A novel nociceptor signaling pathway revealed in protein kinase C epsilon mutant mice

There is great interest in discovering new targets for pain therapy since current methods of analgesia are often only partially successful. Although protein kinase C (PKC) enhances nociceptor function, it is not known which PKC isozymes contribute. Here, we show that epinephrine-induced mechanical and thermal hyperalgesia and acetic acid-associated hyperalgesia are markedly attenuated in PKCepsilon mutant mice, but baseline nociceptive thresholds are normal. Moreover, epinephrine-, carrageenan-, and nerve growth factor- (NGF-) induced hyperalgesia in normal rats, and epinephrine-induced enhancement of tetrodotoxin-resistant Na+ current (TTX-R I(Na)) in cultured rat dorsal root ganglion (DRG) neurons, are inhibited by a PKCepsilon-selective inhibitor peptide. Our findings indicate that PKCepsilon regulates nociceptor function and suggest that PKCepsilon inhibitors could prove useful in the treatment of pain.     

Purification and kinetic characterization of human indoleamine 2,3-dioxygenases 1 and 2 (IDO1 and IDO2) and discovery of selective IDO1 inhibitors

Indoleamine 2,3-dioxygenase (IDO1) catalyzes the first step in tryptophan breakdown along the kynurenine pathway. Therapeutic inhibition of IDO1 is receiving much attention due to its proposed role in the pathogenesis of several diseases including cancer, hypotension and neurodegenerative disorders. A related enzyme, IDO2 has recently been described. We report the first purification and kinetic characterization of human IDO2 using a facile l-tryptophan consumption assay amenable to high throughput screening. We found that the K(m) of human IDO2 for l-tryptophan is much higher than that of IDO1. We also describe the identification and characterization of a new IDO1 inhibitor compound, Amg-1, by high throughput screening, and compare the inhibition profiles of IDO1 and IDO2 with Amg-1 and previously described compounds. Our data indicate that human IDO1 and IDO2 have different kinetic parameters and different inhibition profiles. Docking of Amg-1 and related analogs to the known structure of IDO1 and to homology-modeled IDO2 suggests possible rationales for the different inhibition profiles of IDO1 and IDO2.     

Quorum sensing and silencing in Vibrio parahaemolyticus

The quorum regulatory cascade is poorly characterized in Vibrio parahaemolyticus, in part because swarming and virulence factors--the hallmarks of the organism--are repressed by this scheme of gene control, and quorum sensing seems to be silenced in many isolates. In these studies, we examine a swarming-proficient, virulent strain and identify an altered-function allele of the quorum regulator luxO that is demonstrated to produce a constitutively active mimic of LuxO～P. We find that LuxO* affects the expression of three small regulatory RNAs (Qrrs) and the activity of a translational fusion in opaR, the output regulator. Tests for epistasis showed that luxO* is dominant over luxO and that opaR is dominant over luxO. Thus, information flow through the central elements of the V. parahaemolyticus quorum pathway is proven for the first time. Quorum-sensing output was explored using microarray profiling: the OpaR regulon encompasses ～5.2% of the genome. OpaR represses the surface-sensing and type III secretion system 1 (T3SS1) regulons. One novel discovery is that OpaR strongly and oppositely regulates two type VI secretion systems (T6SS). New functional consequences of OpaR control were demonstrated: OpaR increases the cellular cyclic di-GMP (c-di-GMP) level, positively controls chitin-induced DNA competency, and profoundly blocks cytotoxicity toward host cells. In expanding the previously known quorum effects beyond the induction of the capsule and the repression of swarming to elucidate the global scope of genes in the OpaR regulon, this study yields many clues to distinguishing traits of this Vibrio species; it underscores the profoundly divergent survival strategies of the quorum On/Off phase variants.     

Thermodynamic evaluation of ligand binding in the plant-like phosphoethanolamine methyltransferases of the parasitic nematode Haemonchus contortus

Nematodes are a major cause of disease and the discovery of new pathways not found in hosts is critical for development of therapeutic targets. Previous studies suggest that Caenorhabditis elegans synthesizes phosphocholine via two S-adenosylmethionine (AdoMet)-dependent phosphoethanolamine methyltransferases (PMT). Here we examine two PMT from the parasitic nematode Haemonchus contortus. Sequence analysis suggests that HcPMT1 contains a methyltransferase domain in the N-terminal half of the protein and that HcPMT2 encodes a C-terminal methyltransferase domain, as in the C. elegans proteins. Kinetic analysis demonstrates that HcPMT1 catalyzes the conversion of phosphoethanolamine to phosphomonomethylethanolamine (pMME) and that HcPMT2 methylates pMME to phosphodimethylethanolamine (pDME) and pDME to phosphocholine. The IC(50) values for miltefosine, sinefungin, amodiaquine, diphenhydramine, and tacrine suggest differences in the active sites of these two enzymes. To examine the interaction of AdoMet and S-adenosylhomocysteine (AdoCys), isothermal titration calorimetry confirmed the presence of a single binding site in each enzyme. Binding of AdoMet and AdoCys is tight (K(d) ～2-25 μm) over a range of temperatures (5-25 °C) and NaCl concentrations (0.05-0.5 m). Heat capacity changes for AdoMet and AdoCys binding suggests that each HcPMT differs in interaction surface area. Nonlinear van't Hoff plots also indicate a possible conformational change upon AdoMet/AdoCys binding. Functional analysis of the PMT from a parasitic nematode provides new insights on inhibitor and AdoMet/AdoCys binding to these enzymes.     

HIV-1 infection of human intestinal lamina propria CD4+ T cells in vitro is enhanced by exposure to commensal Escherichia coli

Microbial translocation has been linked to systemic immune activation in HIV-1 disease, yet mechanisms by which microbes may contribute to HIV-associated intestinal pathogenesis are poorly understood. Importantly, our understanding of the impact of translocating commensal intestinal bacteria on mucosal-associated T cell responses in the context of ongoing viral replication that occurs early in HIV-1 infection is limited. We previously identified commensal Escherichia coli-reactive Th1 and Th17 cells in normal human intestinal lamina propria (LP). In this article, we established an ex vivo assay to investigate the interactions between Th cell subsets in primary human LP mononuclear cells (LPMCs), commensal E. coli, and CCR5-tropic HIV-1(Bal). Addition of heat-killed E. coli to HIV-1-exposed LPMCs resulted in increases in HIV-1 replication, CD4 T cell activation and infection, and IL-17 and IFN-γ production. Conversely, purified LPS derived from commensal E. coli did not enhance CD4 T cell infection. E. coli exposure induced greater proliferation of LPMC Th17 than Th1 cells. Th17 cells were more permissive to infection than Th1 cells in HIV-1-exposed LPMC cultures, and Th17 cell infection frequencies significantly increased in the presence of E. coli. The E. coli-associated enhancement of infection was dependent on the presence of CD11c(+) LP dendritic cells and, in part, on MHC class II-restricted Ag presentation. These results highlight a potential role for translocating microbes in impacting mucosal HIV-1 pathogenesis during early infection by increasing HIV-1 replication and infection of intestinal Th1 and Th17 cells.     

Effect of Anionic Salt and Highly Fermentable Carbohydrate Supplementations on Urine pH and on Experimentally Induced Hypocalcaemia in Cows

The objective of this experiment was to determine the effect of dietary grain on calcium homeostasis. Six rumen-fistulated dairy cows with 3 or more previous lactations and no history of parturient paresis were randomly assigned to a sequence of diets in a crossover study with 4 periods of 10 days each. Dietary treatments were: A control ration consisting of wrap grass silage alone (1), the control ration supplemented with ammonium chloride and ammonium sulphate salt solution (2), control ration following a period with supplementation (3) and control ration supplemented with increasing amounts of barley from 4 to 10 kg/cow per day, expected to produce subclinical rumen acidosis (4). Daily intake of the diets was adjusted to 14 kg DM/cow per day. On day 11, the calcium-regulating mechanisms in cows were challenged until recumbency by a standardized intravenous EDTA infusion and cows were left to recover spontaneously. Anion supplementation and the feeding of highly fermentable carbohydrate lowered urine pH below 7.0 due to subclinical acidosis. During spontaneous recovery from EDTA induced hypocalcaemia, the cows more quickly regained a whole blood free calcium concentration of 1.00 mmol/L if they had most recently been supplemented with either anionic salts or with increasing amounts of barley, as compared to the basic ration. It is concluded that so-called slug-feeding or 'steaming up' with highly fermentable carbohydrates before parturition in milk fever susceptible cows enhanced calcium homeostasis similar to the effect seen in cows on anionic diets.     

Evolution of the Sry genes

Existing DNA sequence data on the Sry gene, the mammalian sex- determining locus in the Y chromosome, were analyzed for primates, rodents, and bovids. In all three taxonomic groups, the terminal sequences evolved faster than the HMG (high mobility group) boxes, and this applies both to synonymous (Ks) and nonsynonymous (Ka) nucleotide substitutions. Similar intragenic correlation between synonymous and nonsynonymous substitution rates was not found either in other mammalian genes that contain a conservative box (Sox, Msx) or in the MADS-box genes of plants. The rate of nonsynonymous substitutions exceeds significantly that of synonymous substitutions in the terminal Sry sequences of apes. We did not find good support for the hypothesis that the high evolutionary rate of Sry would be associated with a promiscuous mating system.