Human papilloma virus E6/E7 genes can expand the lifespan of human corneal fibroblasts

Summary Human corneal fibroblasts were infected with a retroviral delivery vector containing the E6 and E7 genes from human Papilloma virus type 16 in order to produce cell lines that have an expanded lifespan in culture. Morphologically, some of the trasfected corneal fibroblast lines appeared to have the normal spindle-shape morphology of diploid fibroblasts, whereas other lines appeared to have a more elongated morphology. All the cell lines were anchorage-dependent. Cells that had a normal morphology grew at a rate similar to normal diploid human corneal fibroblasts and had a population doubling time of 48 h. All E6/E7 expressing cell lines, regardless of morphology, produce types I, III, and V collagen, at levels similar to those observed in the parent corneal diploid fibroblast. These corneal fibroblast lines will be a usefulin vitro system to study collagen expression and fibril formation, as well as normal stroma development. These results also demonstrate that the use of E6/E7 genes to expand a cell’s lifespan can be a powerful tool because it does not appear to alter either the growth rate of the cell or collagen expression.     

Production, Purification, and Characterization of Recombinant Human Hemoglobin Rainier Expressed inSaccharomyces cerevisiae

Hemoglobin Rainier is a naturally occurring hemoglobin variant in which the β145 tyrosine is substituted with cysteine. The α and β^Rainierglobin cDNAs were cloned in a high copy number vector and expressed inSaccharomyces cerevisiaeunder the control of galactose-regulated hybrid promoters. Using this system, we have expressed individual α and β^Rainierglobin chains. Coexpression of both α and β^RainiercDNAs resulted in the production of a functional hemoglobin molecule. Purification of the recombinant protein was accomplished by ion exchange chromatography. The N-termini of the α and β chains were correctly processed, and the molecular mass, as determined by mass spectrometry, indicated amino acid composition identical to that of natural hemoglobin Rainier. The chromatographic properties of the recombinant hemoglobin Rainier were similar to human-derived hemoglobin A₀. The purified recombinant hemoglobin molecule was shown to have an elevated oxygen affinity and a reduced cooperativity as previously reported for natural hemoglobin Rainier. Production of recombinant hemoglobin and especially hemoglobin variants like hemoglobin Rainier has the potential to facilitate use of hemoglobin as a blood substitute as well as in specific applications, such as for use as a therapeutic agent in the treatment of hypotension associated with septic shock.     

Topological model of the division process of cellular and subcellular compartments

The application of the theory of homeomorphic transformations of topological manifolds and the operation of the connected sum of manifolds for a formation of a topological model of membrane transformations during the division process of cellular and subcellular compartments, has been shown. The biological cell and the subcellular structures in the form of vesicles are modelled by an arrangement of two concentric spheres corresponding to the inner and outer layer of the membrane bounding the vesicle. The analysis shows eight succeeding topological stages of membrane transformations during the division process and these stages are characterised. It is concluded that there is a vectorial translocation of lipid molecules from the inner layer of the membrane bounding the vesicle before the division process to the outer layer of the membranes after the division process and there is no lipid translocation from the outer layer to the inner layers during the division process.     

Effect of water-miscible aprotic solvents on kyotorphin synthesis catalyzed by immobilized α-chymotrypsin

Summary Immobilized -chymotrypsin was used as catalyst to synthesize a kyotorphin derivative (Bz-Tyr-Arg-OEt) in the presence of five water-miscible aprotic solvents (dimethylsulphoxide, dimethylformamide, acetonitrile, acetone and tetrahydrofurane) at 30 °C. By using a kinetically-controlled approach, the maximum synthetic activity was obtained when Arg-OEt was used as nucleophile donor at a concentration 1.5-times higher than the acyl-acceptor substrate (Bz-Tyr-OEt). The water-miscible aprotic solvents enhanced greatly the synthetic activity proportionally to their hidrophilicity properties adequately measured by the log P parameter. At the optimum solvent concentration for the enzymatic peptide synthesis, both the water activity (Aw) of the media and the water content of the immobilized derivative showed a saturation profile against the log P parameter. As a function of the solvent hydrophilicity, these water parameters were shown as key parameters for the increase in the synthetic activity of the enzyme by the presence of these solvents.     

Effects of dark- and light-induced proton gradients in thylakoids on the Q and B thermoluminescence bands

Thermoluminescence experiments have been carried out to study the effect of a transmembrane proton gradient on the recombination properties of the S₂ and S₃ states of the oxygen evolving complex with Q_A ^- and Q_B ^-, the reduced electron acceptors of Photosystem II. We first determined the properties of the S₂Q_A ^- (Q band), S₂Q_B ^- and S₃Q_B ^- (B bands) recombinations in the pH range 5.5 to 9.0, using uncoupled thylakoids. The, a proton gradient was created in the dark, using the ATP-hydrolase function of ATPases, in coupled unfrozen thylakoids. A shift towards low temperature of both Q and B bands was observed to increase with the magnitude of the proton gradient measured by the fluorescence quenching of 9-aminoacridine. This downshift was larger for S₃Q_B ^- than for S₂Q_B ^- and it was suppressed by nigericin, but not by valinomycin. Similar results were obtained when a proton gradient was formed by photosystem I photochemistry. When Photosystem II electron transfer was induced by a flash sequence, the reduction of the plastoquinone pool also contributed to the downshift in the absence of an electron acceptor. In leaves submitted to a flash sequence above 0°C, a downshift was also observed, which was supressed by nigericin infiltration. Thus, thermoluminescence provides direct evidence on the enhancing effect of lumen acidification on the S₃S₂ and S₂S₁ reverse-transitions. Both reduction of the plastoquinone pool and lumen acidification induce a shift of the Q and B bands to lower temperature, with a predominance of lumen acidification in non-freezing, moderate light conditions.Abbreviations 9-AA 9-aminoacridine - E_A activation energy - F₀ constant fluorescence level - F_M maximum fluorescence, when all PS-II centers are closed - F_V variable fluorescence (F_M–F₀) - PS I, PS II Photosystem I, photosystem II - PQ plastoquinone - TL thermoluminescence     

Cloning and analysis of structural genes from Streptomyces pristinaespiralis encoding enzymes involved in the conversion of pristinamycin IIB to pristinamycin IIA (PIIA): PIIA synthase and NADH:riboflavin 5'-phosphate oxidoreductase.

In Streptomyces pristinaespiralis, two enzymes are necessary for conversion of pristinamycin IIB (PIIB) to pristinamycin IIA (PIIA), the major component of pristinamycin (D. Thibaut, N. Ratet, D. Bisch, D. Faucher, L. Debussche, and F. Blanche, J. Bacteriol. 177:5199-5205, 1995); these enzymes are PIIA synthase, a heterodimer composed of the SnaA and SnaB proteins, which catalyzes the oxidation of PIIB to PIIA, and the NADH:riboflavin 5'-phosphate oxidoreductase (hereafter called FMN reductase), the SnaC protein, which provides the reduced form of flavin mononucleotide for the reaction. By using oligonucleotide probes designed from limited peptide sequence information of the purified proteins, the corresponding genes were cloned from a genomic library of S. pristinaespiralis. SnaA and SnaB showed no significant similarity with proteins from databases, but SnaA and SnaB had similar protein domains. Disruption of the snaA gene in S. pristinaespiralis led to accumulation of PIIB. Complementation of a S. pristinaespiralis PIIA-PIIB+ mutant with the snaA and snaB genes, cloned in a low-copy-number plasmid, partially restored production of PIIA. The deduced amino acid sequence of the snaC gene showed no similarity to the sequences of other FMN reductases but was 39% identical with the product of the actVB gene of the actinorhodin cluster of Streptomyces coelicolor A(3)2, likely to be involved in the dimerization step of actinorhodin biosynthesis. Furthermore, an S. coelicolor A(3)2 mutant blocked in this step was successfully complemented by the snaC gene, restoring the production of actinorhodin.     

Distribution of the phytoplankton from the Guri Reservoir (Venezuela)

The distribution of phytoplankton species of a tropical blackwater reservoir is discussed on the basis of spatial differences in water composition and of species abundance and diversity. Spatial heterogeneity in water composition identified three different environments within the reservoir itself: (1) strongly colored waters, high turbidity and iron concentrations at the inflow; (2) calcium enriched, nearly uncolored waters at El Pao Bay; (3) lightly colored water, higher transparency and a higher ratio monovalent to divalent cations in the main body of the reservoir. Three corresponding phytoplankton associations were found. Principal Component Analysis helped to explore the relationship of particular species with the abiotic factors. Among them, water color, turbidity, and mineralization proved to be determinant in habitat diversification.     

Regulation of Osteoblast Gene Expression in Intratypic Osteosarcoma Hybrid Cells

Intratypic osteosarcoma hybrids were constructed by fusing the human osteoblast-like osteosarcoma SaOS-2 with the rat osteoblast-like osteosarcoma UMR-106. Both of these osteosarcomas express liver/bone/kidney alkaline phosphatase (ALPL), but only the UMR-106 cell line expresses osteopontin (OPN), a gene expressed during later stages of osteoblast differentiation. Analysis of osteoblast gene expression in these hybrids demonstrated that ALPL continued to be expressed; however, OPN steady-state mRNA levels were dramatically reduced in four hybrids. Quantitative measurements indicated theft OPN steady-state mRNA levels were extinguished by a factor of 20- to 1000-fold. Since SaOS-2 chromosomes are preferentially lost from these hybrids, subclones of extinguished hybrids were isolated that reexpressed OPN mRNA at levels similar to the UMR-106 parental line. These data indicate that trans-acting negative regulatory factors, expressed from the SaOS-2 genome, are responsible for OPN extinction. This report provides the first demonstration of the negative regulation of OPN gene expression and also provides additional evidence that extinction plays a role in the regulation of osteoblast gene expression.