Essential role of phosphatidylinositol 3-kinase in insulin-induced activation and phosphorylation of the cGMP-inhibited cAMP phosphodiesterase in rat adipocytes studies using the selective inhibitor wortmannin

Incubation of rat adipocytes with wortmannin, a potent and selective phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor, completely blocked the antilipolytic action of insulin (IC₅₀≈ 100 nM), the insulin-induced activation and phosphorylation of cGMP-inhibited cAMP phosphodiesterase (cGI-PDE) as well as the activation of the insulin-stimulated cGI-PDE kinase (IC₅₀≈ 10–30 nM). No direct effects of the inhibitor on the insulin-stimulated cGI-PDE kinase, the cGI-PDE and the hormone-sensitive lipase were observed. These data suggest that activation of PI 3-kinase upstream of the insulin-stimulated cGI-PDE kinase in the antilipolytic insulin signalchain has an essential role for insulin-induced cGI-PDE activation/ phosphorylation and anti-lipolysis.     

Molecular cloning of the dog homologue of the lymphocyte antigen CD28

The nucleotide data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number L22178.     

Whole inactivated SIV vaccine grown on human cells fails to protect against homologous SIV grown on simian cells

Several groups have reported protection against experimental SIV infection in macaques immunized with a whole inactivated virus vaccine. The aim of the current study was to investigate whether five macaques vaccinated with whole inactivated SIV and previously shown to be protected against challenge with two divergent strains of SIV grown on human cells could resist challenge with a subsequent homologous SIV grown on macaque cells. We show here that this same vaccine did not protect when the challenge virus was grown on primary cells of monkey origin.     

Cellular Localization of Transforming Growth Factor-α mRNA in Rat Forebrain

Abstract: The cellular localization of transforming growth factor-α (TGFa) mRNA in juvenile and adult rat forebrain was examined using in situ hybridization with a ³⁵S-labeled cRNA probe. TGFα cRNA-labeled neuronal perikarya were distributed across many forebrain regions including the olfactory bulb, caudate-putamen, nucleus accumbens, olfactory tubercle, ventral pallidum, amygdala, hippocam-pal stratum granulosum and CA3 stratum pyramidale, and piriform, entorhinal, and retrosplenial cortices. TGFα cRNA-hybridizing cells were also localized to several thalamic nuclei and to the suprachiasmatic, dorsomedial, and ventromedial nuclei of the hypothalamus. In addition, labeled cells were present in regions of white matter including the corpus callosum, anterior commissure, internal and external capsules, optic tract, and lateral olfactory tract. Thus, both neurons and glia appear to synthesize TGFα in normal brain. Hybridization densities were greater in neuronal fields at 2 weeks of age compared with the adult, suggesting a role for TGFα in the development of several forebrain systems. Our results demonstrating the prominent and widespread expression of TGFα mRNA in forebrain, combined with the extremely low abundance of epidermal growth factor mRNA in brain, support the argument that TGFα is the principal endogenous ligand for the epidermal growth factor receptor in normal brain.     

Enhancing the structural diversity between forest patches—A concept and real-world experiment to study biodiversity,multifunctionality and forest resilience across spatial scales

Intensification of land use by humans has led to a homogenization of landscapes and decreasing resilience of ecosystems globally due to a loss of biodiversity, including the majority of forests. Biodiversity–ecosystem functioning (BEF) research has provided compelling evidence for a positive effect of biodiversity on ecosystem functions and services at the local (α-diversity) scale, but we largely lack empirical evidence on how the loss of between-patch β-diversity affects biodiversity and multifunctionality at the landscape scale (γ-diversity). Here, we present a novel concept and experimental framework for elucidating BEF patterns at α-, β-, and γ-scales in real landscapes at a forest management-relevant scale. We examine this framework using 22 temperate broadleaf production forests, dominated by Fagus sylvatica. In 11 of these forests, we manipulated the structure between forest patches by increasing variation in canopy cover and deadwood. We hypothesized that an increase in landscape heterogeneity would enhance the β-diversity of different trophic levels, as well as the β-functionality of various ecosystem functions. We will develop a new statistical framework for BEF studies extending across scales and incorporating biodiversity measures from taxonomic to functional to phylogenetic diversity using Hill numbers. We will further expand the Hill number concept to multifunctionality allowing the decomposition of γ-multifunctionality into α- and β-components. Combining this analytic framework with our experimental data will allow us to test how an increase in between patch heterogeneity affects biodiversity and multifunctionality across spatial scales and trophic levels to help inform and improve forest resilience under climate change. Such an integrative concept for biodiversity and functionality, including spatial scales and multiple aspects of diversity and multifunctionality as well as physical and environmental structure in forests, will go far beyond the current widely applied approach in forestry to increase resilience of future forests through the manipulation of tree species composition.     

Characterization of anf genes specific for the alternative nitrogenase and identification of nif genes required for both nitrogenases in Rhodobacter capsulatus

To identify Rhodobacter capsulatus nif genes necessary for the alternative nitrogenase, strains carrying defined mutations in 32 genes and open reading frames of nif region A, B or C were constructed. The ability of these mutants to grow on nitrogen-free medium with molybdenum (Nif phenotype) or in a nifHDK deletion background on medium without molybdenum (Anf phenotype) was tested. Nine nif genes and nif-associated coding regions are absolutely essential for the alternative nitrogenase. These genes comprise nifV and nifB, the nif-specific ntr system (nifR1, R2, R4) and four open reading frames, which exhibit no homology to known genes. In addition, a significantly reduced activity of both the alternative nitrogenase and the molybdenum-dependent nitrogenase was found for fdxN mutants. By random Tn5 mutagenesis of a nifHDK deletion strain 42 Anf^? mutants were isolated. Southern hybridization experiments demonstrated that 17 of these Tn5 mutants were localized in at least 13 different restriction fragments outside of known nif regions. Ten different Anf^? Tn5 mutations are clustered on a 6 kb DNA fragment of the chromosome designated anf region A. DNA sequence analysis revealed that this region contained the structural genes of the alternative nitrogenase (anfHDGK). The identification of several Tn5 insertions mapping outside of anf region A indicated that at least 10 genes specific for the alternative nitrogenase are present in R. capsulatus.     

A physical and genetic map of Neisseria meningitidis B1940

A physical map of the chromosome of Neisseria meningitidis B1940 has been constructed by one- and two-dimensional pulsed-field gel electrophoresis techniques. Complete macrorestriction maps for the enzymes Nhe I (16 sites), Sgf I (13 sites), Sfi I (11 sites) and I-Ceu I (4 sites), as well as a partial restriction map for the restriction enzyme Spe I (15 of c. 28 sites) could be established. Altogether, 59 restriction sites were mapped on a single circular chromosome of 2.3 Mbp. By restriction endonuclease digestion and Southern hybridization of cloned genetic markers, 39 genetic loci were assigned to this map. Comparison with the metabolic maps of Neisseria gonorrhoeae MS11-N198 and FA1090 revealed a high degree of conservation in the arrangement of gene loci among these two species, although four out of 24 genetic loci are located at different chromosomal positions, indicating several genomic rearrangements.     

In Situ Microdialysis for Monitoring of Extracellular Glutathione Levels in Normal, Ischemic and Post-Ischemic Skeletal Muscle

Microdialysis probes were inserted into the tibialis anterior muscle and into the femoral vein of anaesthetised Sprague-Dawley rats for monitoring of reduced (GSH) and oxidized (GSSG) extracellular glutathione. The dialysates were analysed using HPLC. The levels of GSH and GSSG were high immediately after implantation in the skeletal muscle and declined to steady state levels after 90 minutes into the same range as that found in the venous dialysate. Total ischemia was induced two hours after implantation of the dialysis probe after steady state levels had been reached. The extracellular levels of GSH increased during total ischemia and had doubled at the end of the ischemic period compared to preischemic values. During the following initial 30 minutes of reperfusion the levels increased further to four-fold the preischemic levels. The levels of GSSG also increased (100%) during the initial 30 minutes of reperfusion. The extracellular GSH levels remained elevated for 1 hour of reperfusion, but the GSSG levels returned to preischemic levels. The results indicate that intermittent hypoxia or anoxia in muscle tissue through hypoperfusion or ischemia decreases intracellular GSH stores by leakage, reducing the intracellular antioxidative capacity and increasing the risk for oxidative reperfusion injury upon final normalization of tissue blood supply.     

The leaf peroxisomal form (MFP IV) of multifunctional protein functioning in fatty-acid β-oxidation

We have purified for the first time from green leaves a multifunctional protein (MFP) involved in fatty acid -oxidation. The protein, designated MFP IV, was extracted from green leaves of three-week-old cucumber (Cucumis sativus L.) plants. Chromatography on cation exchanger, separation on hydroxylapatite, and fast-protein liquid chromatography on Phenylsuperose led to a more than 7000-fold purification and to the isolation of an apparently homogeneous 80-kDa monomeric protein. This protein is immunologically related to the glyoxysomal MFP II, as evidenced by immunodecoration with antiserum raised against MFP II. Comparison of molecular masses of all MFPs presently known revealed that the MFP prepared from green leaves (MFP IV) is distinct from MFP II (76.5 kDa) and MFP I (74 kDa) from dark-grown cotyledons. By including other properties in this comparison, we demonstrated that MFP IV can also be distinguished from the glyoxysomal MFP III (81 kDa) and the bacterially expressed MFP-a (80 kDa). Moreover, MFP IV is a constituent of leaf peroxisomes and contains the activities of 2-enoyl-CoA hydratase (EC 4.2.1.17),l-3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) and 3-hydroxyacyl-CoA epimerase.Abbreviation MFP multifunctional protein This work was supported by grants from the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie.     

Water channels in Chara corallina

Water relations parameters ofChara corallina inter-nodes weremeasured using the single cell pressure probe. The effect ofmercurials, which are recognized as non-specific water channelinhibitors, was examined. HgCl₂ concentrations greater than5 mmol m^–3 were found to inhibit hydraulic conductivity{Lp) close to 90%, whereas pCMPS was found to have no effecton Lp. The activation energy of water flow was increased significantlyfrom 21.0 kJ mol^–1 to 45.6 kJ mol^–1, following theapplication of HgCl₂. These results are in accordance with evidencefor Hg²⁺sensitive water channels in the plasma membrane of charophytes(Henzler and Steudle, 1995; Tazawa et al., 1996). The metaboliceffects must, however, be considered in view of the rapid inhibitionof respiration and the depolarization of the membrane potentialwith HgCl₂ concentrations lower than those found to affect Lp.It was possible to measure simultaneously water relations andmembrane PD, in order to examine the contribution of potassiumchannels to Lp. Cells were induced into a K⁺ permeable state.The K⁺ channels, assumed to be open, were subsequently blockedby various blockers. No significant difference in Lp was foundfor any of these treatments. Finally, the permeability of C.corallina membranes to ethanol was examined. HgCl₂ was foundto cause a decrease in reflection coefficient, coinciding witha decrease in Lp, but there was no change in the ethanol permeabilitycoefficient. This has been interpreted in terms of both thefrictional model and composite model of non-electrolyte membranetransport. Key words: Water channels, Chara, hydraulic, conductivity, membrane transport models, reflection coefficient