Mutual repression between msh and Iro-C is an essential component of the boundary between body wall and wing in Drosophila

During development, the imaginal wing disc of Drosophila is subdivided into territories separated by developmental boundaries. The best characterized boundaries delimit compartments defined by cell-lineage restrictions. Here, we analyze the formation of a boundary that does not rely on such restrictions, namely, that which separates the notum (body wall) and the wing hinge (appendage). It is known that the homeobox genes of the Iroquois complex (Iro-C) define the notum territory and that the distal limit of the Iro-C expression domain demarks the boundary between the notum and the wing hinge. However, it is unclear how this boundary is established and maintained. We now find that msh, a homeobox gene of the Msx family, is strongly expressed in the territory of the hinge contiguous to the Iro-C domain. Loss- and gain-of-function analyses show that msh maintains Iro-C repressed in the hinge, while Iro-C prevents high level expression of msh in the notum. Thus, a mutual repression between msh and Iro-C is essential to set the limit between the contiguous domains of expression of these genes and therefore to establish and/or maintain the boundary between body wall and wing. In addition, we find that msh is necessary for proper growth of the hinge territory and the differentiation of hinge structures. msh also participates in the patterning of the notum, where it is expressed at low levels.     

Synthesis of novel 5-aryl-2-thio-1,3,4-oxadiazoles and the study of their structure-anti-mycobacterial activities

The preparation of novel 5-aryl-2-thio-1,3,4-oxadiazoles 4a-41 and the computer-aided study of their in vitro anti-tubercular activity against Mycobacterium tuberculosis H37Rv (ATCC 27294) are reported. The average accuracy of the electronic-topological method and neural network methods applied to the activity prediction in leave-one-out cross validation is 80%.     

Possible mechanisms underlying copper-induced damage in biological membranes leading to cellular toxicity

It is generally accepted that copper toxicity is a consequence of the generation of reactive oxygen species (ROS) by copper ions via Fenton or Haber-Weiss reactions. Copper ions display high affinity for thiol and amino groups occurring in proteins. Thus, specialized proteins containing clusters of these groups transport and store copper ions, hampering their potential toxicity. This mechanism, however, may be overwhelmed under copper overloading conditions, in which copper ions may bind to thiol groups occurring in proteins non-related to copper metabolism. In this study, we propose that indiscriminate copper binding may lead to damaging consequences to protein structure, modifying their biological functions. Therefore, we treated liver subcellular membrane fractions, including microsomes, with Cu²⁺ ions either alone or in the presence of ascorbate (Cu²⁺/ascorbate); we then assayed both copper-binding to membranes, and microsomal cytochrome P450 oxidative system and GSH-transferase activities. All assayed sub-cellular membrane fractions treated with Cu²⁺ alone displayed Cu²⁺-binding, which was significantly increased in the presence of Zn²⁺, Hg²⁺, Cd²⁺, Ag⁺¹ and As³⁺. Treatment of microsomes with Cu²⁺ in the μM range decreased the microsomal thiol content; in the presence of ascorbate, Cu²⁺ added in the nM concentrations range induced a significant microsomal lipoperoxidation; noteworthy, increasing Cu²⁺ concentration to ≥50 μM led to non-detectable lipoperoxidation levels. On the other hand, μM Cu²⁺ led to the inhibition of the enzymatic activities tested to the same extent in either presence or absence of ascorbate. We discuss the possible significance of indiscriminate copper binding to thiol proteins as a possible mechanism underlying copper-induced toxicity.     

A CAPS marker set for mapping in linkage group III of pea (Pisum sativum L.)

A set of twelve CAPS markers was mapped for linkage group III of pea (Pisum sativum L.). New primers were designed to use a polymerase chain reaction to amplify fragments of sequenced pea genes containing at least one large intron. Amplification products were tested for polymorphism across three pea lines (Chi115, Flagman and WL1238) using eleven four-base restriction endonucleases. Nine STS markers for linkage group III from the literature were also tested for polymorphism, and five of these were used in this mapping study as anchor points. All polymorphic loci were located by genetic analysis of the F(2)population from the cross Chi115 x WL1238, and a map of linkage group III consisting of one morphological and twelve CAPS markers was created. The map covers the full length of the chromosome and is about 162 cM long. All the CAPS markers in a set were used to test for polymorphism among 10 additional pea DNA samples extracted from different marker lines and cultivars.     

A model of the ternary complex of interleukin-10 with its soluble receptors

Background  

Interleukin-10 (IL-10) is a cytokine whose main biological function is to suppress the immune response by induction of a signal(s) leading to inhibition of synthesis of a number of cytokines and their cellular receptors. Signal transduction is initiated upon formation of a ternary complex of IL-10 with two of its receptor chains, IL-10R1 and IL-10R2, expressed on the cell membrane. The affinity of IL-10R1 toward IL-10 is very high, which allowed determination of the crystal structure of IL-10 complexed with the extracellular/soluble domain of IL-10R1, while the affinity of IL-10R2 toward either IL-10 or IL-10/sIL-10R1 complex is quite low. This so far has prevented any attempts to obtain structural information about the ternary complex of IL-10 with its receptor chains.     

Multilayers of a globular protein and a weak polyacid: role of polyacid ionization in growth and decomposition in salt solutions

Thin films obtained from a layer-by-layer deposition of a weak polycarboxylic acid and a positively charged globular protein were studied by in situ ATR-FTIR. The system was chicken egg lysozyme (Lys), bovine pancrease ribonuclease A (RNase), or bovine gamma-globulin (IgG) self-assembled with polycarboxylic acids. When the pH value was lowered below a critical point, the growth of films and their tolerance to decomposition by added sodium chloride improved dramatically. Stabilization of protein/polyacid films in salt solutions at lower pH values occurred due to the onset of nonelectrostatic interactions to intermolecular binding within protein/polyacid multilayers and was controlled by polyacid ionization within the film rather than the pH of the external solution. A fractional ionization of polyacid in the pH-stabilization region was lower with protein-containing films than for polyacid/linear polycation films, reflecting hindrance of the inter-association of protonated carboxylic groups by protein globules. Practical ramifications of the pH-stabilization effect might extend to areas of biotechnology and biomaterials.     

Genetic variation in the HSD17B1 gene and risk of prostate cancer

Steroid hormones are believed to play an important role in prostate carcinogenesis, but epidemiological evidence linking prostate cancer and steroid hormone genes has been inconclusive, in part due to small sample sizes or incomplete characterization of genetic variation at the locus of interest. Here we report on the results of a comprehensive study of the association between HSD17B1 and prostate cancer by the Breast and Prostate Cancer Cohort Consortium, a large collaborative study. HSD17B1 encodes 17β-hydroxysteroid dehydrogenase 1, an enzyme that converts dihydroepiandrosterone to the testosterone precursor Δ5-androsterone-3β,17β-diol and converts estrone to estradiol. The Breast and Prostate Cancer Cohort Consortium researchers systematically characterized variation in HSD17B1 by targeted resequencing and dense genotyping; selected haplotype-tagging single nucleotide polymorphisms (htSNPs) that efficiently predict common variants in U.S. and European whites, Latinos, Japanese Americans, and Native Hawaiians; and genotyped these htSNPs in 8,290 prostate cancer cases and 9,367 study-, age-, and ethnicity-matched controls. We found no evidence that HSD17B1 htSNPs (including the nonsynonymous coding SNP S312G) or htSNP haplotypes were associated with risk of prostate cancer or tumor stage in the pooled multiethnic sample or in U.S. and European whites. Analyses stratified by age, body mass index, and family history of disease found no subgroup-specific associations between these HSD17B1 htSNPs and prostate cancer. We found significant evidence of heterogeneity in associations between HSD17B1 haplotypes and prostate cancer across ethnicity: one haplotype had a significant (p < 0.002) inverse association with risk of prostate cancer in Latinos and Japanese Americans but showed no evidence of association in African Americans, Native Hawaiians, or whites. However, the smaller numbers of Latinos and Japanese Americans in this study makes these subgroup analyses less reliable. These results suggest that the germline variants in HSD17B1 characterized by these htSNPs do not substantially influence the risk of prostate cancer in U.S. and European whites.     

New class of quaternary ammonium salts, derivatives of methyl D-glucopyranosides

Reactions of two aromatic and two aliphatic amines with methyl 6-O-p-toluenesulfonyl-alpha-D-glucopyranoside or methyl 6-O-p-toluenesulfonyl-beta-D-glucopyranoside were performed on a micro-scale. The synthesis and preparative isolation methods have been developed for quaternary N-(methyl 2,3,4-tri-O-acetyl-6-deoxy-alpha- and -beta-D-glucopyranoside-6-yl)ammonium salts derived from three amines: trimethylamine, 2-methylpyridine, and pyridine. The reaction products were examined with 1H, 13C NMR spectroscopy. N-(Methyl 2,3,4-tri-O-acetyl-6-deoxy-beta-d-glucopyranoside-6-yl)trimethylammonium tosylate was additionally analyzed with X-ray crystallography.     

GLUT4 overexpression or deficiency in adipocytes of transgenic mice alters the composition of GLUT4 vesicles and the subcellular localization of GLUT4 and insulin-responsive aminopeptidase

The majority of GLUT4 is sequestered in unique intracellular vesicles in the absence of insulin. Upon insulin stimulation GLUT4 vesicles translocate to, and fuse with, the plasma membrane. To determine the effect of GLUT4 content on the distribution and subcellular trafficking of GLUT4 and other vesicle proteins, adipocytes of adipose-specific, GLUT4-deficient (aP2-GLUT4-/-) mice and adipose-specific, GLUT4-overexpressing (aP2-GLUT4-Tg) mice were studied. GLUT4 amount was reduced by 80-95% in aP2-GLUT4-/- adipocytes and increased approximately 10-fold in aP2-GLUT4-Tg adipocytes compared with controls. Insulin-responsive aminopeptidase (IRAP) protein amount was decreased 35% in aP2-GLUT4-/- adipocytes and increased 45% in aP2-GLUT4-Tg adipocytes. VAMP2 protein was also decreased by 60% in aP2-GLUT4-/- adipocytes and increased 2-fold in aP2-GLUT4-Tg adipocytes. IRAP and VAMP2 mRNA levels were unaffected in aP2-GLUT4-Tg, suggesting that overexpression of GLUT4 affects IRAP and VAMP2 protein stability. The amount and subcellular distribution of syntaxin4, SNAP23, Munc-18c, and GLUT1 were unchanged in either aP2-GLUT4-/- or aP2-GLUT4-Tg adipocytes, but transferrin receptor was partially redistributed to the plasma membrane in aP2-GLUT4-Tg adipocytes. Immunogold electron microscopy revealed that overexpression of GLUT4 in adipocytes increased the number of GLUT4 molecules per vesicle nearly 2-fold and the number of GLUT4 and IRAP-containing vesicles per cell 3-fold. In addition, the proportion of cellular GLUT4 and IRAP at the plasma membrane in unstimulated aP2-GLUT4-Tg adipocytes was increased 4- and 2-fold, respectively, suggesting that sequestration of GLUT4 and IRAP is saturable. Our results show that GLUT4 overexpression or deficiency affects the amount of other GLUT4-vesicle proteins including IRAP and VAMP2 and that GLUT4 sequestration is saturable.     

Influenza hemagglutinins outside of the contact zone are necessary for fusion pore expansion

Current models for membrane fusion in diverse biological processes are focused on the local action of fusion proteins present in the contact zone where the proteins anchored in one membrane might interact directly with the other membrane. Are the fusion proteins outside of the contact zone just bystanders? Here we assess the role of these "outsider" proteins in influenza virus hemagglutinin-mediated fusion between red blood cells and either hemagglutinin-expressing cells or viral particles. To selectively inhibit or enhance the actions of hemagglutinin outsiders, the antibodies that bind to hemagglutinin and proteases that cleave it were conjugated to polystyrene microspheres too large to enter the contact zone. We also involved hemagglutinin outsiders into interactions with additional red blood cells. We find the hemagglutinin outsiders to be necessary and sufficient for fusion. Interfering with the activity of the hemagglutinin outsiders inhibited fusion. Selective conversion of hemagglutinin outsiders alone into fusion-competent conformation was sufficient to achieve fusion. The discovered functional role of fusion proteins located outside of the contact zone suggests a tempting analogy to mechanisms by which proteins mediate membrane fission from outside of the fission site.