A universal expression/silencing vector in plants

A universal vector (IL-60 and auxiliary constructs), expressing or silencing genes in every plant tested to date, is described. Plants that have been successfully manipulated by the IL-60 system include hard-to-manipulate species such as wheat (Triticum duram), pepper (Capsicum annuum), grapevine (Vitis vinifera), citrus, and olive (Olea europaea). Expression or silencing develops within a few days in tomato (Solanum lycopersicum), wheat, and most herbaceous plants and in up to 3 weeks in woody trees. Expression, as tested in tomato, is durable and persists throughout the life span of the plant. The vector is, in fact, a disarmed form of Tomato yellow leaf curl virus, which is applied as a double-stranded DNA and replicates as such. However, the disarmed virus does not support rolling-circle replication, and therefore viral progeny single-stranded DNA is not produced. IL-60 does not integrate into the plant's genome, and the construct, including the expressed gene, is not heritable. IL-60 is not transmitted by the Tomato yellow leaf curl virus's natural insect vector. In addition, artificial satellites were constructed that require a helper virus for replication, movement, and expression. With IL-60 as the disarmed helper "virus," transactivation occurs, resulting in an inducible expressing/silencing system. The system's potential is demonstrated by IL-60-derived suppression of a viral-silencing suppressor of Grapevine virus A, resulting in Grapevine virus A-resistant/tolerant plants.     

Hepatic Mitogen-Activated Protein Kinase Phosphatase 1 Selectively Regulates Glucose Metabolism and Energy Homeostasis

The liver plays a critical role in glucose metabolism and communicates with peripheral tissues to maintain energy homeostasis. Obesity and insulin resistance are highly associated with nonalcoholic fatty liver disease (NAFLD). However, the precise molecular details of NAFLD remain incomplete. The p38 mitogen-activated protein kinase (MAPK) and c-Jun NH₂-terminal kinase (JNK) regulate liver metabolism. However, the physiological contribution of MAPK phosphatase 1 (MKP-1) as a nuclear antagonist of both p38 MAPK and JNK in the liver is unknown. Here we show that hepatic MKP-1 becomes overexpressed following high-fat feeding. Liver-specific deletion of MKP-1 enhances gluconeogenesis and causes hepatic insulin resistance in chow-fed mice while selectively conferring protection from hepatosteatosis upon high-fat feeding. Further, hepatic MKP-1 regulates both interleukin-6 (IL-6) and fibroblast growth factor 21 (FGF21). Mice lacking hepatic MKP-1 exhibit reduced circulating IL-6 and FGF21 levels that were associated with impaired skeletal muscle mitochondrial oxidation and susceptibility to diet-induced obesity. Hence, hepatic MKP-1 serves as a selective regulator of MAPK-dependent signals that contributes to the maintenance of glucose homeostasis and peripheral tissue energy balance. These results also demonstrate that hepatic MKP-1 overexpression in obesity is causally linked to the promotion of hepatosteatosis.     

Mapping of a new quantitative trait locus for resistance to malaria in mice by a comparative mapping approach with human Chromosome 5q31-q33

A number of linkage studies in human populations have identified a locus (pfbi) on Chromosome 5q31-q33 controlling Plasmodiun falciparum blood infection levels. This region contains numerous candidate genes encoding immunological molecules such as cytokines, growth factors and growth-factor receptors. We have used an F₁₁ advance intercross line (AIL) population of mice infected with Plasmodium chabaudi to identify additional mouse quantitative trait loci (QTL) for control of parasitaemia on Chrs 11 and 18, which carry regions homologous to human Chr 5q31-q33. Herein, we report a novel QTL for parasitaemia control (char8) on the mouse Chr 11, linked to marker D11Mit242, and involved in the clearance stages of the parasites from the bloodstream. Strikingly, several Th2 cytokines that are located within char8 have been identified to play a predominant role in the late stages of the infection.     

Consumption of NADPH for 2-HG Synthesis Increases Pentose Phosphate Pathway Flux and Sensitizes Cells to Oxidative Stress

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RAGE mRNA expression in the diabetic mouse kidney

Receptors for advanced glycation end products (RAGE), which bind and internalize AGE-modified proteins formed from oxidation and other products of the nonenzymatic glycation reaction, have been mechanistically implicated in the development of the chronic complications of diabetes. In the present experiments, we sought evidence for the participation of RAGE in diabetic nephropathy by analysis of steady state levels of mRNA encoding RAGE in the renal cortex of a well-defined animal model (the db/db mouse) that develops renal pathology similar to that found in human diabetes. In these animals, increased AGE-product formation was confirmed by measurement of fluorescence in serum and renal cortex proteins. Renal involvement was confirmed by demonstration of increased urine albumin excretion and elevated serum creatinine concentrations relative to nondiabetic (db/m) littermate controls. Despite elevated concentrations of circulating and tissue AGE-modified proteins, the level of RAGE mRNA expression in renal cortex of diabetic mice did not significantly differ from that in nondiabetic littermate controls. The findings militate against changes in RAGE expression in the pathogenesis of renal abnormalities in this animal model.     

Mapping liver fat female-dependent quantitative trait loci in collaborative cross mice

Non-alcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease in the western world, with spectrum from simple steatosis to non-alcoholic steatohepatitis, which can progress to cirrhosis. NAFLD developments are known to be affected by host genetic background. Herein we emphasize the power of collaborative cross (CC) mouse for dissecting this complex trait and revealing quantitative trait loci (QTL) controlling hepatic fat accumulation in mice. 168 female and 338 male mice from 24 and 37 CC lines, respectively, of 18–20 weeks old, maintained on standard rodent diet, since weaning. Hepatic fat content was assessed, using dual DEXA scan in the liver. Using the available high-density genotype markers of the CC line, QTL mapping associated with percentage liver fat accumulation was performed. Our results revealed significant fatty liver accumulation QTL that were specifically, mapped in females. Two significant QTLs on chromosomes 17 and 18, with genomic intervals 3 and 2 Mb, respectively, were mapped. A third QTL, with a less significant P value, was mapped to chromosome 4, with genomic interval of 2 Mb. These QTLs were named Flal1–Flal3, referring to Fatty Liver Accumulation Locus 1–3, for the QTLs on chromosomes 17, 18, and 4, respectively. Unfortunately, no QTL was mapped with males. Searching the mouse genome database suggested several candidate genes involved in hepatic fat accumulation. Our results show that susceptibility to hepatic fat accumulations is a complex trait, controlled by multiple genetic factors in female mice, but not in male.     

The reductive enzymatic cleavage of thiosulfate

Summary Methods presently used for the enzymatic assay of the thiosulfate cleaving reaction in bacterial cell-free systems are critically examined. Conditions under which strong acids are used to terminate the reaction and to release H₂S proved to be unsuitable. A non-enzymatic production of H₂S under such conditions is demonstrated. A reliable procedure for the measurement of H₂S production from the enzymatic cleavage of thiosulfate is described. This method was used to measure the thiosulfate cleaving reaction catalyzed by cell-free extracts of phototrophic bacteria of the genusChromatium. As reductants, hydrogen-hydrogenase/methyl viologen system, reduced glutathione or dihydrolipoate were used. The same extract fraction catalyzed the rhodanese reaction.     

MHD Mixed Convective Peristaltic Motion of Nanofluid with Joule Heating and Thermophoresis Effects

The primary objective of present investigation is to introduce the novel aspect of thermophoresis in the mixed convective peristaltic transport of viscous nanofluid. Viscous dissipation and Joule heating are also taken into account. Problem is modeled using the lubrication approach. Resulting system of equations is solved numerically. Effects of sundry parameters on the velocity, temperature, concentration of nanoparticles and heat and mass transfer rates at the wall are studied through graphs. It is noted that the concentration of nanoparticles near the boundaries is enhanced for larger thermophoresis parameter. However reverse situation is observed for an increase in the value of Brownian motion parameter. Further, the mass transfer rate at the wall significantly decreases when Brownian motion parameter is assigned higher values.     

Exposure of rainbow trout (Oncorhynchus mykiss) to magnetite (Fe3O4) nanoparticles in simplified food chain: Study on ultrastructural characterization

The widespread exposure of metallic nanoparticles to the aquatic ecosystem and its adverse impact on human life is the colossal concern worldwide. In view of this, this context was investigated to analyze microscopically the bioaccumulation and localization of magnetite (Fe₃O₄) nanoparticles in the cellular organelles of rainbow trout (Oncorhynchus mykiss, Walbaum, 1792) in aquatic conditions. Initially, Fe₃O₄ nanoparticles were absorbed on to Elodea (Elodea canadensis) and fed to molluscs (Melanopsis praemorsa). Fish were fed with the same molluscs, and then the intestines and liver were examined using light and transmission electron microscopy. Results showed that nanoparticles were present in the cytoplasm and other organelles of cells (mitochondrion and lysosome) by absorbing through microvilli of the epithelial cells of the tunica mucosa in the intestine. Further, nanoparticles passed through the vessels of the lamina propria of the tunica mucosa and reached to the sinusoids of the liver via blood circulation. It was then accumulated from the endothelium of the sinusoid to the cytoplasm of liver hepatocytes and to mitochondria and lysosome. The accumulation of nanoparticles in the epithelial cells, cytoplasm, mitochondria, and lysosome revealed the degree of transparency of the pattern with slight hesitation. In summary, this investigation contributed towards the understanding of the physiological effects of Fe₃O₄ nanoparticles on O. mykiss, which ascertains essentiality for sustainable development of nanobiotechnology in the aquatic ecosystem.     

Phosphoglycerate mutase from Trypanosoma brucei is hyperactivated by cobalt in vitro, but not in vivo

Production of ATP by the glycolytic pathway in the mammalian pathogenic stage of protists from the genus Trypanosoma is required for the survival of the parasites. Cofactor-independent phosphoglycerate mutase (iPGAM) is particularly attractive as a drug target because it shows no similarity to the corresponding enzyme in humans, and has also been genetically validated as a target by RNAi experiments. It has previously been shown that trypanosomatid iPGAMs require Co(2+) to reach maximal activity, but the biologically relevant metal has remained unclear. In this paper the metal content in the cytosol of procyclic and bloodstream-form T. brucei (analysed by inductively coupled plasma-optical emission spectroscopy) shows that Mg(2+), Zn(2+) and Fe(2+) were the most abundant, whereas Co(2+) was below the limit of detection (<0.035 μM). The low concentration indicates that Co(2+) is unlikely to be the biologically relevant metal, but that instead, Mg(2+) and/or Zn(2+) may assume this role. Results from metal analysis of purified Leishmania mexicana iPGAM by inductively coupled plasma-mass spectrometry also show high concentrations of Mg(2+) and Zn(2+), and are consistent with this proposal. Our data suggest that in vivo cellular conditions lacking Co(2+) are unable to support the maximal activity of iPGAM, but instead maintain its activity at a relatively low level by using Mg(2+) and/or Zn(2+). The physiological significance of these observations is being pursued by structural, biochemical and biophysical studies.