In situ detection of neural progenitor cells including stem-like cells is essential for studying the basic mechanisms of the generation of cellular diversity in the CNS, upon which therapeutic treatments for CNS injuries, degenerative diseases, and brain tumors may be based. We have generated rat monoclonal antibodies (Mab 14H1 and 14B8) that recognize an RNA-binding protein Musashi1, but not a Musashi1-related protein, Musashi2. The amino acid sequences at the epitope sites of these anti-Musashi1 Mabs are remarkably conserved among the human, mouse, and Xenopus proteins. Spatiotemporal patterns of Musashi1 immunoreactivity in the developing and/or adult CNS tissues of frogs, birds, rodents, and humans indicated that our anti-Musashi1 Mabs reacted with undifferentiated, proliferative cells in the CNS of all the vertebrates tested. Double or triple immunostaining of embryonic mouse brain cells in monolayer cultures demonstrated strong Musashi1 expression in Nestin(+)/RC2(+) cells. The relative number of Musashi1(+)/Nestin(+)/RC2(+) cells increased fivefold when embryonic forebrain cells were cultured to form ‘neurospheres’ in which stem-like cells are known to be enriched through their self-renewing mode of growth. Nestin(+)/RC2(–) cells, which included Tα1-GFP(+) neuronal progenitor cells and GLAST(+) astroglial precursor cells, were also Musashi1(+), as were GFAP(+) astrocytes. Young neurons showed a trace of Musashi1 expression. Cells committed to the oligodendroglial lineage were Musashi(–). Musashi1 was localized to the perikarya of CNS stem-like cells and non-oligodendroglial progenitor cells without shifting to cell processes or endfeet, and is therefore advantageous for identifying each cell and counting cells in situ.

In a randomized, double-blind, placebo-controlled study in 64 subjects with Huntington disease (HD), 8 g/day of creatine administered for 16 weeks was well tolerated and safe. Serum and brain creatine concentrations increased in the creatine-treated group and returned to baseline after washout. Serum 8-hydroxy-2'-deoxyguanosine (8OH2'dG) levels, an indicator of oxidative injury to DNA, were markedly elevated in HD and reduced by creatine treatment.

Hydrogen sulfide (H 2 S) was historically recognized as a toxic gas generated by natural resources. However, its enzymatic production from L-cysteine has recently been demonstrated in mammals. Cystathionine ␤-synthase and cystathionine ␥-lyase, both of which can produce H 2 S, were expressed in mouse pancreatic islet cells and the ␤-cell line, MIN6. L-Cysteine and the H 2 S donor NaHS inhibited glucose-induced insulin release from islets and MIN6 cells. These inhibitory effects were reproduced when insulin release was stimulated by ␣-ketoisocaproate, tolbutamide, or high K ؉

a b s t r a c tWe examined the expression of the major H 2 S-producing enzymes, cystathionine-b-synthase (CBS) and cystathionine-c-lyase (CSE). CBS was ubiquitously distributed in the mouse pancreas, but CSE was found only in the exocrine. Freshly isolated islets expressed CBS, while CSE was faint. However, high glucose increased the CSE expression in the beta-cells. L-Cysteine or NaHS suppressed islet cell apoptosis with high glucose, and increased glutathione content in MIN6 beta-cells. Pretreatment with L-cysteine improved the secretory responsiveness following stimulation with glucose. The CSE inhibitor DL-propargylglycine antagonized these L-cysteine effects. We suggest H 2 S may function as an 'intrinsic brake' which protects beta-cells from glucotoxicity.

We generated a series of knockin mouse lines, in which the cytokine receptor gp130-dependent STAT3 and/or SHP2 signals were disrupted, by replacing the mouse gp130 gene with human gp130 mutant cDNAs. The SHP2 signal-deficient mice (gp130F759/F759 were born normal but displayed splenomegaly and lymphadenopathy and an enhanced acute phase reaction. In contrast, the STAT3 signal-deficient mice (gp130FXQ/FXXQ) died perinatally, like the gp130-deficient mice (gp130D/D). The gp130F759/F759 mice showed prolonged gp130-induced STAT3 activation, indicating a negative regulatory role for SHP2. Th1-type cytokine production and IgG2a and IgG2b production were increased in the gp130F759/F759 mice, while they were decreased in the gp130FXXQ/FXXQ immune system. These results indicate that the balance of positive and negative signals generated through gp130 regulates the immune responses.

Rab27a is involved in the control of membrane traffic, a crucial step in the regulated secretion. Typically, the guanosine triphosphate (GTP)-bound form has been considered to be active and, therefore, searching for proteins binding to the GTPform has been attempted to look for their effectors. Here, we have identified the actin-bundling protein coronin 3 as a novel Rab27a effector that paradoxically bound guanosine diphosphate (GDP)-Rab27a in the pancreatic β-cell line MIN6. Coronin 3 directly bound GDP-Rab27a through its β-propeller structure. The most important insulin secretagogue glucose promptly shifted Rab27a from the GTP-to GDP-bound form. Knockdown of coronin 3 by RNAi resulted in the inhibition of phogrin (an insulin-granule-associated protein) internalization and the uptake of FM4-64 (a marker of endocytosis). Similar results were reproduced by disruption of the coronin-3-GDPRab27a interaction with the dominant-negative coronin 3, and coexpression of the GDP-Rab27a mutant rescued these changes. Taken together, our results indicate that interaction of GDPRab27a and coronin 3 is important in stimulus-endocytosis coupling, and that GTP-and GDP-Rab27a regulates insulin membrane recycling at the distinct stages. Supplementary material available online at

The aim of this study was to investigate the in vivo and in vitro effects of exendin-4, a potent glucagon-like peptide 1 agonist, on the protection of the pancreatic b-cells against their cell death. In in vivo experiments, we used b-cell-specific calmodulinoverexpressing mice where massive apoptosis takes place in their b-cells, and we examined the effects of chronic treatment with exendin-4. Chronic and s.c. administration of exendin-4 reduced hyperglycemia. The treatment caused significant increases of the insulin contents of the pancreas and islets, and retained the insulin-positive area. Dispersed transgenic islet cells lived only shortly, and several endoplasmic reticulum (ER) stress-related molecules such as immunoglobulin-binding protein (Bip), inositol-requiring enzyme-1 a, X-box-binding protein-1 (XBP-1), RNA-activated protein kinase-like endoplasmic reticulum kinase, activating transcription factor-4, and C/EBP-homologous protein (CHOP) were more expressed in the transgenic islets. We also found that the spliced form of XBP-1, a marker of ER stress, was also increased in b-cellspecific calmodulin-overexpressing transgenic islets. In the quantitative real-time PCR analyses, the expression levels of Bip and CHOP were reduced in the islets from the transgenic mice treated with exendin-4. These findings suggest that excess of ER stress occurs in the transgenic b-cells, and the suppression of ER stress and resultant protection against cell death may be involved in the anti-diabetic effects of exendin-4.

Kaneko, Yukiko, Tomohisa Ishikawa, Satoshi Amano, and Koichi Nakayama. Dual effect of nitric oxide on cytosolic Ca 2ϩ concentration and insulin secretion in rat pancreatic ␤-cells. Am J Physiol Cell Physiol 284: C1215-C1222, 2003. First published January 15, 2003 10.1152/ajpcell.00223.2002In isolated rat pancreatic ␤-cells, the nitric oxide (NO) donor NOC-7 at 1 M reduced the amplitude of the oscillations of cytosolic Ca 2ϩ concentration ([Ca 2ϩ ]c) induced by 11.1 mM glucose, and at 10 M terminated them. In the presence of N G -nitro-L-arginine (L-NNA), however, NOC-7 at 0.5 and 1 M increased the amplitude of the [Ca 2ϩ ]c oscillations, although the NO donor at 10 M still suppressed them. Aqueous NO solution also had a dual effect on the [Ca 2ϩ ]c oscillations. The soluble guanylate cyclase inhibitor LY-83583 and the cGMP-dependent protein kinase inhibitor KT5823 inhibited the stimulatory effect of NO, and 8-bromo-cGMP increased the amplitude of the [Ca 2ϩ ]c oscillations. Patch-clamp analyses in the perforated configuration showed that 8-bromo-cGMP inhibited whole cell ATP-sensitive K ϩ currents in the isolated rat pancreatic ␤-cells, suggesting that the inhibition by cGMP of ATP-sensitive K ϩ channels is, at least in part, responsible for the stimulatory effect of NO on the [Ca 2ϩ ]c oscillations. In the presence of L-NNA, the glucose-induced insulin secretion from isolated islets was facilitated by 0.5 M NOC-7, whereas it was suppressed by 10 M NOC-7. These results suggest that NO facilitates glucose-induced [Ca 2ϩ ]c oscillations of ␤-cells and insulin secretion at low concentrations, which effects are mediated by cGMP, whereas NO inhibits them in a cGMP-independent manner at high concentrations.islets of Langerhans; calcium oscillations; guanosine 3Ј,5Ј-cyclic monophosphate; NOC-7; glucose NITRIC OXIDE (NO) produced from L-arginine by NO synthase (NOS) is an important regulator of various physiological and pathological functions in various types of cells. In pancreatic islets, a large amount of NO generated by inducible NOS (iNOS) has been postulated to be involved in ␤-cell degeneration in the process of insulin-dependent diabetes mellitus (1,6,22). On the other hand, recent immunostaining studies clearly demonstrated the presence of constitutive NOS (cNOS), which is activated by Ca 2ϩ and calmodulin, in rat and mouse pancreatic islet cells, i.e., neuronal NOS in ␣-, ␤-, and ␦-cells (3, 25) and endothelial NOS in ␣-and ␦-cells (37). Furthermore, direct biochemical evidence for the cNOS enzyme activity was obtained from isolated islets of mice (33). These findings suggest a physiological involvement of NO in the regulation of the hormone secretion from pancreatic islet cells.By binding to iron in the heme at the active site of soluble guanylate cyclase (sGC), NO activates the enzyme, which results in elevation of the cGMP level. Given that the enzyme activities of guanylate cyclase (16) and cGMP-dependent protein kinase have been demonstrated in pancreatic islets (23), it is plausible that NO ...