Mutational analysis identifies functional domains in the influenza A virus PB2 polymerase subunit.

A collection of influenza virus PB2 mutant genes was prepared, including N-terminal deletions, C-terminal deletions, and single-amino-acid insertions. These mutant genes, driven by a T7 promoter, were expressed by transfection into COS-1 cells infected with a vaccinia virus encoding T7 RNA polymerase. Mutant proteins accumulated to levels similar to that of wild-type PB2. Immunofluorescence analyses showed that the C-terminal region of the protein is essential for nuclear transport and that internal sequences affect nuclear localization, confirming previous results (J. Mukaijawa and D. P. Nayak, J. Virol. 65:245-253, 1991). The biological activity of these mutants was tested by determining their capacity to (i) reconstitute RNA polymerase activity in vivo by cotransfection with proteins NP, PB1, and PA and a virion-like RNA encoding the cat gene into vaccinia virus T7-infected COS-1 cells and (ii) complete with the wild-type PB2 activity. In addition, when tested at different temperatures in vivo, two mutant PB2 proteins showed a temperature-sensitive phenotype. The lack of interference shown by some N-terminal deletion mutants and the complete interference obtained with a C-terminal deletion mutant encoding only 124 amino acids indicated that this protein domain is responsible for interaction with another component of the polymerase, probably PB1. To further characterize the mutants, their ability to induce in vitro synthesis of viral cRNA or mRNA was tested by using ApG or beta-globin mRNA as a primer. One of the mutants, 1299, containing an isoleucine insertion at position 299, was able to induce cRNA and mRNA synthesis in ApG-primed reactions but required a higher beta-globin mRNA concentration than wild-type PB2 for detection of in vitro synthesis. This result suggested that mutant I299 has diminished cap-binding activity.     

Mathematical model for the ecosystem response of Lake Ladoga to phosphorus loading

The ecosystem response model described in this paper combines an ecosystem model and a three-dimensional circulation model of Lake Ladoga developed earlier by the authors. The ecosystem model describes the process of Lake Ladoga eutrophication, and its biological submodel describes changes in the phyto- and zooplankton. In the earlier model version, lake circulation was determined using a two-dimensional hydrodynamical model which was not completely adequate. The present model allows calculation of the distributions of phyto- and zooplankton and mineral phosphorus and nitrogen. One of its main advantages is that reliable computations of the ecosystem dynamics over an extended period of time are possible. The response of the ecosystem to different levels of phosphorus pollution loading and to weather conditions is studied.     

Vitamin A supplementation inhibits chemiluminescence and lipid peroxidation in isolated rat liver microsomes and mitochondria

In the present study we investigated if administration of vitamin A could protect rat liver microsomes and mitochondria from in vitro peroxidation. Appreciable decrease of chemiluminescence and lipid peroxidation was measured in microsomal membranes from rats receiving vitamin A, with respect to control animals. In membranes derived from control animals, the fatty acid composition was profoundly modified when subjected to in vitro peroxidation mediated by ascorbate-Fe⁺⁺, with a considerable decrease of 20:4 n6 and 22:6 n3 in mitochondria and 18:2 n6 and 20:4 n6 in microsomes. As a consequence the peroxidizability index, a parameter based on the maximal rate of oxidation of specific fatty acids was higher in supplemented animals than in control group when both kind of membranes were analyzed. These changes were less pronounced in membranes derived from rats receiving vitamin A. These results are in agreement with previous results that indicated that vitamin A may act as an antioxidant protecting membranes from deleterious effects.Abbreviations BHT butylated hydroxytoluene - BSA bovine serum albumin - CL chemiluminescence - PI peroxidizability index Member of Carrera del Investigador Científico, Consejo Nacional de Investigaciones Cientificas y Técnicas de la Republica Argentina     

Increase in heart glutathione redox ratio and total antioxidant capacity and decrease in lipid peroxidation after vitamin e dietary supplementation in guinea pigs

Dietary treatment with three diets differing in vitamin E, Low E (15 mg of vitamin E/kg diet), Medium E (150 mg/kg), or High E (1,500 mg/kg), resulted in guinea pigs with low (but nondeficient), intermediate, or high heart a-tocopherol concentration. Neither the antioxidant enzymes superoxide dismutase, catalase, glutathione peroxidase, and reductase, nor the nonenzymatic antioxidants, GSH, ascorbate, and uric acid were homeostatically depressed by increases in heart a-tocopherol. Protection from both enzymatic (NADPH dependent) and nonenzymatic (ascorbate-Fe²⁺) lipid peroxidation was strongly increased by vitamin E supplementation from Low to Medium E Whereas no additional gain was obtained from the Medium E to the High E group. The GSH/GSSG and GSH/total glutathione ratios increased as a function of the vitamin E dietary concentration closely resembling the shape of the dependence of heart a-tocopherol on dietary vitamin E. The results show the capacity of dietary vitamin E to increase the global antioxidant capacity of the heart and to improve the heart redox status in both the lipid and water-soluble compartments. This capacity occurred at levels six times higher than the minimum daily requirement of vitamin E, even in the presence of optimum dietary vitamin C concentrations and basal unstressed conditions. The need for vitamin E dietary supplementation seems specially important in this tissue due to the low constitutive levels of endogenous enzymatic and nonenzymatic antioxidants present of the mammalian heart in comparison with those of other internal organs.     

Flocculation of industrial and laboratory strains ofSaccharomyces cerevisiae

Summary A comparative study has been made of different laboratory and industrial wild-type strains ofSaccharomyces cerevisiae in relation to their flocculation behavior. All strains were inhibited by mannose and only one by maltose. In regard to the stability of these characters in the presence of proteases and high salt concentrations, a relevant degree of variation was found among the strains. This was to such an extent that it did not allow their inclusion in the Flol or NewFlo phenotypes. Genetic characterization of one wild-type strain revealed that the flocculation-governing gene was allelic toFLO1 found in genetic strains.This paper is dedicated to Professor Herman Jan Phaff in honor of his 50 years of active research which still continues.     

Hydrodynamic characteristics and equilibrium rigidity of pullulan molecules

The hydrodynamic characteristics of the polysaccharide pullulan (polymaltotriose) in water have been investigated and its molecular characteristics have been determined. Experimental values varied over the following ranges: velocity sedimentation coefficient (S): 0.9 < S < 11.2, translational diffusion coefficient (10⁷ cm² s⁻¹): 1.1 < D < 14.7 and intrinsic viscosity (cm³ g⁻¹): 6.7 < [η] < 164, which corresponds to a change in molecular weight (× 10³) in the range 3.9 < M_SD < 644. On the basis of analysis of the literature and our experimental data, excluded volume effects have been shown to have a prevailing influence on the chain length of these polysaccharides. The equilibrium rigidity and hydrodynamic chain diameter of pullulan were evaluated on the basis of the theory of hydrodynamic properties of a wormlike necklace, taking into account excluded volume effects. At low M (< 30 × 10³) the translation friction data (in contrast to viscometric data) cannot be described in the framework of the theory of linear molecules.     

Evidence for the Involvement of Carnitine-Dependent Long-Chain Acyltransferases in Neuronal Triglyceride and Phospholipid Fatty Acid Turnover

Abstract: This study focuses on the potential involvement of carnitine palmitoyltransferase (CRT) on the phospholipid and triglyceride fatty acid turnover in neurons. This category of enzymes, which has been identified in several rat brain tissues, is well known for its role in modulating cellular fatty acid oxidation. Neuronal cell cultures from rat brain cortex incorporated radioactive palmitate or oleate into phospholipids and triglycerides. The largest fraction of radioactive fatty acids was recovered in phosphatidyl- choline followed by triglycerides and, to a lesser extent, phosphatidylethanolamine. CPT activity measured in neuronal lysates obtained from neurons treated with 40 μ M 2-tetradecylglycidic acid (TDGA) was almost completely abolished. Furthermore, between 2 and 10 μ M TDGA CPT activity dropped more rapidly than between 10 and 40 μ M. When the cells were pretreated with TDGA, the incorporation process of either radioactive fatty acid into triglycerides was dose-dependently suppressed. Radioactive fatty acid incorporation into phosphatidylcholine was significantly decreased in cells treated with TDGA. In contrast, phosphatidylethanolamine reacylation was essentially not affected by the CpT inhibitor. Similar results on the fatty acid incorporation into triglycerides and phospholipids were observed with neurons treated with palmitoyl- dl - aminocarnitine (PAC), a reversible CPT inhibitor, which does not consume free CoA. These effects do not seem to be the result of an inhibitory activity toward one of the steps involved in the acylation-deacylation process of triglycerides or phospholipids, as cellular lysates from TDGA-treated cells or lysates containing PAC incorporated radioactive fatty acids at rates comparable to controls. Our results suggest that CRT may be an important partner in the pathway of phospholipid and triglyceride fatty acid turnover in neurons.     

An approximate solution to the periodic bidomain equations in one dimension

An approximate, computationally tractable solution is proposed for the potentials in the bidomain model with periodic intracellular junctions (the periodic bidomain model). This new approach is based on the one-dimensional rigorous spectral method described previously by Trayanova and Pilkington (IEEE Trans. Biomed. Eng., May 1993). The total solution to the one-dimensional periodic bidomain problem is decomposed in the spectral domain into solutions to (1) the single-fiber classical bidomain problem in which the intracellular conductivity value incorporates the average contribution from cytoplasm and junction and (2) the “junctional” potential problem due to the presence of junctions at discrete locations alone. Solving for the junctional term rigorously requires most of the numerical effort in the solution for the periodic bidomain potentials. Here the junctional potential is found approximately with little numerical effort. A comparison between the rigorous and the approximate solutions serves as a justification for the proposed approximate solution procedure. The procedure outlined in this paper is applicable to higher spatial dimensions where both tissue anisotropy and junctional inhomogeneities play a role in establishing the transmembrane potential distribution.     

Studies on the striatal dopamine uptake system of weaver mutant mice and effects of ventral mesencephalic grafts

The dopamine (DA) uptake system was investigated in the mesostriatal system of normal and weaver mutant mice, which lose mesencephalic DA neurons, as well as in weaver mutants with ventral mesencephalic grafts to the striatum. Assays of [³H]DA uptake in striatal synaptosomal fractions in vitro and autoradiography of [³H]mazindol binding in brain sections were carried out in wild-type mice (+/+) and in the two hemispheres of homozygous weaver mutants (wv/wv) that had received unilateral grafts of mesencephalic cell suspensions to the right side. Net [³H]DA uptake, expressed as pmol/mg-protein/2-min, was on the average 50.6 in the striatum of wild-type mice, 7.9 in the non-grafted, and 10.1 in the transplanted striatum of weaver mutants. [³]DA uptake in wild-type mice differed significantly from both the grafted and non-grafted weaver striata (P<0.001). Paired comparisons for [³H]DA uptake between right and left sides of recipient weaver mice showed a significant side effect (P<0.02), the right side being 28–38% higher than the left side [mean of all individual (R-L)/L values]. The results of amphetamine-induced turning behavior tests were compared with the biochemical findings. Mice with grafts to the right side rotated an average of 22 turns to the left and 7 turns to the right during the five one-minute sessions; the mean value L/(L+R) was 64%. A plot of (L-R) rotations against (R-L) [³H]DA uptake gave a correlation coefficient of 0.552 (P<0.05), indicating that animals with a strong rotational bias to the left tended to have higher [³H]DA on the right. Similarly, the animals that were used for [³H]mazindol binding autoradiographic studies displayed on the average 72% rotations to the left side. In the [³H]mazindol binding data, non-grafted weaver mutants showed the severest depletion relative to wild-type in the dorsomedial and dorsolateral caudate-putamen (86% and 87%, respectively). Mice with unilateral grafts to the right side showed an increase in [³H]mazindol binding signal in the transplanted side of 40–64% (depending on dorsoventral topography) over the contralateral, non-grafted side. These findings attest to the functional effects of the grafts at the anatomical, biochemical, and behavioral levels. The parallel measurements of motor performance and DA uptake in the same animals offers an index of behavioral recovery as a function of transmitter-related activity. Furthermore, by conducting measurements of the synaptosomal DA uptake in vitro and of the binding characteristics of mazindol in brain slices by autoradiography, one has the advantage of combining the anatomical resolution of uptake site visualization with a dynamic indicator of function for DA uptake in the nerve terminal.Special issue dedicated to Professor Sidney Ochs     

Characterization of the effectors required for stable inheritance of Streptococcus pyogenes pSM19035-derived plasmids in Bacillus subtilis

The low-copy-number and broad-host-range pSM19035-derived plasmid pBT233 is stably inherited in Bacillus subtilis cells. Two distinct regions, segA and segB, enhance the segregational stability of the plasmid. Both regions function in a replicon-independent manner. The maximization of random plasmid segregation is accomplished by the recombination proficiency of the host or the presence of the pBT233 segA region. The segA region contains two open reading frames (or) [ and ]. Inactivation or deletion of or results in SegA^– plasmids. Better than random segregation requires an active segB region. The segB region contains two ors (or and or). Inactivation of either of the orfs does not lead to an increase in cell death, but or^– plasmids are randomly segregated. These results suggest that pBT233 stabilization relies on a complex system involving resolution of plasmid oligomers (segA) and on the function(s) encoded by the segB region.