Identification of the protein responsible for hepatic system N amino acid transport activity.

In the liver, glutamine utilization may be limited by the rate of transport across the plasma membrane by the System N carrier. System N-mediated transport activity has been solubilized from rat liver plasma membrane, partially purified, and then reconstituted into proteoliposomes. To identify the System N carrier protein, monoclonal antibodies were generated against the protein fraction enriched for System N activity. Two antibodies , 3E1-2 and 1E7-3, inhibited System N activity in hepatocytes. These antibodies also immunoprecipitated System N activity from a mixture of solubilized proteins and were specific for antigen recognition in that neither immunoprecipitated System A activity. The antibody recognized a single protein of molecular size 100 kDa by immunoblot analysis. Recognition of this protein by the antibody increased in parallel with the enrichment of System N activity in solubilized membrane fractions. These data suggest that a 100-kDa plasma membrane protein mediates System N transport activity in rat hepatocytes.     

Alpha-helix stability in proteins. I. Empirical correlations concerning substitution of side-chains at the N and C-caps and the replacement of alanine by glycine or serine at solvent-exposed surfaces.

The importance of amino acid side-chains in helix stability has been investigated by making a series of mutations at the N-caps, C-caps and internal positions of the solvent-exposed faces of the two alpha-helices of barnase. There is a strong positional and context dependence of the effect of a particular amino acid on stability. Correlations have been found that provide insight into the physical basis of helix stabilization. The relative effects of Ala and Gly (or Ser) may be rationalized on the basis of solvent-accessible surface areas: burial of hydrophobic surface stabilizes the protein as does exposure to solvent of unpaired hydrogen bond donors or acceptors in the protein. There is a good correlation between the relative stabilizing effects of Ala and Gly at internal positions with the total change in solvent-accessible hydrophobic surface area of the folded protein on mutation of Ala----Gly. The relationship may be extended to the N and C-caps by including an extra term in hydrophilic surface area for the solvent exposure of the non-intramolecularly hydrogen-bonded main-chain CO, NH or protein side-chain hydrogen bonding groups. The requirement for solvent exposure of the C-cap main-chain CO groups may account for the strong preference for residues having positive phi and psi angles at this position, since this alpha L-conformation results in the largest solvent exposure of the C-terminal CO groups. Glycine in an alpha L-conformation results in the greatest exposure of these CO groups. Further, the side-chains of His, Asn, Arg and Lys may, with positive phi and psi-angles, form a hydrogen bond with the backbone CO of residue in position C -3 (residues are numbered relative to the C-cap). The preferences at the C-cap are Gly much greater than His greater than Asn greater than Arg greater than Lys greater than Ala approximately Ser approximately greater than Asp. The preferences at the N-cap are determined by hydrogen bonding of side-chains or solvent to the exposed backbone NH groups and are: Thr approximately Asp approximately Ser greater than Gly approximately Asn greater than Gln approximately Glu approximately His greater than Ala greater than Val much greater than Pro. These general trends may be obscured when mutation allows another side-chain to become a surrogate cap.(ABSTRACT TRUNCATED AT 400 WORDS)     

Differential regulation by trophic conditions of phosphorylating and non-phosphorylating NADP(+)-dependent glyceraldehyde-3-phosphate dehydrogenases in Chlorella fusca.

The two NADP(+)-dependent glyceraldehyde-3-phosphate dehydrogenases present in the green alga Chlorella fusca, namely, the phosphorylating (chloroplastic) enzyme and the non-phosphorylating (cytosolic) enzyme, are differently affected by the trophic conditions prevailing in the cell cultures. The addition of metabolizable sugars to cell cultures growing in the light promotes a marked decrease of the phosphorylating enzyme activity down to a barely detectable cellular level. In contrast, the cellular level of the non-phosphorylating enzyme is even enhanced in the presence of such sugars. These effects are not observed, however, with a number of non-assimilable sugar analogs. After sugar removal, a recovery of the phosphorylating activity--in a process which is inhibited by cycloheximide but not by lincomycin--is observed in illuminated cells but not in darkness, thus indicating a light-dependent nuclear synthesis of the chloroplastic enzyme. It seems therefore that the two dehydrogenases are adaptative enzymes subject to differential regulation by nutritional conditions.     

Comparison of the folding of 2-Keto-3-deoxy-6-phosphogluconate aldolase,triosephosphate isomerase and pyruvate kinase: Implications in molecular evolution

The 8-fold α/β barrel conformation of 2-keto-3-deoxy-6-phosphogluconate aldolase has been compared to that of triosephosphate isomerase and the A-domain of pyruvate kinase. There are eight supersecondary structure units (α/β) in each of these proteins, and the comparisons were carried out in orientations corresponding to each of the possible congruences, i.e. first to first, first to second,… of the supersecondary structure units. The comparison of the C_α structure of the main chain folding of the three enzymes indicated about 150 equivalences with rootmean-square differences of about 3.1 Å, with no orientational preference, including the aldolase with itself. In addition, there is no sequence homology between the aldolase and the isomerase, and no indication of gene duplication in the former. The lack of orientational preference among the three enzymes suggests convergence to a fold of exceptional stability. However, all three enzymes activate a CH bond adjacent to a carbonyl, and their active sites correspond to the f strand, F helix region of the α/β barrel, thus contradicting the foregoing and suggesting divergent evolution from a common precursor. Other and similar arguments are also presented for and against convergent evolution of these three strikingly similar enzymes.     

Chromosome numbers and karyotypic evolution of Caraboidea

The chromosome numbers of 136 species of the Spanish caraboid fauna were studied. The most frequent karyotypes are 2n=37 (54 species) and 2n=24 (23 species), and the chromosome number ranges from 2n=21 to 2n=69, of which 2n=69 is the highest diploid number hitherto found among the Coleoptera. It is proposed that 2n=37 is the ancestral karyotype of the division Caraboidea and the suborder Adephaga as opposed to that of the suborder Polyphaga, 2n=20. Karyotypic evolution has led to increases and decreases of this number, both tendencies having taken place in four genera. Species of ten genera show a neo-XY bivalent due to an X-autosome fusion. The thirty-three chromosome numbers of Caraboidea reveal that these Coleoptera have a remarkable karyotypical heterogeneity.     

Male achiasmatic meiosis in Caraboidea (Coleoptera,Adephaga)

The meiotic process was studied in fifty-three species belonging to the caraboid families Bembidiidae (48). Trechidae (3), Pogonidae (1), and Harpalidae (1). Males show achiasmatic meiosis patterns of the Callimantis type. In females only early prophase stages were observed, that are similar to those of the males. The characteristics of this abnormal meiosis and its evolutionary and cytotaxonomic significance are discussed.     

PERFUSION FOR MYOCARDIAL REVASCULARIZATION WITHOUT AN ARTIFICIAL OXYGENATOR (New Method to Reduce Surgical Morbidity)

Thirteen patients were submitted to direct myocardial revascularization (saphenous vein graft) without the use of an artificial oxygenator. The perfusion was done by a left ventricle-to-aorta bypass and autogenous oxygenation. Most patients had three grafts implanted plus endarterectomy of the distal right coronary artery. There was one hospital death that was apparently not related to the method used. Perfusion time ranged from 45 minutes to 4 hours. Body temperature during perfusion was kept between 25 and 30 degrees C. Perfusion flow was maintained between 25 to 50 ml per kg of body weight per minute. Ischemic, hypothermic cardiac arrest was employed. We demonstrated for the first time that perfusion for this kind of heart surgery could be done with no artificial oxygenators and, apparently, is safer for the patients. There were no bleeding problems even in perfusions as long as 4 hours. There was no respiratory dysfunction, and artificial respiration was used for only 6 to 12 hours. The patients awoke at the end of surgery with no signs or symptoms of central nervous system damage, and vasopressor drugs were rarely used after surgery. Although the experience is very small, it suggests that many postoperative problems, especially those related to bleeding and respiratory dysfunction may be reduced or eliminated by this new method.     

Synchronous growth of a freshwater diatom Melosira italica under natural environment

Summary A filamentous diatom Melosira italica was collected at the beginning of rainy season from a shallow lake in the tropical savanna region in Brazil. Even the sample taken from surface water contained empty cells in high percentages. The number of cells per filament of M. italica showed a peculiar pulse-like frequency distribution with peak values at 4, 8, 12 and 16. Evidences of the synchronous cell division in this planktonic diatom under natural environment are discussed.     

Preparation and characterization of homogeneous coupling factor 6 from bovine heart mitochondria.

Coupling factor 6 (F₆) and mitochondrial ATPase inhibitor were isolated from the rutamycin-sensitive ATPase complex of bovine heart mitochondria by heating and fractionation with ethanol. F₆ appeared in acrylamide gel electrophoresis in the presence of sodium dodecylsulfate and urea as a single band corresponding to a molecular weight of 8,000. This protein which is required for the ³²P_i-ATP exchange in submitochondrial particles treated with silicotungstate was very sensitive to trypsin.     

PARP-1 Regulates Metastatic Melanoma through Modulation of Vimentin-induced Malignant Transformation

PARP inhibition can induce anti-neoplastic effects when used as monotherapy or in combination with chemo- or radiotherapy in various tumor settings; however, the basis for the anti-metastasic activities resulting from PARP inhibition remains unknown. PARP inhibitors may also act as modulators of tumor angiogenesis. Proteomic analysis of endothelial cells revealed that vimentin, an intermediary filament involved in angiogenesis and a specific hallmark of EndoMT (endothelial to mesenchymal transition) transformation, was down-regulated following loss of PARP-1 function in endothelial cells. VE-cadherin, an endothelial marker of vascular normalization, was up-regulated in HUVEC treated with PARP inhibitors or following PARP-1 silencing; vimentin over-expression was sufficient to drive to an EndoMT phenotype. In melanoma cells, PARP inhibition reduced pro-metastatic markers, including vasculogenic mimicry. We also demonstrated that vimentin expression was sufficient to induce increased mesenchymal/pro-metastasic phenotypic changes in melanoma cells, including ILK/GSK3-β-dependent E-cadherin down-regulation, Snail1 activation and increased cell motility and migration. In a murine model of metastatic melanoma, PARP inhibition counteracted the ability of melanoma cells to metastasize to the lung. These results suggest that inhibition of PARP interferes with key metastasis-promoting processes, leading to suppression of invasion and colonization of distal organs by aggressive metastatic cells.