Careful consideration of the effects induced by glutathione depletion in rat liver and heart. The involvement of cytosolic and mitochondrial glutathione pools

One of the most widely used mechanisms by which the role of glutathione (GSH) in cellular functions has been withdrawn, is to deplete GSH intracellularly. The importance of the procedure and xenobiotic chosen to get it is discussed. Mitochondrial GSH plays certainly an important role in maintaining cellular homeostasis. This contribution varies depending on the tissue and the conclusions obtained about the functions of this GSH pool in one organ may not be applied to others. Original data on the subcellular distribution of GSH in myocardial tissue of the rat are presented, and the effect of phorone on both cardiac GSH pools is compared with the effect in liver. The mechanical failure of myocardium after ischemic or reperfusion damage might involve mitochondrial GSH, in view of the literature data referring to the role of thiol groups in energy transfer from mitochondria to cytosol.     

Enzyme immunoassay of oestrogen and progesterone receptors in uterine and intrauterine tissue during human pregnancy and labour

Oestrogen and progesterone receptors were studied in the non-pregnant state, in early pregnancy and at term using monoclonal antibody enzyme immunoassays. Receptors for both steroids were found in tissues from non-pregnant patients and patients in early pregnancy. At term oestrogen receptors were undetectable in all tissues studied. Progesterone receptors were undetectable in chorion, amnion and placenta at term, while present in extremely low concentrations in decidua and myometrium.     

Ketomethylureas. A new class of angiotensin converting enzyme inhibitors

The design rationale for a new series of tripeptide derived angiotensin converting enzyme (ACE) inhibitors, which we term "ketomethylureas", is described. Analogs of tripeptide substrates (i.e. N-benzoyl-Phe-Ala-Pro) in which the nitrogen atom of the scissile amide bond and the adjacent asymmetric carbon atom of the penultimate amino acid residue are formally transposed give rise to this novel class of inhibitors. The most potent ketomethylureas inhibit ACE with I50 values in the nM range.     

Calcium is necessary for light excitation in barnacle photoreceptors

Summary Illumination of barnacle (Balanus amphitrite) photoreceptors is known to increase the membrane permeability to sodium and Ca²⁺ ions resulting in a depolarizing receptor potential. In this report, we show that lanthanum (La³⁺), a known inhibitor of Ca-binding proteins, reversibly eliminates the receptor potential of barnacle photoreceptors when applied to the extracellular space. Similar reversible elimination of the light response was obtained by removing extracellular Ca²⁺ by application of the calcium chelating agent EGTA. Iontophoretic injection of Ca²⁺, but not K⁺ into the cells protected both the transient and the steady-state phases of the receptor potential from elimination by EGTA while only the transient phase was protected in the presence of La³⁺. The EGTA experiments suggest that internal Ca²⁺ is necessary for light excitation of barnacle photoreceptors while the La³⁺ experiments suggest that La³⁺-sensitive inward current is necessary to maintain excitation during prolonged light.Abbreviations EGTA ethylenglyol-bis-(-aminoethylether) N, N, N¹, N¹-tetraacetate - BAPTA bis-(0-aminophenoxy)-ethane-N, N, N¹, N¹-tetraacetic acid - DMSO dimethyl sulfoxide - trp transient receptor potential - nss no steady state - ASW artificial sea water     

Isolation of cDNA clones and complete amino acid sequence of human erythrocyte glycophorin C

Two cDNA clones for glycophorin C, a transmembrane glycoprotein of the human erythrocyte which carries the blood group Gerbich antigens, have been isolated from a human reticulocyte cDNA library. The clones were identified with a mixture of 32 oligonucleotide probes (14-mer) which have been synthetized according to the amino acid sequence Asp-Pro-Gly-Met-Ala present in the N-terminal tryptic peptide of the molecule. The primary structure of glycophorin C deduced from the nucleotide sequence of the 460 base-pair insert of the pGCW5 clone indicates that the complete protein is a single polypeptide chain of 128 amino acids clearly organized in three distinct domains. The N-terminal part (residues 1-57, approximately) which is N- and O-glycosylated is connected to a hydrophilic C-terminal domain (residues 82-128, approximately) containing 4 tyrosine residues by a hydrophobic stretch of nonpolar amino acids (residues 58-81, approximately) probably interacting with the membrane lipids and permitting the whole molecule to span the lipid bilayer. Northern blot analysis using a 265-base-pair restriction fragment obtained by DdeI digestion of the inserted DNA shows that the glycophorin C mRNA from human erythroblasts is approximately 1.4 kilobases long and is present in the human fetal liver and the human K562 and HEL cell lines which exhibit erythroid features. The glycophorin C mRNA, however, is absent from adult liver and lymphocytes, indicating that this protein represents a new erythrocyte-specific probe which might be useful to study erythroid differentiation.     

Cloning and sequencing of mRNAs coding for two adult alpha globin chains of the salamander Pleurodeles waltlii

A cDNA library of erythrocyte mRNAs was established from immature red blood cells of the adult amphibian, Pleurodeles waltlii (urodele; salamander). The cDNA clones corresponding to the four adult globin chains were first identified and characterized by positive selection and the cDNAs derived from the two (major and minor) alpha-globin chains sequenced. The sequences presented contain both the complete 3'-noncoding region and the coding region of both chains, with the exception of the first nine codons of the minor alpha-chain, and a portion of the 5'-noncoding region of the major chain. The amino acid sequences of the encoded alpha-globin polypeptides have been deduced and compared with those of Xenopus laevis and of man. These comparative studies suggest that the alpha-globins of Pleurodeles waltlii and Xenopus laevis may have diverged from a common ancestral gene at the time when mammalian and amphibian lines diverged, and that they then evolved separately. Duplication of the alpha-gene, which is responsible for the polypeptide heterogeneity, appears to have occurred earlier in Pleurodeles waltlii than in Xenopus laevis.     

New C-nucleoside analogs by dehydration of 1-benzyl-4,5,6,7-tetrahydro-6,6-dimethyl-2-(d-galacto-pentitol-1-yl)-indol-4-one

The reaction between 2-(benzylamino)-2-deoxy-d-glycero-l-gluco-heptose and 5,5-dimethyl-1,3-cyclohexanedione yields 1-benzyl-4,5,6,7-tetrahydro-6,6-dimethyl-2-(d-galacto-pentitol-1-yl)-indol-4-one (2). Acid-catalyzed, intramolecular dehydration of 2 under kinetically controlled conditions gives 1-benzyl-4,5,6,7-tetrahydro-2-α-d-lyxofuranosyl-6,6-dimethylindol-4-one; the anomeric configuration of this compound is only suggested. When the dehydration reaction is conducted under thermodynamically controlled conditions, it produces a 1:1 mixture of the α- and β-d-lyxopyranosyl compounds. The structures of the new compounds were elucidated by chemical and physical methods.     

Low copy number and limited variability of proviral DNA in alveolar macrophages from HIV-1-infected patients: evidence for genetic differences in HIV-1 between lung and blood macrophage populations.

BACKGROUND: We investigated the human immunodeficiency virus (HIV) proviral DNA sequence and copy number in alveolar macrophages (AM) and peripheral blood monocytes (PBM) from 10 HIV-positive patients without any active concurrent pulmonary disease to understand the nature of HIV-1 infection in vivo in the lung microenvironment. MATERIALS AND METHODS: The 10 seropositive patients without active pulmonary disease were selected based on chest roentegenography and pathological/cytological test of bronchoalveolar (BAL) fluid. In order to determine accurate proviral copy numbers, AM and PBM were isolated to 99 and 94% purity, respectively, and quantitative polymerase chain reaction (PCR), with a sensitivity to detect three copies of HIV proviral DNA per 10(5) cells, was applied. For analysis of genetic variation in HIV-1, PCR-amplified HIV-1 DNA from AM and PBM of five patients were subcloned and 2-12 clones from each sample underwent DNA sequence analysis of HIV-1 gp120 V3-V5. Heteroduplex mobility assays were performed to confirm the results of the sequence analysis. RESULTS: The proviral copy number in AM or PBM were less than 20 copies/10(5) cells in all patients, and five patients had less than the detection limit. There was no significant difference in HIV copy number between AM and PBM. No correlation was found between PBM/AM HIV copy number and CD4+ lymphocyte count in the peripheral blood. Sequence analysis revealed that the mean intrapatient genetic similarity in AM was 97.5 +/- 0.18% (n = 107), which was significantly higher than that in PBM (96.2 +/- 0.26% (n = 94), p < 0.001), suggesting that variability of HIV-1 DNA in AM was relatively limited. Divergence occurred when AM derived HIV-1 sequence was compared with PBM derived sequence from the same patient (95.8 +/- 0.17% (n = 223) p < 0.001). Phylogenetic analysis of DNA sequence demonstrated complete separation of HIV lineages from lung and blood in four of five patients. CONCLUSIONS: The results suggest the HIV-1 infection in AM is restricted in vivo with low viral burden and homogenous genotype. We propose that the pulmonary microenvironment may limit the extent of HIV-1 infection.     

Effect of ethanol and fatty acids on malolactic activity ofLeuconostoc oenos

Medium-chain fatty acids (C₆ to C₁₂), produced by yeast metabolism during alcoholic fermentation, are known to be inhibitory to lactic acid bacteria. The purpose of this work was to clarify the effect of both ethanol and decanoic and dodecanoic acids on the growth and malolactic activity of aLeuconostoc oenos strain isolated from Portuguese red wine. Ethanol in concentrations up to 12% had no significant effect on malolactic activity but strongly inhibited cell growth. The fatty acids decanoic acid, in concentrations up to 12.5 mg l^–1, and, dodecanoic acid up to 2.5 mg l^–1 seemed to act as growth factors stimulating also malolactic activity; at higher concentrations they exerted an inhibitory effect. We found clear pH dependence between pH 3.0 and pH 6.0, between decanoic acid concentration and its effect on malolactic activity, indicating that the undissociated molecule is the active form. At pH 3.0 the results can be explained by considering that fatty acids enter the cell as protonated molecules and dissociate in the cytoplasm due to the higher internal pH, leading to increased intracellular hydrogenous concentration. This may be the basis of two different effects that contribute to the observed inhibition: decrease in the intracellular pH and dissipation of the transmembrane proton gradient, thus inhibiting intracellular enzymes and ApH-dependent transport systems.