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To develop somatic gene therapy for diabetes, we studied an animal model with proinsulin-producing fibroblasts with an immunological safety system. Cultured mouse fibroblasts of the [Ltk.sup.-] cell line were transfected [...]

There are currently no therapies that can improve prognosis in cases of heart failure (HF) with preserved ejection fraction (EF). We hypothesized that there is a point of no return after which no response to treatment is noted and that for the prevention of hypertensive HF this point may be determined by left ventricle (LV) strain, in the prevention of hypertensive HF. Here an angiotensin-converting enzyme inhibitor (ACE-I) was initiated based on myocardial strain imaging and its effects were determined in an animal model.

A Goto-Kakizaki (GK) rat is considered as a spontaneous model of type 2 diabetes mellitus without obesity. GK rats show impaired insulin secretion and degeneration of islets of pancreas. On [...]

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor. It was reported that vascular smooth muscle cells (VSMC) produced VEGF more in atherosclerotic plaques than in normal vascular walls [...]

Many growth factors, such as vascular endothelial growth factor (VEGF), are considered 13 participate in pathogenesis of proliferative diabeticretinopathy (PDR). It was previously reported that platelet-derived growth factor induced angiogenesis [...]

Accurate assessment of global and regional right ventricular (RV) systolic function is challenging. The aims of this study were to confirm the reliability and feasibility of a three-dimensional (3D) speckle-tracking echocardiography (STE) system, using comparison with cardiac magnetic resonance imaging (CMR), and to assess the contribution of regional RV function to global function.

Hepatic lipogenesis is nutritionally regulated (i.e., downregulated during fasting and upregulated during the postprandial state) as an adaptation to the nutritional environment. While alterations in the expression level of the transcription factor SREBP-1c are known to be critical for nutritionally regulated lipogenesis, upstream mechanisms governing Srebf1expression remain unclear. Here, we show that the fasting-induced transcription factor KLF15, a key regulator of gluconeogenesis, forms a complex with LXR/RXR, specifically on the Srebf1promoter. This complex recruits the corepressor RIP140 instead of the coactivator SRC1, resulting in reduced Srebf1and thus downstream lipogenic enzyme expression during the early and euglycemic period of fasting prior to hypoglycemia and PKA activation. Through this mechanism, KLF15 overexpression specifically ameliorates hypertriglyceridemia without affecting LXR-mediated cholesterol metabolism. These findings reveal a key molecular link between glucose and lipid metabolism and have therapeutic implications for the treatment of hyperlipidemia.

This study clarified the characteristics of intrarenal Doppler ultrasonography (IRD) profiles and their prognostic implications in heart failure (HF).

In patients with chronic aortic regurgitation (AR), systolic wall stress and volume overload affects left ventricular (LV) systolic function and remodeling. The aim of this study was to assess transmural rearrangements of myocardial deformation to preserve LV ejection performances using speckle-tracking echocardiography in patients with chronic AR.

AbstractThe SDHA, SDHB, SDHC,and SDHDgenes code for subunits of succinate dehydrogenase (SDH), which forms part of the mitochondrial respiratory chain. Germline mutations in the genes encoding SDHB and SDHD have been reported in familial paragangliomas/pheochromocytomas and in apparently sporadic pheochromocytomas. SDHBand SDHDmutations are widely distributed along the genes with no apparent hot spots. SDHBmutations are often detected in malignant and extra-adrenal pheochromocytomas. SDHDmutations are also detected frequently in head and neck paragangliomas. We sequenced the entire coding regions of the SDHBand SDHDgenes in 17 pheochromocytomas. Weidentified novel heterozygous G to A point mutations at the first base of intron 3 of the SDHBgene in a malignant extra-adrenal abdominal pheochromocytoma patient, and at the first base of codon 111 of the SDHDgene in an adrenal pheochromocytoma patient. Further, we confirmed the SDHDmutation by DHPLC. The prevalence of SDHB and SDHDmutations in pheochromocytomas we examined was 12 (2/17). Thus, we identified two novel SDH mutations in Japanese pheochromocytomas. Further studies will investigate the oncogenic potential of these mutations.Copyright © 2007 S. Karger AG, Basel

Pheochromocytoma contains and may synthesize a large number of peptides such as opioids (Leu-, Met-enkephalin), VIP, gastrin, calcitonin gene related peptide (CGRP), somatostatin, and adrenocorticotrophic hormone (ACTH). Also, circulating level of these peptides may be elevated. Although the precise contribution of these peptides secretion to the clinical features are largely unknown, it can be suggested that specific endocrine system could be involved in ectopic hormone production by pheochromocytoma. Indeed, it has occasionally been reported that pheochromocytoma is associated with Cushing's syndrome resulting from excess ACTH production. As there are already many excellent reviews of this area, the discussion that follows will mainly focus on our data, including 1.Opioids, 2.Adrenomedullin (AM) and proadrenomedullin N-terminal 20 peptide (PAMP), 3.Natriuretic peptides (NPs), 4.Leptin, 5.Endothelin 6. Angiotensin II receptor, 8. Chromogranin A and Catestatin.

GM3 ganglioside inhibits tetraspanin CD9-facilitated cell motility in various cell lines (Ono, M., Handa, K., Sonnino, S., Withers, D. A., Nagai, H., and Hakomori, S. (2001) Biochemistry40, 6414–6421). We now report the following: (i) CD9 has the novel feature of being soluble in chloroform/methanol, and classifiable as “proteolipid”; (ii) CD9 and α3integrin were concentrated together in the low-density glycolipid-enriched microdomain (GEM) of ldlD/CD9 cells, and the α3expression ratio (value for cells grown under +Gal condition divided by the value for cells grown under −Gal condition) in GEM of ldlD/CD9 cells was higher than that in control ldlD/moc cells, suggesting that CD9 recruits α3in GEM under +Gal condition, whereby GM3 is present. (iii) Chemical levels of α3and CD9 in the total extract or membrane fractions from cells grown under +Gal versus−Gal condition were nearly identical, whereas α3expressed at the cell surface, probed by antibody binding in flow cytometry, was higher under −Gal than +Gal condition. These results suggest that GM3 synthesized under +Gal condition promotes interaction of α3with CD9, which restricts α3binding to its antibody. A concept of the α3/CD9 interaction promoted by GM3 was further supported by (i) co-immunoprecipitation of CD9 and α3under +Gal but not −Gal condition, (ii) enhanced co-immunoprecipitation of CD9 and α3when GM3 was added exogenously to cells under −Gal condition, and (iii) the co-localization images of CD9 with α3and of GM3 with CD9 in fluorescence laser scanning confocal microscopy. Based on the promotion of α3/CD9 interaction by GM3 and the status of laminin-5 as a true ligand for α3, the laminin-5/α3-dependent motility of ldlD/CD9 cells was found to be greatly enhanced under −Gal condition, but strongly inhibited under +Gal condition. Such a motility difference under +Gal versus−Gal condition was not observed for ldlD/moc cells. The inhibitory effect observed in ldlD/CD9 cells under +Gal condition was reversed upon addition of anti-α3antibody and is therefore based on interaction between α3, CD9, and GM3 in GEM.

Two distinct types of angiotensin II (AngII) receptors, AT1and AT2, have been cloned. We have shown previously that stimulation of AT2reduces intracellular cyclic guanosine monophosphate (cGMP) levels in cultured porcine chromaffin cells in which AT2is the predominantly expressed receptor. However, it has not been determined whether AT1or AT2affects signal transduction pathways involving mitogen-activated protein kinases (MAPKs) and signal transducers and activators of transcription (STATs) in chromaffin cells. Also, it is unclear whether cGMP/protein kinase G (PKG) is involved in the regulation of MAPKs and STATs in these cells.

Hyperinsulinemia has recently been reported as a risk factor for atherosclerotic diseases such as coronary heart disease; however, the effect of insulin on the development of atherosclerosis is not well understood. Here we have investigated the direct effect of insulin on macrophages, which are known to be important in the atherosclerotic process. We treated THP-1 macrophages with insulin (10−7m) and examined the gene expression using nucleic acid array systems. The results of array analysis showed that insulin stimulated gene expression of tumor necrosis factor-α (TNF-α) the most among all genes in the analysis. In addition, insulin administration to macrophages enhanced both mRNA expression and protein secretion of TNF-α in a dose-dependent manner. To determine the signaling pathway involved in this TNF-α response to insulin, we pretreated the cells with three distinct protein kinase inhibitors: wortmannin, PD98059, and SB203580. Only PD98059, which inhibits extracellular signal-regulated kinases, suppressed insulin-induced production of TNF-α mRNA and protein in THP-1 macrophages. These observations indicate that insulin stimulates TNF-α production in macrophages by regulating the expression of TNF-α mRNA and that the extracellular signal-regulated kinase signaling pathway may have a critical role in stimulating the production of TNF-α in response to insulin in macrophages.

Vascular endothelial growth factor (VEGF) is implicated in the pathogenesis of inflammatory joint diseases, including rheumatoid arthritis (RA). To determine the importance of this cytokine in vivo, the effect of administering VEGF-neutralizing antibodies to mice with collagen-induced arthritis (CIA), which has many immunological and pathological similarities to human RA, has been investigated. Either saline, normal rabbit immunoglobulin or anti-human VEGF121 rabbit polyclonal antibody was administered to mice subcutaneously either before the onset of arthritis or after the establishment of clinical disease. Anti-VEGF antibody administered prior to disease onset significantly delayed the development of arthritis and decreased clinical score and paw thickness as well as histological severity. On the other hand, the frequency of occurrence of disease compared to either the control group administered saline or normal rabbit immunoglobulin was not altered. Anti-VEGF antibody also significantly ameliorated clinical and histological parameters even when administered after disease onset. These results indicate a possible therapeutical potential for anti-VEGF treatment in human arthritis.

New orexigenic peptides called orexin-A and -B have recently been described in neurons of the lateral hypothalamus and perifornical area. No orexins have been found in adipose tissues or visceral organs, including the adrenal gland. However, expression of the orexin-receptor 2 (OX2R) in the rat adrenal gland has been reported. To test the effects of orexins on peripheral organs, we investigated their effects on catecholamine synthesis and secretion in the rat pheochromocytoma cell line PC12. Orexin-A and -B (100 nM) significantly reduced basal and PACAP-induced tyrosine hydroxylase (TH) (the rate-limiting enzyme in the biosynthesis of catecholamines) mRNA levels. Orexin-A and -B (100 nM) also significantly inhibited the PACAP-induced increase in the cAMP level, suggesting that the suppressive effect on TH mRNA is mediated, at least in part, by the cAMP/protein kinase A pathway. Furthermore, orexin-A and -B (100 nM) significantly suppressed basal and PACAP-induced dopamine secretion from PC12 cells. Next, we examined whether orexin receptors (OX1R, OX2R) were present in the rat adrenal gland and PC12 cells. In the adrenal glands, OX2R was as strongly expressed as in the hypothalamus, but OX1R was not detected. On the other hand, neither OX1R nor OX2R was expressed in PC12 cells. However, binding assays showed equal binding of orexin-A and -B to PC12 cells, suggesting the existence in these cells of some receptors for orexins. These results indicate that orexins suppress catecholamine release and synthesis, and that the inhibitory effect is mediated by the cAMP/protein kinase A pathway.

It was reported that nicotine‐induced dopamine release in the rat pheochromocytoma cell line, PC12 cells, was inhibited by κ‐opioid. However, it is not known whether inhibition of catecholamine biosynthesis is involved in the inhibitory mechanisms of κ‐opioids in PC12 cells. U‐69593 (a κ‐opioid agonist: ≥100 nM) significantly inhibited the nicotine‐induced increase of tyrosine hydroxylase (TH, a rate‐limiting enzyme in biosynthesis of catecholamine) enzyme activity and TH mRNA levels. These inhibitory effects were completely reversed by naloxone and nor‐binaltorphimine dihydrochloride (nor‐BNI), a specific κ‐antagonist, whereas pertussis toxin (PTX) only partially reversed this inhibitory effect. Also, U‐69593 (≥100 nM) significantly inhibited the nicotine‐induced increase of cAMP production. This inhibitory effect was completely reversed by naloxone and nor‐BNI, whilst only partially reversed by PTX. Moreover, U‐69593 (≥100 nM) significantly inhibited the nicotine‐induced increase of both the TH protein level and intracellular catecholamine levels. These results indicate that the anti‐cholinergic actions of κ‐opioid can be explained partially by its inhibition of both TH enzyme activity and TH synthesis, through suppression of the cAMP/protein kinase A pathway. It would also appear that the PTX‐sensitive G‐protein mediates the inhibitory effect of this pathway, at least in part.

Angiotensin II subtype 2 receptor (AT2-R) is abundantly expressed in adrenal medullary chromaffin cells. However, the physiological roles of AT2-R in chromaffin cells remain to be clarified. Therefore, we investigated the effects of CGP42112 (AT2-R agonist) on catecholamine biosynthesis in cultured porcine adrenal medullary cells. We initially confirmed AT2-R was predominantly expressed in porcine adrenal medullary cells by [125I]-Ang II binding studies. CGP42112 (≧1 nM) significantly inhibited cGMP production from the basal value. Tyrosine hydroxylase (TH) is a rate-limiting enzyme in the biosynthesis of catecholamine, and its activity is regulated by both TH-enzyme activity and TH-synthesis. CGP42112 (≧1 nM) significantly inhibited TH-enzyme activity from the basal value. These inhibitory effects of CGP42112 on TH-enzyme activity and-cGMP production were abolished by PD123319 (AT2-R antagonist) while CV-11974 (AT1-R antagonist) was ineffective. We also tested whether decrease of cGMP is involved in the inhibitory effect of CGP42112 on TH-enzyme activity. Pretreatment of 8-Br-cGMP (membrane-permeable cGMP analogue) prevented the inhibitory effect of CGP 42112 on TH-enzyme activity. Similar to that of TH-enzyme activity, CGP42112 (≧1 nM) significantly reduced TH-mRNA and TH-protein level from the basal value, and these inhibitory effects were abolished by PD123319 but not CV-11974. These findings demonstrate that CGP 42112 reduces both TH-enzyme activity and TH-synthesis and that these inhibitory effects could be mediated by decrease of cGMP production.

Studies in corneal transplant rejection remain important because acute immunologic rejection continues to be the leading cause of human corneal transplant failure. As the permeability of vessels and the neovascularization induce cells infiltration into the graft, we considered the possibility that vascular endothelial growth factor (VEGF), a potent permeability-increasing factor and angiogenesis-mediating factor, could participate in the immune response.

Vascular endothelial growth factor (VEGF) is closely correlated to diabetic retinopathy. Its basal production in three types of cultured retinal cells (endothelial cells, pericytes and retinal pigment epithelial cells; RPE) was examined. RPE production of VEGF was markedly higher than the rest of the cells. VEGF production in RPE was significantly elevated by 10-day, but not by 1- or 3-day exposure to 16.5 mM glucose compared to a 5.5 mM glucose group. Transient deterioration of diabetic retinopathy is frequently observed during rapid correction of glycemic control. To determine whether VEGF is up-regulated following a sharp drop in the glucose concentration or not, we examined the changes in VEGF production in RPE before and after a sudden drop in the glucose concentration. VEGF production was significantly increased by a glucose concentration decrease from 5.5 to 0.5 mM, but not by a decrease from 33 or 16.5 to 5.5 mM. These findings suggest that up-regulation of VEGF may contribute to the development of diabetic retinopathy and its worsening by hypoglycemia.

Reactions between a carotenoid, fucoxanthin and 1,1‐diphenyl‐2‐picrylhydrazyl were investigated both under anoxic and aerobic conditions. Fucoxanthin equimolarly reacted with 1,1‐diphenyl‐2‐picrylhydrazyl under anoxic conditions. Under aerobic conditions, only a part of fucoxanthin consumed 1,1‐diphenyl‐2‐picrylhydrazyl and the degree of reaction fluctuated with repeated trials. β‐Carotene or other carotenoids, β‐cryptoxanthin, zeaxanthin, licopen and lutein, were also examined in the reaction with 1,1‐diphenyl‐2‐picrylhydrazyl under anoxic conditions. All these compounds scarcely reacted with 1,1‐diphenyl‐2‐picrylhydrazyl.

Benzo(a)pyrene (BP) in the sediment of Osaka Bay were determined. High concentration was found at two stations near the mouth of Shin-Yodo and off Kobe respectively (0.98Μg g−1 dry mud basis, 1.1Μg g−1). The concentration decreases regularly from the mouth of the river further into the bay. The other supplemental determination was carried on the sediment and the seawater of Keihin Canal. Notable BP concentration of 89Μg g−1 was observed in the sediment beneath the effluent outlet of a gasworks. The BP in the sediment near the ironworks was also considerable, while the BP was relatively less in the sediment beneath the effluent outlet of refineries. These data suggest that coke plants working in gasworks or ironworks may be the larger sources of BP than refineries. The results of Osaka Bay coincide with this hypothesis.

Bacterial degradation of the hydrocarbons of lubricating oils was investigated by mass spectrometric analysis which gives both total amount and the composition of hydrocarbon types of residual oil. An unused lubricating oil, which mainly consisted of hydrocarbon types with only a small percentage ofn-alkanes, was degraded by marineBacillussp. andPseudomonassp. with 55 % and 25 % decreases in 10 days, respectively. Susceptibility of respective hydrocarbon types to biodegradation was in the following order: alkanes > non-condensed cycloalkanes, mono-aromatics > condensed cycloalkanes. A used lubricating oil of different brand showed a larger decrease than the unused oil. Both species of bacteria degraded large portions of alkane fraction of Arabian light crude oil.

We have cloned multiple extradiol aromatic ring-cleavage dioxygenase (EDO) genes from a gene library of Pseudomonas aeruginosaJI104, which is a benzene degrader isolated from soil near a gasworks. Southern hybridization analysis revealed that P. aeruginosaJI104 possessed three homologous catechol 2,3-dioxygenase (C23O) genes. Nucleotide sequences of the cloned C23O genes, xylEJI104-1,2,3were almost identical to that of the archetypal C23O gene (xylETOL), which is carried on the TOL plasmid, pWW0. We also cloned another EDO gene, bphCJI104, the product of which showed less activity for catechol than did XylEJI104, but higher activity for 2,3-dihydroxy biphenyl. The nucleotide sequence of bphCJI104was identical to that of bphCKF707(2,3-dihydroxybiphenyl dioxygenase gene of Pseudomonas pseudoalcaligenesKF707). The substrate specificities of the four EDOs of P. aeruginosaJI104 were markedly different from each other. Although XylEJI104-1and XylETOLwere 94% homologous, the specificities of the gene products for 4-chlorocatechol were extremely different. Results of a study of the chimeric enzymes composed of XylEJI104-1and XylETOLN- and C-terminal regions showed that the difference in the specificity for 4-chlorocatechol was dependent on the C-terminal amino acid sequences. All of the isofunctional homologous EDOs in P. aeruginosaJI104 seem to have been derived from a common ancestor and evolved into the present forms in which each EDO is involved in a different degradation pathway and they all coexist in one strain.

Summary Secretion of fusion proteins composed of cytoplasmic protein dihydrofolate reductase (DHFR) and the Escherichia coli α-haemolysin (HlyA) C-terminal sequence was examined through the haemolysin secretion machinery of E. coli. DHFR of various lengths was combined with the HlyA C-terminal region, and both secretion and DHFR activity of the fusions were measured. The secretion was found to be inversely correlated with the intracellular DHFR activity. Moreover, when one amino acid (Ile155) in a β-sheet of the DHFR C-terminal region was replaced with Lys, the enzymatically active DHFR fusion protein was secreted into the medium. We discuss the possibility of a relationship between folding and secretion of HlyA-fused protein in the HlyA secretion system.

Messenger RNA of receptor for advanced glycation end products (RAGE) is abundantly expressed in the lung. However, cell types expressing RAGE mRNA in the lung have not been identified. In order to elucidate the function of RAGE in pulmonary tissue, we have identified a cell type expressing RAGE mRNA byin situhybridization and compared its expression level of RAGE mRNA by RNA blot analysis of isolated cells.In situhybridization revealed that RAGE mRNA was intensely and specifically visualized in alveolar epithelial type II (AT-II) cells, and weakly in alveolar macrophages. The expression of RAGE mRNA in the primary culture of AT-II cells was at a high level, but that in alveolar macrophages isolated from alveolar lavage was under the level of detection by RNA blot analysis. These results showed that RAGE mRNA is specifically expressed in AT-II cells, and suggested that RAGE makes a substantial contribution to the function of AT-II cells in the lung.

Expression of major histocompatibility complex (MHC) class 2 molecules on the thyroid follicular cells in human autoimmune thyroid diseases has been reported, and is suggested to play a role in the initiation of the autoimmune responses. Interferon γ is known to induce MHC class 2 molecules on cultured thyrocytes. Therefore, we administered recombinant interferon γ to mice to induce MHC class 2 molecule expression in vivo, and investigated the thyroidal changes following MHC class 2 molecule expression. Recombinant interferon γ3 × 105U, was administered daily, to mice. MHC class 2 molecules, studied by indirect immunohistochemistry, were expressed on the follicular cells on the 2nd to 4th day after the first interferon γ administration and continued thereafter. Serum T4and T3concentrations decreased and anti-thyroglobulin antibodies and anti-microsomal antibodies were detected. Lymphocyte infiltrations were observed in the thyroid glands obtained at 28 days, but they were mild. These results suggest that MHC class 2 molecule expression on thyroid follicular cells induces anti-thyroid antibodies and plays an important role in the initiation of autoimmune thyroiditis. However, the progression of the immune response to thyroiditis with lymphocyte infiltration may require some other factor. This new model of autoimmune thyroid disease in mice might be useful to elucidate the pathogenesis of human autoimmune thyroid diseases.

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Divergence in genital traits between closely related species is occasionally greater in sympatry than in allopatry, possibly because directional selection facilitates differentiation so as to prevent unfit hybridization. Here, we report for the first time that the shape of a functional genital structure, cercus, differs markedly between sympatrically and allopatrically occurring individuals in two brachypterous grasshopper species, Parapodisma setouchiensis Inoue 1979 and Parapodisma subastris Huang 1983 (Orthoptera: Podisminae). Although in areas of allopatry the cerci were straight in both species, in areas of sympatry the cerci of P. setouchiensis had an almost orthogonal bend. Furthermore, the angles of curvature of the cerci of P. setouchiensis varied continuously along a transect through sympatric and allopatric areas. We also observed copulation behavior in pairs and found that the apical part of the cerci was inserted into the space between the posterior edges of the seventh abdominal sternite and the seventh abdominal tergite of the female. We suggest that the orthogonally bent cerci allows the male to obtain a firm grip on the female's abdomen and propose that the conspicuous variation in the cerci of P. setouchiensis contributes to the strength of reinforcement of a premating isolation system.

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