Intramitochondrial pyruvate attenuates hydrogen peroxide-induced apoptosis in bovine pulmonary artery endothelium

In the hydrogen peroxide (H₂O₂) apoptosis model of the murine thymocyte, redox reactant and antioxidant pyruvate prevents programmed cell death. We tested the hypothesis that such protection was mediated, at least in part, via pyruvate handling by mitochondrial metabolism. Cultured bovine pulmonary artery endothelial cells were incubated for 30 min with 0.5 mM H₂O₂ in the absence and presence of 0.5 mM -cyano-3-hydroxycinnamate, as a selective inhibitor of the mitochondrial pyruvate transporter. In controls H₂O₂ decreased cell viability by 30% within 24 h; this was associated with apoptosis-like bodies, nuclear condensation, and biochemical DNA damage consistent with programmed cell death. Pyruvate (0.1–20 mM) enhanced cell viability in a dose-dependent manner, with 85% viable cells at 3 mM and no DNA laddering, no positive nick-end labeling (TUNEL), and no detectable Annexin V or propidium iodide staining. In contrast, using 5 mM L-lactate as a cytosolic reductant or acetate as a redox-neutral substrate, cell death increased to 40%, which was associated with intense DNA laddering, positive TUNEL and Hoechst 33258 assays. -Cyano-3-hydroxycinnamate alone did not significantly decrease endothelial viability but reduced viability from 85 ± 3 to 71 ± 4% (p = 0.023) in presence of 3 mM pyruvate plus H₂O₂; pathological cell morphology and DNA laddering under the same conditions suggested loss of pyruvate protection against apoptosis. Since -cyano-3-hydroxycinnamate re-distributed medium pyruvate and L-lactate consistent with selective blockade of pyruvate uptake into the mitochondria, the findings support the hypothesis that pyruvate protection against H₂O₂ apoptosis is mediated in part via the mitochondrial matrix compartment. Possible mediators include anti-apoptotic bcl-2 and/or products of mitochondrial pyruvate metabolism such as citrate that affect metabolic regulation and anti-oxidant status in the cytoplasm.     

cDNA cloning and expression of the mouse Na/H antiporter (NHE-1) and a potential splice variant

A cDNA for the Mus musculus Na/H exchanger-isoform 1 (NHE-1) was identified in a BALB/c myoblast library by its hybridization to rat NHE-1 sequences. Analysis of the clone showed it to display extensive homology with NHE-1 clones from other mammalian species; however, the region of interspecific homology was abruptly interrupted in the midst of the open reading frame by 166 bp of unrelated sequence. This extra sequence is likely to be an unspliced intron 9. Aside from the retained intron 9, the NHE-1 cDNA clone is otherwise fully processed, with all of the other ten introns removed and containing a poly(A) tract. From PCR results this variant represents a significant but minor population of NHE-1 RNAs. The variant message does associate with polysomes thereby suggesting it to be translated into protein. The location of the retained intron in the carboxy terminus of the protein is such that its translation would produce a protein predicted to be still capable of effecting Na and H translocation but whose regulatory features would be markedly altered.Amino acid sequence comparison of the mouse NHE-1 (derived from the fully processed message) with that of other mammalian species demonstrated two exceptionally divergent regions; the C-terminal cytoplasmic tail (residues 750-790), containing a region of 6-8 contiguous acidic amino acids variably composed of aspartate and glutamate residues, and the N-terminal extracellular domain that includes an N-linked glycosylation site (residues 60-80).     

The self peptide annexin II (208–223) presented by dendritic cells sensitizes autologous CD4+ T lymphocytes to recognize melanoma cells

Annexin II is known to be over-expressed in different types of tumours. We show here that annexin II protein is expressed by melanoma cell lines in various amounts, consistent with previous findings that an annexin II (208-223) peptide could be eluted from isolated HLA-DR molecules of a constitutively MHC class II-positive melanoma line. T cells sensitized to annexin II (208-223) in vitro using peptide-pulsed autologous dendritic cells responded only to the lines which overexpressed annexin II, in a peptide-specific, HLA-DR-restricted fashion. These CD4+ T cells proliferated strongly and secreted large amounts of type 1 cytokines in response to annexin II (208-223) peptide or annexin II protein-positive melanoma cell lines. These results demonstrate that the annexin II (208-223) peptide, corresponding to a non-mutated sequence of a normal protein, induces antigen-specific T cells which can respond to melanoma cells over-expressing the annexin II molecule. This peptide may therefore be useful in immunotherapy for recruiting CD4+ type 1 helper cells active locally in the tumour environment.     

Purification and stability of peroxidase of African oil palm Elaies guineensis

In the previous work, after screening tropical plants (43 species) for peroxidase activity, high activity has been detected in leaves of some palms and especially African oil palm Elaeis guineensis. This palm is widely cultivated in Colombia and presents a promising source for the industrial production of peroxidase. The initial enzyme isolation included homogenization and extraction of pigments using aqueous two phase polymer system. Initially, traditional system, formed by polyethyleneglycol/K₂HPO₄, was used. The replacement of K₂HPO₄ with (NH₄)₂SO₄ allowed direct application of the salt phase with accumulated peroxidase on a Phenyl-Sepharose column. The final purification was carried out by liquid chromatography on Sephacryl S200 and DEAE-Toyopearl columns. The specific activity of the purified peroxidase measured toward guaiacol was 4300 units per mg of protein. The molecular weight and isoelectric point for palm peroxidase were 57.000 and 3.8, respectively. Palm peroxidase possesses uniquely high thermostability and is more stable in organic solvents than horseradish peroxidase is.     

Sensitivity enhanced NMR spectroscopy by quenching scalar coupling mediated relaxation: Application to the direct observation of hydrogen bonds in 13C/15N-labeled proteins

In this paper, we demonstrate that the sensitivity of triple-resonance NMR experiments can be enhanced significantly through quenching scalar coupling mediated relaxation by using composite-pulse decoupling (CPD) or an adiabatic decoupling sequence on aliphatic, in particular alpha-carbons in ¹³C/¹⁵N-labeled proteins. The CPD-HNCO experiment renders 50% sensitivity enhancement over the conventional CT-HNCO experiment performed on a 12 kDa FK506 binding protein, when a total of 266 ms of amide nitrogen–carbonyl carbon defocusing and refocusing periods is employed. This is a typical time period for the direct detection of hydrogen bonds in proteins via trans-hydrogen bond ^3h J _NC couplings. The experimental data fit theoretical analysis well. The significant enhancement in sensitivity makes the experiment more applicable to larger-sized proteins without resorting to perdeuteration.     

5-Membered NH...N hydrogen bonded molecular scaffold in a model dipeptide containing 3-aminophenylacetic acid: Crystal and solution conformations

Crystallographic and spectroscopic studies of a model dipeptidecontaining unusual amino acid residues establish the presence ofan intramolecular, 5-membered NH...N hydrogen bond involvingan amide NH (from 3-amino phenyl acetic acid residue) and anamide N atom from an adjacent amino acid residue in solid stateand in solution. The dipeptide also forms an infinite -sheet ribbon structure in crystals.     

The role of glutamate receptors in antipsychotic drug action

Summary. It has recently been postulated that disturbances in glutamatergic neurotransmission may contribute to the pathophysiology of schizophrenia. Therefore the aim of the present study was to evaluate the role of glutamate NMDA and group II metabotropic receptors in the antipsychotic drug action. To this aim the influence of some well-known neuroleptics on cortical NMDA receptors was examined. Furthermore, their behavioral effects were compared with those of the novel agonist of group II glutamate metabotropic receptors, LY 354740, in some animal models of schizophrenic deficits. We found that long-term administration of the typical neuroleptic haloperidol and the atypical one clozapine increased the number of NMDA receptors labelled with [³H]CGP 39653 in different cortical areas. Long-, but not short-term, treatment with haloperidol and raclopride diminished the deficit of prepulse inhibition produced by phencyclidine, which is a model of sensorimotor gating deficit in schizophrenia. In contrast, neither short- nor long-term treatment with clozapine influenced the phencyclidine effect in that model. Acute treatment with LY 354740 reversed neither (1) the deficit of prepulse inhibition produced by phencyclidine or apomorphine, nor (2) the impairment in a delayed alternation task induced by MK-801, which is commonly used to model the frontal lobe deficits associated with schizophrenia. The present study suggests that an increase in the density of cortical NMDA receptors may be important to a longterm neuroleptic therapy. Conversely, the results do not support the role of group II metabotropic glutamate receptors in the antipsychotic drug action. Received August 31, 1999 Accepted September 20, 1999     

Isolation of several anti-stress genes from the halotolerant green alga Chlamydomonas by simple functional expression screening with Escherichia coli

To isolate anti-stress genes from a halotolerant marine green alga, a simple screening method based on the bacterial expression system was examined. The method consisted of three steps: (i) directional cDNA library construction in a ZAPII vector, (ii) in vivo mass excision of a ZAPII library into phagemid DNA, and (iii) screening for anti-salt-stress and anti-oxidative-stress genes by culturing bacterial cells carrying the in vivo excised phagemid on selection agar plates with a high concentration of NaCl and/or methyl viologen (MV), and by isolating stress-tolerant bacterial colonies. By this method, we screened the cDNA library of halotolerant Chlamydomonas sp. strain W-80, and isolated several stress-related gene homologs, such as glutathione peroxidase, ascorbate peroxidase, bbc1 (breast basic conserved; a low temperature induced gene in Brassica napus), and cyanide-resistant alternative oxidase.     

A peroxidase homologue and novel plastocyanin located by proteomics to the Arabidopsis chloroplast thylakoid lumen

A study by two-dimensional electrophoresis showed that the soluble, lumenal fraction of Arabidopsis thaliana thylakoids can be resolved into 300 protein spots. After subtraction of low-intensity spots and accounting for low-level stromal contamination, the number of more abundant, lumenal proteins was estimated to be between 30 and 60. Two of these proteins have been identified: a novel plastocyanin that also was the predominant component of the total plastocyanin pool, and a putative ascorbate peroxidase. Import studies showed that these proteins are routed to the thylakoid lumen by the Sec- and delta pH-dependent translocation pathways, respectively. In addition, novel isoforms of PsbO and PsbQ were identified.