Prolonged exposure of rodent ␤-cells to combinations of cytokines induces the inducible form of nitric oxide synthase (iNOS) expression and Fas expression, nitric oxide (NO) production, and cell death. It also induces the expression of potential "defense" genes, such as manganese superoxide dismutase (MnSOD) and heat shock protein (hsp) 70. NO is a radical with multifaceted actions. Recent studies have shown that NO, in addition to having cytotoxic actions, may regulate gene transcription. It remains unclear whether NO mediates cytokine-induced gene expression and subsequent ␤-cell death. Previous studies using NO synthase blockers yielded conflicting results, which may be due to nonspecific effects of these agents. In this study, we examined the effects of cytokines on gene expression, determined by reverse transcriptase-polymerase chain reaction (RT-PCR), and viability, determined by nuclear dyes, of pancreatic islets or fluorescence-activated cell sorter (FACS)-purified ␤-cells isolated from iNOS knockout mice (iNOS -/ -, background C57BL/6x129SvEv) or their respective controls (C57BL/6x129SvEv). The combination of cytokines used was interleukin-1␤ (50 U/ml) plus ␥-interferon (1,000 U/ml) plus tumor necrosis factor-␣ (1,000 U/ml). The lack of cytokine-induced iNOS activity in the iNOS -/-islet cells was confirmed by RT-PCR and nitrite determination. Cytokines induced a >3-fold increase in Fas and MnSOD mRNA expression in wild-type (WT) and iNOS -/-islets. On the other hand, hsp 70 was induced in WT but not in iNOS -/-islets. Prolonged (6-9 days) exposure of WT islets to cytokines leads to an 80-90% decrease in islet cell viability, whereas viability decreased by only 10-30% in iNOS -/-islet cells. To determine the mode of cytokine-induced cell death, FACS-purified ␤-cells were exposed to the same cytokines. After 9 days, the apoptosis index was similarly increased in WT (39 ± 3%) and iNOS -/-(33 ± 4%) ␤-cells. On the other hand, cytokines increased necrosis in WT (20 ± 4%) but not in iNOS -/-(7 ± 3%) ␤-cells. From these data, we concluded that 1) NO is required for cytokine-induced hsp 70 mRNA expression but not for Fas and MnSOD expression, 2) cytokines induce both apoptosis and necrosis in mouse ␤-cells, and 3) cytokine-induced apoptosis is mostly NO-independent, whereas necrosis requires NO formation. Diabetes

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Apart from numerous applications, for example in azo dye precursors, explosives, and industrial processes, the nitro group (−NO2) appears on countless molecules in photochemical research owing to its unique characteristics such as a strong electron‐withdrawing ability and facile conversion to the reduced substituent. Although it is well known as a fluorescence quencher, fluorescent chromophores that contain the nitro group have also emerged, with 3‐nitrophenothiazine being recently reported to have 100 % emission quantum yield in nonpolar solvents. The diverse characters of nitro‐containing chromophores motivated us to systematically review those chromophores with nitro substituents, their associated photophysical properties, and applications. In this Review, we succinctly elaborate the advance of the fluorescent nitro chromophores in fields of intramolecular charge transfer, fluorescent probes and nonlinear properties. Special attention is paid to the rationalization of the associated emission spectroscopy, so that the readers can gain insights into the structure‐photophysics relationship and hence gain insights for the strategic design of nitro chromophores.

In type 1 diabetes, autoimmune destruction of pancreatic beta-cells has been attributed to cytokines released from infiltrating immunocytes. Exposure of isolated islets to cytokines leads to nitric oxide (NO) production, which can damage beta-cells. Because ductal cells are closely associated with human beta-cells, we examined whether they can contribute to this process. Isolated human ductal cells were cultured for 48 h with various cytokines. The combination of interleukin-1beta (IL-1beta) plus interferon-gamma (IFN-gamma) increased nitric oxide production 12-fold while stimulating mRNA expression of inducible nitric oxide synthase (iNOS). In this condition, 10-20% of cells positive for the cytokeratin-19 duct marker also stained positive for iNOS protein, whereas no positive cells were found in control preparations. Comparison of the magnitude of iNOS mRNA expression and nitric oxide production in these cells with that in isolated human islets suggests that >50% of total islet nitric oxide production might originate from associated ductal cells. It is concluded that ductal cells are a potential source of nitric oxide production in human islets infiltrated by cytokine-releasing immunocytes.

Type I (insulin-dependent) diabetes mellitus is an autoimmune disease, where T-cells and macrophages invade the islets of Langerhans and lead to specific destruction of pancreatic beta cells [1,2]. Cytokines such as interleukin-1b (IL-1b) and interferon-g are released by these infiltrating mononuclear cells and can contribute to beta-cell death [2,3]. Recent observations indicate that genes and proteins activated by the beta cells are crucial to determine whether these cells will survive or undergo apoptosis when faced with immune-mediated cell damage [4±6].We and others have previously adopted the ªcan-didate geneº approach to characterize beta-cell responses to immune-mediated assaults and observed that cytokines induce the expression of different genes in rodent and human pancreatic islets, encoding both proteins involved in beta-cell damage and Diabetologia (1999) AbstractAims/hypothesis. Interleukin-1b is a putative mediator of pancreatic beta-cell dysfunction and damage in Type I (insulin-dependent) diabetes mellitus. To better understand the molecular mechanisms involved in IL-1b effects, we carried out a differential display of mRNA by RT-PCR to identify novel cytokine-regulated genes. Methods. Fluorescence activated cell sorting-purified rat pancreatic beta-cells were exposed for 6 or 24 h to IL-1b. Differentially expressed cDNA bands were cloned and then identified by comparing their sequences with data from the GenBank. Differential gene expression was confirmed by RT-PCR using specific primers. Results. Interleukin-1b increased the expression of adenine nucleotide translocator-1, phospholipase D-1 and cytokine-induced neutrophil chemoattractant-1 and decreased expression of the protein tyrosine phosphatase-like protein IA-2. Interleukin-1b-induced differential expression of these genes in beta cells was confirmed by RT-PCR. In additional studies, IL-1b was shown to induce chemokines other than cytokine-induced neutrophil chemoattractant-1, including cytokine-induced neutrophil chemoattractant-3 and monocyte chemotactic protein-1. Conclusion/interpretation. Our observations indicate that IL-1b modifies the expression of several genes in pancreatic beta cells. These genes may affect both function, viability and beta-cell recognition by the immune system. Functional characterization of the mRNAs which have been identified could facilitate a better understanding of the mechanisms leading to beta-cell destruction in Type I diabetes. [Diabetologia (1999

Human parvovirus B19 has been found in various tissues in addition to erythroid lineage cells, and non-structural protein (NS1) is reported to induce cytotoxicity and apoptosis in erythroid lineage cells, but the mechanism in non-permissive cells is still unclear. To address this issue, we have constructed the NS1 gene in a cytomegalovirus episomal vector, pEGFP-C1 and transfected it into monkey epithelial cells, COS-7. EGFP-NS1 expression in transfected cells was monitored and assessed by fluorescence microscopy, RT-PCR and Western blot. The flow cytometric analysis showed that the NS1-transfected cells were arrested at G1 phase by paclitaxel treatment and there was increased apoptosis. The expression of p53, an important molecule in apoptosis and cell cycle regulation, and its downstream cell cycle kinase inhibitors p16(INK4) and p21(WAF1/CIP1) were up-regulated in the NS1-transfected cells. Also, increased expression of the pro-apoptotic Bcl-2 members Bax, Bad and activation of caspase 3 and caspase 9, but not the activation of caspase 8 or Fas were detected in the NS1-transfected cells. p53-induced Bax expression and subsequent activation of caspase 9 is probably the apoptotic pathway in NS1-transfected cells since activation of the caspase 9 was suppressed by the p53 inhibitor and apoptosis was significantly inhibited by the caspase 9 inhibitor. Our results suggest that the cell death of the NS1-transfected cells is associated with mitochondria related apoptosis. These findings might provide alternative information for further study and characterization of B19 NS1 protein in B19 non-permissive cells.

These results have shown that urine IL-1beta level may serve as a useful marker for the early detection of acute pyelonephritis in febrile children. Young children are at a risk of the development of renal scarring following acute pyelonephritis.