RESUMO
BACKGROUND: The number of patients living with depression continues to increase in Japan. The economic effects of depression include loss of productivity due to both absenteeism and presenteeism. Gender differences have been reported in prevalence, onset pathways and subjective symptoms of depression. AIMS: To understand how workers with major depressive disorder (MDD) perceive problems in the workplace and examine gender differences in their self-perceived levels of functioning at work, noticed during the initial stages of depression. METHODS: This is a cross-sectional study of Japanese workers with MDD. Participants' self-perceived changes in the level of functioning at work were surveyed after the diagnosis during the first visit. The relationship between gender and changes in the level of functioning at work as initially perceived by the participants themselves was analysed using the chi-square test, supplemented by a residual analysis. RESULTS: We administered the survey to 147 workers with MDD. In terms of gender differences in initial self-perceived changes in the level of functioning at work, the proportion of men reporting reduced work efficiency was significantly higher than that of women, while the proportion of women reporting deterioration in relationships with colleagues and superiors was significantly higher than that of men. CONCLUSIONS: The findings suggest that greater attention to reduced work efficiency by men and to deterioration in work relationships by women with MDD should be essential components of self-care. Managers need to pay attention to the level of functioning and provide adequate social support for employees.
Assuntos
Depressão , Transtorno Depressivo Maior , Absenteísmo , Estudos Transversais , Feminino , Humanos , Japão , Masculino , Caracteres Sexuais , Inquéritos e Questionários , Local de TrabalhoRESUMO
This research was designed to investigate the acoustic characteristics of voluntary expiratory sounds after swallow for detecting dysphagia. Forty-nine patients with complaints of swallow difficulty received a videofluorographic (VF) examination. They were divided into three groups: nine who did not have any apparent disease (Group N), 22 patients with head and neck cancer (Group H&N) and 18 patients with other diseases including cerebrovascular disease (Group OD). After liquid barium swallows, they exhaled voluntarily without voicing. Videofluorographic findings were classified into four groups: normal (Normal), acceptable swallow (Acceptable), swallow with residue (Resid) and swallows with penetration or aspiration (Pen/Asp). The duration of expiratory sounds was measured on the time waveform. Frequency characteristics of expiratory sounds were obtained using one-third octave band analysis ranging from 62·5 to 2000·0 Hz of central frequency. The averaged level of the 1000·0-Hz band was chosen as the reference band level (RB level). The revised averaged level of each band was obtained by subtracting the RB level from the averaged level of each band. Zero decibel of the revised magnitude of the 125·0-Hz band was set as the critical value to differentiate dysphagia (Resid or Pen/Asp) from no dysphagia (Normal or Acceptable). Comparison of this assessment with VF findings showed a significant percentage agreement (85·4%). These results suggest that frequency characteristics of post-swallow expiratory sounds can differentiate dysphagia from no dysphagia among multiple dysphagic patient groups.
Assuntos
Transtornos de Deglutição/diagnóstico , Deglutição/fisiologia , Expiração/fisiologia , Som , Idoso , Idoso de 80 Anos ou mais , Compostos de Bário , Feminino , Fluoroscopia/métodos , Humanos , Masculino , Pessoa de Meia-Idade , Gravação em Vídeo/métodosRESUMO
BACKGROUND: The growth of parotid glands is inhibited by liquid diet intake during growing period, while that of submandibular glands is not affected. This study examined how liquid diet intake affects nerve growth in the parotid and submandibular glands of growing rats, because nerves are closely involved in the maintenance of salivary gland structure. MATERIALS AND METHODS: Male Wistar rats were weaned at 21 days of age. Then, rats were fed a pellet diet and a liquid diet in the control group and experimental group, respectively. At 0, 2, 4, or 8 weeks, they were euthanised by isoflurane overdose, and parotid and submandibular glands were removed. The frozen sections were made and immuno-stained with anti-protein gene product 9.5 (PGP 9.5) antibody (general nerve marker), anti-tyrosine hydroxylase (TH) antibody (sympathetic nerve marker), or anti-neuronal nitric oxide synthase (nNOS) antibody (parasympathetic nerve marker). RESULTS: In control parotid glands, scattered punctate or short linear patterns of PGP 9.5-positive sites were observed at week 0. After 2 weeks, PGP 9.5-positive sites, some of which were arranged in long linear patterns, increased in number. There were some TH-positive sites at week 0. After 2 weeks, there were increasing numbers of TH-positive sites, often in long linear patterns. At week 0, there were very few nNOS-positive sites, and nNOS immunoreactivity increased over time. After week 4, they demonstrated linear patterns. In the experimental parotid glands, there were fewer PGP 9.5- and nNOS-positive sites than in control parotid glands at each time point, although TH immunoreactivity was similar between two groups at each time point. In control submandibular glands, few punctate exhibited PGP 9.5-positive site were observed at week 0. At week 4, PGP 9.5 immunoreaction increased and showed linear patterns. TH-positive sites demonstrated punctate or short linear patterns at week 0, and thereafter TH immunoreactivity increased and were arranged in long linear patterns. There were few nNOS-positive sites at week 0, and they gradually increased after week 4. The immunoreactivities of all antibodies in the experimental submandibular glands were similar to those in the control at each time point. CONCLUSIONS: Parasympathetic nerve growth in rat parotid glands was inhibited by liquid diet intake during the growth period, while liquid diet intake did not affect parasympathetic nerve growth nor sympathetic nerve growth in rat submandibular glands.
Assuntos
Glândula Parótida , Glândulas Salivares , Ratos , Masculino , Animais , Ratos Wistar , Glândula Submandibular , DietaRESUMO
The physiological role of soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE) proteins in insulin exocytosis has been reported in pancreatic beta-cells. To determine whether the beta-cells of GK rats, a nonobese rodent model of type 2 diabetes, exhibit abnormalities in their SNARE proteins, we studied the expression and function of target (t)-SNAREs, syntaxin 1A, and synaptosomal-associated protein of 25 kDa (SNAP-25) in GK rat islets. Although insulin release and insulin content of islets isolated from 12-week-old GK rats were reduced, the proinsulin biosynthetic rate was about twofold higher than that in control rat islets, and no change in the preproinsulin mRNA level was observed. Pulse-chase experiments suggested the increased degradation of insulin in GK rat islets. Immunoblot analysis revealed that protein levels of syntaxin 1A and SNAP-25 in GK rat islets decreased to approximately 60% of the levels in control rat islets. We then examined whether the restoration of the decreased expression of t-SNAREs to the normal level in GK rat islets affected insulin secretion. Restoration was achieved by the overexpression of syntaxin 1A and SNAP-25 via the recombinant adenovirus-mediated gene transduction system, which recovered levels of these proteins to almost control levels. Glucose-stimulated insulin release from AdexlCA syntaxin 1A and Adex1CA SNAP-25-infected GK rat islets increased up to approximately 135 and 200%, respectively, of those from uninfected GK rat islets, although no difference in basal (2.2 mmol/l glucose) insulin release was evident between them. We conclude that decreased expression of t-SNAREs in GK rat islets is in part the defect responsible for impaired insulin secretion.
Assuntos
Antígenos de Superfície/genética , Diabetes Mellitus Tipo 2/fisiopatologia , Insulina/genética , Ilhotas Pancreáticas/metabolismo , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Proteínas de Transporte Vesicular , Animais , Antígenos de Superfície/metabolismo , Glicemia/metabolismo , Células Cultivadas , Regulação da Expressão Gênica , Técnicas de Transferência de Genes , Glucose/farmacologia , Insulina/biossíntese , Insulina/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Masculino , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , RNA Mensageiro/genética , Ratos , Ratos Endogâmicos , Ratos Wistar , Proteínas Recombinantes/metabolismo , Valores de Referência , Proteínas SNARE , Proteína 25 Associada a Sinaptossoma , Sintaxina 1RESUMO
The ontogeny of the GLUT3 glucose transporter gene and protein expression was studied in rat brain. Northern blot analysis using total RNA from rat brains at different developmental stages revealed that the levels of GLUT3 mRNA were very low during the embryonic stage and increased towards the postnatal stage. Immunohistochemistry using a specific antibody showed that the expression of GLUT3 protein was barely detectable in the embryonic stage, but was clearly detected on the plasma membrane of neuronal cells from 10 days after birth to the adult. Expression of GLUT3 mRNA and protein in the cerebral neuronal cell cultures was also examined during the maturation of neurons. GLUT3 glucose transporter of primary neuronal cultured cerebral cortical neurons was only detected in mature neurons after they were cultured for 14 days. These results indicate that GLUT3 plays an important role in glucose homeostasis postnatally in neurons of the rat brain.
Assuntos
Encéfalo/crescimento & desenvolvimento , Proteínas de Transporte de Monossacarídeos/genética , Proteínas do Tecido Nervoso , Sequência de Aminoácidos , Animais , Northern Blotting , Encéfalo/embriologia , Encéfalo/metabolismo , Sondas de DNA , Feminino , Transportador de Glucose Tipo 3 , Técnicas Imunoenzimáticas , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Gravidez , RNA Mensageiro/metabolismo , Ratos , Ratos WistarRESUMO
The identity of the glucose transporters (GLUT) expressed in neurons in situ has yet to be fully established. In the present study we have isolated the GLUT3 (RGT3) cDNA and produced anti RGT3 polyclonal antibody allowing us to investigate the cellular localization and tissue distributions of RGT3 mRNA and protein in the central nervous system of the rat by the methods of in situ hybridization and immunohistochemistry. Here we demonstrate the direct evidence that RGT3 is present in neurons in adult rat brain. In situ hybridization showed the expression of RGT3 mRNA mostly in the regions of hippocampus, cerebral cortex, striatum, and the granule cell layer of the cerebellum, indicating that RGT3 mRNA is predominantly expressed within neurons. Immunohistochemistry showed that RGT3 protein is widely distributed in the rat brain, and concentrated on the plasma membrane of neurons. Double labeling studies with anti-RGT3, glial fibrillary acidic protein (GFAP), and neuron specific enolase (NSE) antibodies revealed the specific expression of RGT3 protein in neurons. Thus, RGT3 is indicated to be a neuron specific glucose transporter isoform (NSGT), and suggested to play a functionally significant role in rat central neurons.
Assuntos
Encéfalo/metabolismo , Proteínas de Transporte de Monossacarídeos/metabolismo , Proteínas do Tecido Nervoso , Neurônios/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA , Transportador de Glucose Tipo 3 , Humanos , Imuno-Histoquímica , Hibridização In Situ , Masculino , Dados de Sequência Molecular , Especificidade de Órgãos , Ratos , Ratos WistarRESUMO
We investigated whether adenovirus-mediated preproinsulin gene transfer into insulin target tissues (adipocytes) ameliorates hyperglycemia in diabetic mice. KKA(y) mice, a genetically obese type 2 diabetic animal model, were treated with a single subcutaneous injection of recombinant adenovirus, Adex1CA-human preproinsulin (Adex1CA-pchi), into the epididymal fat pads. pchi mRNA was expressed only in adipose tissue in which mature insulin was produced. Three days after virus injection these mice showed a marked decrease of blood glucose levels (from about 400 to 200 mg/dl), and an intraperitoneal glucose tolerance test revealed the markedly improved glucose tolerance. There was no significant difference in serum insulin levels between control and recombinant adenovirus-treated KKA(y) mice. The normalized glucose levels in diabetic mice were maintained for at least 2 weeks after the virus injection. This strategy could provide a novel and, most importantly, a simple and convenient gene therapy for obese type 2 diabetes patients.
Assuntos
Adenoviridae/genética , Tecido Adiposo/metabolismo , Diabetes Mellitus Experimental/terapia , Técnicas de Transferência de Genes , Hiperglicemia/terapia , Proinsulina/genética , Precursores de Proteínas/genética , Animais , Southern Blotting , Ensaio de Imunoadsorção Enzimática , Teste de Tolerância a Glucose , Immunoblotting , Imuno-Histoquímica , Insulina/sangue , Camundongos , Camundongos Obesos , Modelos Biológicos , Proinsulina/metabolismo , Precursores de Proteínas/metabolismo , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de TempoRESUMO
Three new chlorinated marine steroids, yonarasterols G, H and I, were isolated from the Okinawan soft coral, Clavularia viridis. Their structures including the absolute configuration were determined based on the results of spectroscopic analysis and chemical conversion.
Assuntos
Cnidários/química , Esteroides/química , Esteroides/isolamento & purificação , Animais , Japão , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Espectrometria de Massas por Ionização por Electrospray , EstereoisomerismoRESUMO
The presence of water-soluble vitamin D and 25-OH-D sulfates in human breast and cow's milk was studied. We first confirmed that synthetic vitamin D2 and D3 sulfates could not be hydrolyzed by alkali but by acid. Breast or cow's milk was separated into milk whey containing water-soluble components and milk curd containing crude proteins and lipophilic components. The separated milk whey and curd were hydrolyzed by acid or alkali and each lipid extract was subjected to HPLC analysis. Neither peak due to vitamin D and 25-OH-D was observed in the chromatograms of acid- and alkali-hydrolyzed milk whey, whereas the peaks due to vitamin D3 and 25-OH-D3 were found in the chromatograms of both acid- and alkali-hydrolyzed milk curd and there was no significant difference between the respective peak heights. The eluates corresponding to the respective peaks observed on the latter's chromatograms were collected and subjected to UV, HPLC, GC-MS and GLC to identify the existence of vitamin D3 and 25-OH-D3, respectively. We concluded from these results that neither breast nor cow's milk contained water-soluble vitamin D and 25-OH-D sulfates, whereas they contained fat-soluble vitamin D3 and 25-OH-D3. The concentrations of vitamin D3 and 25-OH-D3 in breast milk were about 125 and 350 ng/liter, while those in cow's milk were about 420 and 270 ng/liter, respectively. The experiments on the transfer of 3H-D3 and 3H-25-OH-D3 perorally dosed to lactating rats into suckling pups through their milk also supported the above conclusion.
Assuntos
Calcifediol/análise , Ergocalciferóis/análogos & derivados , Leite Humano/análise , Leite/análise , Vitamina D/análise , 25-Hidroxivitamina D 2 , Animais , Bovinos , Cromatografia Líquida de Alta Pressão , Ergocalciferóis/análise , Feminino , Cromatografia Gasosa-Espectrometria de Massas , Concentração de Íons de Hidrogênio , Hidrólise , Gravidez , Ratos , Ratos Endogâmicos , Solubilidade , Ácidos Sulfúricos/análiseRESUMO
Odontoclasts and cementoclasts are considered to play major roles in the internal resorption of dentin and the external resorption of tooth roots. In this study, we evaluated the osteoclast-inducing ability of human dental pulp and periodontal ligament cells, which are mesenchymal cells in dental tissues. These cells expressed RANKL and OPG mRNA constitutively. As osteoclast precursors, CD14(+) monocytes derived from human peripheral blood were isolated, and incubated together with human dental pulp or periodontal ligament cells. Both cell types spontaneously induced the differentiation of CD14(+) monocytes into osteoclasts without osteotropic factors. These results suggest that dental pulp and periodontal ligament cells are involved in regulating the differentiation and function of osteoclasts.
Assuntos
Polpa Dentária/fisiologia , Osteoclastos/citologia , Ligamento Periodontal/fisiologia , Adulto , Diferenciação Celular , Células Cultivadas , Criança , Técnicas de Cocultura , Polpa Dentária/citologia , Polpa Dentária/metabolismo , Feminino , Humanos , Receptores de Lipopolissacarídeos , Masculino , Monócitos , Osteoclastos/fisiologia , Osteoprotegerina/biossíntese , Ligamento Periodontal/citologia , Ligamento Periodontal/metabolismo , Ligante RANK/biossíntese , Reabsorção de Dente/metabolismoRESUMO
To explore alpha-SNAP function in insulin exocytosis from either immature or mature secretory granules in pancreatic beta cells, we studied the effects of overexpression of adenovirus-mediated wild-type alpha-SNAP and C-terminally deleted alpha-SNAP mutant (1-285) on newly synthesized proinsulin and insulin release by rat islets and MIN6 cells. Rat islets overexpressing alpha-SNAP and mutant alpha-SNAP were pulse-chased. Exocytosis from immature and mature insulin secretory granules was measured as fractional (%) labeled-proinsulin release immediately after the pulse-labeling and percentage labeled-insulin release after a 3-h chase period, respectively. There was no difference in percentage labeled-proinsulin release between the control and alpha-SNAP or mutant alpha-SNAP-overexpressed islets. Although percentage labeled-insulin release after a 3-h chase period was significantly increased in alpha-SNAP-overexpressed islets, it was decreased in mutant alpha-SNAP-overexpressed islets. Thus, the results demonstrated that alpha-SNAP overexpression in rat islets primarily increased exocytosis from mature, but not immature insulin secretory granules. On the other hand, in MIN6 cells, alpha-SNAP overexpression scarcely affected glucose-stimulated insulin release; therefore, we examined the effect of mutant alpha-SNAP overexpression as the dominant-negative inhibitor on the newly synthesized proinsulin/insulin release using the same protocol as in the rat islet experiments. alpha-SNAP mutant (1-285) overexpression in MIN6 cells decreased the percentage labeled insulin release from mature secretory granules, but not percentage labeled proinsulin release from immature secretory granules. Thus, our data demonstrate that alpha-SNAP functions mainly in the mature insulin secretory granules in pancreatic beta cells.
Assuntos
Proteínas de Transporte/fisiologia , Grânulos Citoplasmáticos/metabolismo , Exocitose , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Proteínas de Membrana/fisiologia , Proteínas de Transporte Vesicular , Adenoviridae/genética , Animais , Linhagem Celular , Relação Dose-Resposta a Droga , Glucose/farmacologia , Ilhotas Pancreáticas/ultraestrutura , Masculino , Ratos , Ratos Wistar , Proteínas SNARE , Proteínas de Ligação a Fator Solúvel Sensível a N-EtilmaleimidaRESUMO
It was previously reported that insulin biosynthesis in mouse beta TC3 cells was regulated by glucose (Nagamatsu, S., and D. F. Steiner. Endocrinology 130: 748-754, 1992). In the present study, we examined the effect of glucose on the glucose transporter expression and hexokinase activities and determined the relationship between them and glucose-stimulated insulin biosynthesis in beta TC3 cells. Reverse transcriptase-polymerase chain reaction and Northern blot analysis revealed that beta TC3 cells expressed GLUT-1 and GLUT-3 glucose transporter mRNAs, but not GLUT-2. The levels of GLUT-1 and GLUT-3 mRNAs were not affected by glucose (0 or 11 mM glucose) over a period of 48 h. Immunoprecipitation of metabolically labeled beta TC3 cells with specific antibodies against GLUT-1 or GLUT-3 proteins revealed no effect of glucose on the biosynthesis of glucose transporters. Hexokinase [low Michaelis constant (Km) hexokinase] activity from cells incubated in 11 mM glucose for 48 h increased nearly twofold compared with cells maintained in 0 mM glucose, although the amount of cellular hexokinase protein detected by immunoblot analysis was unchanged between 0 and 11 mM glucose conditions. Glucokinase (high Km hexokinase) activity, in contrast, was not affected by glucose. Preincubation of beta TC3 cells with 2-deoxyglucose to inhibit hexokinase, thereby inhibiting all glycolysis, resulted in the decrease of glucose-stimulated insulin biosynthesis. Thus, in mouse beta TC3 cells that do not express GLUT-2, there is a close relationship between hexokinase activity and glucose-stimulated insulin biosynthesis, but not between the glucose transporter and glucose-stimulated insulin biosynthesis.
Assuntos
Hexoquinase/metabolismo , Insulina/biossíntese , Ilhotas Pancreáticas/metabolismo , Proteínas de Transporte de Monossacarídeos/metabolismo , Animais , Linhagem Celular , Expressão Gênica/efeitos dos fármacos , Glucoquinase/metabolismo , Glucose/farmacologia , Isomerismo , Camundongos , Camundongos Transgênicos , Proteínas de Transporte de Monossacarídeos/genética , Proinsulina/biossínteseRESUMO
Recent studies have revealed that soluble N-ethylmaleimide sensitive factor attachment receptor (SNARE)-related proteins, originally identified in neural tissues, are also expressed in pancreatic beta cells. In this study, we investigated the effect of glucose on syntaxin 1 and alpha/beta SNAP biosynthesis in pancreatic beta cells and we demonstrated that syntaxin 1, but not alpha/beta SNAP biosynthesis by rat isolated pancreatic islets was stimulated specifically by glucose nearly in parallel with proinsulin biosynthesis. Stimulation of syntaxin 1 and proinsulin biosynthesis by glucose was dose-dependent (Km = approximately 8 mmol/l) and reached the maximum (about 8-12 fold) at concentrations over 11 mmol/l. In contrast, 22 mmol/l glucose increased alpha/beta SNAP biosynthesis about 2-fold only, similar to the increase in total protein synthesis. Stimulation of syntaxin 1 biosynthesis by glucose was also time-dependent, taking around 3 h to reach the maximum, and was not affected by actinomycin-D, suggesting regulation at the translational level. On the other hand, glucose had a similar stimulating effect on both syntaxin 1 and alpha/beta SNAP biosynthesis by mouse insulinoma betaTC3 cells as it did on proinsulin biosynthesis. The evidence showing coordinated stimulation of syntaxin 1 and proinsulin biosynthesis by glucose in rat islets suggested the critical functional role of syntaxin 1 in the insulin exocytotic mechanism.
Assuntos
Proteínas de Transporte/biossíntese , Glucose/farmacologia , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Proteínas de Membrana/biossíntese , Proinsulina/biossíntese , Proteínas de Transporte Vesicular , Animais , Northern Blotting , Células Cultivadas , Dactinomicina/farmacologia , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Glucose/administração & dosagem , Técnicas de Imunoadsorção , Masculino , Proteínas de Membrana/genética , Inibidores da Síntese de Ácido Nucleico/farmacologia , Proteínas Qa-SNARE , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida , Sintaxina 1RESUMO
The simultaneous analysis of the molecular weight and concentration of hyaluronic acid in biological samples using high-performance liquid chromatography with two gel permeation columns is described. The elution volumes of various molecular weights of hyaluronic acids were linearily related to the logarithms of their molecular weights up to 600,000. The concentration of hyaluronic acid could be determined in the range from 20 to 100 micrograms/ml, i.e., from 4 to 20 micrograms per 200 microliter injected. The method was applied to the analysis of several animal skin extracts and rabbit synovial fluid. Skin extracts from mouse, rat, guinea-pig and rabbit could be chromatographed without prior isolation and purification. Hyaluronic acids in skin were separated clearly from chondroitin sulphates and their concentrations were determined. The molecular weights were estimated simultaneously to be more than 10(6). Rabbit synovial fluids from intact joints and saline- and carrageenin-treated joints could be chromatographed directly. The chromatograms showed that the concentration of hyaluronic acid in carrageenin-treated synovial fluid is lower than that in saline-treated fluid and the molecular weight distribution is broader. This technique enabled the rapid analysis of hyaluronic acid present at low levels in biological samples.
Assuntos
Ácido Hialurônico/análise , Pele/análise , Líquido Sinovial/análise , Animais , Cromatografia em Gel , Sangue Fetal/análise , Cobaias , Humanos , Masculino , Camundongos , Peso Molecular , Coelhos , Ratos , Espectrofotometria UltravioletaRESUMO
The concentration and degradation of hyaluronic acid in the synovial fluid of carrageenin-induced arthritic joints of rabbits was studied. A 0.5-ml volume of 1% lambda-carrageenin was intra-articularly injected three times into a right knee joint, and saline into a left. After 5 d from the last injection, inflammatory changes were observed in the synovial membrane and synovial fluid, but not in the articular cartilage. In the inflammatory synovial fluid, lipid peroxide content, phosphatase activity and cell counts were significantly increased, but the copper concentration was not changed. Concentration of polymeric hyaluronic acid and total hyaluronic acid were determined by high-performance liquid chromatography using gel-permeation columns. Total hyaluronic acid was appreciably decreased in the inflammatory fluid. The polymeric hyaluronic acid determined was 38% of the total hyaluronic acid in the inflammatory fluid and 74% in the control fluid. This suggests that in the inflammatory fluid, molecular weights of hyaluronic acid are distributed in the broader range. The concentration of chondroitin sulphates was similar in both the inflammatory fluid and the control fluid, but the content ratio of chondroitin sulphates to hyaluronic acid was higher in the inflammatory fluid. In the inflamed synovial membrane, synthesis of hyaluronic acid as measured by incorporation of [14C]glucosamine into glycoconjugates was increased by about twice that in the control membrane.
Assuntos
Artrite Experimental/metabolismo , Ácido Hialurônico/análise , Líquido Sinovial/química , Animais , Carragenina , Cartilagem Articular/metabolismo , Joelho , Masculino , Peso Molecular , Coelhos , Membrana Sinovial/metabolismoRESUMO
AIMS/HYPOTHESIS: The interaction of syntaxin-1 and SNAP-25 with insulin exocytosis was examined using the diabetic Goto-Kakizaki (GK) rat and a total internal reflection fluorescence (TIRF) imaging system. METHODS: Primary rat pancreatic beta cells were immunostained with anti-syntaxin-1A, anti-SNAP-25 and anti-insulin antibodies, and then observed by TIRF microscopy. The real-time image of GFP-labelled insulin granules motion was monitored by TIRF. RESULTS: The number of syntaxin-1A and SNAP-25 clusters, and the number of docked insulin granules on the plasma membrane were reduced in GK beta cells. When GK rats were treated with daily insulin injection for 2 weeks, the number of syntaxin-1 and SNAP-25 clusters was restored, along with the number of docked insulin granules. The infection of GK beta cells with Adex1CA SNAP-25 increased the number of docked insulin granules. TIRF imaging analysis demonstrated that the decreased number of fusion events from previously docked insulin granules in GK beta cells was restored when the number of docked insulin granules increased by insulin treatment or Adex1CA SNAP-25 infection. CONCLUSIONS/INTERPRETATION: There was a close correlation between the number of syntaxin-1 and SNAP-25 clusters and the number of docked insulin granules, which is associated with the fusion of insulin granules.
Assuntos
Antígenos de Superfície/metabolismo , Grânulos Citoplasmáticos/ultraestrutura , Insulina/análise , Ilhotas Pancreáticas/patologia , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Diabetes Mellitus Experimental/patologia , Insulina/química , Insulina/farmacologia , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/efeitos dos fármacos , Fusão de Membrana , Ratos , Ratos Endogâmicos , Ratos Wistar , Proteínas SNARE , Proteína 25 Associada a Sinaptossoma , Sintaxina 1 , Proteínas de Transporte Vesicular/metabolismoRESUMO
We examined the action of mastoparan on beta cell exocytosis. Mastoparan stimulated GABA and insulin release from MIN6 beta cells. On the other hand, mastopraran-induced GABA release was decreased by expressing the tetanus toxin C1 light chain in MIN6 cells. We have then investigated the relationship between SNARE proteins and mastoparan action using adenovirus-mediated gene transfer system. Overexpression of t-SNAREs, syntaxin 1A, and SNAP-25 inhibited the mastoparan-induced insulin release by approximately half-fold of control levels, however, the mastoparan-induced GABA release was not affected by these t-SNAREs overexpression. The overexpression of mutant alpha-SNAP (1-285), which inhibits the wild-type alpha-SNAP function in a dominant negative manner, did not influence either mastoparan-induced GABA or insulin release in spite of its marked inhibition of glucose-stimulated insulin release. Our data indicate that mastoparan stimulates GABA exocytosis via vesicular transport; however, SNARE proteins are differently involved in the exocytosis of insulin and GABA.
Assuntos
Proteínas de Membrana/fisiologia , Proteínas de Transporte Vesicular , Venenos de Vespas/farmacologia , Ácido gama-Aminobutírico/metabolismo , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/fisiologia , Linhagem Celular , Exocitose/efeitos dos fármacos , Exocitose/fisiologia , Insulina/metabolismo , Secreção de Insulina , Peptídeos e Proteínas de Sinalização Intercelular , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/fisiologia , Proteínas de Membrana/genética , Camundongos , Mutação , Peptídeos , Proteínas SNARE , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida , Sintaxina 1RESUMO
We have previously established a stable beta TC3 cell line that overexpresses syntaxin 1A, designated beta TC-hpc1 cells, in which glucose-stimulated insulin release was decreased. Using beta TC-hpc1 cells, we aimed to determine whether syntaxin 1A functions in the regulatory or constitutive pathway of insulin release. We therefore examined the secretion of phorbol-12-myristate-13-acetate (TPA)-stimulated newly synthesized proinsulin/insulin and total immunoreactive insulin. beta TC3 and beta TC-hpc1 cells were simultaneously pulse-labeled with 3H-leucine for 30 min in 11 mM glucose and chased for 1 h in one of a number of different concentrations of TPA in 11 mM glucose. Total immunoreactive insulin release (IRI) by both cell types during the chase period was markedly increased by the addition of TPA in a dose-dependent manner; however, the IRI from beta TC-hpc1 cells was lower than that from beta TC3 cells. The secretion of newly synthesized proinsulin/insulin from both cell types, which in beta TC3 cells is thought to occur via a constitutive pathway, was in the same range under any condition. Thus, the evidence indicates that syntaxin 1A preferentially functions in the regulated insulin release pathway in beta TC3 cells. In order to clarify the effect of overexpressed syntaxin 1A on glucose metabolism and intracellular Ca2+ we analyzed the glucose transport system, glucose phosphorylation activity, and cytosolic concentration of free Ca2+ ([Ca2+]i). 2-Deoxy-glucose uptake and the content of GLUT1 protein in the plasma membrane fractions of beta TC-hpc1 cells were not different from those of beta TC3 cells. Radiometric assays of glucose phosphorylation activity showed that there were no differences in hexokinase activity and glucokinase activity between beta TC3 and beta TC-hpc1 cells. [Ca2+]i measured by using fura 2 demonstrated that there was no difference in [Ca2+]i between beta TC3 and beta TC-hpc 1 cells under glucose-stimulated conditions. The present experiments indicate that syntaxin 1A plays a central role in a late step of the regulatory insulin release pathway without a change in glucose metabolism and [Ca2+]i in beta TC3 cells.