In Vitro and In Vivo Effects of Natural Putative Secretagogues of Glucagon-Like Peptide-1 (GLP-1)

Glucagon-like peptide-1 (GLP-1) is an intestinal hormone with well-established glucose-lowering activity. The in vitro and in vivo actions of natural putative secretagogues of GLP-1 were investigated. The acute GLP-1 releasing activity of olive leaf extract (OLE), glutamine (GLN), alpha casein (ACAS), beta casein (BCAS) and chlorogenic acid (CGA) were assessed in STC-1 cells and C57BL/6 mice. All compounds except ACAS significantly increased acute in vitro GLP-1 secretion (66-386%; P

AN IMMUNOCYTOCHEMICAL STUDY OF PUTATIVE NEUROTRANSMITTERS IN THE METACERCARIAE OF 2 STRIGEOID TREMATODES FROM RAINBOW-TROUT (ONCORHYNCHUS-MYKISS)

Whole mounts of the metacercariae of Diplostomum sp. and Cotylurus erraticus from rainbow trout have been treated cytochemically for the demonstration of cholinergic, serotoninergic (5-hydroxytryptamine) and peptidergic elements in the nervous system. Antisera directed against four vertebrate (pancreatic polypeptide, peptide YY, substance P and peptide histidine isoleucine) and two invertebrate peptides (neuropeptide F and FMRFamide) were used in an indirect immunofluorescence procedure in conjunction with confocal scanning laser microscopy (CSLM). Of the seven antisera tested, all except peptide histidine isoleucine showed significant immunoreactivity. Cholinergic and serotoninergic staining was found primarily in the central nervous system (CNS) and in cell bodies associated with the ventral and dorsal nerve cords in both trematodes. Peptidergic immunoreactivity was localised in the CNS and PNS of both genera, revealing an extensive innervation within the holdfast organ and in and around the oral and ventral suckers.

Synthesis of the putative structures of homopumiliotoxins 235C and 233F

A novel approach to diene based quinolizidines, using an intramolecular Heck reaction in which the vinyl bromide double bond undergoes inversion of configuration, is reported. These quinolizidines have previously been proposed as tentative structures for homopumiliotoxin alkaloids 233F and 235C. The mass spectral data of the synthetic materials were different to those of the natural products confirming that the original structures need to be revised. (C) 2004 Elsevier Ltd. All rights reserved.

Suppressors of cytokine signalling (SOCS): putative modulators of cytokine bioactivity in health and disease

Cytokines are important modulators of homeostatic processes such as development, haematopoiesis and host defence, A recently identified family of proteins, the supressors of cytokine signalling (SOCS) act as negative regulators of the key cytokine-activated signalling pathway, the Janus kinase/signal transducers and activators of transcription (JAK/STAT) cascade, In the current review, the discovery, structural features, regulation of expression, mechanisms of JAK/STAT inhibition and putative role in health and disease of the SOCS family are discussed.

Selective extracellular vesicle-mediated export of an overlapping set of microRNAs from multiple cell types

Background: MicroRNAs (miRNAs) are a class of small RNA molecules that regulate expression of specific mRNA targets. They can be released from cells, often encapsulated within extracellular vesicles (EVs), and therefore have the potential to mediate intercellular communication. It has been suggested that certain miRNAs may be selectively exported, although the mechanism has yet to be identified. Manipulation of the miRNA content of EVs will be important for future therapeutic applications. We therefore wished to assess which endogenous miRNAs are enriched in EVs and how effectively an overexpressed miRNA would be exported.

Results: Small RNA libraries from HEK293T cells and vesicles before or after transfection with a vector for miR-146a overexpression were analysed by deep sequencing. A subset of miRNAs was found to be enriched in EVs; pathway analysis of their predicted target genes suggests a potential role in regulation of endocytosis. RT-qPCR in additional cell types and analysis of publicly available data revealed that many of these miRNAs tend to be widely preferentially exported. Whilst overexpressed miR-146a was highly enriched both in transfected cells and their EVs, the cellular:EV ratios of endogenous miRNAs were not grossly altered. MiR-451 was consistently the most highly exported miRNA in many different cell types. Intriguingly, Argonaute2 (Ago2) is required for miR-451 maturation and knock out of Ago2 has been shown to decrease expression of other preferentially exported miRNAs (eg miR-150 and miR-142-3p).

Conclusion: The global expression data provided by deep sequencing confirms that specific miRNAs are enriched in EVs released by HEK293T cells. Observation of similar patterns in a range of cell types suggests that a common mechanism for selective miRNA export may exist.

Aminoarabinose is essential for lipopolysaccharide export and intrinsic antimicrobial peptide resistance in Burkholderia cenocepacia

One common mechanism of resistance against antimicrobial peptides in Gram-negative bacteria is the addition of 4-amino-4-deoxy-l-arabinose (l-Ara4N) to the lipopolysaccharide (LPS) molecule. Burkholderia cenocepacia exhibits extraordinary intrinsic resistance to antimicrobial peptides and other antibiotics. We have previously discovered that unlike other bacteria, B. cenocepacia requires l-Ara4N for viability. Here, we describe the isolation of B. cenocepacia suppressor mutants that remain viable despite the deletion of genes required for l-Ara4N synthesis and transfer to the LPS. The absence of l-Ara4N is the only structural difference in the LPS of the mutants compared with that of the parental strain. The mutants also become highly sensitive to polymyxin B and melittin, two different classes of antimicrobial peptides. The suppressor phenotype resulted from a single amino acid replacement (aspartic acid to histidine) at position 31 of LptG, a protein component of the multi-protein pathway responsible for the export of the LPS molecule from the inner to the outer membrane. We propose that l-Ara4N modification of LPS provides a molecular signature required for LPS export and proper assembly at the outer membrane of B. cenocepacia, and is the most critical determinant for the intrinsic resistance of this bacterium to antimicrobial peptides.

A putative gene cluster for aminoarabinose biosynthesis is essential for Burkholderia cenocepacia viability

Using a conditional mutagenesis strategy we demonstrate here that a gene cluster encoding putative aminoarabinose (Ara4N) biosynthesis enzymes is essential for the viability of Burkholderia cenocepacia. Loss of viability is associated with dramatic changes in bacterial cell morphology and ultrastructure, increased permeability to propidium iodide, and sensitivity to sodium dodecyl sulfate, suggesting a general cell envelope defect caused by the lack of Ara4N.

Wzx proteins involved in biosynthesis of O antigen function in association with the first sugar of the O-specific lipopolysaccharide subunit

One of the most common pathways for the export of O-specific lipopolysaccharide (LPS) across the plasma membrane requires the participation of the Wzx protein. Wzx belongs to a family of integral membrane proteins that share little conservation in their primary amino acid sequence, making it difficult to delineate functional domains. This paper reports the cloning and expression in Escherichia coli K-12 of various Wzx homologues from different bacteria as FLAG epitope-tagged protein fusions. A reconstitution system for O16 LPS synthesis was used to assess the ability of each Wzx protein to complement an E. coli K-12 Deltawzx mutant. The results demonstrate that Wzx proteins from O-antigen systems that use N-acetylglucosamine or N-acetylgalactosamine for the initiation of the biosynthesis of the O repeat can fully complement the formation of O16 LPS. Partial complementation was seen with Wzx from Pseudomonas aeruginosa, a system that uses N-acetylfucosamine in the initiation reaction. In contrast, there was negligible complementation with the Wzx protein from Salmonella enterica, a system in which galactose is the initiating sugar. These results support a model whereby the first sugar of the O repeat can be recognized by the O-antigen translocation machinery.

Export of O-specific lipopolysaccharide

The O antigen is the most surface-exposed component of the lipopolysaccharide (LPS) molecule and its biogenesis involves several complex mechanisms not completely well understood. All of these mechanisms involve biochemical reactions that occur on the cytoplasmic side of the plasma membrane as well as several different translocation pathways that deliver the nascent O antigens in a glycolipid form to the periplasmic side of the plasma membrane. This article discusses our current understanding of the mechanisms operating in the biogenesis of the O-specific LPS.

The activity of a putative polyisoprenol-linked sugar translocase (Wzx) involved in Escherichia coli O antigen assembly is independent of the chemical structure of the O repeat

During O antigen lipopolysaccharide (LPS) synthesis in bacteria, transmembrane migration of undecaprenylpyrophosphate (Und-P-P)-bound O antigen subunits occurs before their polymerization and ligation to the rest of the LPS molecule. Despite the general nature of the translocation process, putative O-antigen translocases display a low level of amino acid sequence similarity. In this work, we investigated whether complete O antigen subunits are required for translocation. We demonstrate that a single sugar, GlcNAc, can be incorporated to LPS of Escherichia coli K-12. This incorporation required the functions of two O antigen synthesis genes, wecA (UDP-GlcNAc:Und-P GlcNAc-1-P transferase) and wzx (O-antigen translocase). Complementation experiments with putative O-antigen translocases from E. coli O7 and Salmonella enterica indicated that translocation of O antigen subunits is independent of the chemical structure of the saccharide moiety. Furthermore, complementation with putative translocases involved in synthesis of exopolysaccharides demonstrated that these proteins could not participate in O antigen assembly. Our data indicate that recognition of a complete Und-P-P-bound O antigen subunit is not required for translocation and suggest a model for O antigen synthesis involving recognition of Und-P-P-linked sugars by a putative complex made of Wzx translocase and other proteins involved in the processing of O antigen.

Weighing China’s export basket: The domestic content and technology intensity of Chinese exports

In this paper we use new, detailed, and comprehensive linked firm-transaction data to measure the domestic content and technology intensity of Chinese exports over the period 2000–2007. We evaluate the extent of value-added in China’s exports, using a modification of a method proposed by Hummels et al. (2001) which takes into account the prevalence of processing firms. In addition, we provide new estimates of the skill-and technology-intensity of China’s exports. Our estimates of value-added suggest that the domestic content of China’s exports increased from only 53% to about 60% over the period 2003–2006. Our cross-firm analysis reveals that processing exporters have value-added shares approximately 50% lower than non-processing exporters, even after accounting for ownership, location, and industry. We also show that Chinese exports have become increasingly sophisticated, largely driven by skill and technology improvement within industries.