Characteristic elevation of soluble TNF receptor II : I ratio in macrophage activation syndrome with systemic juvenile idiopathic arthritis.

To investigate the clinical significance of soluble tumour necrosis factor receptor (sTNF-R) II/I ratio as an indicator of the diagnosis of macrophage activation syndrome (MAS) complicating systemic juvenile idiopathic arthritis (s-JIA), we measured the serum sTNF-RI and II levels in 117 patients with s-JIA, including 29 patients with MAS, 15 with Epstein-Barr virus-induced haemophagocytic lymphohistiocytosis (EBV-HLH), 15 with Kawasaki disease (KD) and 28 healthy controls (HCs). We determined their correlation with measurements of disease activity and severity. Furthermore, we measured serum interleukin (IL)-18 levels in patients with EBV-HLH and compared these in levels in patients with MAS. The sTNF-RII/I ratio was elevated significantly in MAS and EBV-HLH patients compared with those in the acute phase of s-JIA and KD patients, whereas there were no significant differences between HCs and those in the acute phase of s-JIA. The sTNF-RII/I ratio increased profoundly as MAS developed and correlated positively with disease activity. Serum IL-18 levels were elevated significantly in MAS patients compared with EBV-HLH patients. The monitoring of serum IL-18 and sTNF-RII/I might be useful for the diagnosis of MAS and the differentiation between MAS and EBV-HLH.

Telmisartan attenuates fatty-acid-induced oxidative stress and NAD(P)H oxidase activity in pancreatic beta-cells.

AIM: Angiotensin II receptor blockers (ARB) have been shown to lower insulin resistance in obese diabetic animal models and to reduce the risk of new-onset diabetes in hypertensive patients. In the present study, we studied whether telmisartan, an ARB with partial peroxisome proliferator-activated receptor-gamma (PPARgamma) activity, can exert a direct effect against fatty-acid-induced oxidative stress in pancreatic beta-cells. METHODS: The effect of telmisartan on lipotoxicity was evaluated using mouse insulin-secreting clonal MIN6 and isolated mouse pancreatic islet cells. Reactive oxygen species, protein kinase-C (PKC) activity and NAD(P)H oxidase activity were examined to clarify the underlying mechanisms. RESULT: Telmisartan decreased the accumulation of palmitate-induced reactive oxygen species in MIN6 cells by 25% and in mouse islet cells by 55%. Telmisartan also decreased palmitate-induced PKC activity by 36% and NAD(P)H oxidase activity by 32% in MIN6 cells. CONCLUSION: These findings indicate that telmisartan attenuated fatty-acid-induced oxidative stress and NAD(P)H oxidase activity in pancreatic beta-cells. Our observations pave the way to the possible use of ARB as a means of protecting beta-cell survival and preserving insulin secretion capacity in patients with diabetes mellitus.

Pioglitazone attenuates fatty acid-induced oxidative stress and apoptosis in pancreatic beta-cells.

AIMS: Thiazolidinediones (TZDs), ligands for peroxisome proliferator-activated receptor gamma, are antidiabetic agents that improve hyperglycemia by decreasing insulin resistance in obese diabetic animal models and patients with type 2 diabetes. We have studied whether pioglitazone, a TZD, can exert a direct effect against pancreatic beta-cell lipoapoptosis. METHODS: MIN6 cells were cultured in medium containing either 5.6 (low glucose) or 25 mM glucose (high glucose) in the presence or absence of 0.5 mM palmitate for 48 h. We examined the effect of 10 microM pioglitazone on MIN6 cells on glucose-stimulated insulin secretion, cellular ATP, uncoupling protein-2 (UCP-2) mRNA expression, intracellular triglyceride content, reactive oxygen species production, the number of apoptotic cells and nuclear factor-kappaB (NF-kappaB) activity. RESULTS: Pioglitazone recovered partly impaired glucose-stimulated insulin secretion and cellular ATP in MIN6 cell exposed to high glucose with 0.5 mM palmitate. Pioglitazone suppressed intracellular triglyceride accumulation in cells exposed to high glucose with 0.5 mM palmitate. Palmitate-induced upregulation of UCP-2 mRNA levels was suppressed by pioglitazone in a dose-dependent manner. Pioglitazone decreased palmitate-induced reactive oxygen species production in MIN6 cells by 24% and in mouse islet cells by 53%. Pioglitazone also decreased palmitate-induced NF-kappaB activity by 40% and protected beta-cells from palmitate-induced apoptosis by 22% in MIN6 cell. CONCLUSIONS: Pioglitazone attenuated fatty acid-induced oxidative stress and apoptosis in pancreatic beta-cells. TZDs might be used as a mean for maintaining beta-cell survival and preserving capacity of insulin secretion in patients with diabetes mellitus.

Muonium as a shallow center in GaN.

A paramagnetic muonium (Mu) state with an extremely small hyperfine parameter was observed for the first time in single-crystalline GaN below 25 K. It has a highly anisotropic hyperfine structure with axial symmetry along the <0001> direction, suggesting that it is located either at a nitrogen-antibonding or a bond-centered site oriented parallel to the c axis. Its small ionization energy (

Early events involved in the development of insulin resistance in Zucker fatty rat.

AIM: To clarify the mechanism by which insulin resistance develops in obesity, Zucker fatty rats (ZFR) and lean litter mates (ZLR) were temporally subjected to oral glucose tolerance tests (OGTT) at 6 and 15 weeks of age. METHOD: As candidates for causative factors of insulin resistance, plasma leptin, free fatty acids (FFA) and tumor necrosis factor (TNF)-alpha levels were evaluated. RESULTS: There was no difference in the body weight between the two groups at 6 weeks of age, but ZFR were significantly heavier than ZLR at 15 weeks of age. At 6 weeks of age, blood glucose levels and area under the curve of glucose (AUCg) during OGTT were not significantly different between the two groups, while plasma insulin levels and area under the curve of insulin (AUCi) in the ZFR group were significantly higher than those in the ZLR group. At 15 weeks of age, the blood glucose levels and AUCg as well as plasma insulin levels and AUCi in the ZFR group during OGTT were significantly higher than those in the ZLR group. The ratio of fasting insulin to glucose in the ZFR group was significantly higher than that in the ZLR group at 6 and 15 weeks of age. Peripheral and portal plasma leptin and FFA levels were significantly higher in ZFR than ZLR both at 6 weeks and 15 weeks of age. Meanwhile, at 6 weeks, plasma TNF-alpha levels and expression of TNF-alpha protein in subcutaneous and visceral fat tissues were similar in both groups; however at 15 weeks, these were significantly higher in the ZFR group than the ZLR group. CONCLUSION: These results suggest that FFA rather than TNF-alpha may play an important role in early events involved in the development of insulin resistance and TNF-alpha accelerates insulin resistance together with FFA in the later stage.

Humoral autoreactivity to an alternatively spliced variant of ICA512/IA-2 in Type I diabetes.

AIMS/HYPOTHESIS: The receptor tyrosine phosphatase like-protein ICA512/IA-2 occurs as a proteolytically-processed 65,000 Mr type 1 transmembrane glycoprotein in beta cells and is a major autoantigen of Type I (insulin-dependent) diabetes mellitus. We investigated whether alternative splicing could affect humoral autoreactivity to the molecule. METHODS: Genomic and cDNA sequence analysis showed the presence of a ICA512 variant in islets and lymphoid tissues with an in-frame deletion of exon 13 which produces a secreted form lacking aa 557-629 including the transmembrane domain (aa 577 to 600). The alternatively spliced protein is detectable by western blotting in normal islets and translated into a protein that is processed to a series of soluble forms of 25,000-35,000 Mr Radioimmuno-precipitation assays for anti-ICA512 autoantibodies were developed with the widely used ICA512.bdc construct (which has exon 13 deleted) and a series of full-length and modified ICA512/IA-2 molecules. RESULTS: The assays showed that ICA512.bdc and ICA512604-979 gave the best discrimination between diabetic and control sera. With ICA512604-979 a somewhat greater proportion of patients expressing antibodies were detected than with ICA512.bdc in the groups studied (70.5 % vs 63.2 % of prediabetic/new-onset and 25.0 vs 13.9% in patients with diabetes > 20 years). Conversely, a small proportion (3 % recent-onset and 6% > 20 years) had antibodies to ICA512.bdc but not ICA512(604-979). CONCLUSION/INTERPRETATION: Important epitopes lie within the exon 13 region and others can be generated by the alternative splicing. As the deltaexon 13 variant is probably secreted by the beta cell, it could be recognized by the cellular and humoral arm of the immune system in the absence of cellular damage.

Detection of interstitial Ga in GaN

We report the direct detection of interstitial Ga by optical detection of electron paramagnetic resonance (ODEPR) in the photoluminescence of n-type GaN after irradiation in situ at 4.2 K with 2.5 MeV electrons. It is stable upon annealing until room temperature, where it becomes mobile and trapped to form a new defect which is observed to emerge as the interstitial disappears. The time constant of the process at room temperature is approximately 200 min. The emergence of another ODEPR center beginning at approximately 135 K suggests even easier migration of one of the other intrinsic defects in the GaN lattice.

Localization and functional role of synaptotagmin III in insulin secretory vesicles in pancreatic beta-cells.

Pancreatic beta-cells secrete insulin by Ca2+-triggered exocytosis of insulin-containing large dense-core vesicles. Synaptotagmin is a Ca2+/phospholipid-binding protein and is a good candidate for the Ca2+ sensor for exocytosis of synaptic vesicles in neurons. In the present study, we generated a polyclonal antibody against synaptotagmin III, and found that synaptotagmin III immunoreactivity was present at high levels in insulin-containing pancreatic islet cells and insulin-secreting clonal MIN6 cells. In subcellular fractionations of MIN6 cells, synaptotagmin III was recovered in the vesicular fractions containing both insulin and vesicle-associated membrane protein-2 (VAMP-2), but not in synaptophysin-positive fractions. The secretory vesicles immunoprecipitated by anti-VAMP-2 antibody contained synaptotagmin III and insulin. In addition, treatment of streptolysin-O-permeabilized MIN6 cells with anti-synaptotagmin III antibody significantly inhibited Ca2+-triggered insulin secretion. These results indicate that synaptotagmin III is localized in insulin-containing dense-core vesicles in pancreatic beta-cells, and further strongly suggest that synaptotagmin III is the Ca2+ sensor in the exocytosis of insulin secretory vesicles.

Noc2, a putative zinc finger protein involved in exocytosis in endocrine cells.

We have cloned a cDNA encoding a novel protein of 302 amino acids (designated Noc2, no C2 domain) that has 40.7% amino acid identity with and 77.9% similarity to the N-terminal region of rabphilin-3A, a target molecule of Rab3A. However, unlike rabphilin-3A, Noc2 lacks two C2 domains that are thought to interact with Ca2+ and phospholipids. Noc2 is expressed predominantly in endocrine tissues and hormone-secreting cell lines and at very low levels in brain. Immunoblot analysis of subcellular fractions of the insulin-secreting cell line MIN6 and immunocytochemistry reveal that Noc2 is a 38-kDa protein present in the cytoplasm. Overexpression of Noc2 in PC12 cells cotransfected with growth hormone enhances high K+-induced growth hormone secretion. Screening a mouse embryonic cDNA library with the yeast two-hybrid system shows that Noc2 interacts with the LIM domain-containing protein zyxin, a component of the cytoskeleton, and this interaction is further confirmed by the coimmunoprecipitation experiment. Accordingly, Noc2 is probably involved in regulated exocytosis in endocrine cells by interacting with the cytoskeleton.

A candidate case for lymphocytic infundibulo-neurohypophysitis mimicking a neurohypophysial tumor.

A 56-year-old Japanese man presented with a 2-month duration of polyuria and polydipsia. The diagnosis of diabetes insipidus was confirmed by water deprivation and vasopressin injection. The secretory function of the adenohypophysis was estimated as normal by a variety of provocative tests. Magnetic resonance imaging (MRI) displayed the loss of the hyperintense signal of the neurohypophysis and a tumor-like lesion confined to the neurohypophysis. The tissue specimen resected at transsphenoidal surgery showed diffuse lymphocytic infiltration. These findings suggest that this is a candidate case for lymphocytic infundibuloneurohypophysitis (LIN) that is not identical to classical lymphocytic hypophysitis. This patient will be followed up to determine whether this case simply represents an early stage of classical hypophysitis or a different clinical entity.

[Characteristics of home parenteral nutrition and effectiveness of 3 way valved PICC in regional medical care].

Home medical care is becoming a greater matter of concern along with the increasing population of elderly persons. The various difficulties in home care are also found in regional care, and it could be said that in Japan regional care serves as an "advanced model" for home medical care. Ohya Town is located in the mountains of northern Hyogo Prefecture. It has a population of about 5,000 of which 32% is elderly people. There is no railroad, and it takes around 30 minutes to travel to the central hospital from the area. The three town clinics are in contact with each other and with the central hospital concerning medical information. The characteristics of Home Parenteral Nutrition in this underpopulated district are discussed and reported in reference mainly to cases in which the 3 way valved peripherally inserted central venous catheter (Groshong Catheter) is used, in comparison with HPN cases in the city university hospital.

Fluorometric detection of glycosphingolipids on thin-layer chromatographic plates.

A microdetection system for glycosphingolipid analysis has been developed using 5-hydroxy-1-tetralone as the fluorescent labeling reagent. The reagents in H2SO4 permit the fluorometric detection of acidic and neutral glycosphingolipids both in test tube and on thin-layer chromatographic plates. Glycosphingolipids can be detected at concentrations as low as 5 pmol on the thin-layer chromatographic plate. The method is a rapid and simple, and feasible for determination of glycosphingolipid from small amounts of biological samples.

Cellular localization of synaptotagmin I, II, and III mRNAs in the central nervous system and pituitary and adrenal glands of the rat.

Three isoforms of synaptotagmin, a synaptic vesicle protein involved in neurotransmitter release, have been characterized in the rat, although functional differences between these isoforms have not been reported. In situ hybridization was used to define the localization of synaptotagmin I, II, and III transcripts in the rat CNS and pituitary and adrenal glands. Each of the three synaptotagmin genes has a unique expression pattern. The synaptotagmin III gene is expressed in most neurons, but transcripts are much less abundant than the products of the synaptotagmin I and II genes. A majority of neurons in the forebrain expressed both synaptotagmin I and III mRNAs while synaptotagmin II gene expression was confined to subsets of neurons in layers IV-VI of the cerebral cortex, in the dentate granule cell region, the hilus, and the CA1-CA3 areas of the hippocampus. In the cerebellum, all three transcripts were visualized in the granule cell layer. Furthermore, synaptotagmin I probes revealed striking differences between distinct populations of neurons, as in addition to moderate labeling of granule cells, much more prominent hybridization signals were detected on scattered cell bodies likely to be Golgi interneurons. In the most caudal part of the brain, synaptotagmin II transcripts were abundant and were coexpressed with synaptotagmin III mRNAs. This pattern was found in putative motoneurons of the spinal cord, suggesting that the two isoforms might be involved in exocytosis at the neuromuscular junction. Only synaptotagmin I mRNAs were detected in the anterior and intermediate pituitary and in adrenal medullary cells. These data reveal an unexpectedly subtle segregation of the expression of synaptotagmin genes and the existence of multiple combinations of synaptotagmin isoforms which may provide diversity in the regulation of neurosecretion.

A mathematical model of the volume effect which postulates cell migration from unirradiated tissues.

PURPOSE: In order to simulate the large variation in tolerance doses for very small treatment volumes, we introduce a model which assumes the presence of cells which have migrated from unirradiated tissues. METHODS AND MATERIALS: In order to represent serial architecture, the new model adds a new parameter to the familiar expression for serial architecture. Data derived from the model is fitted to the dose-response data developed by Hopewell et al. (Hopewell, J.W., Morris, A.D. and Dixon-Brown, A. The influence of field size on the late tolerance of the rat spinal cord to single doses of X rays. Br. J. Radiol. 60: 1099-1108, 1987) using white matter necrosis of rat spinal cord. RESULTS: The new model with a cell-migration term more accurately describes the large differences in threshold doses for a very small treatment volume than a model without a cell-migration term. CONCLUSION: Although these results do not prove that cell migration is the mechanism behind the volume effect for very small volume, they do suggest that the probability of normal tissue complication is more accurately predicted by the new model.

Cloning and functional characterization of a novel ATP-sensitive potassium channel ubiquitously expressed in rat tissues, including pancreatic islets, pituitary, skeletal muscle, and heart.

ATP-sensitive K+ (KATP) channels play a crucial role in coupling metabolic energy to the membrane potential of cells. We have isolated a cDNA encoding a novel member (uKATP-1) of the inward rectifier K+ channel family from a rat pancreatic islet cDNA library. Rat uKATP-1 is a 424-amino acid residue protein (M(r) = 47,960). Electrophysiological studies of uKATP-1 expressed in Xenopus laevis oocytes show that uKATP-1 is a weak rectifier and is blocked with Ba2+ ions. Single-channel patch clamp study of clonal human kidney epithelial cells (HEK293) transfected with uKATP-1 cDNA reveals that uKATP-1 closes in response to 1 mM ATP and has a single channel conductance of 70 +/- 2 picosiemens (n = 6), indicating that uKATP-1 is an ATP-sensitive inward rectifier K+ channel. In addition, uKATP-1 is activated by the KATP channel opener, diazoxide. RNA blot analysis shows that uKATP-1 mRNA is expressed ubiquitously in rat tissues, including pancreatic islets, pituitary, skeletal muscle, and heart, suggesting that uKATP-1 may play a physiological role as a link between the metabolic state and membrane K+ permeability of cells in almost every normal tissue. Since uKATP-1 shares only 43-46% amino acid identity with members of previously reported inward rectifier K+ channel subfamilies, including ROMK1, IRK1, GIRK1, and cKATP-1, uKATP-1 is not an isoform of these subfamilies and, therefore, represents a new subfamily of the inward rectifier K+ channel family having two transmembrane segments.

Confirmation of the local nature of the plasma grating photorefractive effect.

Two-wave mixing measurements made during the encoding of plasma gratings in AlGaAs:Te indicate that there is no phase shift between the illumination intensity pattern and the encoded refractive-index grating. These results support our previously proposed photorefractive mechanism, which is based on persistent plasma gratings and does not require charge transport.

Cloning of a mouse Rabphilin-3A expressed in hormone-secreting cells.

Rab3A, a ras p21-related small GTP-binding protein, is implicated in the exocytosis of neurotransmitters. Recently, Rabphilin-3A, a putative target protein for Rab3A, was identified and its cDNA has been cloned from bovine brain. In this study, we isolated a cDNA encoding a mouse Rabphilin-3A homolog from the insulin-secreting cell line, MIN6. Mouse Rabphilin-3A is a protein of 681 amino acids exhibiting overall 88.5% identity with bovine Rabphilin-3A. The amino acid identity between mouse and bovine Rabphilin-3A is highest in their carboxyl-terminal halves (97.8% identity) and amino-termini (93.0% identity), which contain the region of the two internal repeats homologous to the regulatory domain (C2 domain) of protein kinase C and the putative Rab3A-binding region, respectively. RNA blot analysis revealed that Rabphilin-3A mRNA is expressed in endocrine and hormone-secreting clonal cells, including rat adrenal glands, MIN6, the hamster insulin-secreting cell line, HIT-T15, and the rat catecholamine-secreting cell line, PC12, as well as rat brain. These results suggest that Rabphilin-3A might be involved in the exocytosis of secretory vesicles in hormone-secreting cells as well as in neurons.

Synaptotagmin III is a novel isoform of rat synaptotagmin expressed in endocrine and neuronal cells.

Synaptotagmin (p65), an integral membrane protein of synaptic vesicles, is thought to be involved in calcium-dependent exocytosis of synaptic vesicles. Here, we report the cloning and tissue distribution of a novel isoform of synaptotagmin, designated synaptotagmin III. The cDNA clones encoding synaptotagmin III have been isolated from a rat brain cDNA library. Rat synaptotagmin III is a protein of 588 amino acids having 40.5, 38.3, and 64.0% identity with rat synaptotagmin I, rat synaptotagmin II, and o-p65-C, a third synaptotagmin isoform of marine ray Discopyge ommata, respectively. The region of the two internal repeats homologous to the regulatory domain (C2 domain) of protein kinase C is highly conserved among synaptotagmin I, II, and III. RNA blotting studies reveal that synaptotagmin III mRNA is expressed in brain, various endocrine tissues, and hormone-secreting clonal cells. These results suggest that rat synaptotagmin III is a mammalian homolog of o-p65-C and is involved in Ca(2+)-dependent exocytosis of secretory vesicles in endocrine cells, as well as in neurons.