Enzymatic degradation of β(1 → 4) xylan single crystals

The enzymatic degradation of β(1 → 4) xylan single crystals with xylanases was investigated by electron microscopy and electron diffraction. The enzyme attack takes place at the edge of the crystals and progresses towards their centers. This is consistent with an endo-enzyme mechanism, where the enzyme interacts essentially with the accessible xylan chains located at the crystal periphery.     

Leptin, skeletal muscle lipids, and lipid-induced insulin resistance

Leptin-induced increases in insulin sensitivity are well established and may be related to the effects of leptin on lipid metabolism. However, the effects of leptin on the levels of lipid metabolites implicated in pathogenesis of insulin resistance and the effects of leptin on lipid-induced insulin resistance are unknown. The current study addressed in rats the effects of hyperleptinemia (HL) on insulin action and markers of skeletal muscle (SkM) lipid metabolism in the absence or presence of acute hyperlipidemia induced by an infusion of a lipid emulsion. Compared with controls (CONT), HL increased insulin sensitivity, as assessed by hyperinsulinemic-euglycemic clamp ( approximately 15%), and increased SkM Akt ( approximately 30%) and glycogen synthase kinase 3 alpha ( approximately 52%) phosphorylation. These improvements in insulin action were associated with decreased SkM triglycerides (TG; approximately 61%), elevated ceramides ( approximately 50%), and similar diacylglycerol (DAG) levels in HL compared with CONT. Acute hyperlipidemia in CONT decreased insulin sensitivity ( approximately 25%) and increased SkM DAG ( approximately 33%) and ceramide ( approximately 60%) levels. However, hyperlipidemia did not induce insulin resistance or SkM DAG and ceramide accumulation in HL. SkM total fatty acid transporter CD36, plasma membrane fatty acid binding protein, acetyl Co-A carboxylase phosphorylation, and fatty acid oxidation were similar in HL compared with CONT. However, HL decreased SkM protein kinase C theta (PKC theta), a kinase implicated in mediating the detrimental effects of lipids on insulin action. We conclude that increases in insulin sensitivity induced by HL are associated with decreased levels of SkM TG and PKC theta and increased SkM insulin signaling, but not with decreases in other lipid metabolites implicated in altering SkM insulin sensitivity (DAG and ceramide). Furthermore, insulin resistance induced by an acute lipid infusion is prevented by HL.     

Molecular and immunohistochemical analyses of cardiac troponin T during cardiac development in the Mexican axolotl, Ambystoma mexicanum

The Mexican axolotl, Ambystoma mexicanum, is an excellent animal model for studying heart development because it carries a naturally occurring recessive genetic mutation, designated gene c, for cardiac nonfunction. The double recessive mutants (c/c) fail to form organized myofibrils in the cardiac myoblasts resulting in hearts that fail to beat. Tropomyosin expression patterns have been studied in detail and show dramatically decreased expression in the hearts of homozygous mutant embryos. Because of the direct interaction between tropomyosin and troponin T (TnT), and the crucial functions of TnT in the regulation of striated muscle contraction, we have expanded our studies on this animal model to characterize the expression of the TnT gene in cardiac muscle throughout normal axolotl development as well as in mutant axolotls. In addition, we have succeeded in cloning the full-length cardiac troponin T (cTnT) cDNA from axolotl hearts. Confocal microscopy has shown a substantial, but reduced, expression of TnT protein in the mutant hearts when compared to normal during embryonic development.     

Composition and three-dimensional EM structure of double affinity-purified, human prespliceosomal A complexes

Little is known about the higher-order structure of prespliceosomal A complexes, in which pairing of the pre-mRNA's splice sites occurs. Here, human A complexes were isolated under physiological conditions by double-affinity selection. Purified complexes contained stoichiometric amounts of U1, U2 and pre-mRNA, and crosslinking studies indicated that these form concomitant base pairing interactions with one another. A complexes contained nearly all U1 and U2 proteins plus approximately 50 non-snRNP proteins. Unexpectedly, proteins of the hPrp19/CDC5 complex were also detected, even when A complexes were formed in the absence of U4/U6 snRNPs, demonstrating that they associate independent of the tri-snRNP. Double-affinity purification yielded structurally homogeneous A complexes as evidenced by electron microscopy, and allowed for the first time the generation of a three-dimensional structure. A complexes possess an asymmetric shape (approximately 260 x 200 x 195 angstroms) and contain a main body with various protruding elements, including a head-like domain and foot-like protrusions. Complexes isolated here are well suited for in vitro assembly studies to determine factor requirements for the A to B complex transition.     

Ectopic expression and dynamics of TPM1alpha and TPM1kappa in myofibrils of avian myotubes

From the four known vertebrate tropomyosin genes (designated TPM1, TPM2, TPM3, and TPM4) over 20 isoforms can be generated. The predominant TPM1 isoform, TPM1alpha, is specifically expressed in both skeletal and cardiac muscles. A newly discovered alternatively spliced isoform, TPM1kappa, containing exon 2a instead of exon 2b contained in TPM1alpha, was found to be cardiac specific and developmentally regulated. In this work, we transfected quail skeletal muscle cells with green fluorescent proteins (GFP) coupled to chicken TPM1alpha and chicken TPM1kappa and compared their localizations in premyofibrils and mature myofibrils. We used the technique of fluorescence recovery after photobleaching (FRAP) to compare the dynamics of TPM1alpha and TPM1kappa in myotubes. TPM1alpha and TPM1kappa incorporated into premyofibrils, nascent myofibrils, and mature myofibrils of quail myotubes in identical patterns. The two tropomyosin isoforms have a higher exchange rate in premyofibrils than in mature myofibrils. F-actin and muscle tropomyosin are present in the same fibers at all three stages of myofibrillogenesis (premyofibrils, nascent myofibrils, mature myofibrils). In contrast, the tropomyosin-binding molecule nebulin is not present in the initial premyofibrils. Nebulin is gradually added during myofibrillogenesis, becoming fully localized in striated patterns by the mature myofibril stage. A model of thin filament formation is proposed to explain the increased stability of tropomyosin in mature myofibrils. These experiments are supportive of a maturing thin filament and stepwise model of myofibrillogenesis (premyofibrils to nascent myofibrils to mature myofibrils), and are inconsistent with models that postulate the immediate appearance of fully formed thin filaments or myofibrils.     

Microencapsulation of Zanthoxylum limonella oil (ZLO) in glutaraldehyde crosslinked gelatin for mosquito repellent application

Glutaraldehyde (GA) crosslinked gelatin (G) microcapsules containing Zanthoxylum limonella oil (ZLO) were prepared by coacervation technique. The effect of various parameters such as variation of oil-loading, gelatin concentration and degree of crosslinking on release rate of oil were studied. Scanning electron microscopy (SEM) was used to understand the surface characteristics of microcapsules. FTIR-results indicated the absence of any significant interaction between polymer and oil.     

Single nucleotide polymorphisms in the interferon gamma gene are associated with distinct types of retinochoroidal scar lesions presumably caused by Toxoplasma gondii infection

The association of single nucleotide polymorphisms (SNPs) in the interferon (IFN)-γ gene ( IFNG ) with different types of retinal scar lesions presumably caused by toxoplasmosis were investigated in a cross-sectional population-based genetic study. Ten SNPs were investigated and after Bonferroni correction, only the associations between SNPs rs2069718 and rs3181035 with retinal/retinochoroidal scar lesions type A (most severe scar lesions) and C (least severe scar lesions), respectively, remained significant. The associations of two different IFNG SNPs with two different types of retinal lesions attributable to toxoplasmosis support the hypothesis that different inflammatory mechanisms underlie the development of these lesions. The in vitro analysis of IFN-γ secretion by peripheral blood mononuclear cells stimulated with Toxoplasma gondii antigens was also investigated. The association between SNP rs2069718 and type A scar lesions revealed that differential IFN-γ levels are correlated with distinct genotypes. However, no correlation was observed with IFN-γ secretion levels and the SNP rs3181035 , which was significantly associated with type C scar lesions. Our findings strongly suggest that immunogenetic studies of individuals with congenital or postnatally acquired infection are needed to better understand the role of IFN-γ and its polymorphisms in the pathogenesis of ocular toxoplasmosis.