Zygotic Wnt/beta-catenin signaling preferentially regulates the expression of Myf5 gene in the mesoderm of Xenopus

Zygotic Wnt signaling has been shown to be involved in dorsoventral mesodermal patterning in Xenopus embryos, but how it regulates different myogenic gene expression in the lateral mesodermal domains is not clear. Here, we use transient exposure of embryos or explants to lithium, which mimics Wnt/beta-catenin signaling, as a tool to regulate the activation of this pathway at different times and places during early development. We show that activation of Wnt/beta-catenin signaling at the early gastrula stage rapidly induces ectopic expression of XMyf5 in both the dorsal and ventral mesoderm. In situ hybridization analysis reveals that the induction of ectopic XMyf5 expression in the dorsal mesoderm occurs within 45 min and is not blocked by the protein synthesis inhibitor cycloheximide. By contrast, the induction of XMyoD is observed after 2 h of lithium treatment and the normal expression pattern of XMyoD is blocked by cycloheximide. Analysis by RT-PCR of ectodermal explants isolated soon after midblastula transition indicates that lithium also specifically induces XMyf5 expression, which takes place 30 min following lithium treatment and is not blocked by cycloheximide, arguing strongly for an immediate-early response. In the early gastrula, inhibition of Wnt/beta-catenin signaling blocks the expression of XMyf5 and XMyoD, but not of Xbra. We further show that zygotic Wnt/beta-catenin signaling interacts specifically with bFGF and eFGF to promote XMyf5 expression in ectodermal cells. These results suggest that Wnt/beta-catenin pathway is required for regulating myogenic gene expression in the presumptive mesoderm. In particular, it may directly activate the expression of the XMyf5 gene in the muscle precursor cells.     

X-ray structure and ligand binding study of a moth chemosensory protein

Chemosensory proteins (CSPs) are believed to be involved in chemical communication and perception. Such proteins, of M(r) 13,000, have been isolated from several sensory organs of a wide range of insect species. Several CSPs have been identified in the antennae and proboscis of the moth Mamestra brassicae. One of them, CSPMbraA6, a 112-amino acid antennal protein, has been expressed in large quantities and is soluble in the Escherichia coli periplasm. X-ray structure determination has been performed in parallel with ligand binding assays using tryptophan fluorescence quenching. The protein has overall dimensions of 25 x 30 x 32 A and exhibits a novel type of alpha-helical fold with six helices connected by alpha-alpha loops. A narrow channel extends within the protein hydrophobic core. Fluorescence quenching with brominated alkyl alcohols or fatty acids and modeling studies indicates that CSPMbraA6 is able to bind such compounds with C12-18 alkyl chains. These ubiquitous proteins might have the role of extracting hydrophobic linear compounds (pheromones, odors, or fatty acids) dispersed in the phospholipid membrane and transporting them to their receptor.     

Design of peptide-based inhibitors of human islet amyloid polypeptide fibrillogenesis

Human islet amyloid polypeptide (IAPP) is the major component of amyloid deposits found in the pancreas of over 90% of all cases of type-2 diabetes. We have generated a series of overlapping hexapeptides to target an amyloidogenic region of IAPP (residues 20-29) and examined their effects on fibril assembly. Peptide fragments corresponding to SNNFGA (residues 20-25) and GAILSST (residues 24-29) were strong inhibitors of the beta-sheet transition and amyloid aggregation. Circular dichroism indicated that even at 1:1 molar ratios, these peptides maintained full-length IAPP (1-37) in a largely random coil conformation. Negative stain electron microscopy revealed that co-incubation of these peptides with IAPP resulted in the formation of only semi-fibrous aggregates and loss of the typical high density and morphology of IAPP fibrils. This inhibitory activity, particularly for the SNNFGA sequence, also correlated with a reduction in IAPP-induced cytotoxicity as determined by cell culture studies. In contrast, the peptide NFGAIL (residues 22-27) enhanced IAPP fibril formation. Conversion to the amyloidogenic beta-sheet was immediate and the accompanying fibrils were more dense and complex than IAPP alone. The remaining peptide fragments either had no detectable effects or were only weakly inhibitory. Specificity of peptide activity was illustrated by the fragments, SSNNFG and AILSST. These differed from the most active inhibitors by only a single amino acid residue but delayed the random-to-beta conformational change only when used at higher molar ratios. This study has identified internal IAPP peptide fragments which can regulate fibrillogenesis and may be of therapeutic use for the treatment of type-2 diabetes.     

Activity of Sinorhizobium meliloti NodAB and NodH enzymes on thiochitooligosaccharides

Rhizobium bacteria synthesize signal molecules called Nod factors that elicit responses in the legume root during nodulation. Nod factors, modified N-acylated beta-(1,4)-N-acetylglucosamine, are synthesized by the nodulation (nod) gene products. We tested the ability of three Sinorhizobium meliloti nod gene products to modify Nod factor analogs with thio linkages instead of O-glycosidic bonds in the oligosaccharide backbone.     

Involvement of DNA polymerase beta in protection against the cytotoxicity of oxidative DNA damage

We had shown previously that DNA polymerase beta (beta-pol) null mouse fibroblasts, deficient in base excision repair (BER), are hypersensitive to monofunctional methylating agents but not to hydrogen peroxide (H2O2). This is surprising because beta-pol is thought to be involved in BER of oxidative as well as methylated DNA damage. We confirm these findings here in early-passage cells. However, with time in culture, beta-pol null cells become hypersensitive to H2O2 and other reactive oxygen species-generating agents. Analysis of in vitro BER reveals a strong deficiency in single-nucleotide BER of 8-oxoguanine (8-oxoG) by both early- and late-passage beta-pol null cell extracts. Therefore, in early-passage wild-type and beta-pol null cells, the capacity for single-nucleotide BER of 8-oxoG does not correlate with cellular sensitivity to H2O2. Expression of beta-pol protein in the late-passage null cells almost completely reverses the H2O2-hypersensitivity phenotype. Methoxyamine (MX) treatment sensitizes late-passage wild-type cells to H2O2 as expected for beta-pol-mediated single-nucleotide BER; however in beta-pol null cells, MX has no effect. The data indicate a role(s) of beta-pol-dependent repair in protection against the cytotoxicity of oxidative DNA damage in wild-type cells.     

Role of resident alveolar macrophages in leukocyte traffic into the alveolar air space of intact mice

Intratracheal instillation of the monocyte chemoattractant JE/monocyte chemoattractant protein (MCP)-1 in mice was recently shown to cause increased alveolar monocyte accumulation in the absence of lung inflammation, whereas combined JE/MCP-1/lipopolysaccharide (LPS) challenge provoked acute lung inflammation with early alveolar neutrophil and delayed alveolar monocyte influx. We evaluated the role of resident alveolar macrophages (rAM) in these leukocyte recruitment events and related phenomena of lung inflammation. Depletion of rAM by pretreatment of mice with liposomal clodronate did not affect the JE/MCP-1-driven alveolar monocyte accumulation, despite the observation that rAM constitutively expressed the JE/MCP-1 receptor CCR2, as analyzed by flow cytometry and immunohistochemistry. In contrast, depletion of rAM largely suppressed alveolar cytokine release as well as neutrophil and monocyte recruitment profiles upon combined JE/MCP-1/LPS treatment. Despite this strongly attenuated alveolar inflammatory response, increased lung permeability was still observed in rAM-depleted mice undergoing JE/MCP-1/LPS challenge. Lung leakage was abrogated by codepletion of circulating neutrophils or administration of anti-CD18. Collectively, rAM are not involved in JE/MCP-1-driven alveolar monocyte recruitment in noninflamed lungs but largely contribute to the alveolar cytokine response and enhanced early neutrophil and delayed monocyte influx under inflammatory conditions (JE/MCP-1/LPS deposition). Loss of lung barrier function observed under these conditions is rAM independent but involves circulating neutrophils via beta(2)-integrin engagement.     

Carbon flux analysis in a pantothenate overproducing Corynebacterium glutamicum strain

Carbon flux analysis during a pseudo-stationary phase of metabolite accumulation in a genetically engineered strain of Corynebacterium glutamicum, containing plasmids leading to over-expression of the ilvBNCD and panBC operons, has identified the basic metabolic constraints governing the potential of this bacterium to produce pantothenate. Carbon flux converging on pyruvate (75% of glucose uptake) is controlled by anabolic precursor requirements and NADPH demand provoking high carbon loss as CO₂ via the pentose pathway. Virtually all the flux of pyruvate is directed into the branched pathway leading to both valine and pantothenate production, but flux towards valine is tenfold higher than that transformed to pantothenate, indicating that significant improvements will only be obtained if carbon flux at the ketoisovalerate branchpoint can be modulated.     

Beta 2-adrenergic receptor stimulated,G protein-coupled receptor kinase 2 mediated,phosphorylation of ribosomal protein P2

G protein-coupled receptor kinases are well characterized for their ability to phosphorylate and desensitize G protein-coupled receptors (GPCRs). In addition to phosphorylating the beta2-adrenergic receptor (beta2AR) and other receptors, G protein-coupled receptor kinase 2 (GRK2) can also phosphorylate tubulin, a nonreceptor substrate. To identify novel nonreceptor substrates of GRK2, we used two-dimensional gel electrophoresis to find cellular proteins that were phosphorylated upon agonist-stimulation of the beta2AR in a GRK2-dependent manner. The ribosomal protein P2 was identified as an endogenous HEK-293 cell protein whose phosphorylation was increased following agonist stimulation of the beta2AR under conditions where tyrosine kinases, PKC and PKA, were inhibited. P2 along with its other family members, P0 and P1, constitutes a part of the elongation factor-binding site connected to the GTPase center in the 60S ribosomal subunit. Phosphorylation of P2 is known to regulate protein synthesis in vitro. Further, P2 and P1 are shown to be good in vitro substrates for GRK2 with K(M) values approximating 1 microM. The phosphorylation sites in GRK2-phosphorylated P2 are identified (S102 and S105) and are identical to the sites known to regulate P2 activity. When the 60S subunit deprived of endogenous P1 and P2 is reconstituted with GRK2-phosphorylated P2 and unphosphorylated P1, translational activity is greatly enhanced. These findings suggest a previously unrecognized relationship between GPCR activation and the translational control of gene expression mediated by GRK2 activation and P2 phosphorylation and represent a potential novel signaling pathway responsible for P2 phosphorylation in mammals.     

The cyanide hydratase enzyme of Fusarium lateritium also has nitrilase activity

The filamentous fungus Fusarium lateritium produces cyanide hydratase when grown in the presence of cyanide. The cyanide hydratase protein produced at a high level in Escherichia coli shows a low but significant nitrilase activity with acetonitrile, propionitrile and benzonitrile. The nitrilase activity is sufficient for growth of the recombinant strain on acetonitrile, propionitrile or benzonitrile as the sole source of nitrogen. The recombinant enzyme shows highest nitrilase activity with benzonitrile. Site-directed mutagenesis of the F. lateritium cyanide hydratase gene indicates that mutations leading to a loss of cyanide hydratase activity also lead to a loss of nitrilase activity. This suggests that the active site for cyanide hydratase and nitrilase activity in the protein is the same. This is the first evidence of cyanide hydratase having nitrilase activity.