Vergleichende Untersuchungen an HNO2-Inaktivierten und UV-Inaktivierten Bakteriophagen T4

Summary The inactivation of phage T4 by nitrous acid (HNO₂) is essentially an exponential function of time of treatment. HNO₂-inactivated T4 is able to undergo multiplicity reactivation, and genetic markers may be rescued by live phage, however, the extent of both effects is appreciably less than after UV-inactivation. Also, the survival of phenotypic function of the cistronsr II-A andr II-B is lower with HNO₂-treatment than with a UV-irradiation of a corresponding number of hits.The reduced effects are quantitatively accounted for by the assumption of lethal hits blocking early steps of infection. These early-step damages amount to approximately 1/6 of the total hit number; it is still unknown whether they occur in DNA or in protein. Some indication for the occurrence in protein comes from the result that the host-killing efficiency of HNO₂-inactivated phage is reduced at a similar rate as these early-step damages occur. However, at least 5/6 of the lethal hits are due to chemical changes within the DNA, as can be calculated from the results of multiplicity reactivation, marker rescue, and phenotypic survival of therII-cistrons.

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A mitochondrial protein from Neurospora crassa detected both on ribosomes and in membrane fractions. Analysis of the gene, the message, and the protein

We have isolated clones representing at least three nuclear genes for mitochondrial ribosomal proteins from Neurospora crassa by screening a lambda gt11 cDNA library with an antiserum against a mixture of these proteins. The cDNA and genomic DNA sequence for one of these genes, mrp-3, was determined. The MRP3 protein was purified by immune-affinity chromatography, using a monoclonal antibody probe, and subjected to amino acid sequence analysis to identify the mature amino terminus and a prospective mitochondrial-targeting presequence. MRP3 was identified as the largest, least basic protein detected from the small subunit of ribosomes which had been salt-washed and fractionated on sucrose gradients. However, the mRNA and protein products of mrp-3 were found to be present in excess over those of other N. crassa mitoribosomal protein genes. Using a solution hybridization/S1 nuclease assay, we found three-fold- more mRNA for mrp-3 than for another mito-ribosomal protein gene. In addition, a 30- to 50-fold excess of non-ribosomal MRP3 protein was discovered. The additional protein was localized in mitochondrial membrane fractions; none was detected in matrix fractions after removal of the ribosomes. An immunologically related protein was detected in ribosome and membrane fractions of mitochondria from Saccharomyces cerevisiae. The functional significance of this dual localization remains an enigma.     

Lactate dehydrogenase from the extreme halophilic archaebacterium Halobacterium marismortui

D-Lactate dehydrogenase from the extreme halophilic archaebacterium Halobacterium marismortui has been partially purified by ammonium-sulfate fractionation, hydrophobic and ion exchange chromatography. Catalytic activity of the enzyme requires salt concentrations beyond 1M NaCl: optimum conditions are 4M NaCl or KCl, pH 6-8, 50 degrees C. Michaelis constants for NADH and pyruvate under optimum conditions of enzymatic activity are 0.070 and 4.5mM, respectively. As for other bacterial D-specific lactate dehydrogenases, fructose 1,6-bisphosphate and divalent cations (Mg2+, Mn2+) do not affect the catalytic activity of the enzyme. As shown by gel-filtration and ultracentrifugal analysis, the enzyme under the conditions of the enzyme assay is a dimer with a subunit molecular mass close to 36 kDa. At low salt concentrations (less than 1M), as well as high concentrations of chaotropic solvent components and low pH, the enzyme undergoes reversible deactivation, dissociation and denaturation. The temperature dependence of the enzymatic activity shows non-linear Arrhenius behavior with activation energies of the order of 90 and 25 kJ/mol at temperatures below and beyond ca. 30 degrees C. In the presence of high salt, the enzyme exhibits exceptional thermal stability; denaturation only occurs at temperatures beyond 55 degrees C. The half-time of deactivation at 70 and 75 degrees C is 300 and 15 min, respectively. Maximum stability is observed at pH 7.5-9.0.     

Population genetics ofEulemur macaco macaco (Primates: Lemuridae) on the islands of Nosy-Be and Nosy-Komba and the Peninsula of Ambato (Madagascar)

Analysis for genetic variation of insular and mainland populations ofEulemur macaco has revealed: (1) a different degree of genetic variation between populations; and (2) the phylogenetic relationships between groups, on the islands of Nosy-Be and Nosy-Komba, and in the Peninsula of Ambato (Madagascar). Eleven systems of blood proteins from 157 animals were used as genetic markers. The genetic variation was lower on the island of Nosy-Komba than in the mainland of Ambato. This is consistent with the expectation that genetic variation is lower on islands than on mainlands. In contrast, the genetic variation on the island of Nosy-Be was the highest of the three populations. This finding can best be explained by assuming that the sample of Nosy-Be consists of individuals of several small isolated groups, where genetic drift computation showed the population of Nosy-Be to be distinct, and the populations of Nosy-Komba and Ambato to be close within the same branch of the dendrogram. These findings give an insight into the population history of the island of Nosy-Komba, which might have been populated by mainland groups from Ambato.     

Localized delivery of epidermal growth factor improves the survival of transplanted hepatocytes

Hepatocyte transplantation may provide a new approach for treating a variety of liver diseases if a sufficient number of the transplanted cells survive over an extended time period. In this report, we describe a technique to deliver growth factors to transplanted hepatocytes to improve their engraftment. Epidermal growth factor (EGF) was incorporated (0.11%) into microspheres (19 +/- 12 mum) fabricated from a copolymer of lactic and glycolic acid using a double emulsion technique. The incorporated EGF was steadily released over 1 month in vitro, and it remained biologically active, as determined by its ability to stimulate DNA synthesis, cell division, and long-term survival of cultured hepatocytes. EGF-containing microspheres were mixed with a suspension of hepatocytes, seeded onto porous sponges, and implanted into the mesentery of two groups of Lewis rats. The first group of animals had their portal vein shunted to the inferior vena cava prior to cell transplantation (portal-caval shunt = PCS), and the second group of animals did not (non-PCS). This surgical procedure improves the survival of transplanted hepatocytes. The engraftment of transplanted hepatocytes in PCS animals was increased two-fold by adding EGF microspheres, as compared to adding control microspheres that contained no growth factors. Devices implanted into non-PCS animals had fewer engrafted hepatocytes than devices implanted into PCS animals, regardless of whether blank or EGF-containing microspheres were added. These results first indicate that it is possible to design systems which can alter the microenvironment of transplanted hepatocytes to improve their engraftment. They also suggest that hepatocyte engraftment is not improved by providing single growth factors unless the correct environment (PCS) is provided for the transplanted cells. (c) 1996 John Wiley & Sons, Inc.     

Entomopathogenic fungi in predatory beetles (Col.: Carabidae andStaphylinidae) from agricultural fields

Prevalence of entomopathogenic fungi was studied in overwintering ground beetles (Col.: Carabidae) and rove beetles (Col.: Staphylinidae) collected from fields of lucerne, white cabbage and white cabbage undersown with white clover. In general infection levels in adult ground beetles and rove beetles were low (Carabidae: max. 7.6%,Staphylinidae: max. 7.0%). In comparison, prevalence of entomopathogenic fungi in carabid larvae was high (19–50%). At one study site an epizootic ofBeauveria bassiana was observed, infecting 67% of the staphylinidAnotylus rugosus and 37% of the staphylinidGyrohypnus angustatus. Beauveria bassiana was the predominant fungus isolated from ground beetles and rove beetles from all studied sites. Other fungal species included the hyphomycetesMetarhizium anisopliae, Paecilomyces farinosus andVerticillium lecanii as well asZoophthora radicans andZoophthora philonthi (Zygomycetes: Entomophthorales). Two individuals ofAnotylus rugosus were found to have a dual infection ofZoophthora philonthi andBeauveria bassiana.

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Résumé La présence de champignons entomopathogènes a été recherchée chez des carabes (Col.: Carabidae) et des staphylins (Col.: Staphylinidae) récoltés dans des champs de luzerne, de chou et d'une culture mélangée chou-trèfle blanc. Chez les adultes, le taux d'infection dans les deux groupes d'insectes est en général très faible (Carabidae: max. 7,6%, Staphylinidae: max. 7%). Chez les larves de carabes par contre, le taux d'infection par les champignons est élevé (19–50%). Sur l'un des sites de l'étude, une épizootie àBeauveria bassiana a été observée, l'infection portant sur 67% des individus récoltés appartenant à l'espèceAnotylus rugosus et 37% desGyrohypnus angustatus. B. bassiana est le champignon prédominant isolé à partir des coléoptères de tous les sites étudiés. D'autres espèces fongiques ont été relevées: ce sont les hyphomycètesMetarhizium anisopliae, Paecilomyces farinosus etVerticillium lecanii ainsi queZoophthora radicans etZoophthora philonthi. Deux individus d'A. rugosus ont été retrouvés infectés à la fois parErynia etB. bassiana.

     

Molecular chaperones cooperate with PIM1 protease in the degradation of misfolded proteins in mitochondria.

ATP dependent proteolytic degradation of misfolded proteins in the mitochondrial matrix is mediated by the PIM1 protease and depends on the molecular chaperone proteins mt-hsp70 and Mdj1p. Chaperone function is essential to maintain misfolded proteins in a soluble state, a prerequisite for their degradation by PIM1 protease. In the absence of functional mt-hsp70 or Mdj1p misfolded proteins either remain associated with mt-hsp70 or form aggregates and thereby are no longer substrates for PIM1 protease. Mdj1p is shown to regulate the ATP dependent association of an unfolded polypeptide chain with mt-hsp70 affecting binding to as well as release from mt-hsp70. These findings establish a central role of molecular chaperone proteins in the degradation of misfolded proteins by PIM1 protease and thereby demonstrate a functional interrelation between components of the folding machinery and the proteolytic system within mitochondria.     

Effects of decreased atmospheric deposition on the sulfur budgets of two Dutch moorland pools

The chemical composition of surface waters of two Dutch moorland pools and of incident precipitation, was monitored from 1982 to 1990. For this period, sulfur and water budgets were calculated using a hydrochemical model developed for well-mixed non-stratifying lakes. Total atmospheric deposition of S decreased significantly after 1986 at both locations. A model describing the sulfur budget in terms of input, output and reduction/oxidation processes predicted a fast decrease of pool water SO₄ ²⁻ concentrations after a decrease of atmospheric input. However, SO₄ ²⁻ concentrations in the surface water was lowered only slightly or remained constant. Apparently a source within the lake caused the unexpectedly high SO₄ ²⁻ concentrations. The possible supply of SO₄ ²⁻ from the sediment through regulation by (K-)Al-SO₄ containing minerals or desorption of SO₄ ²⁻ from positively charged surfaces in the sediment was evaluated. Solubility calculations of pore water with respect to alunite, basaluminite and jurbanite indicated that SO₄ ²⁻ concentration was not regulated by these minerals. It is suggested here (1) that desorption of SO₄ ²⁻ from peaty sediments may account for the estimated SO₄ ²⁻ supply provided that the adsorption complex is periodically recharged by partial oxidation of the upper bottom sediments and (2) that because of exposure of a part of the pool bottom to the atmosphere during dry summers and subsequent oxidation of reduced S, the amount of SO₄ ²⁻ may be provided which complements the decreasing depositional SO₄ ²⁻ input. In future research these two mechanisms need to be investigated.     

Extracellular matrix controls tubulin monomer levels in hepatocytes by regulating protein turnover.

Cells have evolved an autoregulatory mechanism to dampen variations in the concentration of tubulin monomer that is available to polymerize into microtubules (MTs), a process that is known as tubulin autoregulation. However, thermodynamic analysis of MT polymerization predicts that the concentration of free tubulin monomer must vary if MTs are to remain stable under different mechanical loads that result from changes in cell adhesion to the extracellular matrix (ECM). To determine how these seemingly contradictory regulatory mechanisms coexist in cells, we measured changes in the masses of tubulin monomer and polymer that resulted from altering cell-ECM contacts. Primary rat hepatocytes were cultured in chemically defined medium on bacteriological petri dishes that were precoated with different densities of laminin (LM). Increasing the LM density from low to high (1-1000 ng/cm2), promoted cell spreading (average projected cell area increased from 1200 to 6000 microns2) and resulted in formation of a greatly extended MT network. Nevertheless, the steady-state mass of tubulin polymer was similar at 48 h, regardless of cell shape or ECM density. In contrast, round hepatocytes on low LM contained a threefold higher mass of tubulin monomer when compared with spread cells on high LM. Furthermore, similar results were obtained whether LM, fibronectin, or type I collagen were used for cell attachment. Tubulin autoregulation appeared to function normally in these cells because tubulin mRNA levels and protein synthetic rates were greatly depressed in round cells that contained the highest level of free tubulin monomer. However, the rate of tubulin protein degradation slowed, causing the tubulin half-life to increase from approximately 24 to 55 h as the LM density was lowered from high to low and cell rounding was promoted. These results indicate that the set-point for the tubulin monomer mass in hepatocytes can be regulated by altering the density of ECM contacts and changing cell shape. This finding is consistent with a mechanism of MT regulation in which the ECM stabilizes MTs by both accepting transfer of mechanical loads and altering tubulin degradation in cells that continue to autoregulate tubulin synthesis.