Quantitative association between altered plasma esterified omega-6 fatty acid proportions and psychological stress.

Medical students (MS) tested during the first year of medical school showed both greater stress on the Brief Symptom Inventory and lower plasma proportions of total esterified arachidonic acid (AA, C20:4n-6), and its omega-6 fatty acid (FA) precursor, linoleic acid (C18:2n-6) than control laboratory workers. This association suggests that omega-6 FA metabolism may be affected during stress. Low AA values might result from depletion of plasma stores for immunoregulatory prostenoids formation or from modification of metabolic pathways by cortisol or other cytokine compounds implicated in stress. Values for other major FA and the omega-3 neuronal metabolic substrate, docosahexaenoic acid (DHA, C22:6n-3) were similar between students and controls. The clear preservation of the omega-3 FA pathway suggests their programmed availability for neuronal function during stress. Since plasma FA proportions may affect immune cell membrane function(s), we suggest that altered values of plasma FAs may be an important component of the physiological effects of psychological stress.     

Cellulose-bound Peptide Arrays: Preparation and Applications

Spatial organization of metabolic enzymes may represent a general cellular mechanism to regulate metabolic flux. One recent example of this type of cellular phenomenon is the purinosome, a newly discovered multi-enzyme metabolic assembly that includes all of the enzymes within the de novo purine biosynthetic pathway. Our understanding of the components and regulation of purinosomes has significantly grown in recent years. This paper reviews the purine de novo biosynthesis pathway and its regulation, and presents the evidence supporting the purinosome assembly and disassembly processes under the control of G-protein-coupled receptor (GPCR) signaling. This paper also discusses the implications of purinosome and GPCR regulation in drug discovery.     

A novel spliced variant of the type 1 corticotropin-releasing hormone receptor with a deletion in the seventh transmembrane domain present in the human pregnant term myometrium and fetal membranes

CRH exerts its actions via activation of specific G protein-coupled receptors, which exist in two types, CRH-R1 and CRH-R2, and arise from different genes with multiple spliced variants. RT-PCR amplification of CRH receptor sequences from human myometrium and fetal membranes yielded cDNAs that encode a novel CRH-R type 1 spliced variant. This variant (CRH-R1d) is present in the human pregnant myometrium at term only, which suggests a physiologically important role at the end of human pregnancy and labor. The amino acid sequence of CRH-R1d is identical to the CRH-R1alpha receptor except that it contains an exon deletion resulting in the absence of 14 amino acids in the predicted seventh transmembrane domain. Binding studies in HEK-293 cells stably expressing the CRH-R1d or CRH-R1alpha receptors revealed that the deletion does not change the binding characteristics of the variant receptor. In contrast, studies on the G protein activation demonstrated that CRH-R1d is not well coupled to the four subtypes of G proteins (G(s), G(i), G(o), G(q)) that CRH-R1alpha can activate. These data suggest that although the deleted segment is not important for CRH binding, it plays a crucial role in CRH receptor signal transduction. Second messenger studies of the variant receptor showed that CRH and CRH-like peptides can stimulate the adenylate cyclase system, with reduced sensitivity and potency by 10-fold compared with the CRH-R1alpha. Furthermore, CRH failed to stimulate inositol trisphosphate production. Coexpression studies between the CRH-R1d or CRH-R1alpha showed that this receptor does not play a role as a dominant negative receptor for CRH.     

Potential signalling pathways underlying corticotrophin-releasing hormone-mediated neuroprotection from excitotoxicity in rat hippocampus

In several neurological disorders including cerebral ischaemia, glutamate has been implicated as a neurotoxic agent in the mechanisms leading to neuronal cell death. The role of corticotrophin-releasing hormone (CRH), the 41-amino acid peptide, which activates the HPA axis in response to stressful stimuli, remains controversial. In this study, we report that CRH in low physiological concentrations (2 pM), prevented glutamate-induced neurotoxicity via receptor-mediated mechanisms when administered to organotypic hippocampal cultures both during and after the glutamate-induced insult. Detailed investigations on the mechanisms mediating this neuroprotective effect showed that activation of the adenylate cyclase pathway and induction of MAP kinase phosphorylation mediate the CRH action. In addition we showed that CRH can inhibit the phosphorylation of JNK/SAPK by glutamate. Most importantly, we showed that CRH can afford neuroprotection against neurotoxicity up to 12 h following the insult, suggesting that CRH is acting at a late stage in the neuronal death cycle, and this might be important in the development of novel neuroprotective agents in order to improve neuronal survival following the insult.     

Corticotropin-releasing hormone receptors

Corticotropin-releasing hormone (CRH) and related peptides (urocortins, sauvagine, urotensin) play a central role in the co-ordination of autonomic, behavioural, cardiovascular, immune and endocrine responses to stressful stimuli. Their actions are mediated through activation of two types of G-protein-coupled receptors encoded by separate genes. In this review we focus on the diverse structural and functional characteristics of the family of CRH-like peptides and their receptors.     

Quantitative trait loci in a bacterially induced model of inflammatory bowel disease

Inflammatory bowel diseases (IBDs) are complex disorders caused by a combination of environmental, microbial, and genetic factors. Genome-wide association studies in humans have successfully identified multiple genes and loci associated with disease susceptibility, but the mechanisms by which these loci interact with each other and/or with environmental factors (i.e., intestinal microbiota) to cause disease are poorly understood. Helicobacter hepaticus-induced intestinal inflammation in mice is an ideal model system for elucidating the genetic basis of IBD susceptibility in a bacterially induced system, as there are significant differences in H. hepaticus-induced disease susceptibility among inbred mouse strains. Infected A/J mice develop acute overexpression of proinflammatory cytokines followed 2?C3?months later by chronic cecal inflammation, whereas infected C57BL/6 mice fail to develop cecal inflammation or increased cytokine expression. The goal of this project was to use quantitative trait locus (QTL) mapping to evaluate genetic factors that contribute to the differential disease susceptibility between these two mouse strains. Using acute cecal IL-12/23p40 expression as a biomarker for disease susceptibility, QTL analysis of H. hepaticus-infected F₂ mice revealed involvement of multiple loci. The loci with the strongest association were located on Chromosome 3 and Chromosome 17, with logarithm of odds (LOD) scores of 6.89 and 3.09, respectively. Cecal expression of IL-12/23p40 in H. hepaticus-infected C57BL/6J-Chr3^A/J/NaJ chromosome substitution mice had an intermediate phenotype, significantly higher than in resistant C57BL/6 but lower than in susceptible A/J mice, confirming the importance of this locus to the immune response to H. hepaticus infection.     

A type VII secretion system of Streptococcus gallolyticus subsp. gallolyticus contributes to gut colonization and the development of colon tumors

Streptococcus gallolyticus subspecies gallolyticus (Sgg) has a strong clinical association with colorectal cancer (CRC) and actively promotes the development of colon tumors. However, the molecular determinants involved in Sgg pathogenicity in the gut are unknown. Bacterial type VII secretion systems (T7SS) mediate pathogen interactions with their host and are important for virulence in pathogenic mycobacteria and Staphylococcus aureus. Through genome analysis, we identified a locus in Sgg strain TX20005 that encodes a putative type VII secretion system (designated as SggT7SS^T05). We showed that core genes within the SggT7SS^T05 locus are expressed in vitro and in the colon of mice. Western blot analysis showed that SggEsxA, a protein predicted to be a T7SS secretion substrate, is detected in the bacterial culture supernatant, indicating that this SggT7SS^T05 is functional. Deletion of SggT7SS^T05 (TX20005Δesx) resulted in impaired bacterial adherence to HT29 cells and abolished the ability of Sgg to stimulate HT29 cell proliferation. Analysis of bacterial culture supernatants suggest that SggT7SS^T05-secreted factors are responsible for the pro-proliferative activity of Sgg, whereas Sgg adherence to host cells requires both SggT7SS^T05-secreted and bacterial surface-associated factors. In a murine gut colonization model, TX20005Δesx showed significantly reduced colonization compared to the parent strain. Furthermore, in a mouse model of CRC, mice exposed to TX20005 had a significantly higher tumor burden compared to saline-treated mice, whereas those exposed to TX20005Δesx did not. Examination of the Sgg load in the colon in the CRC model suggests that SggT7SS^T05-mediated activities are directly involved in the promotion of colon tumors. Taken together, these results reveal SggT7SS^T05 as a previously unrecognized pathogenicity determinant for Sgg colonization of the colon and promotion of colon tumors.     

One-step refolding and purification of disulfide-containing proteins with a C-terminal MESNA thioester

Background  

Expression systems based on self-cleavable intein domains allow the generation of recombinant proteins with a C-terminal thioester. This uniquely reactive C-terminus can be used in native chemical ligation reactions to introduce synthetic groups or to immobilize proteins on surfaces and nanoparticles. Unfortunately, common refolding procedures for recombinant proteins that contain disulfide bonds do not preserve the thioester functionality and therefore novel refolding procedures need to be developed.