Possible role of nitric oxide in radiation-induced salivary gland dysfunction

In this study, we developed a murine model of xerostomia to elucidate the mechanism of radiation-induced salivary gland dysfunction and determined the levels of nitric oxide (NO) in the salivary glands to assess its involvement in the salivary dysfunction induced by radiation. In addition, an inhibitor of NO synthesis was administered to the model in vivo, and its effect on saliva secretion was investigated. Salivary gland irradiation at a dose of 15 Gy caused a significant decrease in secretion compared to unirradiated salivary glands. There were no marked differences between the irradiated mice and unirradiated mice in water or food consumption or in body weight changes. The NO levels in the cultured salivary gland epithelial cells were increased by treatment with a combination of interferon gamma (Ifng), interleukin 1-beta (Il1b), and tumor necrosis factor alpha (Tnfa). Irradiation increased the NO level in the salivary gland tissue. The presence of N(G)-monomethyl-l-arginine acetate (l-NMMA), an inhibitor of NO synthesis, caused a decrease in the NO level in cultured salivary gland tissues after irradiation. Administration of l-NMMA to irradiated mice improved saliva secretion. These results suggest that excessive production of NO induced by radiation is involved in the formation of radiation-induced xerostomia. The finding that administration of an inhibitor of NO synthesis ameliorated the dysfunction of irradiated salivary glands indicates that NO plays a role as a mediator of the dry mouth symptoms that occur after irradiation.     

Tumor necrosis factor-alpha from bone marrow-derived cells is not essential for the expression of adhesion molecules in lipopolysaccharide-induced nasal inflammation

Both the role and source of tumor necrosis factor (TNF-alpha) in lipopolysaccharide (LPS)-induced nasal inflammation were investigated using TNF-alpha gene deficient (TNF-alpha -/-) mice and chimeric mice that are TNF-alpha gene deficient only in bone marrow-derived cells. In the present study, intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) mRNA expression levels in the nasal mucosa were significantly decreased following intranasal instillation of LPS in TNF-alpha gene deficient mice compared to those in wild type mice. In contrast, ICAM-1 and VCAM-1 mRNA expressions were not significantly decreased although TNF-alpha mRNA expression was dramatically decreased in TNF-alpha gene deficient bone marrow-transplanted-chimeric (TNF-alpha -/--->+/+) mice compared to those in wild type bone marrow-transplanted-control (TNF-alpha +/+-->+/+) mice. These results indicate that the elevation of TNF-alpha mRNA in the nasal mucosa is mainly originated from bone marrow-derived cells. However, even low expression of TNF-alpha at local inflammation sites is sufficient to induce the expression of adhesion molecules in acute LPS-induced experimental rhinitis.     

Expression and function of the Ror-family receptor tyrosine kinases during development: lessons from genetic analyses of nematodes,mice, and humans

Receptor tyrosine kinases (RTKs) play crucial roles in various developmental processes. Ror-family RTKs are characterized by the intracellular tyrosine kinase domains, highly related to those of the Trk-family RTKs, and by the extracellular Frizzled-like cysteine-rich domains (CRDs) and Kringle domains. Rors are evolutionally conserved among Caenorhabditis elegans, Aplysia, Drosophila melanogaster, Xenopus, mice, and humans. In D. melanogaster and mammals, pairs of structurally related Rors are found, while a single Ror protein is identified in C. elegans or Aplysia. In Aplysia and D. melanogaster, Rors are expressed exclusively in developing nervous systems. On the other hand, rather widespread expression of Rors was observed in C. elegans and mammals. Mutations in Ror of C. elegans cause inappropriate axon outgrowth as well as defects in cell migration and asymmetric cell division. It has also been reported that the nematode Ror possesses kinase-dependent and kinase-independent functions. Mouse Rors, Ror1, and Ror2, are expressed mainly in migrating neural crest cells and mesenchymal cells, and Ror2-deficient mice exhibit skeletal abnormalities and ventricular septal defects in the heart. Although Ror1-deficient mice exhibit no apparent skeletal or cardiac abnormalities, Ror1/Ror2 double mutant mice show markedly enhanced skeletal and cardiac abnormalities compared with Ror2 mutant mice, indicating genetic interaction of Ror1 and Ror2. In humans, mutations within Ror2 have been found in two genetic skeletal disorders, recessive Robinow syndrome and dominant Brachydactyly type B (BDB), further emphasizing critical functions of Ror2 during developmental morphogenesis. In this article, we also discuss the signaling machinery mediated by Ror-family RTKs with a particular emphasis on our recent structure-function analyses of Ror-family RTKs.     

A linkage map of the basidiomycete Coprinus cinereus based on random amplified polymorphic DNAs and restriction fragment length polymorphisms

A genetic linkage map of the basidiomycete Coprinus cinereus was constructed on the basis of the segregation of 219 RAPD markers, 28 RFLP markers and the A and B mating-type loci among 40 random basidiospore progeny from a single cross between a wild-type homokaryon, KF(3)#2, and an AmutBmut strain, #326. Thirteen linkage groups covering a total of 1346cM were identified and correlated to the 13 chromosomes of this fungus by hybridization of RFLP and RAPD marker probes to CHEF blots. These probes also revealed chromosome length polymorphisms (CLP), which could be associated with haplotype plots of the progeny. The average kb/cM ratio in this cross was approximately 27.9kb/cM. The AmutBmut strain undergoes sexual development without mating, because of mutations in both A and B mating-type loci, and has been used to identify mutations affecting developmental processes such as dikaryosis, fruit body morphogenesis, and meiosis. The markers in the map, especially the RAPD ones, would facilitate mapping of genes responsible for such mutations induced in the AmutBmut strain.     

Beta 3-adrenergic receptor is involved in feeding regulation in chicks

We examined whether the brain beta 3-adrenergic receptor (B3-AR) is involved in the feeding regulation of chicks. Intracerebroventricular (ICV) injection of BRL37344, a B3-AR agonist, reduced food intake of chicks under ad libitum, but not fasting, feeding conditions. The ICV injection of BRL37344 did not affect chick posture or locomotion activity suggesting that BRL37344 inhibited feeding without induction of sleep-like behavior as caused by norepinephrine. Furthermore, the rectal temperature increased following the ICV injection of BRL37344. Intraperitoneal administration of BRL37344 did not reduce food intake under ad libitum feeding condition. The present study demonstrated that the brain B3-AR is involved in the inhibition of feeding in chicks. We also suggested that activation of the brain affects the energy metabolism in chicks.     

Anterograde axonal transport of endopeptidase 24.15 in rat sciatic nerves

Axonal transport of endopeptidase 24.15 (EP24.15), a putative neuropeptide degrading-enzyme, was examined in the proximal, middle, and distal segments of rat sciatic nerves using a double ligation technique. At 48h after ligation, a significant amount of the axonal transport of EP24.15 activity was found in the proximal segment, while axonal transport of deamidase activity, a lysosomal enzyme, increased in both proximal and distal segments. Western blot analysis of EP24.15 showed that EP24.15 immunoreactivity in the proximal segment was 1.8-fold higher than that in the middle segment. The immunohistochemical analysis of the segments also showed an increase in the immunoreactive EP24.15 in the proximal segment in comparison with that in the middle segment. In the distal segment, no axonal transport of EP24.15 was found in all methods examined, indicating that EP24.15 is mainly transported by an anterograde axonal flow. These observations suggest that EP24.15 may be involved in the metabolism of neuropeptides in nerve terminals or synaptic clefts.     

Mammalian twisted gastrulation is essential for skeleto-lymphogenesis

Dorsoventral patterning depends on the local concentrations of the morphogens. Twisted gastrulation (TSG) regulates the extracellular availability of a mesoderm inducer, bone morphogenetic protein 4 (BMP-4). However, TSG function in vivo is still unclear. We isolated a TSG cDNA as a secreted molecule from the mouse aorta-gonad-mesonephros region. Here we show that TSG-deficient mice were born healthy, but more than half of the neonatal pups showed severe growth retardation shortly after birth and displayed dwarfism with delayed endochondral ossification and lymphopenia, followed by death within a month. TSG-deficient thymus was atrophic, and phosphorylation of SMAD1 was augmented in the thymocytes, suggesting enhanced BMP-4 signaling in the thymus. Since BMP-4 promotes skeletogenesis and inhibits thymus development, our findings suggest that TSG acts as both a BMP-4 agonist in skeletogenesis and a BMP-4 antagonist in T-cell development. Although lymphopenia in TSG-deficient mice would partly be ascribed to systemic effects of runtiness and wasting, our findings may also provide a clue for understanding the pathogenesis of human dwarfism with combined immunodeficiency.     

Collection and analysis of hematopoietic progenitor cells from cynomolgus macaques (Macaca fascicularis): assessment of cross-reacting monoclonal antibodies

Previous studies have shown that hematopoietic progenitor cells can be isolated from human or nonhuman primate bone marrow (BM) cells. In the present study, we studied the cross-reactivity of 13 anti-human CD34, two anti-human c-Kit, and one anti-human CD133 monoclonal antibodies (mAbs) with cynomolgus macaque (Macaca fascicularis) BM cells, using flow cytometric analysis, cell enrichment, and clonogenic assay. Among the 13 anti-human CD34 mAbs assessed, six cross-reacted as previously reported by other groups. However, only three of these six mAbs (clones 561, 563, and 12.8) recognized cynomolgus CD34+ cells that formed progenitor colonies when grown in methylcellulose culture. Similarly, of the two anti-human c-Kit mAbs (clones NU-c-kit and 95C3) that were previously reported to cross-react with cynomolgus BM cells, only one (clone NU-c-kit) resulted in a similar outcome. The anti-human CD133 mAb (clone AC133) also cross-reacted with cynomolgus BM cells, although these cells did not give rise to colonies when grown in culture. These results suggest that antibodies that cross-react with nonhuman primate cells may not identify the hematopoietic cells of interest. In addition, while the CD34 mAb (clone 561) results in the selection of hematopoietic progenitor cells of all lineages when assessed in methylcellulose culture, the c-Kit(high) fraction (NU-c-kit) exclusively identifies erythroid-specific progenitor cells after growth in culture. It is important to consider these findings when selecting cross-reacting mAbs to identify cells of hematopoietic lineages in macaque species.     

Does enhanced expression of the Na+-CA2+ exchanger increase myocardial vulnerability to ischemia/reperfusion injury in rabbit hearts?

The present study focused on examining the efficacy of feeding a rutin-glucose derivative (G-rutin) to inhibit glycation reactions that can occur in muscle, kidney and plasma proteins of diabetic rats. Both thiobarbituric acid-reactive substance levels and protein carbonyl contents in muscle and kidney were significantly (p < 0.05) reduced in streptozotocin-induced diabetic rats fed G-rutin supplemented diet, compared to diabetic rats fed control diet. The N -fructoselysine content in muscle and kidney, a biomarker of early glycation reaction, was markedly (p < 0.05) increased by diabetes, but significantly (p < 0.05) reduced in diabetic rats fed G-rutin. Advanced glycation end-products (AGEs) in serum and kidney protein were measured by immunoblot using anti-AGE antibody, and were also reduced in diabetic rats fed dietary G-rutin. Feeding G-rutin also slightly inhibited aldose reductase activity in these animals. These results demonstrate for the first time that dietary G-rutin consumption can provide potential health benefits that are related to the inhibition of tissue glycation reactions common to diabetes.