in the perinatal mouse neocortex and tectum, whereas the Gli genes, which are targets and mediators of SHH signaling, are expressed in proliferative zones. In vitro and in vivo assays show that SHH is a mitogen for neocortical and tectal precursors and that it modulates cell proliferation in the dorsal brain. Together with its role in the cerebellum, our findings indicate that SHH signaling unexpectedly controls the development of the three major dorsal brain structures. We also show that a variety of primary human brain tumors and tumor lines consistently express the GLI genes and that cyclopamine, a SHH signaling inhibitor, inhibits the proliferation of tumor cells. Using the in vivo tadpole assay system, we further show that misexpression of GLI1 induces CNS hyperproliferation that depends on the activation of endogenous Gli1 function. SHH-GLI signaling thus modulates normal dorsal brain growth by controlling precursor proliferation, an evolutionarily important and plastic process that is deregulated in brain tumors.

Animals↗
PubMed2001

Antibodies to assess phosphorylation of spinach leaf nitrate reductase on serine 543 and its binding to 14-3-3 proteins.

To monitor site-specific phosphorylation of spinach leaf nitrate reductase (NR) and binding of the enzyme to 14-3-3 proteins, serum antibodies were raised that select for either serine 543 phospho- or dephospho-NR. The dephospho-specific antibodies blocked NR phosphorylation on serine 543. The phospho-specific antibodies prevented NR binding to 14-3-3s, NR inhibition by 14-3-3s, NR dephosphorylation on serine 543, and did not precipitate 14-3-3s together with NR. Together, this confirms that 14-3-3s bind to NR at hinge 1 after it has been phosphorylated on serine 543. The amounts of individual NR forms were determined in leaf extracts by immunoblotting and immunoprecipitation. The phosphorylation state of NR on serine 543 increased 2-3-fold in leaves upon a light/ dark transition. Before the transition, one-third of NR was already phosphorylated on serine 543 but was not bound to 14-3-3s. Phosphorylation of serine 543 seems not to be enough to bind to 14-3-3s in leaves.

14-3-3 Proteins↗
PubMed2000

Basis for half-of-the-site reactivity and the dominance of the K487 oriental subunit over the E487 subunit in heterotetrameric human liver mitochondrial aldehyde dehydrogenase.

Human liver mitochondrial aldehyde dehydrogenase is a tetrameric enzyme composed of 4 identical 500 amino acid containing subunits arranged such that the protein is a dimer of dimers. No kinetic evidence for subunit interactions has been reported. However, the enzyme exhibits half-of-the-site reactivity in that there is a pre-steady-state burst of 2 mol of NADH per mole of enzyme. A variant of the enzyme, found in Asian people, contains a lysine rather than a glutamate at position 487. This enzyme has a high K(M) for NAD(+) and a low specific activity. In heterotetramers composed of both subunit types, it appeared that the lysine-containing subunit was dominant over the glutamate-containing subunits. To allow for the separation of various heterotetrameric forms of the enzyme, surface residues were changed. Each of the five possible tetrameric forms of the modified enzyme was isolated and characterized with respect to steady-state kinetics and pre-steady-state burst magnitudes. The data best fit a model where in each dimer pair there is one functioning and one nonfunctioning subunit. Further, the lysine subunit affects the properties only of its dimer partner. Residue 487 is located at the dimer interface, and the glutamate forms salt bonds with two arginine residues. One is to Arg(264) in the same subunit; the other is to Arg(475) located in the other subunit. Most likely the presence of a lysine affects these salt bonds so the lysine subunit can cause the other subunit to become essentially nonfunctional.

Aldehyde Dehydrogenase↗