Isolation and characterisation of insulin secretory granules from a rat islet cell tumour

Summary Density gradient centrifugation techniques, using iso-osmotic colloidal silica suspensions (Percoll), were developed for the isolation of insulin secretory granules from a transplantable rat islet cell tumour. These procedures were readily completed within 7 h and from each animal yielded approximately 1 mg of granule protein. The isolated granules were essentially free of other subcellular organelles as evaluated by their contents of marker proteins, electron microscopy and by electrophoretic analyses. Their susceptibilities to lysis at low osmotic strength, at pH values above 7 or in media containing sodium ions were similar to those of granules partially purified from islets. Insulin comprised 50–60% of the total granule protein when determined by immunoassay or by densitometry of electrophoretic profiles. The proinsulin content was marginally higher than that of islets, as was the ratio of insulins I to II. Electrophoretic analyses revealed that the secretory granules contained 150 or more proteins besides insulin-related peptides. The majority of these had acidic isoelectric points and were located both within the granule interior and its enveloping membrane.     

Long-term effects of exposure of pancreatic islets to nicotinamide in vitro on DNA synthesis,metabolism and B-cell function

Summary Using³H-thymidine labeling techniques, we found that rates of DNA synthesis in islet cells doubled when mouse pancreatic islets were cultured for 1 week with 10 mmol/1 nicotinamide, a potent poly(ADP-ribose) synthetase inhibitor. Culture with nicotinamide partially inhibited glucose-stimulated insulin release, whereas the islet insulin content and rate of (pro)insulin biosynthesis remained unchanged. Long-term exposure to nicotinamide decreased glucose oxidation and ATP content in the islets. The findings support the view that poly(ADP-ribose)synthetase inhibitors stimulate islet cell replication, but may be accompanied by significant inhibitory effects on islet cell function.     

大鼠哮喘模型Clara细胞及其分泌蛋白的表达

目的　观察大鼠哮喘模型Clara细胞及其分泌蛋白 (CCSP)的表达。方法　SD大鼠 2 4只 ,分为哮喘模型组和健康对照组。哮喘模型组大鼠用卵白蛋白 (OVA)致敏激发建立大鼠哮喘模型。用逆转录 聚合酶链反应、斑点免疫印迹、免疫组化及图像分析方法检测 2组大鼠肺组织CCSPmRNA表达、支气管肺泡灌洗液(BALF)中CCSP蛋白水平、细小支气管Clara细胞比率及气道形态学参数。结果　哮喘模型组大鼠OVA激发2周的支气管总管壁面积、内壁面积及平滑肌面积均较健康对照组和哮喘模型组OVA激发 1周时的增加 (P <0 0 1) ,并与CCSP及其mRNA呈负相关 (r分别为 - 0 5 9、- 0 72、- 0 6 5、- 0 6 3、- 0 78及 - 0 73,P <0 0 1)。哮喘模型组大鼠细支气管、终末细支气管及呼吸性细支气管Clara细胞数量减少 (P <0 0 1或 0 0 5 )。哮喘模型组大鼠肺组织CCSPmRNA表达较健康对照组降低 ,BALF中CCSP蛋白含量降低。结论　CCSPmRNA表达降低 ,Clara细胞数量减少。     

Immunocytochemical localization of heat-shock protein 60-related protein in Beta-cell secretory granules and its altered distribution in non-obese diabetic mice

Summary Immuno-electron microscopy technique was employed to investigate the cellular distribution of 60 kDa heat-shock protein (HSP60) in pancreatic Beta cells of control and non-obese diabetic mice. In thin sections prepared from control mice, antibody to mammalian HSP60 cross-reacted with protein(s) located to mitochondria and secretory granules. In particular, prominent binding of the antibody was seen to the insulin core of the mature insulin-secreting granules. In comparison, very little immunoreactivity was observed with immature secretory granules or with the Golgi apparatus. No binding to secretory granules or mitochondria was observed with normal mouse serum or with unrelated sera. On Western blots, HSP60 antibody specifically interacted with a single 62 kDa islet cell protein. These results suggest the existence of an HSP60-related protein with a novel location in mature secretory granules of Beta cells. The preferential association of the HSP60-related protein with the insulin core was gradually lost in Beta cells of pre-diabetic non-obese diabetic mice, and correlated with the progression of insulitis. The decrease in the granular binding of the HSP60 antibody was accompanied by an increase in cytoplasm staining, and was concomitant with a significant expansion of the insulin core diameter. The altered distribution of the HSP60-related protein in prediabetic mice, together with our observation that immature secretory granules accumulate in these animals indicate that the presence of HSP60-related protein in secretory granules might be associated with the secretory function of Beta cells.     

Structural domains and molecular lifestyles of insulin and its precursors in the pancreatic Beta cell

Summary Insulin is both produced and degraded within the pancreatic Beta cell. Production involves the synthesis of the initial insulin precursor preproinsulin, which is converted to proinsulin shortly after (or during) translocation into the lumen of the rough endoplasmic reticulum. Proinsulin is then transported to the trans-cisternae of the Golgi complex where it is directed towards nascent secretory granules. Conversion of proinsulin to insulin and C-peptide arises within secretory granules, and is dependent upon their acidification. Granule contents are discharged by exocytosis in response to an appropriate stimulus. This represents the regulated secretory pathway to which more than 99% of proinsulin is directed in Beta cells of a healthy individual. An alternative route also exists in the Beta cell, the constitutive secretory pathway. It involves the rapid transfer of products from the Golgi complex to the plasma membrane for immediate release, with, it is supposed, little occasion for prohormone conversion. Even if delivered appropriately to secretory granules, not all insulin is released; some is degraded by fusion of granules with lysosomes (crinophagy). Each event in the molecular lifestyles of insulin and its precursors in the Beta cell will be seen to be governed by their own discrete functional domains. The identification and characterisation of these protein domains will help elucidate the steps responsible for delivery of proinsulin to secretory granules and conversion to insulin. Understanding the molecular mechanism of these steps may, in turn, help to explain defective insulin production in certain disease states including diabetes mellitus.Presented in part as the 25th Minkowski Prize Lecture at the EASD Annual Meeting. Copenhagen, September, 1990     

PARP-1和caspase-3在胰腺癌及癌旁组织中的表达及意义

目的探讨聚(腺苷酸二磷酸核糖)聚合酶(PARP)1和半胱氨酸天冬氨酸蛋白酶(caspase)3在胰腺癌及胰腺癌旁组织中的表达。方法收集2013年1月-2014年6月于兰州大学第一附属医院行手术治疗并经术后病理证实为胰腺癌患者的癌组织标本66例及癌旁组织标本113例,行免疫组化检查,分别检测标本中PARP-1及caspase-3的表达。计数资料组间比较采用χ2检验。结果 66例胰腺癌标本中53例有不同程度PARP-1表达,阳性率为80.3%;113例癌旁组织中有39例PARP-1表达,阳性率为34.5%;PARP-1在胰腺癌组织中的阳性表达率明显高于癌旁组织,差异具有统计学意义(χ2=34.79,P0.01)。在中分化(18/25,72.0%)和低分化(10/14,71.4%)的胰腺癌组织中PARP-1的表达强度明显高于高分化(3/14,21.4%)肿瘤组织,三者间差异具有统计学意义(χ2=10.76,P0.01)。66例胰腺癌标本中47例有不同程度caspase-3表达,阳性率为71.2%;113例癌旁组织中有71例caspase-3表达,阳性率为62.8%。高分化(11/18,61.1%)的胰腺癌组织中caspase-3的表达强度明显高于中分化(4/20,20.0%)和低分化(0/9,0)肿瘤组织,三者间差异具有统计学意义(χ2=11.44,P0.01)。结论胰腺癌组织中PARP-1表达水平明显高于癌旁组织,其强阳性率随肿瘤分化程度降低而升高;caspase-3在胰腺癌中的表达与癌旁组织相似,其强阳性率随肿瘤分化程度的降低而降低。PARP-1、caspase-3的表达水平可能与胰腺癌的发生发展有关。     

糖皮质激素对哮喘大鼠Clara细胞分泌蛋白表达的影响

目的　观察吸入糖皮质激素对大鼠支气管哮喘 (简称哮喘 )模型肺组织Clara细胞分泌蛋白 (CCSP)mRNA表达的影响。方法　用卵白蛋白 (OVA)致敏、雾化建立大鼠哮喘模型 ,以逆转录 聚合酶链反应 (RT PCR)、斑点免疫印迹法 ,分别检测大鼠激发哮喘 3天后肺组织CCSPmRNA表达水平、支气管肺泡灌洗液 (BALF)CCSP蛋白浓度及雾化吸入布地奈德的影响。结果　哮喘组大鼠肺组织CCSPmRNA表达量为 0 .5 6± 0 .0 5 ,与正常对照组 (0 .6 5± 0 .0 4 )比较差异有显著性 (P <0 .0 1) ;激素治疗组CCSPmRNA表达量为 0 .6 3± 0 .0 4 ,与哮喘组 (0 .5 6± 0 .0 5 )比较差异也有显著性 (P <0 .0 5 )。哮喘组大鼠BALF中CCSP蛋白含量为 4 9± 5 ,与正常对照组 (6 0± 5 )比较 ,差异有显著性 (P <0 .0 1) ;激素治疗组CCSP蛋白含量 (5 7± 5 )与哮喘组比较 (P <0 .0 5 ) ,BALF中嗜酸细胞比例降低 ,气道炎症反应减轻。结论　CCSPmRNA表达减少导致CCSP水平下降 ,从而促进气道炎症而参与哮喘发病。雾化吸入糖皮质激素可增加CCSPmRNA表达 ,抑制哮喘气道炎症。     

Aspects of novel sites of regulation of the insulin stimulus-secretion coupling in normal and diabetic pancreatic islets

Noninsulin-depenent diabetes mellitus (NIDDM), a major health care problem in the Western world, is a disease typified by a relative deficiency of insulin, leading to vast derangements in glucose and lipid homeostasis with disastrous vascular complications. Despite immense research efforts aimed at a clear understanding of the etiology of this complex disease, the molecular mechanisms causing the disorder still remain elusive. This article reviews extant data from recent publications implicating novel signal transduction pathways as important regulators of the insulin stimulus-secretion coupling in the pancreatic β-cell. The significance of nitric oxide and serine/threonine protein phosphatases, and their inactivation by insulin secretagogues, glucose metabolites, ATP, GTP, glutamate, and inositol hexaphosphate in this arena is scrutinized. Additionally, also presented is the growing concept that an important signal for insulin secretion may reside in the inextricable interplay between glucose and lipid metabolism, specifically the generation of malonyl-CoA, which inhibits carnitine palmitoyl-transferase 1 with the attendant accumulation of long-chain acyl CoA esters. Moreover, attention is directed towards novel intracellular actions of hypoglycemic sulfonylureas in the β-cell. Finally, the importance of “lipotoxicity” and aberrations in glucose uptake and metabolism in β-cell dysfunction is given consideration. Future research efforts should aim at further characterization of effects of second messengers on protein phosphorylation elements in β-cells. Additionally, long-term regulation by glucose and the diabetic state (e.g., fatty acids and ketones) on β-cell protein phosphatases, pyruvate dehydrogenase, and carnitine palmitoyltransferase 1 needs to be explored in greater depth. Clearly, the detrimental impact of diabetic hyperlipidemia on β-cell function has been a relatively neglected area, but future pharmacological approaches directed at preventing lipotoxicity may prove beneficial in the treatment of diabetes.     

Effect of islet activating protein on glucose tolerance,insulin secretion and insulin responsiveness in the NZO mouse

Summary The effect of islet activating protein on glucose tolerance, insulin secretion and insulin responsiveness was studied in the NZO mouse, a model of non-insulin dependent diabetes and obesity. A single IV injection of 5 ng/g body weight islet activating protein markedly lowered plasma glucose and the glucose response to IP glucose administration, measured 5 days later (mean±SEM, plasma glucose levels 0, 10, 30 and 60 min after glucose 6.0±0.9, 14.6±1.3, 14.1±1.3 and 13.2±1.7 mmol/l in islet activating protein-treated NZO mice versus 12.8±1.6, 27.8±3.4, 34.7±4.1, 39.1±3.8 mmol/l in carrier-treated NZO mice). There was no difference in fasting plasma insulin levels between islet activating protein and carrier-treated mice. No response of plasma insulin to glucose occurred in the carrier-treated mice, but a highly significant insulin response to glucose was seen in the islet activating protein-treated mice. The in vitro responsiveness of pancreatic islets of islet activating protein-treated NZO mice to glucose was improved, and the inhibitory effect of adrenaline on insulin secretion was reduced. The in vivo hypoglycaemic response to exogenous insulin was not improved by islet activating protein and a demonstrated defect in the insulin sensitivity and responsiveness of glucose utilization by isolated soleus muscle was not reversed by islet activating protein treatment. It is concluded that islet activating protein is highly effective in improving glucose tolerance and insulin secretion in NZO mice, and that the improvement in glucose tolerance occurs without demonstrable improvement in the responsiveness to exogenous insulin or sensitivity of soleus muscle to insulin.     

Comparison of the effects of D-mannoheptulose and its hexaacetate ester on D-glucose metabolism and insulinotropic action in rat pancreatic islets

Summary It was recently, and surprisingly, found that D-mannoheptulose did not affect D-glucose metabolism and insulinotropic action in pancreatic islets incubated at a low concentration of D-glucose. To explain this finding, the metabolism and secretory response to the hexose were investigated in rat islets exposed to D-mannoheptulose hexaacetate, which was recently found to inhibit D-glucose catabolism in cells that are otherwise fully resistant to the heptose. At a high concentration of D-glucose (16.7 mmol/l), the utilisation of D-[5-³H]glucose and oxidation of D-[U-¹⁴C]glucose, as well as the insulinotropic action of the hexose, were affected less by D-mannoheptulose tetraacetate than by unesterified D-mannoheptulose. This coincided with a reduced uptake of the ester by intact islets and a lower rate of hydrolysis of the ester in islet homogenates compared with findings in other monosaccharide esters such as D-glucose pentaacetate. At a low concentration of D-glucose (2.8 mmol/l), D-mannoheptulose hexaacetate was slightly more efficient than the unesterified heptose in reducing D-glucose catabolism, but still failed to suppress the secretory response to the hexose. These findings do not necessarily mean that unesterified D-mannoheptulose enters beta-cells more efficiently at high than at low extracellular D-glucose concentrations, especially if possible differences in the respective contributions of distinct islet cell types to the overall catabolism of D-glucose by whole islets is allowed for. These data do not rule out the possibility that D-glucose phosphorylation is more resistant to D-mannoheptulose in beta cells incubated at a low than a high concentration, independently of any difference in the intracellular concentration of the heptose. However, the mechanism of this resistance is still not explained. [Diabetologia (1998) 41: 1109–1113] Received: 29 January 1998 and in revised form: 1 April 1998     

不同潮气量机械通气对大鼠肺组织Clara细胞分泌蛋白及分泌型磷脂酶A2的影响

目的 通过观察不同潮气量机械通气大鼠肺组织Clara细胞分泌蛋白(CC16)及分泌型磷脂酶A2(sPLA2)的表达,探讨CC16和sPLA2在呼吸机所致肺损伤(VILI)发生中的作用.方法 24只雄性Wistar大鼠随机分为对照组、小潮气量组和大潮气量组,测定各组大鼠支气管肺泡灌洗液(BALF)总蛋白含量及中性粒细胞计数,采用免疫组织化学染色法检测肺组织Clara细胞计数和CC16蛋白表达,采用逆转录聚合酶链反应法检测肺组织sPLA2 mRNA的表达.结果 大潮气量组大鼠BALF总蛋白含量、中性粒细胞的计数和肺组织sPLA2 mRNA表达水平均明显高于对照组和小潮气量组(P值均＜0.01),而肺组织Clara细胞计数和CC16蛋白表达水平则明显低于对照组和小潮气量组(P值均＜0.01);小潮气量组各项指标与对照组比较差异无统计学意义(P＞0.05).结论 大潮气量机械通气通过诱导肺组织sPLA2 mRNA高表达,使Clara细胞合成及分泌CC16减少,导致肺组织炎症反应扩大,可能是VILI发生的重要因素之一.     

Influence of a transplantable insulinoma on the pancreatic status of insulin and pancreatic polypeptide in the rat

Summary The effect of endogenous hyperinsulinaemia, produced by syngeneic transplantation of rat insulinoma at an extrapancreatic site, on pancreatic insulin and pancreatic polypeptide has been examined by radioimmunoassay and immunohistochemistry. Twenty days after subcutaneous transplantation, tumour-bearing rats exhibited marked hyperinsulinaemia and hypoglycaemia, with plasma pancreatic polypeptide concentrations similar to controls. Immunoreactive insulin levels in the head and tail of pancreas of tumour-bearing rats were reduced by 90–95% compared with control animals. Immunoreactive pancreatic polypeptide levels in the head of the pancreas were reduced by 70%, but the relatively low levels of peptide in the pancreatic tail were similar in tumour-bearing and control rats. Insulin and pancreatic polypeptide cells were weakly immunofluorescent in tumour-bearing rat pancreas. In conclusion, the presence of an insulinoma at an extrapancreatic site resulted in a severe depletion of endogenous insulin and pancreatic polypeptide, suggesting that there is a functional relationship between the beta and pancreatic polypeptide cell.     

Abnormal insulin secretion and glucose metabolism in pancreatic islets from the spontaneously diabetic GK rat

Summary Insulin secretion and islet glucose metabolism were compared in pancreatic islets isolated from GK/Wistar (GK) rats with spontaneous Type 2 (non-insulin-dependent) diabetes mellitus and control Wistar rats. Islet insulin content was 24.5±3.1 U/ng islet DNA in GK rats and 28.8±2.5 U/ng islet DNA in control rats, with a mean (±SEM) islet DNA content of 17.3±1.7 and 26.5±3.4 ng (p < 0.05), respectively. Basal insulin secretion at 3.3 mmol/l glucose was 0.19±0.03 · ng islet DNA^–1· h^–1 in GK rat islets and 0.40±0.07 in control islets. Glucose (16.7 mmol/l) stimulated insulin release in GK rat islets only two-fold while in control islets five-fold. Glucose utilization at 16.7 mmol/l glucose, as measured by the formation of ³H₂O from [5-³ H]glucose, was 2.4 times higher in GK rat islets (3.1±0.7 pmol · ng islet DNA^–1 · h^–1) than in control islets (1.3±0.1 pmol · ng islet DNA^–1 · h^–1; p<0.05). In contrast, glucose oxidation, estimated as the production of ¹⁴CO₂ from [U-¹⁴C]glucose, was similar in both types of islets and corresponded to 15±2 and 30±3 % (p<0.001) of total glucose phosphorylated in GK and control islets, respectively. Glucose cycling, i. e. the rate of dephosphorylation of the total amount of glucose phosphorylated, (determined as production of labelled glucose from islets incubated with ³H₂O) was 16.4±3.4% in GK rat and 6.4±1.0% in control islets, respectively (p<0.01). We conclude that insulin secretion stimulated by glucose is markedly impaired in GK rat islets. Glucose metabolism is also altered in GK rat islets, with diminished ratio between oxidation and utilization of glucose, and increased glucose cycling, suggesting links between impaired glucose-induced insulin release and abnormal glucose metabolism.     

老年上皮性卵巢癌患者血清人附睾上皮分泌蛋白4的变化及临床意义

目的 探讨老年上皮性卵巢癌患者血清中人附睾上皮分泌蛋白4(human epididymis protein 4,HE4)的改变及其临床意义.方法 用ELISA法检测老年上皮性卵巢癌33例、良性卵巢疾病17例及老年健康女性20例血清HE4水平,另选同期非老年上皮性卵巢癌患者31例及健康女性20例为对照,同时测定糖类抗原125(CA125).结果 健康人群中≥60岁组血清HE4水平(32.25±13.15)pmol/L高于＜60岁组(24.59±8.60)pmol/L,血清HE4水平与年龄呈显著正相关(r=0.525,P＜0.01).老年组中上皮性卵巢癌患者血清HE4水平明显高于卵巢良性疾病患者(中位数81.50 pmol/L对45.60 pmol/L,U=168.5,P＜0.05)和健康对照者(中位数81.50 pmol/L对33.30 pmo1/L,U=76.5,P＜0.01);根据受试者操作特性曲线(ROC曲线)分析,HE4检测老年上皮性卵巢癌的临界值为65.43 pmol/L时,其敏感度为66.67%,特异性为86.49%,与CA125相比曲线下面积为0.799比0.782,HE4诊断价值稍高于CA125;老年与非老年上皮性卵巢癌患者血清HE4水平比较,差异无统计学意义(U=404.2,P＞0.05).结论 年龄是影响人血清HE4水平的重要生理因素,HE4测定有助于老年人上皮性卵巢癌的诊断,与CA125联合检测,可显著提高诊断的准确性.     

Co-localization of islet amyloid polypeptide and insulin in the B cell secretory granules of the human pancreatic islets

Summary Islet amyloid polypeptide is a novel 37 amino-acid-residues polypeptide which has been isolated from amyloid deposits in an insulinoma, and in human and cat islets of Langerhans. The molecule has 46% homology with the calcitonin gene-related peptide. Light microscopy examination of the pancreas shows that islet amyloid polypeptide immunoreactivity is restricted to the islet B cells. The present study utilized a rabbit antiserum against a synthetic peptide corresponding to positions 20–29 of islet amyloid polypeptide, a sequence without any amino-acid identity with calcitonin gene-related peptide. By applying the immunogold technique at the ultrastructural level, it was shown that both insulin and islet amyloid polypeptide immunoreactivity occurs in the central granular core of the human B cell secretory granules, while the A cells remain unlabelled. The demonstration that islet amyloid polypeptide is a granular protein of the B cells may indicate that it is released together with insulin. Further studies are necessary to evaluate the functional role of islet amyloid polypeptide.     

Expression of the human phosphatases of regenerating liver (PRLs) in colonic adenocarcinoma and its correlation with lymph node metastasis

Background Human phosphatases of regenerating liver (PRLs) can induce cell growth, differentiation, and malignant transformation. In this study, we used specific polyclonal antibodies against PRLs to investigate their expression in colonic adenocarcinomas and its correlation with patient gender, age, tumor differentiation, localization, invasion, and metastasis. Materials and methods The polyclonal antibodies against PRL-1, PRL-2, and PRL-3 were produced and purified. The expression of PRLs in human colorectal carcinoma cell lines (SW480 and SW620) was examined by Western blotting. We also examined their expression in normal and pathologic tissues from the human colon. The tissues included 49 primary colonic adenocarcinomas, 14 cases with lymph node metastases, 15 colonic adenomas, and 12 normal colon samples. Hematoxylin and eosin staining, immunohistochemistry, and semiquantitative morphological analysis were used to evaluate the sections. Results PRLs were widely expressed in SW480 and SW620. PRL-1, PRL-2, and PRL-3 were expressed, respectively, in 16, 10, and 16% of primary colonic adenocarcinomas. In contrast, PRLs were strongly expressed in all lymph node metastases. There were no significant correlations between the expression of PRLs and patient gender, age, tumor differentiation, depth of invasion, or localization of tumor within the different sections of the colon. PRLs were not expressed in normal colon tissues or in colonic adenomas. PRLs were mainly expressed in the cytoplasm and at the cytoplasmic membranes of the colonic adenocarcinoma cells as well as in the endothelial cells and the surrounding smooth muscle cells of larger vessels in the lymph node metastases. Conclusion Colonic adenocarcinoma cells have the ability to produce PRLs, which may relate to the lymph node metastasis of colonic adenocarcinoma. Y. Wang and Z.-F. Li contributed equally to this work.     

A method for the simultaneous measurement of insulin release and B cell membrane potential in single mouse islets of langerhans

Summary A method has been developed for the simultaneous measurement of insulin release and electrical activity in single micro-dissected mouse islets of Langerhans. The effects of D-glucose have been studied in individual islets. Each islet was exposed to 0, 5.6, 11.1, 16.7, 22.2, 27.8 and 33.3 mmol/l glucose in a stepwise fashion. The minimum glucose concentration required to elicit spike activity is lower than that required to stimulate insulin release above basal levels and the maximum spike frequency occurs at a lower glucose concentration than does maximum insulin release. Following a reduction in glucose from 27.8 (or 33.3) to 5.6 mmol/l, membrane potentials returned to resting values within 2 min whereas insulin returned to basal values after 20 min. Increasing glucose from 5.6 to 27.8 mmol/l induced spike activity within 10 s; the insulin response was detected within 40 s. Thus, it is possible to use the single mouse islet for simultaneous measurements of insulin release and electrical activity.     

Effects of feeding and fasting on the insulin secretory response to glucose and sulfonylureas in intact rats and isolated perfused rat pancreas

Summary In intact rats 16 h of fasting reduced the plasma insulin response to i.v. stimulation with either glucose, tolbutamide or glibenclamide by 50–80 %, without affecting the extractable insulin content of the pancreas. In subsequent studies with the isolated perfused rat pancreas two distinct patterns of insulin release could be discerned during the secondary phase. In thefed state, glucose 1.5 mg/ml induced a more or less constant elevation of the insulin secretion rate over 30 min (type I). At glucose concentrations of 2 and 3 mg/ml the release pattern was characterized by progressively increasing secretion rates (type II pattern). Infusion of tolbutamide (0.2 mg/ml) lowered the threshold for glucose stimulation and induced both patterns of secretion at lower glucose concentrations.Fasting for 24 h caused a 70–80 % inhibition of insulin secretion per 30 min at glucose levels of 1.5 and 2 mg/ml. Decreased glucose sensitivity was indicated by a shift to the right of the entire dose-response curve for glucose and by reduced inhibition (30 %) at a glucose level of 3 mg/ml. The effect of tolbutamide was also strongly diminished. The percent inhibition of the response to tolbutamide at different levels of glucose showed a pattern of inhibition similar to that observed with glucose alone. These findings suggest that the glucose-dependent release mechanism is highly sensitive to relatively short periods of fasting.Presented in part at the Sixth Annual Meeting of the European Association for the Study of Diabetes, Warsaw, September 1970.Supported by a grant from the Netherlands Organization for the Advancement of Pure Research (Z.W.O.).     

弓形虫致密颗粒蛋白基因的表达及其重组蛋白免疫反应性的评价

目的 将克隆入pGEX-4T-1的致密颗粒蛋白基因进行表达并对表达产物的免疫反应性进行评价。方法 将重组表达质粒pGEX-4T-1／GRA7转入大肠埃希菌BL21，经IPTG诱导进行SDS变性蛋白质电泳，分别以Anti-GSTAn-tibody、免抗弓形虫阳性血清和人抗弓形虫阳性血清为一抗进行Western Blot分析。用GSTrap FF HiTrap affinity columns纯化重组蛋白，以此蛋白作为包被抗原，BLISA法检测抗弓形虫阴性、阳性血清。结果 SDS变性蛋白质电泳显示在43KDa～66KDa蛋白条带之间有特异蛋白的表达，蛋白分子量大小与理论值相符。Western Blot分析表明该重组蛋白为GST融合蛋白，且该蛋白能被人抗弓形虫阳性血清、免抗弓形虫阳性血清所识别。ELISA结果表明该蛋白能与人抗弓形虫阳性血清、免抗弓形虫阳性血清特异结合，而与抗弓形虫阴性血清无反应。结论 弓形虫致密颗粒蛋白基因在大肠埃希菌中得到表达；该重组蛋白具有良好的免疫反应性。     

Interleukin 1 dose-dependently affects the biosynthesis of (pro)insulin in isolated rat islets of Langerhans

Summary Human crude and recombinant interleukin 1 (IL-1) was found to dose- and time-dependently affect the biosynthesis of (pro)insulin in isolated rat islets of Langerhans. Incubation of rat islets with either 0.5 U/ml or 5 U/ml of crude IL-1 for 1 h had no detectable effect on (pro)insulin biosynthesis. After 24 hours of exposure 0.5 U/ml of crude or 0.6 ng/ml of recombinant IL-1 (beta) increased the (pro)insulin biosynthesis by 42% and 58%, respectively, whereas a 10-fold greater concentration of IL-1 decreased the (pro)insulin biosynthesis by 74% and 89%, respectively. The increase in (pro)insulin biosynthesis was accompanied by an increase in total protein biosynthesis indicating a nonspecific stimulatory action of low IL-1 concentrations. In contrast, high IL-1 concentrations caused a more selective decrease of the (pro)insulin biosynthesis when compared to the total protein biosynthesis. In addition, low IL-1 concentrations were found to increase and high concentrations to decrease the relative levels of pre-proinsulin mRNA suggesting that IL-1 may act both at a pre- and post-translational level of insulin biosynthesis.