Biphasic modulation by mGlu5 receptors of TRPV1-mediated intracellular calcium elevation in sensory neurons contributes to heat sensitivity.

BACKGROUND AND PURPOSE: Elevation of glutamate, an excitatory amino acid, during inflammation and injury plays a crucial role in the reception and transmission of sensory information via ionotropic and metabotropic receptors. This study aimed to investigate the mechanisms underlying the biphasic effects of metabotropic glutamate mGlu5 receptor activation on responses to noxious heat. EXPERIMENTAL APPROACH: We assessed the effects of intraplantar quisqualate, a non-selective glutamate receptor agonist, on heat and mechanical pain behaviours in mice. In addition, the effects of quisqualate on the intracellular calcium response and on membrane currents mediated by TRPV1 channels, were examined in cultured dorsal root ganglion neurons from mice. KEY RESULTS: Activation of mGlu5 receptors in hind paw transiently increased, then decreased, the response to noxious heat. In sensory neurons, activation of mGlu5 receptors potentiated TRPV1-mediated intracellular calcium elevation, while terminating activation of mGlu5 receptors depressed it. TRPV1-induced currents were potentiated by activation of mGlu5 receptors under voltage clamp conditions and these disappeared after washout. However, voltage-gated calcium currents were inhibited by the mGlu5 receptor agonist, even after washout. CONCLUSIONS AND IMPLICATIONS: These results suggest that, in sensory neurons, mGlu5 receptors biphasically modulate TRPV1-mediated intracellular calcium response via transient potentiation of TRPV1 channel-induced currents and persistent inhibition of voltage-gated calcium currents, contributing to heat hyper- and hypoalgesia.

Restoration of innate and adaptive immune responses by HCV viral inhibition with an induction approach using natural interferon-beta in chronic hepatitis C.

Chronic hepatitis C (CHC) is a serious medical problem necessitating more effective treatment. This study investigated the hypothesis that an induction approach with nIFN-beta for 24 weeks followed by PEG-IFN-alpha+ribavirin (standard of care: SOC) for 48 weeks (novel combination treatment: NCT) would increase the initial virologic response rate and restore innate and adaptive immune responses in CHC. Seven CHC patients with a high viral load and genotype 1b were treated with NCT. Serum cytokine and chemokine levels were evaluated during NCT. NCT prevented viral escape and breakthrough resulting in persistent viral clearance of HCVRNA. IL-15 was increased at the end of induction therapy in both early virologic responders (EAVRs) and late virologic responders (LAVRs); CXCL-8, CXCL-10, and CCL-4 levels were significantly decreased (P < 0.05) in EAVR but not in LAVR during NCT, and IL-12 increased significantly (P < 0.05) and CXCL-8 decreased significantly (P < 0.05) after the end of NCT in EAVR but not in LAVR. NCT prevented viral breakthrough with viral clearance leading to improvement of innate and adaptive immunity resulting in a sustained virologic response (SVR). NCT (n = 8) achieved a higher SVR rate than SOC (n = 8) in difficult-to-treat CHC patients with genotype 1 and high viral loads.

Distinctive role of integrin-mediated adhesion in TNF-induced PKB/Akt and NF-kappaB activation and endothelial cell survival.

Tumor necrosis factor (TNF) is a pro-inflammatory cytokine exerting pleiotropic effects on endothelial cells. Depending on the vascular context it can induce endothelial cell activation and survival or death. The microenvironmental cues determining whether endothelial cells will survive or die, however, have remained elusive. Here we report that integrin ligation acts permissive for TNF-induced protein kinase B (PKB/Akt) but not nuclear factor (NF)-kappaB activation. Concomitant activation of PKB/Akt and NF-kappaB is essential for the survival of endothelial cells exposed to TNF. Active PKB/Akt strengthens integrin-dependent endothelial cell adhesion, whereas disruption of actin stress fibers abolishes the protective effect of PKB/Akt. Integrin-mediated adhesion also represses TNF-induced JNK activation, but JNK activity is not required for cell death. The alphaVbeta3/alphaVbeta5 integrin inhibitor EMD121974 sensitizes endothelial cells to TNF-dependent cytotoxicity and active PKB/Akt attenuates this effect. Interferon gamma synergistically enhanced TNF-induced endothelial cell death in all conditions tested. Taken together, these observations reveal a novel permissive role for integrins in TNF-induced PKB/Akt activation and prevention of TNF-induced death distinct of NF-kappaB, and implicate the actin cytoskeleton in PKB/Akt-mediated cell survival. The sensitizing effect of EMD121974 on TNF cytotoxicity may open new perspectives to the therapeutic use of TNF as anticancer agent.

Free radical scavenging action of the medicinal herb Limonium wrightii from the Okinawa islands.

Free radical scavenging action of Limonium wrightii O. kunthe was examined in vitro and in vivo by using electron spin resonance spectrometer and chemiluminescence analyzer. A water extract of L. wrightii showed a strong scavenging action for the 1,1-diphenyl-2-picrylhydrazyl, or superoxide anion and moderate for hydroxyl radical. The extract also depressed production of reactive oxygen species from polymorphonuclear leukocytes stimulated by phorbor-12-mysistate acetate and inhibited lipid peroxidation in rat liver microsomes. When the extract was given intraperitoneally to mice prior to carbon tetrachloride (CCl4) treatment, CCl4-induced liver toxicity, as seen by an elevation of serum aspartate aminotransferase and alanine aminotransferase activities, was significantly reduced. Gallic acid was identified as the active component of L. wrightii with a strong free radical scavenging action. Our results demonstrate the free radical scavenging action of L. wrightii and that gallic acid contributes to these actions.

NO(x) contamination in laboratory ware and effect of countermeasures.

Contamination of various types of laboratory wares with NO(x) (NO(-)(2) and NO(-)(3)) was assessed systematically and the effect of extensive washing as a countermeasure was evaluated. Mean NO(x) contamination arising from a model procedure for NO(x) determination in plasma was 0.93 microM (range, 0.35-1.49 microM). The major source of contamination included conical tubes (54.8%) and pipette tips used for transfer of solution (12.3-16.3%). Except for soft glassware, most NO(x) contamination could be washed out by pure water. Although NO(x) contamination in respective laboratory wares could be reduced below detection levels by extensive washing, summation of the contamination through the model procedure could not be completely abolished (but the effect of washing persisted at least 10 days). Heavy contamination was noted in glassware (especially soft glass) and ultrafiltration units, which was difficult to remove. Several types of vacuum blood sampling tubes contained various levels of NO(x). Our results indicated that a small but significant amount of contamination remained in laboratory ware even after extensive washing, and that it is advisable to avoid the use of glassware (soft glass), ultrafiltration units, and vacuum blood sampling tubes during the processing of clinical sampling for the measurement of NO(x).

Involvement of tyrosine kinase in citrate-stimulated aldosterone production in bovine glomerulosa cells.

The present study was designed to assess whether citrate stimulates aldosterone production by isolated bovine adrenal glomerulosa cells in vitro. When the cells were incubated with graded concentrations of citrate up to 4.0 mM, basal aldosterone production was significantly elevated, with a gradual reduction of extracellular ionized calcium concentration. Without citrate, however, adding increasing amounts of calcium chloride to a calcium-free medium did not reproduce the citrate's effect on basal aldosterone production. Genistein, an inhibitor of tyrosine kinases, inhibited the citrate (4 mM)-induced aldosterone production in a dose-dependent manner, with 89.8% of inhibition at a concentration of 10 microM. When the cells were exposed to citrate (4 mM) for 5, 10, and 30 min, tyrosine in Mr 105,000 endogenous protein was dominantly phosphorylated. This study demonstrates for the first time that citrate stimulates aldosterone production in bovine adrenal glomerulosa cells in vitro and also suggests a crucial involvement of protein tyrosine kinase in the steroidogenic action of citrate in the cells.

Adenoma arising from the cystic duct and extending to the confluence of the extrahepatic biliary tract.

We describe a rare case of adenoma with a few foci of severe atypia arising from the cystic duct in a 68-year-old woman, initially diagnosed by the presence of intracholecystic tumefactive sludge on abdominal ultrasonography. Endoscopic retrograde cholangiography (ERC) disclosed a tuberous filling defect at the orifice of the cystic duct. Pathology examination of the biopsied specimen obtained from ERC disclosed not a cancerous but an adenomatous lesion. Macroscopically, the resected specimen showed a superficially spreading and shallowly elevated lesion extending from the cystic duct to the common bile duct. Although a few sporadic foci of severe atypia were observed, microscopy did not reveal any cancer findings, but confirmed the tumorous lesion as benign adenoma, showing mild-to-moderate atypia. Postoperative immunohistochemistry revealed no expression of p53 protein. We briefly refer to the rarity of adenoma in the biliary tract and discuss the difficulty of differential diagnosis of neoplastic lesions in the extrahepatic biliary tract.

Mitochondrial DNA mutations are associated with both decreased insulin secretion and advanced microvascular complications in Japanese diabetic subjects.

To assess the roles of various mitochondrial (Mt) DNA mutations in diabetic and nondiabetic subjects, we screened Mt DNAs at the 3243 base pair (bp) and its adjacent portion in unrelated Japanese diabetic and nondiabetic subjects. Furthermore, to clarify the clinical features of diabetic subjects harboring a Mt DNA mutation, we evaluated the ability of insulin secretion and microvascular complications in diabetic subjects. Five hundred thirty-seven diabetic patients and 612 unrelated nondiabetic subjects were recruited into this study. In Mt DNA analyses, Mt DNA was isolated from peripheral leukocytes of the subjects, and then an Mt DNA fragment surrounding the tRNA(Leu(UUR)) site was amplified by the polymerase chain reaction (PCR) using two sets of primers. These fragments were further digested with three kinds of restriction endonucleases and were subjected to agarose gel electrophoresis. When a mutation was present, Mt DNA fragments were directly sequenced with an autosequencer. Baseline characteristics in all subjects were examined, and microvascular complications and insulin secretory capacity in diabetic subjects were newly evaluated. Eight kinds of Mt DNA mutations, which were point mutations, were found in 74 subjects. Each affected subject had only one mutation in the Mt DNA examined. Among them, the mutations at np 3316, 3394, 3593, and 3391 were accompanied by amino acid replacement. Thirty-eight diabetic patients were affected (7.1%), including two subjects with a point mutation at np 3243, and 26 nondiabetic subjects were affected (4.2%). Thus, there was a higher prevalence in diabetic subjects than in nondiabetic subjects. There was no significant difference in the prevalence of maternally inherited diabetes between these two groups. The mean level of urinary C-peptide excretion was lower in diabetic subjects with an Mt DNA mutation (DM+) than in those without it (DM-). Although the prevalence of hypertension in DM+ was higher than that in DM-, diabetic retinopathy and nephropathy in DM+ were problematic, in comparison with those in DM-, when statistical corrections were performed for the effect of hypertension. Furthermore, a strategy based on logistic regression analysis revealed that advanced retinopathy and decreased urinary C-peptide excretion in all diabetic subjects studied were strongly related to the presence of Mt DNA mutation. Our results suggest that Mt DNA mutations in Japanese diabetic subjects are related to the development of diabetes, and also that these mutations are associated with not only a decrease in insulin secretion but also advanced diabetic microvascular complications.

Transgenic Japanese lawngrass (Zoysia japonica Steud.) plants regenerated from protoplasts.

Transgenic Japanese lawngrass (Zoysia japonica Steud.) plants were generated by means of polyethylene glycol (PEG)-mediated direct gene transfer into protoplasts. The plasmid pBC1 was used to deliver the hygromycin phosphotransferase (hph) and ß-glucuronidase (gus) genes into protoplasts. Selection with a high concentration (400 mg/l) of hygromycin yielded a number of resistant calli and about 400 plants were generated. Polymerase chain reaction (PCR) and Southern hybridization analyses revealed that all of then plants tested contained introduced genes. The gus gene regulated by the maize alcohol dehydrogenase-1 (Adh 1) promoter was expressed in the leaves and roots of transgenic Japanese lawngrass plants.

Characterization of the gene for pyruvate,orthophosphate dikinase from rice, a C3 plant, and a comparison of structure and expression between C3 and C4 genes for this protein.

To investigate the molecular changes that might have occurred in genes for pyruvate,orthophosphate dikinase (PPDK) during the evolution of C4 plants from C3 plants, we isolated a full-length cDNA and the corresponding gene for a C4-like PPDK from rice, a C3 gramineous plant and compared their structures and promoter activities to those of the corresponding gene from maize, a C4 gramineous plant. As in maize, there are at least two ppdk genes in rice and one of them was very similar to the maize C4-type ppdk. The deduced amino acid sequence of the rice PPDK was 88% homologous to the maize C4-type PPDK in the mature peptide region and 56% homologous in the transit peptide region. The C4-like ppdk in rice contained 21 exons, which were interrupted by twenty introns, and the positions of the introns were essentially the same as those in the gene from maize, with the except in that the gene from rice had two extra introns. Such extra introns were also found in the C4-type ppdk from a dicot, Flaveria, at the same positions. These results strongly suggest that the two introns were present in an ancestral gene before the divergence of monocot and dicot plants. The C4-like ppdk in rice contained two functionally independent promoters had generated a larger transcript with the transit peptide region and a smaller transcript without this region. The unusual dual-promoter system for transcription has been conserved in the C4-type ppdk gene from maize, indicating that the dual-promoter system is a common feature of ppdk genes in both C3 and C4 plants. The patterns of expression of the two transcripts in rice were different: the larger transcript was expressed exclusively in green leaves at a low level whereas the smaller transcript was expressed in some reproductive organs at a high level. Essentially the same patterns of expression were observed in maize, but the level of expression of the larger transcript in maize green leaves was much higher than that in green leaves of rice. The promoter activities of the rice and maize genes for PPDK were examined directly in a transient expression assay in maize mesophyll protoplasts after electroporation with promoter::beta-glucuronidase chimeric genes. The rice promoter for the smaller transcript was very active in the protoplasts but the rice promoter for the larger transcript had relatively low activity. By contrast, both promoters of the maize gene had high activity. Taken together, these results demonstrate that the rice C4-like ppdk is very similar to the maize C4-type ppdk, not only in terms of primary structure but also in terms of the regulation of expression, with the exception that the strength of the maize promoter for the larger transcript is higher. The results strongly suggest that the genetic alterations required to give rise to the C4-type ppdk gene were relatively limited.

Enhancement of potassium-, and angiotensin II-stimulated aldosterone production by the calcium chelator EGTA in bovine adrenal glomerulosa cells in vitro.

The present study was designed to assess the effect of the calcium chelator EGTA (ethylenglycolbis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid; 1.0 mM) on potassium (8 mM)- and angiotensin II (AII; 10 nM)-stimulated aldosterone production in bovine adrenal glomerulosa cells in vitro. The combined administration of EGTA and potassium, or of EGTA and AII, yielded a significant increase in the levels of aldosterone production. The net increment in aldosterone production after the combined administration of EGTA and potassium, or that after the combined administration of EGTA and AII was also significantly higher than the sum of that evoked by EGTA and potassium alone, or the sum of that evoked by EGTA and AII alone, respectively. At similar concentrations of extracellular ionized calcium ([Ca2+]out) or magnesium, the levels of agonist-stimulated aldosterone production were markedly elevated by the administration of EGTA. These results indicate that lowering [Ca2+]out concentrations with EGTA enhances potassium- and AII-stimulated aldosterone production in bovine adrenal glomerulosa cells in vitro. This enhancement may be predominantly due to another effect of EGTA, in addition to the stimulation of calcium entry into the cells.

cDNA cloning and tissue specific expression of a gene for sucrose transporter from rice (Oryza sativa L.).

We describe the cloning and expression analysis of a sucrose transporter cDNA from a monocot (the rice plant, Oryza sativa L.). The cDNA clone (OsSUT1) encoded an open reading frame of 1,611 bp (537 amino acids) and showed 76.8 to 79.7% similarity at the amino acid level to other sucrose transporters of dicot species. The predicted membrane topology of OsSUT1 protein is made up of 12 transmembrane helices which is consistent with most of the mono- and disaccharide transporters previously identified. When OsSUT1 cDNA was introduced into yeast and expressed, the cells rapidly accumulated sucrose demonstrating that OsSUT1 does, in fact, encode a sucrose transporter. From genomic Southern hybridization OsSUT1 appeared to be a single copy gene. OsSUT1 was expressed in source organs such as leaf blade, leaf sheath and germinating seed whereas little or no expression was observed in some sink organs such as the panicles before heading and the roots. Transcript was observed at high levels in panicles after heading, particularly in the portion containing endosperm and embryo. In addition, expression of OsSUT1 was high in etiolated seedlings and decreased during light-induced greening.

Gas-forming liver abscess following transcatheter hepatic arterial embolization for an iatrogenic intrahepatic pseudoaneurysm: report of a case.

We herein describe a case of gas-forming pyogenic liver abscess following transcatheter hepatic arterial embolization (THAE) for an iatrogenic intrahepatic pseudoaneurysm in a 74-year-old woman. Hemobilia developed 19 days after percutaneous transhepatic cholangio-drainage was performed for the purpose of percutaneous cholangioscopic lithotripsy for the treatment of post-gastrectomy choledocholithiasis. Celiac arteriography disclosed a saccular aneurysm in the right hepatic artery. The pseudoaneurysm was successfully occluded by THAE with gelatin powder and a stainless steel coil of the Gianturco type. Ten days after successful THAE, abdominal computed tomography revealed a gas-containing cavity, which suggested the presence of a gas-forming abscess, in the posterior hepatic segment, and percutaneous transhepatic abscess drainage was performed. Thus, impaired hepatic perfusion following effective THAE and coexisting cholangitis may play an important role in the development of a gas-forming pyogenic liver abscess.

Enhancement of aortic cholesterol deposition by dietary linoleic acid in cholesterol-fed mice: an animal model for primary screening of antiatherosclerotic agents.

We tried to develop an experimental model using mice for the primary screening of antiatherosclerotic agents. Male ICR strain mice were given a high-cholesterol diet supplemented with 10% linoleic acid for 14 weeks. Throughout the experimental period, weight gain of these mice was significantly inhibited as compared to that of control mice given a basal diet, but displayed a steady increase comparable to that of the high-cholesterol diet without linoleic acid. The cholesterol and linoleic acid-fed mice showed increased serum cholesterol and phospholipid levels, and decreased serum triglyceride and high-density lipoprotein-(HDL) cholesterol levels and lecithin/cholesterol acyltransferase (LCAT) activity, as well as a markedly increased lipid peroxide level which was a characteristic appearance in the serum of this mouse model. At the end of the experiment, uniform and significant increases in cholesterol, notably cholesteryl ester, were observed in the aorta. Also found were marked decreases in the aorta contents of desmosine and isodesmosine, which are cross-linking amino acids present only in the elastin. Histological observations showed accumulations of fatty droplets in the intima. These changes were much less in mice receiving a high-cholesterol diet without linoleic acid. In this mouse model, probucol prevented elevation of serum cholesterol, phospholipid, and cholesterol accumulation in the aorta. Increases in lipid peroxide level and decreases in LCAT activity were also prevented. These findings indicate that this mouse model is useful for primary screening of antiatherosclerotic agents with antioxidative activity.

Pharmacological analysis of receptors involved in the late, tachykininergic contractile response to electrical transmural stimulation in isolated rabbit iris sphincter muscle.

We characterized the pharmacological nature of the tachykinin receptor subtype mediating the contractile response to electrical transmural stimulation (ETS) in the isolated rabbit iris sphincter muscle preparation by using selective NK1-receptor antagonists, spantide and L-668,169, and a selective NK2-receptor antagonist, L-659,877. ETS caused a biphasic contraction in this preparation: a rapidly developing cholinergic component followed by a slowly decaying tachykininergic component. The tachykininergic contractile response to ETS was effectively attenuated by spantide and L-668,169, but only slightly by L-659,877, indicating that the tachykinin receptors mediating ETS-induced contraction are of the NK1 type. In the same preparation, the contractile activity of substance P (SP) was slightly more potent than that of neurokinin A (NKA). Unlike in other tissues rich in NK1-receptor subtypes, spantide and L-668,169 antagonized the contractile response to NKA more effectively than that to SP, and the reverse was observed for L-659,877. These results strongly suggest that the tachykininergic contraction induced by ETS in the rabbit iris sphincter preparation is mediated by NK1-receptors which are activated by endogenously released NKA.

Altered vasoconstrictor responsiveness in vitamin D-induced arteriosclerotic rat aortas.

This investigation was undertaken to characterize the vasoconstrictor responsiveness in aortas isolated from a rat model of arteriosclerosis induced by vitamin D2 (VD) administration followed by feeding with a high-cholesterol diet. Cumulative contractile responses to KCl, noradrenaline and serotonin in thoracic aortic strips isolated from arteriosclerotic rats were slightly augmented in concentrations lower than the EC50 value of each agent and rather attenuated in their higher concentrations as compared with those from normal rats. Maximum contractions to the agonists were markedly attenuated in arteriosclerotic aortas; the degree of attenuation was greater in rats treated with a combination of VD and cholesterol than in those treated with VD alone. There was a significant negative correlation between the maximum contraction to KCl, noradrenaline or serotonin and the content of calcium or cholesterol ester in aortas. Removal of endothelium markedly enhanced sensitivity and contractility to the agonists in aortic strips from normal rats, whereas the same procedure only slightly enhanced them in aortic strips from arteriosclerotic rats. These results indicate that in arteriosclerotic rat aortas, contractile responsiveness to agonists of vascular smooth muscle cells is impaired with deposition of calcium and cholesterol, and they suggest that augmentation of contractile responses to the agonists in lower concentrations is due to impairment of endothelial function.

Effects of cholesterol loading on autoimmune MRL-lpr/lpr mice: susceptibility to hypercholesterolemia and aortic cholesterol deposition.

Autoimmune MRL-lpr/lpr (MRL/l) mice, with a systemic lupus erythematosus-like disease, were shown to spontaneously develop hyperlipidemia and yet be susceptible to diet-induced hypercholesterolemia and aortic cholesterol deposition. Control animals on a basal diet showed significant increases in the serum total cholesterol, phospholipids, triglycerides, high density lipoprotein (HDL)-cholesterol and lipid peroxide levels, but a significant decline in the serum lecithin: cholesterol acyltransferase (LCAT) activity compared to those of 5-week-old mice. Animals on the high-cholesterol diet showed a rapid rise in serum total cholesterol to a plateau level (800 mg/100 ml) that was approximately 2.5 times higher than that in the control animals on a basal diet. However, the levels of serum triglycerides, HDL-cholesterol and lipid peroxides significantly decreased (by 61%, 23% and 53%, respectively) compared to those of the control animals, whereas LCAT activity and phospholipid level were not affected. The aortic contents of total cholesterol, free cholesterol and cholesteryl ester were significantly higher (by 35%, 36% and 31%, respectively) in animals fed the high-cholesterol diet than the control animals. These findings suggest that MRL/l mice are susceptible to diet-induced hypercholesterolemia and aortic cholesterol deposition.

[Intermittent intra-arterial infusion chemotherapy using implantable reservoir for the treatment of hepatic metastasis].

Intermittent intra-arterial infusion chemotherapy using implantable reservoir was performed for hepatic metastases and the therapeutic effects were evaluated. We treated 21 patients with hepatic metastases of gastric cancer in 8 cases, rectal cancer in 6 cases, colon cancer in 5 cases and breast cancer in 2 cases. The reduction rate of the tumor diameter as seen by CT scan was used as a criteria for antitumor effectiveness. Only 1 case was PR, for an efficacy rate of 5%. Changes in serum CEA level were related to antitumor effectiveness.