Thermal stabilities of globular proteins

Statistical thermodynamic theory has recently been developed to account for the stabilities of globular proteins. Here we extend that work to predict the dependence on temperature. Folding is assumed to be driven by solvophobic interactions and opposed by the conformational entropy. The temperature dependence of the solvophobic interaction is taken from the transfer experiments on amino acids by Tanford and Nozaki and on model solutes by Gill and Wads?. One long-standing puzzle has been why proteins denature upon heating, since the solvophobic force to fold strengthens with increasing temperature. This is resolved by the theory, which predicts two first-order phase transitions. "Cold denaturation" is driven principally by the weakening of the solvophobic interaction, but normal denaturation is driven principally by the gain of conformational entropy of the chain. Predictions of the thermodynamic state functions are in reasonable agreement with the calorimetric experiments of Privalov and Khechinashvili. Comparison of the theory with experiments suggests that there may be an additional enthalpic driving force toward folding which is not due to the solvophobic interactions.     

Membrane lipids composition and metabolism during early embryonic development. Phospholipid subcellular distribution and 32P labeling

Phospholipid composition and 32P metabolism were studied in oocytes and early developing embryos of the toad, Bufo arenarum, Hensel. The content and distribution of phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidic acid, sphingomyelin, phosphatidylserine, and diphosphatidylglycerol in embryos, whole oocytes, and the subcellular fractions of both were determined. Phosphatidylcholine and phosphatidylethanolamine were the major constituents of yolk platelet. Diphosphatidylglycerol was confined to the mitochondrial fraction, where it represented about 7% of the total phosphoacylglycerols. Relatively large amounts of sphingomyelin were found in microsomal and postmicrosomal supernatants. After in vivo labeling with 32P, the early development of individual phospholipids in subcellular fractions and in whole eggs was followed. The greatest uptake was found in mitochondrial and yolk platelet fractions. A steady increase in the amount of 32P present in phosphatidylethanolamine, phosphatidylcholine, and phosphatidylinositol was seen in the whole embryo from oocyte to late gastrula stage and in all subcellular fractions. Phosphatidic acid exhibited a slight decrease in specific activity, except in the yolk platelet fraction. This high 32P incorporation would indicate a rapid and uneven polar headgroup turnover determined by phospholipid class and subcellular fraction. At the same time, the phospholipid content of the subcellular fractions studied remained unchanged during early embryogenesis. Moreover, 32P was actively incorporated into the individual phospholipids in the absence of measurable net synthesis.     

Translation of capped viral mRNAs in poliovirus-infected HeLa cells.

HeLa cells doubly infected with Semliki Forest virus (SFV) and poliovirus synthesize either more poliovirus proteins or more SFV late proteins depending on the time of super-infection with poliovirus. Under some conditions, the infected cells translate uncapped poliovirus mRNA and capped 26S mRNA from SFV simultaneously, even though host protein synthesis has been shut down. Vesicular stomatitis virus (VSV) protein synthesis is depressed drastically when VSV-infected cells are super-infected with poliovirus. In cells doubly infected with VSV and encephalomyocarditis (EMC) virus or with VSV and SFV, dominance of one of the viruses depends on the time of addition of the challenge virus. The influence of external conditions on the relative translation of capped or uncapped viral mRNA in doubly infected cells has also been analysed.     

Methionine-defective mutants inCandida utilis

Ethionine-resistant mutants ofCandida utilis CCY-158 overproducing methionine have been isolated. In these mutants the intracellular methionine concentration decreased significantly during the stationary phase. The wild-type strain CCY-158 and the ethionine-resistant mutants isolated were able to use methionine as the nitrogen source but not as the carbon source. From these ethionine-resistant mutants we isolated mutants unable to use methionine as nitrogen source (Mec^- mutants), the principal alteration being at the level of methionine uptake. Some of the Mec mutants lost also the ability to use other amino acids as nitrogen source.     

Specific Detection of Arcobacter and Campylobacter Strains in Water and Sewage by PCR and Fluorescent In Situ Hybridization

The aim of this study was to evaluate PCR and fluorescent in situ hybridization (FISH) techniques for detecting Arcobacter and Campylobacter strains in river water and wastewater samples. Both 16S and 23S rRNA sequence data were used to design specific primers and oligonucleotide probes for PCR and FISH analyses, respectively. In order to assess the suitability of the methods, the assays were performed on naturally and artificially contaminated samples and compared with the isolation of cells on selective media. The detection range of PCR and FISH assays varied between 1 cell/ml (after enrichment) to 10³ cells/ml (without enrichment). According to our results, both rRNA-based techniques have the potential to be used as quick and sensitive methods for detection of campylobacters in environmental samples.     

Spatial and Temporal Variation in the Ant Occupants of a Facultative Ant-Plant1

Striking variation in ant occupation of a facultative ant-plant, Conostegia setosa (Melastomataceae), was found at three scales: local spatial, geographic, and temporal. C. setosa provides housing for ants and grows in groups of stems (clones). The ant occupants of 14 C. setosa clones were censused four times over a 14-mo period at the La Selva Biological Station, Costa Rica, and twice over a 9-mo period at the Nusagandi Station, Panama. Twelve facultative ant species occupied C. setosa stems at La Selva, compared to six facultative and one obligate species at Nusagandi. Occupancy (as % of stems ever occupied/clone) was higher at Nusagandi (median = 89%) compared to La Selva (65%). Occupancy varied among clones at La Selva but not at Nusagandi. C. setosa clones differed between sites, with larger clones and more small stems/clone at La Selva. Occupancy was influenced by clone structure; larger clones contained more ant species at both sites and had lower occupancy at La Selva. Occupancy was highest in larger stems and lowest in small stems at both sites. Temporally, percent occupation/clone did not differ among censuses at either site, but overall occupancy was lower in the dry season at La Selva. Turnover in ant occupants was higher at La Selva than at Nusagandi. The variation observed in this study is likely due to a number of factors, including differences between sites in plant population structure and history, differences between and within sites in ant faunas and their nesting requirements, and changes over space and time in microclimatic variables. Such high variation at multiple scales draws attention to the importance of long-term comparative studies of facultative animal-plant interactions.     

Analysis of the mechanisms involved in NK resistance induced by a new tumor factor NK-RIF

The mechanisms involved in susceptibility or resistance of neoplasic cells to lysis by NK cells are not well known. We have recently described a 12-kDa factor (NK-RIF), produced and released by different tumor cell lines, making K562 resistant to NK lysis without affecting the cytotoxic function of NK effector cells. In this paper we further study the mechanism involved in NK resistance of K562 mediated by NK-RIF and its biological implications. The results show that NK-RIF does not affect the binding capacity of target and effector cells nor the levels of HLA class I antigen expression on the target cells, as a proof that resistance to NK-mediated lysis is not always associated with a defect in target effector binding or with an increased MHC class I antigen expression. However NK-RIF-treated K562 loses its capacity to induce NK cell activation and the subsequent capacity to release NKCF and makes K562 resistant to lysis by NKCF. Therefore our results show that induction of resistance to NK cytotoxicity can be the result of the modulation of target structures responsible for inducing effector cell activation without affecting target/effector binding molecules. This indicates that the structures involved in adherence and activation of NK cells have a different nature and that molecules other than HLA participate in NK resistance.     

The heat shock protein hsp70 binds in vivo to subregions 2-48BC and 3-58D of the polytene chromosomes ofDrosophila hydei

Multiple interactions of members of the hsp70 family with cellular components have already been described. We present, however, the first evidence that upon heat shock treatment hsp70 molecules interact with specific chromosomal subdivisions of the polytene chromosomes ofDrosophila hydei. After a heat shock treatment of 20 min the protein binds to subdivision 3-58D₁ and to the heat shock inducible subdivisions 2-48B_3–6 and 2-48C_1–2. Hsp70 molecules were also observed in subdivision 3-58D₁ during recovery at 25°C but not in subdivisions 2-48B_3–6 and 2-48C_1–2. Our data suggest that this interaction is stress specific. DNase and RNase experiments suggest, moreover, that the hsp70 molecules bind to RNA from ribonucleoproteins (RNPs) in subdivisions 2-48B_3–6 and 2-48C_1–2 and to DNA in subdivision 3-58D₁. The DNA sequences in subdivision 3-58D₁ seem to have the potential to adopt the Z-DNA conformation.     

Trypanosomatidae codon usage and GC distribution.

A study of Trypanosomatidae GC distribution and codon usage is presented. The codon usage patterns in coincidence with the phylogenetical data are similar in Crithidia and Leishmania, whereas they are more divergent in Trypanosoma brucei and T. cruzi. The analysis of the GC mutational pressure in these organisms reveals that T. brucei, and to a lesser extent T. cruzi, have evolved towards a more balanced use of all bases, whereas Leishmania and Crithidia retain features of a primeval genetic apparatus. Tables with the approximated GC mutational pressure in homologous genes, and codon usage in Trypanosomatidae are presented.