Extremely small-diameter, high-density, radio frequency, plasma sources and central gas feeding for next-generation electrodeless plasma thrusters.

Radio frequency (RF) waves including helicon waves can readily produce high-electron-density (ne up to 1013 cm-3) plasmas with a broad range of external operating parameters. Various featured RF and helicon sources in a wide range of scales are suitable for plasma propulsion schemes. Electrodeless RF plasmas have no direct contact between electrodes and antennas, which enables long-life operation. However, one of the crucial problems is to reduce the plasma size for future applications in nano- and pico-satellites. Diagnostics of the plasma parameters in a small area should also be improved. Furthermore, to increase plasma performance, it is important to consider the radial electron density (ne) profile with an increasing upper limit, observed in high-density helicon sources due to the depletion of neutrals. This problem may be controlled by the location of neutral gas feeding and knowledge of the gas pressure distribution. Here, production of RF plasmas, with extremely small diameters from 3-mm down to 0.5-mm including 1-mm, was demonstrated, and characterization of ne and the electron temperature was performed with a collisional radiative model. Finally, to improve plasma performance such as ne and the thrust force, internal gas feeding was demonstrated using a developed Pirani gauge to measure neutral density.

Durability of protective effect of dulaglutide on pancreatic ß-cells in diabetic mice: GLP-1 receptor expression is not reduced despite long-term dulaglutide exposure.

AIMS: It is well-known that chronic exposure to large amounts of ligand leads to downregulation of its receptor. It is not known, however, whether a GLP-1R agonist downregulates its receptor. For this reason, our study examined whether GLP-1R expression is reduced after long-term exposure to dulaglutide (Dula) in non-diabetic and diabetic mice. METHODS: Seven-week-old male db/db and db/m mice were given either Dula (0.6mg/kg×2/week) or a control vehicle (CTL) for 17 weeks. Various metabolic parameters, such as glucose-stimulated insulin secretion (GSIS), insulin and TG content in islets, were evaluated after the intervention. ß-cell-related gene expression was also analyzed by real-time RT-PCR. RESULTS: In db/m mice, GLP-1R expression in ß-cells did not decrease, not even after long-term administration of Dula, compared with control mice, while GLP-1R expression in 24-week-old db/db mice treated with Dula was augmented, rather than downregulated, compared with 24-week-old CTL db/db mice. This was probably due to improved glycaemic control. In db/db mice treated with Dula, food intake and blood glucose levels were significantly decreased up to 24 weeks of age compared with CTL db/db mice, and their expression levels of various ß-cell-related genes, insulin content and GSIS were also enhanced. In contrast, oxidative and endoplasmic reticulum stress, inflammation, fibrosis and apoptosis were suppressed with Dula treatment. CONCLUSION: Dula exerts beneficial effects on glycaemic control and has long-lasting protective effects on pancreatic ß-cells. GLP-1R expression levels were not reduced at all in non-diabetic as well as diabetic mice despite long-term dulaglutide exposure.

Influence of adrenal subclinical hypercortisolism on hypertension in patients with adrenal incidentaloma.

OBJECTIVE: The purpose of our study was to clarify whether subtle cortisol-producing tumors, such as not only subclinical Cushing's syndrome (SubCS) but also subclinical hypercortisolism (SH), influence the prevalence of hypertension, since numerous basic research studies have noted that glucocorticoid excess influences blood pressure. METHODS: 80 patients with adrenocortical adenomas (39 women and 41 men; mean age 62.1 years) were enrolled. SubCS was diagnosed using a diagnostic criteria, and SH was diagnosed as the presence of a serum cortisol level greater than 50 nmol/L following 1-mg dexamethasone suppression test (DST). RESULTS: SubCS, SH, or non-functioning adrenocortical adenoma (NF) was diagnosed in 14, 13, or 53 patients, respectively. The prevalence of hypertension differed significantly among the diagnoses (SubCS, 78.6%; SH, 84.6%; NF, 39.6%; P=0.002), whereas no differences in other clinical characteristics such as age, sex, or waist girth were observed. The patients with SH had an 11.7-fold increased risk (95% confidence interval: 1.9-72.7, P=0.009) and those with SubCS had a 9.5-fold increased risk (95% confidence interval: 1.9-48.3, P=0.007) for hypertension compared to those with NF using a multivariate analysis. CONCLUSION: We demonstrated that subtle cortisol-producing tumors, such as SH as well as SubCS, were an independent risk factor for hypertension. The cut-off value of the 1-mg DST would be appropriate to predict the development of hypertension.

Two cases of nephrotic syndrome (NS)-induced acute kidney injury (AKI) associated with renal hypouricemia.

Renal hypouricemia is a clinical disorder attributed to an increased renal urate excretion rate and is well known to involve a high risk of urolithiasis and exercise-induced acute kidney injury (AKI). This report concerns two interesting cases of nephrotic syndrome (NS)-induced AKI associated with renal hypouricemia. A 64-year-old female (Case 1) and a 37-year-old male (Case 2) were hospitalized because of AKI (serum creatinine: 2.07 mg/dl and 3.3 mg/dl, respectively), oliguria and NS. They were treated with prednisolone and temporary hemodialysis. Renal function improved, but hypouricemia persisted during hospitalization. Histological findings in both cases led to a diagnosis of minimal change nephrotic syndrome and identification of the diuretic phase of tubulointerstitial damage because of findings such as acute tubular necrosis. Furthermore, distal tubules of Case 2 showed an amorphous mass, possibly a uric acid crystal. Analysis of the two cases with the URAT1 gene, encoded by SLC22A12, found a homozygous mutation in exon 4 (W258stop) of each one. Our cases show that patients with renal hypouricemia may be susceptible to AKI without involvement of exercise if they possess some facilitators. Renal hypouricemic patients should therefore be carefully examined for all complications from renal hypouricemia because of high risk of AKI.

Hepatitis C virus-related kidney disease: various histological patterns.

BACKGROUND: Although hepatitis C virus (HCV) infection is known to be associated with Type 2 cryoglobulinemic glomerulopathy (CG), only a few reports about other types of nephropathy have been published. METHODS: 68 HCV antibody positive patients in whom renal biopsy had been performed for persistent proteinuria, hematuria, and/or renal dysfunction between 1992 and 2008 at our institute were included. The histological, clinical and laboratory characteristics including the age, gender, hypertension, diabetes mellitus, liver histology (chronic hepatitis or liver cirrhosis), HCV-RNA, HCV genotype, splenomegaly, gastroesophageal varices, serum creatinine, hemoglobin, platelet count, rheumatoid factor, cryoglobulin, IgG, IgA, IgM, CH50, C3, C4, creatinine clearance, 24-h protein excretion, and hematuria, between their nephropathy with and without immune deposition were compared. RESULTS: Nephropathy was classified into two groups based on the detection of immune deposits by immunofluorescence microscopy: i.e., a positive group (n = 39) and a negative group (n = 29). The former group was further classified into three types of nephropathy: IgG dominant group (n = 10) (including membranous nephropathy (MN)), IgA dominant group (n = 20) (including IgA nephropathy (IgAN)), membranoproliferative glomerulonephritis (MPGN) (IgA type)), and IgM dominant group (n = 9) (MPGN apart from the IgA type). The latter group included diabetic nephropathy (n = 13), focal glomerular sclerosis (n = 4), and benign nephrosclerosis (n = 3), malignant nephrosclerosis (n = 1), tubulointerstitial nephritis (TIN) (n = 2), minimal change nephrotic syndrome (n = 1), cast nephropathy (n = 1), granulomatous TIN (n = 1), and others (n = 3). An increased serum IgM level, hypocomplementemia, splenomegaly, thrombocytopenia, liver cirrhosis, hematuria, and a high HCV RNA level were features of patients with MPGN of IgM dominant group (consistent with "CG"). CONCLUSIONS: Our results showed various histological patterns of HCV-related kidney disease and the specificity of CG, and revealed that a minority of HCV patients (n = 7) presented typical CG, while IgAN, MN, and diabetic nephropathy were more frequent.

A patient with pregnancy-related acute abdomen after hemodialysis for over 18 years.

Because pregnancy is rare in women with end-stage renal disease, dialysis patients have not been reported to present with acute abdominal symptoms related to pregnancy including ectopic pregnancy. A 41-year-old woman treated with hemodialysis for over 18 years was brought to the emergency room at our institution because of acute abdominal pain. Ultrasonography detected an abdominal fluid collection, and her anemia had worsened (hematocrit 18%). Emergency laparoscopic exploration disclosed a hemorrhagic corpus luteum of pregnancy, causing ovarian bleeding on the left. Coagulation of bleeding points was carried out. At this time, pregnancy at 7 weeks of gestation was discovered. After the procedures, hemodialysis frequency was increased to 5 times weekly, and an erythropoietin derivative was administered to maintain a hematocrit above 30%. The patient developed no hypertension. At 33 weeks of gestation, cesarean section was performed because of a decrease in amniotic fluid and frequent late deceleration of the fetal heart rate. A live baby girl weighing 1,422 g was born. The successful pregnancy reflects remarkable progress in dialysis technology. Pregnancy, then, can underlie an acute abdomen in childbearing-age women (14 - 44 years old) undergoing long-term dialysis.

A pediatric occurrence of crescentic glomerulonephritis associated with antineutrophil cytoplasmic antibodies and mesangial IgA deposits.

Antineutrophil cytoplasmic antibody-(ANCA) associated glomerulonephritis usually shows histopathologic features of pauciimmune crescentic glomerulonephritis and occurs late in life. We report a 14-year-old Japanese girl presenting with proteinuria, hematuria and mildly elevated serum creatinine. A renal biopsy specimen demonstrated crescentic glomerulonephritis, immunofluorescence showed mesangial IgA staining. Electron microscopic examination disclosed paramesangial deposits. Serum ANCA against myeloperoxidase (MPO) were detected at high titers. Myeloperoxidase-ANCA-related nephritis accompanied by IgA nephropathy is considered rare in childhood and teen years. Yet, if ANCA assays and detailed electron microscopic examination of renal specimens were performed routinely in patients with rapidly progressive glomerulonephritis, the diagnosis might be more frequent in young patients.

Basic and clinical aspects of parathyroid hyperplasia in chronic kidney disease.

Marked parathyroid hyperplasia develops in patients with chronic kidney disease, especially those with long dialysis vintage. Although progression of hyperplasia is associated with downregulation of vitamin D receptor and calcium-sensing receptor, initial abnormality that triggers and maintains parathyroid cell proliferation, as well the critical abnormality for the progression of diffuse hyperplasia to nodular hyperplasia, still remains to be elucidated. It is quite important for the optimal management of renal osteodystrophy to recognize the development of nodular hyperplasia, because the cells in nodular hyperplasia are usually resistant to medical therapy and further treatment of such patients often leads to vascular calcification. For this purpose, size and blood supply of enlarged parathyroid glands have been used as good clinical markers. Furthermore, we have recently shown that the serum fibroblast growth factor 23 level can be used for predicting refractory hyperparathyroidism. Once nodular hyperplasia develops in any of the enlarged parathyroid glands, such patients need to be treated by parathyroid intervention including percutaneous ethanol injection therapy. In addition, as direct vitamin D injection therapy has been shown to induce regression of hyperplasia, it may become possible to reverse or normalize established nodular hyperplasia if we can develop new agents with such effects in the near future.

Relationship between HbA(1)c and mortality in a Japanese population.

AIM/HYPOTHESIS: HbA(1)c concentrations are known to be associated with all-cause excess mortality risk in Caucasians. However, the relationship has not been clarified well in the Japanese. In addition, studies of the relationship between HbA(1)c and mortality from malignant neoplasms are scarce. METHODS: HbA(1)c was measured for 3,710 people of a cohort composed of A-bomb survivors and controls. At baseline they were divided into five groups: a normal HbA(1)c group of 1,143 individuals with HbA(1)c of <5.5%, a slightly high but normal HbA(1)c group of 1,341 individuals with HbA(1)c > or =5.5% to <6.0%, a slightly high HbA(1)c group of 589 individuals with HbA(1)c > or =6.0% to <6.5%, a high HbA(1)c group of 259 individuals with HbA(1)c > or =6.5%, and a group of 378 individuals known to have type 2 diabetes. Using a Cox proportional hazards model, hazard ratios based on comparisons with the normal HbA(1)c group were obtained. RESULTS: During the observation period there were 754 deaths. For all-cause and cardiovascular disease mortality, a significant increase of the hazard ratio was observed for the slightly high HbA(1)c group. A similar increase in malignant neoplasm-related mortality was observed for both the high HbA(1)c group and the diabetes group. CONCLUSIONS/INTERPRETATION: Our results suggest that individuals in the Japanese population with HbA(1)c levels of 6% or more might have increased mortality risk. The results indicate that HbA(1)c measurements should be sought even for people who have not been diagnosed with diabetes.

A comparison between Japanese-Americans living in Hawaii and Los Angeles and native Japanese: the impact of lifestyle westernization on diabetes mellitus.

We have been conducting the Hawaii-Los Angeles-Hiroshima Study since 1970, mainly to determine the effects of environmental changes on various diseases by comparing Japanese-Americans with native Japanese subjects. Japanese-Americans living in Hawaii and Los Angeles are originated mainly from Hiroshima, Japan and are genetically identical with native Japanese. Through this study, we made several clear observations about Japanese-Americans. First, Japanese-Americans were highly exposed to a westernized lifestyle ; in other words, a relatively high fat and simple carbohydrate diet with low physical activity as compared to native Japanese. Second, the prevalence of type 2 diabetes among Japanese-Americans and death from ischemic heart disease among Japanese-American diabetic patients were higher. Third, the serum fasting insulin level as well as the insulin level after a glucose load, was higher among Japanese-Americans, even when the serum glucose levels were not statistically different as compared to native Japanese. Accordingly, Japanese-Americans were thought to have a high insulin resistance status. However, the initial insulin response after a glucose load was low, which was more similar to Japanese people than to Caucasians. Fourth, the total cholesterol and triglyceride levels were higher among Japanese-Americans. These results are supposed to be derived from the insulin resistant status by the westernization of lifestyle, as well as from the weakness of pancreatic beta cell function that is supposed to be genetically regulated among Japanese. In conclusion, it appears that for genetically Japanese people, environmental factors are important for the development of metabolic diseases such as diabetes mellitus and cardiovascular disease.

The effect of polymorphism in the intestinal fatty acid-binding protein 2 gene on fat metabolism is associated with gender and obesity amongst non-diabetic Japanese-Americans.

AIM: The role of the codon 54 polymorphism of the fatty acid-binding protein 2 (FABP2) gene on fat metabolism has been controversial. Assuming that the effects of the polymorphism were modulated by gender and obesity which were related to lipid and glucose metabolism, we investigated this polymorphism and its effect on fat metabolism according to such factors. METHODS: Subjects were Japanese-Americans (123 men and 126 women) who were diagnosed as non-diabetic by a 75 g oral glucose tolerance test at the baseline. RESULTS: During approximately 7.8 years, 49 (24 men and 25 women) were diagnosed with type 2 diabetes. In a Cox proportional hazards model, this polymorphism was not a significant variable in the incidence of diabetes in either gender. Amongst non-obese men with the Thr54 allele, there was a significant elevation of triglycerides (TGs) (p=0.033) compared with alanine (Ala) homozygotes. Women with the Thr54 allele had significantly elevated total cholesterol (p=0.033) and low-density lipoprotein-cholesterol (LDL-C) (p=0.023) compared with Ala54 homozygotes. CONCLUSIONS: These results therefore suggested that the effects of the FABP2 polymorphism on TG, LDL-C and body mass index were associated with gender difference and obesity amongst non-diabetic Japanese-American subjects.

Erectile dysfunction is strongly linked with decreased libido in diabetic men.

Erectile dysfunction frequently occurs with diabetes mellitus. A survey of diabetic men was conducted by anonymous questionnaire to investigate the associations of erectile dysfunction with various predictive factors. A total of 112 diabetic males without an obvious history of erectile dysfunction were available for analyses. The mean age and duration of diabetes were 53.7 +/- 12.2 years and 10.2 +/- 8.6 years (mean +/- standard deviation), respectively. The questionnaire included questions on the presence or absence of smoking, hypertension, libido and subjective symptoms of diabetic neuropathy that may be associated with erectile dysfunction. Analysis of the answers to the questionnaire revealed that 40% of the patients complained of erectile dysfunction (erection 'always insufficient'). Erectile dysfunction was significantly correlated with age (p = 0.005), but not with duration of diabetes (p = 0.25), adjusted for age. Erectile dysfunction was also associated with sensory neuropathy and reduced libido, independently of age. The logistic regression analysis revealed that erectile dysfunction was positively associated with reduced libido and age. The odds ratio of erectile dysfunction for reduced compared to unreduced libido was 18.21, suggesting that psychogenic factors have a marked influence on erectile dysfunction. It is concluded that the presence of erectile dysfunction should be considered when symptoms related to diabetic neuropathy are observed; psychological approaches, such as sexual counseling, could be applied for the treatment of erectile dysfunction.

RP-1776, a novel cyclic peptide produced by Streptomyces sp., inhibits the binding of PDGF to the extracellular domain of its receptor.

RP-1776, a novel cyclic peptide, was isolated from the culture broth of Streptomyces sp. KY11784. RP-1776 selectively inhibited the binding of PDGF BB to the extracellular domain of the PDGF beta-receptor with an IC50 value of 11 +/- 6 microM. Detailed binding experiments suggested that RP-1776 directly interacts with PDGF BB. RP-1776 inhibited the phosphorylation of the PDGF beta-receptor induced by PDGF BB. These results suggested that RP-1776 antagonizes the signaling of PDGF BB probably through the inhibition of PDGF BB binding to the PDGF beta-receptor.

Evaluation of the general suitability of the rat for the micronucleus assay: the effect of cyclophosphamide in 14 strains.

To evaluate the general suitability of the rat for the micronucleus assay, we conducted the assay in males of 14 different strains, 13 inbred (ACI, BN, BUF, COP, DRH, F344, IS, LEW, RCS, SHR, WAG, WKYO, WTC) and 1 outbred (SD), using cyclophosphamide as the test chemical. Cyclophosphamide at 0 (vehicle), 5, 10, or 20mg/kg per day was administered orally twice, 24h apart, to five rats per dosage group. Bone marrow and peripheral blood were collected 24h after the second treatment. All 14 strains showed a positive response to cyclophosphamide, with slight differences in sensitivity. We concluded that the rat is suitable for the micronucleus assay regardless of strain.

Synthesis, SARs, and pharmacological characterization of 2-amino-3 or 6-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic acid derivatives as potent, selective, and orally active group II metabotropic glutamate receptor agonists.

(+)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid (4, LY354740), a highly selective and orally active group II metabotropic glutamate receptor (mGluR) agonist, has increased interest in the study of group II mGluRs. Our interest focused on a conformationally constrained form of compound 4, because it appeared that the rigid form resulted in not only selectivity for group II mGluR but was orally active. Therefore, we introduced a fluorine atom to compound 4, based on the molecular size (close resemblance to hydrogen atom) and electronegativity (effects on the electron distribution in the molecule) of this atom and carbon-fluorine bond energy. Compound (+)-7 (MGS0008), the best compound among 3-fluoro derivatives 7-10, retained the agonist activity of compound 4 for mGluR2 and mGluR3 ((+)-7: EC(50) = 29.4 +/- 3.3 nM and 45.4 +/- 8.4 nM for mGluR2 and mGluR3, respectively; 4: EC(50) = 18.3 +/- 1.6 nM and 62.8 +/- 12 nM for mGluR2 and mGluR3, respectively) and increased the oral activity of compound 4 ((+)-7: ED(50) = 5.1 mg/kg and 0.26 mg/kg for phencyclidine (PCP)-induced hyperactivity and PCP-induced head-weaving behavior, respectively; 4: ED(50) = >100 mg/kg and 3.0 mg/kg for PCP-induced hyperactivity and PCP-induced head-weaving behavior, respectively). In addition, a compound [(3)H]-(+)-7 binding study using mGluR2 or 3 expressed in CHO cells was successful ((+)-7: K(i) = 47.7 +/- 17 nM and 65.9 +/- 7.1 nM for mGluR2 and mGluR3, respectively; 4: K(i) = 23.4 +/- 7.1 nM and 53.5 +/- 13 nM for mGluR2 and mGluR3, respectively). On the basis of a successful result of compound 7, we focused on the introduction of a fluorine atom on the C6 position of compound 4. (1R,2S,5R, 6R)-2-amino-6-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic acid ((-)-11) exhibited a high degree of agonist activity for group II mGluRs equal to that of compound 4 or 7 ((-)-11: K(i) = 16.6 +/- 5.6 and 80.9 +/- 31 nM for mGluR2 and mGluR3, respectively). Our interest shifted to modification on CH(2) at C4 position of compound 11, since replacement of the CH(2) group with either an oxygen atom or sulfur atom yielded compound 5 or 6, resulting in increased agonist activity. We selected a carbonyl group instead of CH(2) at the C4 position of compound 11. The carbonyl group might slightly change the relative conformation of three functional groups, the amino group and two carboxylic acids, which have important roles in mediating the interaction between group II mGluRs and their ligand, compared with the CH(2) group of 4, oxygen atom of 5, and sulfur atom of 6. (1R,2S,5S,6S)-2-Amino-6-fluoro-4-oxobicyclo[3.1. 0]hexane-2,6-dicarboxylic acid monohydrate ((+)-14, MGS0028) exhibited a remarkably high degree of agonist activity for mGluR2 (K(i) = 0.570 +/- 0.10 nM) and mGluR3 (K(i) = 2.07 +/- 0.40 nM) expressed in CHO cells but not mGluR4, 6, 7, 1a, or 5 expressed in CHO cells (K(i) = >100 000 nM). Furthermore, compound (+)-14 strongly inhibited phencyclidine (PCP)-induced head-weaving behavior (ED(50) = 0.090 microg/kg) and hyperactivity (ED(50) = 0.30 mg/kg) in rats. Thus, (+)-7 and (+)-14 are potent, selective, and orally active group II mGluR agonists and might be useful not only for exploring the functions of mGluRs but in the treatment of schizophrenia.

Induction of midbrain dopaminergic neurons from ES cells by stromal cell-derived inducing activity.

We have identified a stromal cell-derived inducing activity (SDIA) that promotes neural differentiation of mouse ES cells. SDIA accumulates on the surface of PA6 stromal cells and induces efficient neuronal differentiation of cocultured ES cells in serum-free conditions without use of either retinoic acid or embryoid bodies. BMP4, which acts as an antineuralizing morphogen in Xenopus, suppresses SDIA-induced neuralization and promotes epidermal differentiation. A high proportion of tyrosine hydroxylase-positive neurons producing dopamine are obtained from SDIA-treated ES cells. When transplanted, SDIA-induced dopaminergic neurons integrate into the mouse striatum and remain positive for tyrosine hydroxylase expression. Neural induction by SDIA provides a new powerful tool for both basic neuroscience research and therapeutic applications.

[Cost analysis on stomach cancer treatment in Japanese hospitals].

BACKGROUND: In an effort to further the discussion of DRG/PPS, we performed a coat analysis on stomach cancer cases in Japanese hospitals. We analyzed individual in-patient data (both clinical and financial) to research the amount of variation in treatment and costs. METHODS: The data was taken from two hospitals. In order to compare the daily cost/revenue of each episode of operation/hospitalization, we analyzed the data that was stored for reimbursement purposes by using patient ID numbers. We also simulated the cost/revenue in cases where the length of stay could be shortened with clinical pathways created by Japanese hospitals. RESULTS: (1) There is a common pattern for the operation/hospitalization of stomach cancer cases if the patient's condition, like severity, is well controlled. However, there is still a noticeable difference in the length of stay for patients both before and after the operation. (2) Hospitals are currently losing money by having extended patients' lengths of stay. Simulation results indicate that hospitals can reduce losses, even become profitable, by shortening the patient's length of stay. CONCLUSIONS: The potentiality of implementing a standard treatment process and/or a fixed payment system, like DRG/PPS, is high, because there is a common a pattern among the treatment process. More research must be completed in this area, specifically on the significant variations in lengths of stay and the methodology for standardization of treatment. Furthermore, clinical outcomes research must also be done.

G protein-coupled receptor kinase-mediated desensitization of metabotropic glutamate receptor 1A protects against cell death.

Metabotropic glutamate receptors (mGluRs) constitute a unique subclass of G protein-coupled receptors (GPCRs) that bear little sequence homology to other members of the GPCR superfamily. The mGluR subtypes that are coupled to the hydrolysis of phosphoinositide contribute to both synaptic plasticity and glutamate-mediated excitotoxicity in neurons. In the present study, the expression of mGluR1a in HEK 293 cells led to agonist-independent cell death. Since G protein-coupled receptor kinases (GRKs) desensitize a diverse variety of GPCRs, we explored whether GRKs contributed to the regulation of both constitutive and agonist-stimulated mGluR1a activity and thereby may prevent mGluR1a-mediated excitotoxicity associated with mGluR1a overactivation. We find that the co-expression of mGluR1a with GRK2 and GRK5, but not GRK4 and GRK6, reduced both constitutive and agonist-stimulated mGluR1a activity. Agonist-stimulated mGluR1a phosphorylation was enhanced by the co-expression of GRK2 and was blocked by two different GRK2 dominant-negative mutants. Furthermore, GRK2-dependent mGluR1a desensitization protected against mGluR1a-mediated cell death, at least in part by blocking mGluR1a-stimulated apoptosis. Our data indicate that as with other members of the GPCR superfamily, a member of the structurally distinct mGluR family (mGluR1a) serves as a substrate for GRK-mediated phosphorylation and that GRK-dependent "feedback" modulation of mGluR1a responsiveness protects against pathophysiological mGluR1a signaling.

Cryptic dimer interface and domain organization of the extracellular region of metabotropic glutamate receptor subtype 1.

Previously, we produced the whole extracellular region of metabotropic glutamate receptor subtype 1 (mGluR1) in a soluble form. The soluble receptor retained a ligand affinity comparable with that of the full-length membrane-bound receptor and formed a disulfide-linked dimer. Here, we have identified a cysteine residue responsible for the intermolecular disulfide bond and determined domain organization of the extracellular region of mGluR1. A mutant, C140A, was a monomer under nonreduced conditions by SDS-polyacrylamide gel electrophoresis; however, C140A was eluted at the position similar to that of mGluR113, the wild type soluble receptor, by size exclusion column chromatography. Furthermore, C140A bound a ligand, [(3)H]quisqualate, with an affinity similar to that obtained by mGluR113. Oocytes injected with RNA for full-length mGluR1 containing C140A mutation showed responses to ligands at magnitudes similar to those with wild type full-length RNA. Thus, elimination of the disulfide linkage did not perturb the dimer formation and ligand signaling, suggesting that cryptic dimer interface(s) possibly exist in mGluR1. Limited proteolysis of the whole extracellular fragment (residue 33-592) revealed two trypsin-sensitive sites, after the residues Arg(139) and Arg(521). A 15-kDa NH(2)-terminal proteolytic fragment (residue 33-139) was associated with the downstream part after the digestion. Arg(521) was located before a cysteine-rich stretch preceding the transmembrane region. A new shorter soluble receptor (residue 33-522) lacking the cysteine-rich region was designed based on the protease-sensitive boundary. The purified receptor protein gave a K(d) value of 58.1 +/- 0.84 nm, which is compatible to a reported value of the full-length receptor. The B(max) value was 7.06 +/- 0. 82 nmol/mg of protein. These results indicated that the ligand-binding specificity of mGluR1 is confined to the NH(2)-terminal 490-amino acid region of the mature protein.