Cyclic ADP-ribose (cADPR) is a natural compound that mobilizes calcium ions in several eukaryotic cells. Although it can lead to the release of calcium ions in T lymphocytes, it has not been firmly established as a second messenger in these cells. Here, using high-performance liquid chromatography analysis, we show that stimulation of the T-cell receptor/CD3 (TCR/CD3) complex results in activation of a soluble ADP-ribosyl cyclase and a sustained increase in intracellular levels of cADPR. There is a causal relation between increased cADPR concentrations, sustained calcium signalling and activation of T cells, as shown by inhibition of TCR/CD3-stimulated calcium signalling, cell proliferation and expression of the early- and late-activation markers CD25 and HLA-DR by using cADPR antagonists. The molecular target for cADPR, the type-3 ryanodine receptor/calcium channel, is expressed in T cells. Increased cADPR significantly and specifically stimulates the apparent association of [3H]ryanodine with the type-3 ryanodine receptor, indicating a direct modulatory effect of cADPR on channel opening. Thus we show the presence, causal relation and biological significance of the major constituents of the cADPR/calcium-signalling pathway in human T cells.

It was the aim of this study to further explore the functional role of vitamin D in the endocrine pancreas. By gene targeting, we have recently generated mice in which a lacZ reporter gene is driven by the endogenous vitamin D receptor (VDR) promoter. These mice express a functionally inactive mutant VDR. Pancreatic islets but not exocrine pancreas cells showed strong lacZ reporter gene expression in mutant mice. To rule out possible influences of hypocalcemia on pancreatic endocrine function, a rescue diet enriched with calcium, phosphorus, and lactose was fed to wild-type (WT) and VDR mutant mice. The rescue diet normalized body weight and mineral homeostasis in VDR mutants. In glucose tolerance tests, baseline blood glucose levels were unchanged in fasting VDR mutants. However, blood glucose was elevated after oral or subcutaneous glucose loading, and maximum serum insulin levels were reduced by approximately 60% in VDR mutants vs. WT mice on either diet. In addition, insulin mRNA levels were decreased in VDR mutant mice on both diets, whereas pancreatic beta cell mass, islet architecture, and islet neogenesis were normal. These findings clearly establish a molecular role of the vitamin D-responsive elements in pancreatic insulin synthesis and secretion in vivo.

The vitamin D hormone 1,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)], the biologically active form of vitamin D, is essential for an intact mineral metabolism. Using gene targeting, we sought to generate vitamin D receptor (VDR) null mutant mice carrying the reporter gene lacZ driven by the endogenous VDR promoter. Here we show that our gene-targeted mutant mice express a VDR with an intact hormone binding domain, but lacking the first zinc finger necessary for DNA binding. Expression of the lacZ reporter gene was widely distributed during embryogenesis and postnatally. Strong lacZ expression was found in bones, cartilage, intestine, kidney, skin, brain, heart, and parathyroid glands. Homozygous mice are a phenocopy of mice totally lacking the VDR protein and showed growth retardation, rickets, secondary hyperparathyroidism, and alopecia. Feeding of a diet high in calcium, phosphorus, and lactose normalized blood calcium and serum PTH levels, but revealed a profound renal calcium leak in normocalcemic homozygous mutants. When mice were treated with pharmacological doses of vitamin D metabolites, responses in skin, bone, intestine, parathyroid glands, and kidney were absent in homozygous mice, indicating that the mutant receptor is nonfunctioning and that vitamin D signaling pathways other than those mediated through the classical nuclear receptor are of minor physiological importance. Furthermore, rapid, nongenomic responses to 1,25-(OH)(2)D(3) in osteoblasts were abrogated in homozygous mice, supporting the conclusion that the classical VDR mediates the nongenomic actions of 1,25-(OH)(2)D(3).

Ca 2ϩ is a universal messenger from bacterial to mammalian cells since its concentration gradients across both organelle and plasma membranes can be efficiently used to communicate biological signals. Therefore, the control of the intracellular free Ca 2ϩ concentration [Ca 2ϩ ] i 3 is of crucial importance for the regulation of many cellular functions, including proliferation, contraction, fertilization, motility, apoptosis, and cell death (1). Receptor-mediated Ca 2ϩ influx from the extracellular space is one important mechanism to control [Ca 2ϩ ] i in non-excitable cells, e.g. leukocytes (2). Although the molecular machinery underlying Ca 2ϩ entry is still poorly defined, cation channels of the transient receptor potential (TRP) family that includes several subfamilies (3-5) are likely candidates for Ca 2ϩ entry pathways under the control of membrane receptors. TRPM2 (formerly LTRPC2 and TRPC7) is a member of the TRPM subfamily. TRPM2 forms non-selective Ca 2ϩ -permeable cation channels and is mainly expressed in brain and in cells of the immune system (6 -8). Opening of the channel is induced by intracellular ADP-ribose (ADPR; Refs. 6 and 7) and enhanced by increased cytosolic Ca 2ϩ (9). Whether NAD also activates TRPM2 currents is still controversial (6, 7, 10, 11). The nudix box in the cytosolic C-terminal region of TRPM2, a conserved motif of enzymes with nucleotide pyrophosphatase activity, seems to be responsible for gating of TRPM2 by ADPR and possibly by NAD (6,7,12). An involvement of TRPM2 in cellular signaling processes has been proposed since the expression of TRPM2 confers susceptibility to oxidant-induced cell death (11).A key question in the field relates to the potential role of the NAD metabolite ADPR as a second messenger. A function of NAD or ADPR as the missing link between specific extracellular signals and Ca 2ϩ influx mediated by TRPM2 has been hypothesized (8,13,14), but experimental proofs are missing so far. To test this hypothesis directly, we developed a method to measure intracellular levels of ADPR in Jurkat T cells (15). We report that cytosolic ADPR concentrations are raised in response to concanavalin A (ConA) and induce Ca 2ϩ entry through TRPM2, thereby significantly increasing [Ca 2ϩ ] i . Inhibition of intracellular ADPR formation or gene silencing of TRPM2 efficiently diminished receptor-mediated Ca 2ϩ influx carried by TRPM2. Moreover, blockade of ADPR formation also efficiently blocked ConA-induced cell death. EXPERIMENTAL PROCEDURESElectrophysiology-Membrane currents were recorded in the wholecell configuration of the patch clamp technique (16) or the perforatedpatch configuration with nystatin (17). An EPC9 patch clamp amplifier was used in conjunction with the PULSE stimulation and data acquisition software (HEKA Elektronik, Lamprecht, Germany). The patch electrodes were made from 1.5-mm diameter borosilicate glass capillaries and filled with intracellular solution. Data were low pass-filtered at 1 kHz and compensated for both fast and slow capacity transients. Se...

Canine infectious respiratory disease (CIRD) is an acute, highly contagious disease complex caused by a variety of infectious agents. At present, the role of viral and bacterial components as primary or secondary pathogens in CIRD is not fully understood. The aim of this study was to investigate the prevalence of canine parainfluenza virus (CPIV), canine adenovirus type 2 (CAV-2), canine influenza virus (CIV), canine respiratory coronavirus (CRCoV), canine herpes virus-1 (CHV-1), canine distemper virus (CDV) and Bordetella bronchiseptica in dogs with CIRD and to compare the data with findings in healthy dogs. Sixty-one dogs with CIRD and 90 clinically healthy dogs from Southern Germany were prospectively enrolled in this study. Nasal and pharyngeal swabs were collected from all dogs and were analysed for CPIV, CAV-2, CIV, CRCoV, CHV-1, CDV, and B. bronchiseptica by real-time PCR. In dogs with acute respiratory signs, 37.7% tested positive for CPIV, 9.8% for CRCoV and 78.7% for B. bronchiseptica. Co-infections with more than one agent were detected in 47.9% of B. bronchiseptica-positive, 82.6% of CPIV-positive, and 100% of CRCoV-positive dogs. In clinically healthy dogs, 1.1% tested positive for CAV-2, 7.8% for CPIV and 45.6% for B. bronchiseptica. CPIV and B. bronchiseptica were detected significantly more often in dogs with CIRD than in clinically healthy dogs (P < 0.001 for each pathogen) and were the most common infectious agents in dogs with CIRD in Southern Germany. Mixed infections with several pathogens were common. In conclusion, clinically healthy dogs can carry respiratory pathogens and could act as sources of infection for susceptible dogs.

No abstract

The role of bacterial communities in canine nasal disease has not been studied so far using next generation sequencing methods. Sequencing of bacterial 16S rRNA genes has revealed that the canine upper respiratory tract harbors a diverse microbial community; however, changes in the composition of nasal bacterial communities in dogs with nasal disease have not been described so far. Aim of the study was to characterize the nasal microbiome of healthy dogs and compare it to that of dogs with histologically confirmed nasal neoplasia and chronic rhinitis. Nasal swabs were collected from healthy dogs (n = 23), dogs with malignant nasal neoplasia (n = 16), and dogs with chronic rhinitis (n = 8). Bacterial DNA was extracted and sequencing of the bacterial 16S rRNA gene was performed. Data were analyzed using Quantitative Insights Into Microbial Ecology (QIIME). A total of 376 Operational Taxonomic Units out of 26 bacterial phyla were detected. In healthy dogs, Moraxella spp. was the most common species, followed by Phyllobacterium spp., Cardiobacteriaceae, and Staphylococcus spp. While Moraxella spp. were significantly decreased in diseased compared to healthy dogs (p = 0.005), Pasteurellaceae were significantly increased (p = 0.001). Analysis of similarities used on the unweighted UniFrac distance metric (p = 0.027) was significantly different when nasal microbial communities of healthy dogs were compared to those of dogs with nasal disease. The study showed that the canine nasal cavity is inhabited by a highly species-rich bacterial community, and suggests significant differences between the nasal microbiome of healthy dogs and dogs with nasal disease.

N1-[(5' '-O-Phosphorylethoxy)methyl]-5'-O-phosphorylinosine 5',5''-cyclicpyrophosphate (cIDPRE 2a) and the 8-substituted derivatives 8-bromo-, 8-azido-, 8-amino-, and 8-Cl-cIDPRE (2b-e) were synthesized from N1-[(5''-acetoxyethoxy)methyl]-2',3'-O-isopropylideneinosine (5) in good yields. The pharmacological activities of cIDPRE and the 8-substituted derivatives (2a-e) were analyzed in intact and permeabilized human Jurkat T-lymphocytes. The results indicate that cIDPRE permeates the plasma membrane, releases Ca2+ from an intracellular, cADPR-sensitive Ca2+ store, and subsequently initiates Ca2+ release-activated Ca2+ entry. The Ca(2+)-releasing activity of cIDPRE was confirmed directly in permeabilized cells. Using time-resolved confocal Ca2+ imaging at the single cell level, the development of global Ca2+ signals starting from local small Ca2+ signals evoked by cIDPRE was observed. 8-N3-cIDPRE 2c and 8-NH2-cIDPRE 2d were similarly effective in their agonistic activity as compared to cIDPRE 2a, showing almost indistinguishable concentration-response curves for 2a, 2c, and 2d and very similar kinetics of Ca2+ signaling. In contrast, the halogenated derivatives 8-Br- and 8-Cl-cIDPRE (2b and 2e) did not significantly elevate [Ca2+]i. Therefore, cIDPRE 2a, 8-N3-cIDPRE 2c, and 8-NH2-cIDPRE 2d are novel membrane permeant cADPR mimic and may provide important novel tools to study cADPR-mediated Ca2+ signaling in intact cells.