Congenic analysis of the NKT cell control gene Nkt2 implicates the peroxisomal protein Pxmp4

Type 1 NKT cells play a critical role in controlling the strength and character of adaptive and innate immune responses. We have previously reported deficiencies in the numbers and function of NKT cells in the NOD mouse strain, which is a well-validated model of type 1 diabetes and systemic lupus erythematosus. Genetic control of thymic NKT cell numbers was mapped to two linkage regions: Nkt1 on distal chromosome 1 and Nkt2 on chromosome 2. Herein, we report the production and characterization of a NOD.Nkrp1(b).Nkt2b(b) congenic mouse strain, which has increased thymic and peripheral NKT cells, a decreased incidence of type 1 diabetes, and enhanced cytokine responses in vivo and increased proliferative responses in vitro following challenge with alpha-galactosylceramide. The 19 highly differentially expressed candidate genes within the congenic region identified by microarray expression analyses included Pxmp4. This gene encodes a peroxisome-associated integral membrane protein whose only known binding partner is Pex19, an intracellular chaperone and component of the peroxisomal membrane insertion machinery encoded by a candidate for the NKT cell control gene Nkt1. These findings raise the possibility that peroxisomes play a role in modulating glycolipid availability for CD1d presentation, thereby influencing NKT cell function.     

Density and population estimate of gibbons (Hylobates albibarbis) in the Sabangau catchment, Central Kalimantan, Indonesia

We demonstrate that although auditory sampling is a useful tool, this method alone will not provide a truly accurate indication of population size, density and distribution of gibbons in an area. If auditory sampling alone is employed, we show that data collection must take place over a sufficient period to account for variation in calling patterns across seasons. The population of Hylobates albibarbis in the Sabangau catchment, Central Kalimantan, Indonesia, was surveyed from July to December 2005 using methods established previously. In addition, auditory sampling was complemented by detailed behavioural data on six habituated groups within the study area. Here we compare results from this study to those of a 1-month study conducted in 2004. The total population of the Sabangau catchment is estimated to be about in the tens of thousands, though numbers, distribution and density for the different forest subtypes vary considerably. We propose that future density surveys of gibbons must include data from all forest subtypes where gibbons are found and that extrapolating from one forest subtype is likely to yield inaccurate density and population estimates. We also propose that auditory census be carried out by using at least three listening posts (LP) in order to increase the area sampled and the chances of hearing groups. Our results suggest that the Sabangau catchment contains one of the largest remaining contiguous populations of Bornean agile gibbon.     

Histoplasma capsulatum cyclophilin A mediates attachment to dendritic cell VLA-5

Histoplasma capsulatum (Hc) is a pathogenic fungus that replicates in macrophages (Mphi). In dendritic cells (DC), Hc is killed and fungal Ags are processed and presented to T cells. DC recognize Hc yeasts via the VLA-5 receptor, whereas Mphi recognize yeasts via CD18. To identify ligand(s) on Hc recognized by DC, VLA-5 was used to probe a Far Western blot of a yeast freeze/thaw extract (F/TE) that inhibited Hc binding to DC. VLA-5 recognized a 20-kDa protein, identified as cyclophilin A (CypA), and CypA was present on the surface of Hc yeasts. rCypA inhibited the attachment of Hc to DC, but not to Mphi. Silencing of Hc CypA by RNA interference reduced yeast binding to DC by 65-85%, but had no effect on binding to Mphi. However, F/TE from CypA-silenced yeasts still inhibited binding of wild-type Hc to DC, and F/TE from wild-type yeasts depleted of CypA also inhibited yeast binding to DC. rCypA did not further inhibit the binding of CypA-silenced yeasts to DC. Polystyrene beads coated with rCypA or fibronectin bound to DC and Mphi and to Chinese hamster ovary cells transfected with VLA-5. Binding of rCypA-coated beads, but not fibronectin-coated beads, was inhibited by rCypA. These data demonstrate that CypA serves as a ligand for DC VLA-5, that binding of CypA to VLA-5 is at a site different from FN, and that there is at least one other ligand on the surface of Hc yeasts that mediates binding of Hc to DC.     

Differential gene expression of TRPM1, the potential cause of congenital stationary night blindness and coat spotting patterns (LP) in the Appaloosa horse (Equus caballus)

The appaloosa coat spotting pattern in horses is caused by a single incomplete dominant gene (LP). Homozygosity for LP (LP/LP) is directly associated with congenital stationary night blindness (CSNB) in Appaloosa horses. LP maps to a 6-cM region on ECA1. We investigated the relative expression of two functional candidate genes located in this LP candidate region (TRPM1 and OCA2), as well as three other linked loci (TJP1, MTMR10, and OTUD7A) by quantitative real-time RT-PCR. No large differences were found for expression levels of TJP1, MTMR10, OTUD7A, and OCA2. However, TRPM1 (Transient Receptor Potential Cation Channel, Subfamily M, Member 1) expression in the retina of homozygous appaloosa horses was 0.05% the level found in non-appaloosa horses (R = 0.0005). This constitutes a >1800-fold change (FC) decrease in TRPM1 gene expression in the retina (FC = -1870.637, P = 0.001) of CSNB-affected (LP/LP) horses. TRPM1 was also downregulated in LP/LP pigmented skin (R = 0.005, FC = -193.963, P = 0.001) and in LP/LP unpigmented skin (R = 0.003, FC = -288.686, P = 0.001) and was downregulated to a lesser extent in LP/lp unpigmented skin (R = 0.027, FC = -36.583, P = 0.001). TRP proteins are thought to have a role in controlling intracellular Ca(2+) concentration. Decreased expression of TRPM1 in the eye and the skin may alter bipolar cell signaling as well as melanocyte function, thus causing both CSNB and LP in horses.     

A facile reporter system for the experimental identification of twin-arginine translocation (Tat) signal peptides from all kingdoms of life

We have developed a reporter protein system for the experimental verification of twin-arginine signal peptides. This reporter system is based on the Streptomyces coelicolor agarase protein, which is secreted into the growth medium by the twin-arginine translocation (Tat) pathway and whose extracellular activity can be assayed colorimetrically in a semiquantitative manner. Replacement of the native agarase signal peptide with previously characterized twin-arginine signal peptides from other Gram-positive and Gram-negative bacteria resulted in efficient Tat-dependent export of agarase. Candidate twin-arginine signal peptides from archaeal proteins as well as plant thylakoid-targeting sequences were also demonstrated to mediate agarase translocation. A naturally occurring variant signal peptide with an arginine-glutamine motif instead of the consensus di-arginine was additionally recognized as a Tat-targeting sequence by Streptomyces. Application of the agarase assay to previously uncharacterized candidate Tat signal peptides from Bacillus subtilis identified two further probable Tat substrates in this organism. This is the first versatile reporter system for Tat signal peptide identification.     

A minimal Tat system from a gram-positive organism: a bifunctional TatA subunit participates in discrete TatAC and TatA complexes

The Tat system transports folded proteins across bacterial and thylakoid membranes. In Gram-negative organisms, a TatABC substrate-binding complex and separate TatA complex are believed to coalesce to form an active translocon, with all three subunits essential for translocation. Most Gram-positive organisms lack a tatB gene, indicating major differences in organization and possible differences in mode of action. Here, we have studied Tat complexes encoded by the tatAdCd genes of Bacillus subtilis. Expression of tatAdCd in an Escherichia coli tat null mutant results in efficient export of a large, cofactor-containing E. coli Tat substrate, TorA. We show that the tatAd gene complements E. coli mutants lacking either tatAE or tatB, indicating a bifunctional role for this subunit in B. subtilis. Second, we have identified and characterized two distinct Tat complexes that are novel in key respects: a TatAdCd complex of approximately 230 kDa that is significantly smaller than the analogous E. coli TatABC complex (approximately 370 kDa on BN gels) and a separate TatAd complex. The latter is a discrete entity of approximately 270 kDa as judged by gel filtration chromatography, very different from the highly heterogeneous E. coli TatA complex that ranges in size from approximately 50 kDa to over 600 kDa. TatA heterogeneity has been linked to the varying size of Tat substrates being translocated, but the singular nature of the B. subtilis TatAd complex suggests that discrete TatAC and TatA complexes may form a single form of translocon.     

Distribution of beta-glucosidase and beta-glucuronidase activity and of beta-glucuronidase gene gus in human colonic bacteria

beta-Glycosidase activities present in the human colonic microbiota act on glycosidic plant secondary compounds and xenobiotics entering the colon, with potential health implications for the human host. Information on beta-glycosidases is currently limited to relatively few species of bacteria from the human colonic ecosystem. We therefore screened 40 different bacterial strains that are representative of dominant bacterial groups from human faeces for beta-glucosidase and beta-glucuronidase activity. More than half of the low G+C% Gram-positive firmicutes harboured beta-glucosidase activity, while beta-glucuronidase activity was only found in some firmicutes within clostridial clusters XIVa and IV. Most of the Bifidobacterium spp. and Bacteroides thetaiotaomicron carried beta-glucosidase activity. A beta-glucuronidase gene belonging to family 2 glycosyl hydrolases was detected in 10 of the 40 isolates based on degenerate PCR. These included all nine isolates that gave positive assays for beta-glucuronidase activity, suggesting that the degenerate PCR could provide a useful assay for the capacity to produce beta-glucuronidase in the gut community. beta-Glucuronidase activity was induced by growth on d-glucuronic acid, or by addition of 4-nitrophenol-glucuronide, in Roseburia hominis A2-183, while beta-glucosidase activity was induced by 4-nitrophenol-glucopyranoside. Inducibility varied between strains.     

Long-term high-fat feeding induces greater fat storage in mice lacking UCP3

Uncoupling protein-3 (UCP3) is a mitochondrial inner-membrane protein highly expressed in skeletal muscle. While UCP3's function is still unknown, it has been hypothesized to act as a fatty acid (FA) anion exporter, protecting mitochondria against lipid peroxidation and/or facilitating FA oxidation. The aim of this study was to determine the effects of long-term feeding of a 45% fat diet on whole body indicators of muscle metabolism in congenic C57BL/6 mice that were either lacking UCP3 (Ucp3(-/-)) or had a transgenically induced approximately twofold increase in UCP3 levels (UCP3tg). Mice were fed the high-fat (HF) diet for a period of either 4 or 8 mo immediately following weaning. After long-term HF feeding, UCP3tg mice weighed an average of 15% less than wild-type mice (P < 0.05) and were 20% less metabolically efficient than both wild-type and Ucp3(-/-) mice (P < 0.01). Additionally, wild-type mice had 21% lower, whereas UCP3tg mice had 36% lower, levels of adiposity compared with Ucp3(-/-) mice (P < 0.05 and P < 0.001, respectively), indicating a protective effect of UCP3 against fat gain. No differences in whole body oxygen consumption were detected following long-term HF feeding. Glucose and insulin tolerance tests revealed that both the UCP3tg and Ucp3(-/-) mice were more glucose tolerant and insulin sensitive compared with wild-type mice after short-term HF feeding, but this protection was not maintained in the long term. Findings indicate that UCP3 is involved in protection from fat gain induced by long-term HF feeding, but not in protection from insulin resistance.