Isolation of zymogen granules from rat pancreas and characterization of their membrane proteins

A zymogen granule fraction has been isolated from rat pancreas, and its purity has been assessed by biochemical and morphological criteria. Specific activities of two marker enzymes, amylase and chymotrypsin, are increased by 4.6 and 5.4-fold, respectively, as compared to the homogenate. The purified fraction is devoid of detectable RNA, DNA and 5'-nucleotidase, glucose-6-phosphatase, and cytochrome c oxidase activities. Electron micrographs confirm the absence of mitochondria, lysosomes, and rough endoplasmic reticulum fragments. Zymogen granule membranes were isolated from this fraction on a sucrose gradient following lysis in alkaline buffer. Secretory contaminants were efficiently removed from the membranes as indicated by experiments in which labeled secretory proteins were added during the isolation procedure and secondly by measuring residual levels of amylase and chymotrypsin. Three enzyme activities were found in the membranes: thiamine pyrophosphatase, ATP-diphosphohydrolase, and low levels of acid phosphatase. Membrane proteins were solubilized by urea-Triton X-100 and separated in double-dimension (isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Isoelectric point and molecular weight of each protein band were determined.     

Sequence composition similarities with the 7SL RNA are highly predictive of functional genomic features

Transposable elements derived from the 7SL RNA gene, such as Alu elements in primates, have had remarkable success in several mammalian lineages. The results presented here show a broad spectrum of functions for genomic segments that display sequence composition similarities with the 7SL RNA gene. Using thoroughly documented loci, we report that DNaseI-hypersensitive sites can be singled out in large genomic sequences by an assessment of sequence composition similarities with the 7SL RNA gene. We apply a root word frequency approach to illustrate a distinctive relationship between the sequence of the 7SL RNA gene and several classes of functional genomic features that are not presumed to be of transposable origin. Transposable elements that show noticeable similarities with the 7SL sequence include Alu sequences, as expected, but also long terminal repeats and the 5′-untranslated regions of long interspersed repetitive elements. In sequences masked for repeated elements, we find, when using the 7SL RNA gene as query sequence, distinctive similarities with promoters, exons and distal gene regulatory regions. The latter being the most notoriously difficult to detect, this approach may be useful for finding genomic segments that have regulatory functions and that may have escaped detection by existing methods.     

The ostrich pituitary contains a major peptide homologous to mammalian chromogranin A(1-76)

A major peptide related to the NH2-terminal fragment (position 1 to 76) of mammalian chromogranin A was isolated from ostrich adenohypophyses following acid-acetone extraction. The complete amino acid sequence of the homogenous peptide was deduced following automatic Edman degradation of the native peptide as well as of CNBr-, tryptic- and Lysobacter-derived peptides. The 76 amino acid sequence is strikingly homologous to bovine (80.3% sequence identity), porcine (79.0%), human (79.0%) and rat (72.4%) corresponding sequences, but much less so to human chromogranin B (22.4%). As this peptide is followed in bovine, porcine and human structure by a pair of basic residues (Lys-Lys), it could conceivably be produced during maturation in secretory granules. Finally, its structure appears to contain two potential amphipathic helices joined by the single disulfide bridge present in all chromogranin A and B molecules.     

Synthesis of oligophosphoseryl sequences occurring in casein. Identification of beta-elimination during phosphorylation

The protected oligophosphoseryl peptides from bovine caseins, Z-Xxx-(Ser[PO(OPh)2])3-Glu(OBzl)-OBzl for Xxx = Ile, Val, Gly, Leu and Ph = phenyl, were synthesized in high yields by stepwise lengthening using Boc-Ser[PO(OPh)2]-OH as acylating carboxyl component and N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride as coupling reagent. The hydrogenolytic deprotection (PtO2) was carried out with the valine derivative and with the tetrapeptide Ser[PO(OPh)2]3-Glu(OBz)-OBzl. Phosphorylation of oligoseryl peptides failed to give the expected products. Large scale phosphorylation of protected serine was carried out in the presence of triethylamine using absolute ether as a solvent. 2,2,2-Trichloroethyl group (Tc) was shown to be a useful phosphorus protecting moiety in phosphopeptide synthesis: Boc-Ser[PO(OTc)2]-OBzl, Z-Ser[PO(OTc)2]-OBzl and Boc-Glu(OBzl)-Ser[PO(OTc)2]-OBzl were synthesized in high yields using bis-(2,2,2-trichloroethyl) phosphochloridate.     

Synthetic glycolipid-based TLR4 antagonists negatively regulate TRIF-dependent TLR4 signalling in human macrophages

TLRs, including TLR4, play a crucial role in inflammatory-based diseases, and TLR4 has been identified as a therapeutic target for pharmacological intervention. In previous studies, we investigated the potential of FP7, a novel synthetic glycolipid active as a TLR4 antagonist, to inhibit haematopoietic and non-haematopoietic MyD88-dependent TLR4 pro-inflammatory signalling. The main aim of this study was to investigate the action of FP7 and its derivative FP12 on MyD88-independent TLR4 signalling in THP-1 derived macrophages. Western blotting, Ab array and ELISA approaches were used to explore the effect of FP7 and FP12 on TRIF-dependent TLR4 functional activity in response to LPS and other endogenous TLR4 ligands in THP-1 macrophages. A different kinetic in the inhibition of endotoxin-driven TBK1, IRF3 and STAT1 phosphorylation was observed using different LPS chemotypes. Following activation of TLR4 by LPS, data revealed that FP7 and FP12 inhibited TBK1, IRF3 and STAT1 phosphorylation which was associated with down-regulation IFN-β and IP-10. Specific blockage of the IFN type one receptor showed that these novel molecules inhibited TRIF-dependent TLR4 signalling via IFN-β pathways. These results add novel information on the mechanism of action of monosaccharide FP derivatives. The inhibition of the TRIF-dependent pathway in human macrophages suggests potential therapeutic uses for these novel TLR4 antagonists in pharmacological interventions on inflammatory diseases.     

Radiation-induced risks at low dose: moving beyond controversy towards a new vision

The paper recently published by Mothersill and Seymour (Radiat Environ Biophys 2013, doi: 10.1007/s00411-013-0472-y) is commented upon by emphasizing on the recommendation not to confound the fields of radiation protection and radiobiological science as a source of controversy. Instead, these authors are proposing a new vision which suggests novel lines of scientific investigations to be addressed. At the moment, these include moving beyond the conceptual approach of DNA alteration through energy deposition in cells, and exploring the striking parallel currently existing between the ongoing individual/population debate in radioecology and that for cells/tissues in radiobiology. These interesting issues are briefly discussed and supported.     

Suppression of Hepcidin Expression and Iron Overload Mediate Salmonella Susceptibility in Ankyrin 1 ENU-Induced Mutant

Salmonella, a ubiquitous Gram-negative intracellular bacterium, is a food borne pathogen that infects a broad range of hosts. Infection with Salmonella Typhimurium in mice is a broadly recognized experimental model resembling typhoid fever in humans. Using a N-ethyl-N-nitrosurea (ENU) mutagenesis recessive screen, we report the identification of Ity16 (Immunity to Typhimurium locus 16), a locus responsible for increased susceptibility to infection. The position of Ity16 was refined on chromosome 8 and a nonsense mutation was identified in the ankyrin 1 (Ank1) gene. ANK1 plays an important role in the formation and stabilization of the red cell cytoskeleton. The Ank1^Ity16/Ity16 mutation causes severe hemolytic anemia in uninfected mice resulting in splenomegaly, hyperbilirubinemia, jaundice, extramedullary erythropoiesis and iron overload in liver and kidneys. Ank1^Ity16/Ity16 mutant mice demonstrated low levels of hepcidin (Hamp) expression and significant increases in the expression of the growth differentiation factor 15 (Gdf15), erythropoietin (Epo) and heme oxygenase 1 (Hmox1) exacerbating extramedullary erythropoiesis, tissue iron deposition and splenomegaly. As the infection progresses in Ank1^Ity16/Ity16, the anemia worsens and bacterial load were high in liver and kidneys compared to wild type mice. Heterozygous Ank1^+/Ity16 mice were also more susceptible to Salmonella infection although to a lesser extent than Ank1^Ity16/Ity16 and they did not inherently present anemia and splenomegaly. During infection, iron accumulated in the kidneys of Ank1^+/Ity16 mice where bacterial loads were high compared to littermate controls. The critical role of HAMP in the host response to Salmonella infection was validated by showing increased susceptibility to infection in Hamp-deficient mice and significant survival benefits in Ank1 ^+/Ity16 heterozygous mice treated with HAMP peptide. This study illustrates that the regulation of Hamp and iron balance are crucial in the host response to Salmonella infection in Ank1 mutants.     

The Positive and Negative Syndrome Scale (PANSS): A Three-Factor Model of Psychopathology in Marginally Housed Persons with Substance Dependence and Psychiatric Illness

Rates of psychopathology are elevated in marginalized and unstably housed persons, underscoring the need for applicable clinical measures for these populations. The Positive and Negative Syndrome Scale (PANSS) is a clinical instrument principally developed for use in schizophrenia to identify the presence and severity of psychopathology symptoms. The current study investigates whether a reliable and valid PANSS factor structure emerges in a marginally housed, heterogeneous sample recruited from the Downtown Eastside of Vancouver where substance use disorders and psychiatric illness are pervasive. Participants (n = 270) underwent structured clinical assessments including the PANSS and then were randomly assigned to either exploratory (EFA) or confirmatory factor analytic (CFA) subsamples. EFA pointed to a novel three factor PANSS. This solution was supported by CFA. All retained items (28 out of 30) load significantly upon hypothesized factors and model goodness of fit analyses are in the acceptable to good range. Each of the three first-order factor constructs, labeled Psychosis/Disorganized, Negative Symptoms/Hostility, and Insight/Awareness, contributed significantly to measurement of a higher-order psychopathology construct. Further, the latent structure of this 3-factor solution appears temporally consistent over one-year. This PANSS factor structure appears valid and reliable for use in persons with multimorbidity, including substance use disorders. The structure is somewhat distinct from existing solutions likely due to the unique characteristics of this marginally housed sample.     

Communal roosting,thermoregulatory benefits and breeding group size predictability in cooperatively breeding sociable weavers

Extreme temperatures impose energy costs on endotherms through thermoregulation and different adaptations help individuals to cope with these conditions. In social species, communal roosting and huddling are thought to decrease the energetic requirement of thermoregulation under low temperatures. This is likely to represent an important mechanism by which individuals save energy during the coldest parts of the year and hence to represent a non‐breeding benefit of sociality. Here, we investigate the potential thermoregulatory benefits of group living in roosting groups of sociable weavers Philetairus socius, a colonial cooperatively breeding passerine that builds communally a massive nest structure with several independent chambers wherein individuals breed and roost throughout the year. To investigate the benefits of sociality during the non‐breeding season, we studied the thermal environment during roosting in relation to group size. In addition, to understand the link between non‐breeding and breeding sociality in this species we studied group size stability between the pre‐breeding and breeding periods. As expected, we found that the nest chamber's night‐time temperature is strongly related to the number of birds roosting together, especially during cold nights. Specifically, birds in larger groups spent less time below the critical thermal minimum temperature (i.e. the temperature below which energy expenditure increases substantially). They were less exposed to external temperature variations. We also found a positive relationship between the number of birds roosting during winter and the breeding group size, indicating breeding group size predictability. In cooperative breeders such as the sociable weaver, the costs and benefits of sociality are usually studied during the breeding period. This study shows that a better understanding of non‐breeding benefits of group membership and group dynamics between the non‐breeding and breeding periods are necessary for a comprehensive understanding of the benefits of sociality.