Purification and functional characterisation of rhiminopeptidase A, a novel aminopeptidase from the venom of Bitis gabonica rhinoceros

This study describes the discovery and characterisation of a novel aminopeptidase A from the venom of B. g. rhinoceros and highlights its potential biological importance. Similar to mammalian aminopeptidases, rhiminopeptidase A might be capable of playing roles in altering the blood pressure and brain function of victims. Furthermore, it could have additional effects on the biological functions of other host proteins by cleaving their N-terminal amino acids. This study points towards the importance of complete analysis of individual components of snake venom in order to develop effective therapies for snake bites

Phospholipase C Isozymes Are Deregulated in Colorectal Cancer – Insights Gained from Gene Set Enrichment Analysis of the Transcriptome

Colorectal cancer (CRC) is one of the most common cancer types in developed countries. To identify molecular networks and biological processes that are deregulated in CRC compared to normal colonic mucosa, we applied Gene Set Enrichment Analysis to two independent transcriptome datasets, including a total of 137 CRC and ten normal colonic mucosa samples. Eighty-two gene sets as described by the Kyoto Encyclopedia of Genes and Genomes database had significantly altered gene expression in both datasets. These included networks associated with cell division, DNA maintenance, and metabolism. Among signaling pathways with known changes in key genes, the “Phosphatidylinositol signaling network”, comprising part of the PI3K pathway, was found deregulated. The downregulated genes in this pathway included several members of the Phospholipase C protein family, and the reduced expression of two of these, PLCD1 and PLCE1, were successfully validated in CRC biopsies (n = 70) and cell lines (n = 19) by quantitative analyses. The repression of both genes was found associated with KRAS mutations (P = 0.005 and 0.006, respectively), and we observed that microsatellite stable carcinomas with reduced PLCD1 expression more frequently had TP53 mutations (P = 0.002). Promoter methylation analyses of PLCD1 and PLCE1 performed in cell lines and tumor biopsies revealed that methylation of PLCD1 can contribute to reduced expression in 40% of the microsatellite instable carcinomas. In conclusion, we have identified significantly deregulated pathways in CRC, and validated repression of PLCD1 and PLCE1 expression. This illustrates that the GSEA approach may guide discovery of novel biomarkers in cancer

The Generic Short Patient Experiences Questionnaire (GS-PEQ): identification of core items from a survey in Norway

<p>Abstract</p> <p>Background</p> <p>Questionnaires are commonly used to collect patient, or user, experiences with health care encounters; however, their adaption to specific target groups limits comparison between groups. We present the construction of a generic questionnaire (maximum of ten questions) for user evaluation across a range of health care services.</p> <p>Methods</p> <p>Based on previous testing of six group-specific questionnaires, we first constructed a generic questionnaire with 23 items related to user experiences. All questions included a "not applicable" response option, as well as a follow-up question about the item's importance. Nine user groups from one health trust were surveyed. Seven groups received questionnaires by mail and two by personal distribution. Selection of core questions was based on three criteria: applicability (proportion "not applicable"), importance (mean scores on follow-up questions), and comprehensiveness (content coverage, maximum two items per dimension).</p> <p>Results</p> <p>1324 questionnaires were returned providing subsample sizes ranging from 52 to 323. Ten questions were excluded because the proportion of "not applicable" responses exceeded 20% in at least one user group. The number of remaining items was reduced to ten by applying the two other criteria. The final short questionnaire included items on outcome (2), clinician services (2), user involvement (2), incorrect treatment (1), information (1), organisation (1), and accessibility (1).</p> <p>Conclusion</p> <p>The Generic Short Patient Experiences Questionnaire (GS-PEQ) is a short, generic set of questions on user experiences with specialist health care that covers important topics for a range of groups. It can be used alone or with other instruments in quality assessment or in research. The psychometric properties and the relevance of the GS-PEQ in other health care settings and countries need further evaluation.</p

Effects of Hydrogen Peroxide on Wound Healing in Mice in Relation to Oxidative Damage

10.1371/journal.pone.0049215PLoS ONE711

The Base Excision Repair System of Salmonella enterica serovar Typhimurium Counteracts DNA Damage by Host Nitric Oxide

Intracellular pathogens must withstand nitric oxide (NO·) generated by host phagocytes. Salmonella enterica serovar Typhimurium interferes with intracellular trafficking of inducible nitric oxide synthase (iNOS) and possesses multiple systems to detoxify NO·. Consequently, the level of NO· stress encountered by S. Typhimurium during infection in vivo has been unknown. The Base Excision Repair (BER) system recognizes and repairs damaged DNA bases including cytosine and guanine residues modified by reactive nitrogen species. Apurinic/apyrimidinic (AP) sites generated by BER glycosylases require subsequent processing by AP endonucleases. S. Typhimurium xth nfo mutants lacking AP endonuclease activity exhibit increased NO· sensitivity resulting from chromosomal fragmentation at unprocessed AP sites. BER mutant strains were thus used to probe the nature and extent of nitrosative damage sustained by intracellular bacteria during infection. Here we show that an xth nfo S. Typhimurium mutant is attenuated for virulence in C3H/HeN mice, and virulence can be completely restored by the iNOS inhibitor L-NIL. Inactivation of the ung or fpg glycosylase genes partially restores virulence to xth nfo mutant S. Typhimurium, demonstrating that NO· fluxes in vivo are sufficient to modify cytosine and guanine bases, respectively. Mutants lacking ung or fpg exhibit NO·–dependent hypermutability during infection, underscoring the importance of BER in protecting Salmonella from the genotoxic effects of host NO·. These observations demonstrate that host-derived NO· damages Salmonella DNA in vivo, and the BER system is required to maintain bacterial genomic integrity

The PTEN Phosphatase Controls Intestinal Epithelial Cell Polarity and Barrier Function: Role in Colorectal Cancer Progression

The PTEN phosphatase acts on phosphatidylinositol 3,4,5-triphosphates resulting from phosphatidylinositol 3-kinase (PI3K) activation. PTEN expression has been shown to be decreased in colorectal cancer. Little is known however as to the specific cellular role of PTEN in human intestinal epithelial cells. The aim of this study was to investigate the role of PTEN in human colorectal cancer cells.Caco-2/15, HCT116 and CT26 cells were infected with recombinant lentiviruses expressing a shRNA specifically designed to knock-down PTEN. The impact of PTEN downregulation was analyzed on cell polarization and differentiation, intercellular junction integrity (expression of cell-cell adhesion proteins, barrier function), migration (wound assay), invasion (matrigel-coated transwells) and on tumor and metastasis formation in mice. Electron microscopy analysis showed that lentiviral infection of PTEN shRNA significantly inhibited Caco-2/15 cell polarization, functional differentiation and brush border development. A strong reduction in claudin 1, 3, 4 and 8 was also observed as well as a decrease in transepithelial resistance. Loss of PTEN expression increased the spreading, migration and invasion capacities of colorectal cancer cells in vitro. PTEN downregulation also increased tumor size following subcutaneous injection of colorectal cancer cells in nude mice. Finally, loss of PTEN expression in HCT116 and CT26, but not in Caco-2/15, led to an increase in their metastatic potential following tail-vein injections in mice.Altogether, these results indicate that PTEN controls cellular polarity, establishment of cell-cell junctions, paracellular permeability, migration and tumorigenic/metastatic potential of human colorectal cancer cells

Identification of calcium-binding proteins associated with the human sperm plasma membrane

<p>Abstract</p> <p>Background</p> <p>The precise composition of the human sperm plasma membrane, the molecular interactions that define domain specific functions, and the regulation of membrane associated proteins during the capacitation process, still remain to be fully understood. Here, we investigated the repertoire of calcium-regulated proteins associated with the human sperm plasma membrane.</p> <p>Methods</p> <p>Surface specific radioiodination was combined with two-dimensional gel electrophoresis, a 45Ca-overlay assay, computer assisted image analysis and mass spectrometry to identify calcium-binding proteins exposed on the human sperm surface.</p> <p>Results</p> <p>Nine acidic 45Ca-binding sperm proteins were excised from stained preparative 2D gels and identified by mass spectrometry. Five of the calcium binding proteins; HSPA2 (HSP70-1), HSPA5 (Bip), HYOU1 (ORP150), serum amyloid P-component (SAP) and protein kinase C substrate 80K-H (80K-H) were found to be accessible to Iodo-Bead catalyzed 125I-labelling on the surface of intact human sperm. Agglutination and immunofluorescence analysis confirmed that SAP is situated on the plasma membrane of intact, motile sperm as well as permeabilized cells. Western blot analysis showed increased phosphorylation of human sperm 80K-H protein following in vitro capacitation. This is the first demonstration of the 80K-H protein in a mammalian sperm.</p> <p>Conclusion</p> <p>The presence of SAP on the surface of mature sperm implies that SAP has a physiological role in reproduction, which is thought to be in the removal of spermatozoa from the female genital tract via phagocytosis. Since 80K-H is a Ca2+-sensor recently implicated in the regulation of both inositol 1,4,5-trisphosphate receptor and transient receptor potential (TRP) cation channel activities, its detection in sperm represents the first direct signaling link between PKC and store-operated calcium channels identified in human sperm.</p

Alopecia in a Viable Phospholipase C Delta 1 and Phospholipase C Delta 3 Double Mutant

BACKGROUND: Inositol 1,4,5trisphosphate (IP(3)) and diacylglycerol (DAG) are important intracellular signalling molecules in various tissues. They are generated by the phospholipase C family of enzymes, of which phospholipase C delta (PLCD) forms one class. Studies with functional inactivation of Plcd isozyme encoding genes in mice have revealed that loss of both Plcd1 and Plcd3 causes early embryonic death. Inactivation of Plcd1 alone causes loss of hair (alopecia), whereas inactivation of Plcd3 alone has no apparent phenotypic effect. To investigate a possible synergy of Plcd1 and Plcd3 in postnatal mice, novel mutations of these genes compatible with life after birth need to be found. METHODOLOGY/PRINCIPAL FINDINGS: We characterise a novel mouse mutant with a spontaneously arisen mutation in Plcd3 (Plcd3(mNab)) that resulted from the insertion of an intracisternal A particle (IAP) into intron 2 of the Plcd3 gene. This mutation leads to the predominant expression of a truncated PLCD3 protein lacking the N-terminal PH domain. C3H mice that carry one or two mutant Plcd3(mNab) alleles are phenotypically normal. However, the presence of one Plcd3(mNab) allele exacerbates the alopecia caused by the loss of functional Plcd1 in Del(9)olt1Pas mutant mice with respect to the number of hair follicles affected and the body region involved. Mice double homozygous for both the Del(9)olt1Pas and the Plcd3(mNab) mutations survive for several weeks and exhibit total alopecia associated with fragile hair shafts showing altered expression of some structural genes and shortened phases of proliferation in hair follicle matrix cells. CONCLUSIONS/SIGNIFICANCE: The Plcd3(mNab) mutation is a novel hypomorphic mutation of Plcd3. Our investigations suggest that Plcd1 and Plcd3 have synergistic effects on the murine hair follicle in specific regions of the body surface