Advanced intercross line mapping of Eae5 reveals Ncf-1 and CLDN4 as candidate genes for experimental autoimmune encephalomyelitis

Eae5 in rats was originally identified in two F(2) intercrosses, (DA x BN) and (E3 x DA), displaying linkage to CNS inflammation and disease severity in experimental autoimmune encephalomyelitis (EAE), respectively. This region overlaps with an arthritis locus, Pia4, which was also identified in the (E3 x DA) cross. Two congenic strains, BN.DA-Eae5 and BN.DA-Eae5.R1, encompassing the previously described Eae5 and Pia4, were established. DA alleles within the chromosome 12 fragment conferred an increase in disease susceptibility as well as increased inflammation and demyelination in the CNS as compared with BN alleles. To enable a more precise fine mapping of EAE regulatory genes, we used a rat advanced intercross line between the EAE-susceptible DA strain and the EAE-resistant PVG.1AV1 strain. Linkage analysis performed in the advanced intercross line considerably narrowed down the myelin oligodendrocyte glycoprotein-EAE regulatory locus (Eae5) to a approximately 1.3-megabase region with a defined number of candidate genes. In this study we demonstrate a regulatory effect of Eae5 on MOG-EAE by using both congenic strains as well as fine mapping these effects to a region containing Ncf-1, a gene associated with arthritis. In addition to structural polymorphisms in Ncf-1, both sequence polymorphisms and expression differences were identified in CLDN4. CLDN4 is a tight junction protein involved in blood-brain barrier integrity. In conclusion, our data strongly suggests Ncf-1 to be a gene shared between two organ-specific inflammatory diseases with a possible contribution by CLDN4 in encephalomyelitis.     

Insulin-facilitated increase of muscle protein synthesis after resistance exercise involves a MAP kinase pathway

Recent studies have implicated the mTOR-signaling pathway as a primary component for muscle growth in mammals. The purpose of this investigation was to examine signaling pathways for muscle protein synthesis after resistance exercise. Sprague-Dawley rats (male, 6 mo old) were assigned to either resistance exercise or control groups. Resistance exercise was accomplished in operantly conditioned animals using a specially designed flywheel apparatus. Rats performed two sessions of resistance exercise, separated by 48 h, each consisting of 2 sets of 25 repetitions. Sixteen hours after the second session, animals were killed, and soleus muscles were examined for rates of protein synthesis with and without insulin and/or rapamycin (mTOR inhibitor) and/or PD-098059 (PD; MEK kinase inhibitor). Results of this study demonstrated that rates of synthesis were higher (P < 0.05) with insulin after exercise compared with without insulin, or to control muscles, regardless of insulin. Rapamycin lowered (P < 0.05) rates of synthesis in controls, with or without insulin, and after exercise without insulin. However, insulin was able to overcome the inhibition of rapamycin after exercise (P < 0.05). PD had no effect on protein synthesis in control rats, but the addition of PD to exercised muscle resulted in lower (P < 0.05) rates of synthesis, and this inhibition was not rescued by insulin. Western blot analyses demonstrated that the inhibitors used in the present study were selective and effective for preventing activation of specific signaling proteins. Together, these results suggest that the insulin-facilitated increase of muscle protein synthesis after resistance exercise requires multiple signaling pathways.     

Dipeptidyl peptidases 8 and 9: specificity and molecular characterization compared with dipeptidyl peptidase IV

Dipeptidyl peptidases 8 and 9 have been identified as gene members of the S9b family of dipeptidyl peptidases. In the present paper, we report the characterization of recombinant dipeptidyl peptidases 8 and 9 using the baculovirus expression system. We have found that only the full-length variants of the two proteins can be expressed as active peptidases, which are 882 and 892 amino acids in length for dipeptidyl peptidase 8 and 9 respectively. We show further that the purified proteins are active dimers and that they show similar Michaelis-Menten kinetics and substrate specificity. Both cleave the peptide hormones glucagon-like peptide-1, glucagon-like peptide-2, neuropeptide Y and peptide YY with marked kinetic differences compared with dipeptidyl peptidase IV. Inhibition of dipeptidyl peptidases IV, 8 and 9 using the well-known dipeptidyl peptidase IV inhibitor valine pyrrolidide resulted in similar K(i) values, indicating that this inhibitor is non-selective for any of the three dipeptidyl peptidases.     

Alteration of the Canine Small-Intestinal Lactic Acid Bacterium Microbiota by Feeding of Potential Probiotics

Five potentially probiotic canine fecal lactic acid bacterium (LAB) strains, Lactobacillus fermentum LAB8, Lactobacillus salivarius LAB9, Weissella confusa LAB10, Lactobacillus rhamnosus LAB11, and Lactobacillus mucosae LAB12, were fed to five permanently fistulated beagles for 7 days. The survival of the strains and their potential effects on the indigenous intestinal LAB microbiota were monitored for 17 days. Denaturing gradient gel electrophoresis (DGGE) demonstrated that the five fed LAB strains survived in the upper gastrointestinal tract and modified the dominant preexisting indigenous jejunal LAB microbiota of the dogs. When the LAB supplementation was ceased, DGGE analysis of jejunal chyme showed that all the fed LAB strains were undetectable after 7 days. However, the diversity of the intestinal indigenous microbiota of the dogs, as characterized from jejunal chyme plated on Lactobacillus selective medium without acetic acid, was reduced and did not return to the original level during the study period. In all but one dog, an indigenous Lactobacillus acidophilus strain emerged as the dominant LAB strain. In conclusion, strains LAB8 to LAB12 have potential as probiotic strains for dogs as they survive in and dominate the jejunal LAB microbiota during feeding and have the ability to modify the intestinal microbiota.     

Different types of biological proton transfer reactions studied by quantum chemical methods

Different types of proton transfer occurring in biological systems are described with examples mainly from ribonucleotide reductase (RNR) and cytochrome c oxidase (CcO). Focus is put on situations where electron and proton transfer are rather strongly coupled. In the long range radical transfer in RNR, it is shown that the presence of hydrogen atom transfer (HAT) is the most logical explanation for the experimental observations. In another example from RNR, it is shown that a transition state for concerted motion of both proton and electron can be found even if the donors are separated by a quite long distance. In CcO, the essential proton transfer for the OO bond cleavage, and the most recent modelings of proton translocation are described, indicating a few remaining major problems.     

Purification and characterization of two protein kinases acting on the aquaporin SoPIP2;1

Aquaporins are water channel proteins that facilitate the movement of water and other small solutes across biological membranes. Plants usually have large aquaporin families, providing them with many ways to regulate the water transport. Some aquaporins are regulated post-translationally by phosphorylation. We have previously shown that the water channel activity of SoPIP2;1, an aquaporin in the plasma membrane of spinach leaves, was enhanced by phosphorylation at Ser115 and Ser274. These two serine residues are highly conserved in all plasma membrane aquaporins of the PIP2 subgroup. In this study we have purified and characterized two protein kinases phosphorylating Ser115 and Ser274 in SoPIP2;1. By anion exchange chromatography, the Ser115 kinase was purified from the soluble protein fraction isolated from spinach leaves. The Ca²⁺-dependent Ser274 kinase was purified by peptide affinity chromatography using plasma membranes isolated from spinach leaves. When characterized, the Ser115 kinase was Mg²⁺-dependent, Ca²⁺-independent and had a pH-optimum at 6.5. In accordance with previous studies using the oocyte expression system, site-directed mutagenesis and kinase and phosphatase inhibitors, the phosphorylation of Ser274, but not of Ser115, was increased in the presence of phosphatase inhibitors while kinase inhibitors decreased the phosphorylation of both Ser274 and Ser115. The molecular weight of the Ser274 kinase was approximately 50 kDa. The identification and characterization of these two protein kinases is an important step towards elucidating the signal transduction pathway for gating of the aquaporin SoPIP2;1.     

Purification and characterization of two protein kinases acting on the aquaporin SoPIP2;1

Aquaporins are water channel proteins that facilitate the movement of water and other small solutes across biological membranes. Plants usually have large aquaporin families, providing them with many ways to regulate the water transport. Some aquaporins are regulated post-translationally by phosphorylation. We have previously shown that the water channel activity of SoPIP2;1, an aquaporin in the plasma membrane of spinach leaves, was enhanced by phosphorylation at Ser115 and Ser274. These two serine residues are highly conserved in all plasma membrane aquaporins of the PIP2 subgroup. In this study we have purified and characterized two protein kinases phosphorylating Ser115 and Ser274 in SoPIP2;1. By anion exchange chromatography, the Ser115 kinase was purified from the soluble protein fraction isolated from spinach leaves. The Ca2+-dependent Ser274 kinase was purified by peptide affinity chromatography using plasma membranes isolated from spinach leaves. When characterized, the Ser115 kinase was Mg2+-dependent, Ca2+-independent and had a pH-optimum at 6.5. In accordance with previous studies using the oocyte expression system, site-directed mutagenesis and kinase and phosphatase inhibitors, the phosphorylation of Ser274, but not of Ser115, was increased in the presence of phosphatase inhibitors while kinase inhibitors decreased the phosphorylation of both Ser274 and Ser115. The molecular weight of the Ser274 kinase was approximately 50 kDa. The identification and characterization of these two protein kinases is an important step towards elucidating the signal transduction pathway for gating of the aquaporin SoPIP2;1.     

A hydrophobic patch in the competence-stimulating Peptide, a pneumococcal competence pheromone, is essential for specificity and biological activity

Induction of competence for natural genetic transformation in Streptococcus pneumoniae depends on pheromone-mediated cell-cell communication and a signaling pathway consisting of the competence-stimulating peptide (CSP), its membrane-embedded histidine kinase receptor ComD, and the cognate response regulator ComE. Extensive screening of pneumococcal isolates has revealed that two major CSP variants, CSP1 and CSP2, are found in members of this species. Even though the primary structures of CSP1 and CSP2 are about 50% identical, they are highly specific for their respective receptors, ComD1 and ComD2. In the present work, we have investigated the structural basis of this specificity by determining the three-dimensional structure of CSP1 from nuclear magnetic resonance data and comparing the agonist activity of a number of CSP1/CSP2 hybrid peptides toward the ComD1 and ComD2 receptors. Our results show that upon exposure to membrane-mimicking environments, the 17-amino-acid CSP1 pheromone adopts an amphiphilic alpha-helical configuration stretching from residue 6 to residue 12. Furthermore, the pattern of agonist activity displayed by the various hybrid peptides revealed that hydrophobic amino acids, some of which are situated on the nonpolar side of the alpha-helix, strongly contribute to CSP specificity. Together, these data indicate that the identified alpha-helix is an important structural feature of CSP1 which is essential for effective receptor recognition under natural conditions.     

Glycosylation of the self-recognizing Escherichia coli Ag43 autotransporter protein

Glycosylation is a common modulation of protein function in eukaryotes and is biologically important. However, in bacteria protein glycosylation is rare, and relatively few bacterial glycoproteins are known. In Escherichia coli only two glycoproteins have been described to date. Here we introduce a novel member to this exclusive group, namely, antigen 43 (Ag43), a self-recognizing autotransporter protein. By mass spectrometry Ag43 was demonstrated to be glycosylated by addition of heptose residues at several positions in the passenger domain. Glycosylation of Ag43 by the action of the Aah and TibC glycosyltransferases was observed in laboratory strains. Importantly, Ag43 was also found to be glycosylated in a wild-type strain, suggesting that Ag43-glycosylation may be a widespread phenomenon. Glycosylation of Ag43 does not seem to interfere with its self-associating properties. However, the glycosylated form of Ag43 enhances bacterial binding to human cell lines, whereas the nonglycosylated version of Ag43 does not to confer this property.     

Determination of the toxic potential of Bacillus cereus isolates by quantitative enterotoxin analyses

Haemolysin BL (HBL) and non-haemolytic enterotoxin (Nhe), each consisting of three components, represent the major enterotoxins produced by Bacillus cereus. To evaluate the expression of these toxins, a set of 100 B. cereus strains was examined. Molecular biological characterization showed that 42% of the strains harboured the genes for HBL and 99% for Nhe. The production of all Nhe and HBL components were analyzed using specific antibodies and, in culture supernatants, detectable levels of HBL and Nhe were found for 100% of hbl-positive and 96% of nhe-positive strains. The concentrations of the HBL-L(2) and NheB component ranged from 0.02 to 5.6 microg mL(-1) and from 0.03 to 14.2 microg mL(-1), respectively. Comparison of the amount of NheB produced by food poisoning and food/environmental strains revealed that the median value for all food poisoning strains was significantly higher than for the food/environmental isolates. The data presented in this study provide evidence that specific and quantitative determination of the enterotoxins is necessary to evaluate the toxic potential of B. cereus. In particular, the level of Nhe seems to explain most of the cytotoxic activity of B. cereus isolates and may indicate a highly diarrheic potential.