Islet amyloid polypeptide/amylin in pancreatic beta-cell line derived from transgenic mouse insulinoma.

We examined the production and secretion of IAPP in a beta-cell line, MIN6, which is derived from an insulinoma obtained by targeted expression of the SV40 T-antigen gene in a transgenic mouse. RNA blot analysis revealed an abundance of IAPP and insulin II mRNA in the cells, findings comparable with those in the pancreas of a normal mouse. The presence of IAPP and insulin was confirmed immunohistochemically and by RIA. Analysis of the reverse-phase HPLC identified IAPP in cells with authentic mouse IAPP. Raising the glucose concentration from 5.6 to 25 mM failed to induce increments in IAPP and insulin II mRNAs. The cells secrete IAPP and insulin for short- and long-term incubations in response to concentration of glucose in the medium. These features resemble those of islet cells from normal animals. This beta-cell line will aid in analyzing the regulation of IAPP gene expression and the mechanisms of IAPP biosynthesis and secretion.

Dual mechanism involved in the hydrolysis of polyphosphoinositides in rat pancreatic islets.

We investigated insulin secretion and inositol phosphate formation in intact and permeabilized rat pancreatic islets, the objective being to elucidate mechanisms of activation of phospholipase-C in pancreatic islets. The intact islets prelabeled with myo-[3H]inositol were incubated in Krebs-Ringer bicarbonate buffer containing 10 mM LiCl and 1 mM myoinositol. Glucose, alpha-ketoisocaproate (KIC), and sulfated cholecystokinin (CCK8S) increased insulin secretion and formation of [3H]inositol phosphate, [3H]inositol bisphosphate, and [3H] inositol trisphosphate. Mannoheptulose, a glucokinase inhibitor, inhibited glucose-induced insulin secretion and [3H]inositol phosphate formation; however, it did not inhibit KIC- and CCK8S-induced secretion and formation. Both glucose- and KIC-induced insulin secretion and [3H]inositol phosphate formation were blocked by 2,4-dinitrophenol, an uncoupler of oxidative phosphorylation in the mitochondria. The islets prelabeled with myo-[3H]inositol were permeabilized by digitonin and then incubated in intracellular mimicking medium containing 1 microM Ca2+ and 2.5 mM ATP. Glucose had no effect on [3H]inositol phosphate formation in the permeabilized islets, and CCK8S increased the formation of [3H]inositol phosphates. Thus, phospholipase-C in pancreatic islets is activated not only via ligand-receptor interaction in the plasma membrane in the case of hormone stimulation, but also by metabolic product(s) in the case of fuel stimulation.

Hypersecretion of islet amyloid polypeptide from pancreatic islets of ventromedial hypothalamic-lesioned rats and obese Zucker rats.

To investigate the possible role of islet amyloid polypeptide (IAPP) in the development of type 2 diabetes mellitus, we examined the IAPP content and secretion in pancreatic islets isolated from ventromedial hypothalamic (VMH)-lesioned rats and genetically obese Zucker rats, using a specific RIA for IAPP. Obesity and hyperinsulinemia were observed in rats 21 days after VMH lesioning. IAPP content was increased in the islets of VMH-lesioned rats compared with findings in the sham-operated controls (100.9 +/- 6.6 vs. 72.8 +/- 3.85 fmol/islet; P less than 0.01). Isolated islets of VMH-lesioned rats secreted larger amounts of IAPP in the presence of 2.8 mM and 16.7 mM glucose (2.99 +/- 0.98 and 11.2 +/- 1.29 fmol.islet(-1).3 h-1) than was noted in sham-operated rats (ND and 6.65 +/- 0.78 fmol.islet(-1).3 h-1). In the obese Zucker rats, aged 14 weeks, IAPP concentrations in the islets were elevated compared with lean rats (133.3 +/- 10.6 vs. 84.4 +/- 8.5 fmol/islet; P less than 0.01). The isolated islets secreted larger amounts of IAPP in response to 2.8 mM and 16.7 mM glucose (2.83 +/- 0.88 and 15.81 +/- 1.35 fmol.islet(-1).3 h-1) than did those from lean control rats (0.36 +/- 0.19 and 12.49 +/- 1.20 fmol.islet(-1).3 h-1). These results strongly suggest that overproduction and hypersecretion of IAPP occur in animals with obesity and hyperinsulinemia.

Secretion of islet amyloid polypeptide in response to glucose.

The content of islet amyloid polypeptide (IAPP) in isolated rat pancreatic islets was determined by a radioimmunoassay. Reverse-phase high-performance liquid chromatography analysis revealed that a main peak of IAPP immunoreactivity in the extracts from the islets corresponded to a synthetic rat IAPP. Secretion of IAPP from the cells is regulated by the extracellular glucose concentration. Thus, IAPP may be a novel regulator for glucose homeostasis and changes in the secretion perhaps relate to insular amyloid deposits and impaired glucose tolerance in type 2 diabetes mellitus.

Preheparin serum lipoprotein lipase mass is negatively related to coronary atherosclerosis.

In preheparin serum, there exists lipoprotein lipase (LPL) mass with little activity. The clinical significance of this preheparin serum LPL mass (preheparin LPL mass) is unclear. We studied the levels of preheparin LPL mass in patients with coronary atherosclerosis, comparing the results with those in healthy men. We also evaluated the correlation between preheparin LPL mass and the severity of coronary atherosclerosis by comparing with other risk factors such as age, smoking, family history, hypertension, hyperuricemia, diabetes mellitus, total cholesterol, triglyceride, high density lipoprotein-cholesterol and body mass index. The subjects, 70 men presenting with symptoms of coronary artery disease, underwent coronary angiographic examination. Significant narrowness was defined as > or = 75%. Control group comprised 77 men who had annual health checks and showed no abnormal findings. Preheparin LPL mass in the stenosis group was lower than normal coronary group and also than the control group. Multivariate analysis showed that preheparin LPL mass had the highest t-value (-2.53) for the number of lesions among the risk factors listed above. These results suggest that low preheparin LPL mass may be deeply involved in the progression of coronary atherosclerosis.

Evidence for direct effect of tolbutamide on hepatic glycogenolysis induced by Ca2+-dependent hormones.

The effects of tolbutamide and glibenclamide on hepatic glycogenolysis in perfused rat liver were investigated. Tolbutamide per se did not influence glucose output from the liver, but at therapeutic concentrations (about 350 microM) it significantly inhibited the glycogenolysis induced by phenylephrine, vasopressin and angiotensin II, while glibenclamide did not. Neither tolbutamide nor glibenclamide inhibited the glycogenolysis induced by glucagon. Tolbutamide potentiated the inhibitory effect of submaximal concentrations of insulin on glycogenolysis induced by phenylephrine. This effect of tolbutamide was elicitable even in the absence of calcium in the perfusate, and was additive to that of trifluoperazine. However, tolbutamide did not potentiate the inhibitory effect of insulin on glucagon-induced glycogenolysis. Tolbutamide inhibited the glycogenolysis induced by A23187, a calcium ionophore. These results indicate that, in addition to its known effect on insulin secretion, tolbutamide has a direct effect on the liver to inhibit glycogenolysis induced by Ca2+-dependent hormones (catecholamines, vasopressin and angiotensin II) and A23187. Thus, it is likely that tolbutamide inhibits the effect of Ca2+ mobilized by Ca2+-dependent hormones to stimulate glycogenolysis.

Formation of islet amyloid fibrils in beta-secretory granules of transgenic mice expressing human islet amyloid polypeptide/amylin.

To investigate the relationship between human islet amyloid polypeptide (IAPP)/amylin expression and islet amyloid deposits in the pathogenesis of human non-insulin-dependent diabetes mellitus (NIDDM), we developed transgenic mice using a human IAPP cDNA connected to an insulin promoter. Ribonucleic acid blotting and immunohistochemistry revealed the expression of the transgene in the pancreatic beta cells. Immunogold electron microscopy showed that beta-secretory granules contained the human C-terminal flanking peptide of the IAPP precursor. Reverse-phase HPLC demonstrated human and mouse IAPP amide in the pancreas. Electron microscopy showed the accumulation of fibril-like material in a considerable number of beta-secretory granules. These results suggest that in transgenic mice, the human IAPP precursor is expressed in beta cells and becomes normally sorted into beta-secretory granules in which normal conversion to mature human IAPP takes place. The human IAPP molecules, because of their amyloidogenesis, aggregate into amyloid fibrils in secretory granules. Glucose tolerance was normal at 7 months old and islet amyloid was not observed. A longer time may be required for islet amyloid deposits and hyperglycemia to develop in mice. Our working hypothesis is that in human NIDDM, IAPP aggregates into amyloid fibrils in beta-secretory granules, and that the fibrils are released into the extracellular space and islet amyloid deposits become substantial with time.

Somatocardiovascular reflexes in anesthetized rats with the central nervous system intact or acutely spinalized at the cervical level.

The effects of noxious mechanical stimulation of various segmental areas on heart rate and mean arterial blood pressure (MAP), as well as cardiac and renal sympathetic nerve activities were examined in anesthetized rats with the central nervous system (CNS) intact or acutely spinalized at the cervical level. In CNS-intact rats, pinching for 20 s applied to any segmental skin area, but particularly that of the paw, produced an increase in heart rate, blood pressure and the sympathetic nerve activities. In acutely spinalized rats, pinching the chest, abdomen and back of the body produced large increases, while hindlimb and perineum stimulation induced only a small increase or no increase in heart rate, blood pressure and the sympathetic nerve activities. Stimulation of the right side produced particularly large responses in heart rate and stimulation of the ipsilateral side produced large responses in cardiac and renal sympathetic nerve activities in spinalized rats. These results suggest the existence of the two types of reflex responses, supraspinal and propriospinal, in the somatocardiovascular reflex. The supraspinal one has characteristics of diffuse reflex organization, while the propriospinal one has strong segmental and lateral organization.

Cutaneous afferents producing a reflex pupil dilation in anesthetized rats.

Cutaneous afferents producing a reflex pupil dilation were examined using natural mechanical stimulation of the hindlimb skin and electrical stimulation of a sural nerve in anesthetized, artificially ventilated rats. Pupil diameter was continuously recorded after magnification using a microscope connected to a charge coupled device camera. Innocuous brushing, or weak pressing of the skin, did not have any effect on pupil diameter, while pressing the skin more than 720 g/cm2 produced a pressure-dependent pupil dilation. Pinching rather than pressing the skin induced the larger pupil dilation. Electrical stimulation of a sural afferent nerve with weak intensity, which was supra-threshold for Abeta-afferents and sub-threshold for Asigma-afferents, induced a reflex pupil dilation. This dilation continued to increase with further increases in stimulus intensity which involved excitation of Asigma afferents and C afferents. It is concluded that Abeta, Asigma and C afferents in the skin can work as afferents in eliciting reflex pupil dilation in anesthetized rats.

Hypersecretion of IAPP from the islets of VMH-lesioned rats and obese Zucker rats.

To investigate the possible role of islet amyloid polypeptide (IAPP) in the development of type 2 diabetes mellitus, we examined the IAPP content and secretion in pancreatic islets isolated from ventromedial hypothalamic (VMH)-lesioned rats and genetically obese Zucker rats, using a specific radioimmunoassay for IAPP. Obesity and hyperinsulinemia were observed in rats 21 days after VMH lesioning. IAPP content was increased in the islets of VMH-lesioned rats compared with findings in the sham-operated controls (100.9 +/- 6.6 vs 72.8 +/- 3.85 fmol/islet; P less than 0.01). Isolated islets of VMH-lesioned rats secreted larger amounts of IAPP in the presence of 2.8 and 16.7 mM glucose (2.99 +/- 0.98 and 11.2 +/- 0.29 fmol islet-1 3 h-1) than was noted in sham-operated rats (ND and 6.65 +/- 0.78 fmol islet-1 3 h-1). In the obese Zucker rats, aged 14 weeks, IAPP concentrations in the islets were elevated compared with lean rats (133.3 +/- 10.6 vs 84.4 +/- 8.5 fmol/islet; P less than 0.01). The isolated islets secreted larger amounts of IAPP in response to 2.8 and 16.7 mM glucose (2.83 +/- 0.88 and 15.81 +/- 1.35 fmol islet-1 3 h-1) than did those from lean control rats (0.36 +/- 0.19 and 12.49 +/- 1.20 fmol islet-1 3 h-1). These results strongly suggest that overproduction and hypersecretion of IAPP occur in animals with obesity and hyperinsulinemia.

Islet amyloid polypeptide-derived amyloid deposition increases along with the duration of type 2 diabetes mellitus.

To investigate the involvement of islet amyloid polypeptide (IAPP) and amyloid deposits in the pathophysiology of this disease, we studied the relationship between IAPP-derived amyloid deposition and the clinical features in type 2 diabetes mellitus. We examined pancreata obtained from 37 type 2 diabetic subjects and 12 non-diabetic ones by immunohistochemical techniques using two specific antibodies to IAPP. IAPP-derived deposits occurred in 1 of the 12 (8.3%) non-diabetic subjects and 28 of the 37 (75.7%) diabetics. When diabetic patients were divided into categories according to the presence of the deposits, the duration of the disease was significantly longer in patients with amyloid than that in the patients without it. The odds ratio of type 2 diabetes mellitus of at least 14-years-duration to the deposition was significantly high, and a body weight of at least 120% maximal ideal body weight was relatively high. In conclusion, IAPP-derived amyloid deposition increases along with the duration of type 2 diabetes mellitus and obesity may further enhance these deposits, hence hypersecretion of IAPP may be involved in the progression of this disease.

Effect of chronic treatment with haloperidol on vasopressin release and behavioral changes by osmotic stimulation of the supraoptic nucleus.

Chronic treatment with dopamine D2 blockers in schizophrenic patients has been proposed as one of the causes of polydipsia and water intoxication, but this conclusion is still controversial. To investigate the relationship between dopamine D2 blockers and these syndromes, we designed a behavioral and neurochemical study using hyperosmotic stimulation in the supraoptic nucleus (SON) by microdialysis after chronic treatment with haloperidol in rats. Animals were injected with haloperidol decanoate (20 mg/kg, i.m.) or sesame oil at 2-week intervals for 8 successive weeks. During the 7th week, water-intake was increased 30-60 min after the hyperosmotic stimulation in both groups, but more so in haloperidol-treated animals compared to that in the control group. Moreover, arginine vasopressin (AVP) was released by the hyperosmotic stimulation in SON, but was not significantly different between groups. In addition, striatal dopamine levels 3-4 days after the microdialysis study showed a significant decrease in the haloperidol-treated animals. These results suggest that chronic treatment with haloperidol enhances water-intake produced by hyperosmotic stimulation in the SON but does not increase AVP levels in dialysates following hyperosmotic stimulation. Thus, these symptoms may be mediated by dopaminergic systems in brain.

CT is useful for identifying patients with complicated appendicitis.

BACKGROUND AND AIMS: We often come across patients with complicated appendicitis (perforation, abscess formation, or peritonitis) and it is essential to get accurate and detailed information on these patients preoperatively. In this study, we investigated whether or not preoperative computed tomography is useful for identifying these patients. PATIENTS AND METHODS: Plain and intravenously-contrasted helical computed tomography was obtained preoperatively in 94 (75%) of 125 patients who underwent appendectomy. Twenty-eight (30%) of the 94 patients had complicated appendicitis (Compli(+) group). We compared clinical factors and computed tomography findings of the Compli(+) group with those of 66 other patients (Compli(-) group). RESULTS: There was no significant difference between the Compli(+) and Compli(-) groups in gender, white blood cell count, the present rate of an enlarged appendix, or appendicolith. Fat stranding and free fluid on computed tomography were significantly associated with complicated appendicitis by both univariate and multilogistic regression analysis. Fourteen (70%) of the 20 patients with fat stranding and free fluid on computed tomography had complicated appendicitis and only 1 (4%) of the 28 Compli(+) patients had neither fat stranding nor free fluid on computed tomography. CONCLUSION: Our study has indicated that fat stranding and free fluid on computed tomography are significant for complicated appendicitis and helical computed tomography is a powerful tool for identifying patients with complicated appendicitis preoperatively.

Interlaboratory validation of the in vitro eye irritation tests for cosmetic ingredients. (5) Skin(2TM) ZK1100 and tissue equivalent assay.

Skin(2TM) ZK1100 (ZK1100) assay and tissue equivalent assay (TEA, Skin(2TM) ZK1200) are human dermal models. These assays were evaluated as alternatives to the Draize eye irritation test (Draize test) in rabbits. Thirty-nine cosmetic ingredients were selected and used as test substances. The ZK1100 assay was conducted according to an original protocol provided by Advanced Tissue Sciences, a kit supplier. The TEA assay followed a protocol developed by Osborne et al., (1995a). Coefficients of variation (CV) ranged from 11.7 to 133 in results from the ZK1100 assay; three test substances showed the CVs more than 100. These were cetyltrimethylammonium chloride (S3-7), domiphen bromide (S3-11) and di(2-ethylhexyl) sodium sulfosuccinate (S3-14). Acid Red 92 (S2-3) was excluded from data analysis because its absorbance interfered with the endpoint of ZK1100 assay. The CVs from the TEA assay ranged from 31.8 to 119; two test substances showed the CVs more than 100. These were acetic acid and glycolic acid (S3-13). Butanol (S3-9) was excluded from the analysis because it was assumed to volatilize during a sample preparation. Pearson's coefficient of correlation with maximum average Draize total score (MAS) and 24hr score from the Draize tests were -0.71 and -0.72 for the ZK1100 results and -0.63 and -0.60 for the TEA results. When a MAS of 15 was set as a breakpoint for the classification of eye irritancy on Cooper's plots comparing the in vitro and the Draize data, the ZK1100 results showed five false positives and four false negatives; the TEA results showed three false positives and no false negatives.

Malignant schwannoma of the heart.

Primary cardiac tumor is an extremely rare disease entity. Only three cases of primary malignant cardiac schwannoma, the subject of this report, have been recorded in Japan. Recently, we encountered a case of malignant schwannoma in which retention of pericardial effusion was the first clinical finding. This case was a 30-year-old female, who had dyspnea at work, general fatigue, and fever. Striking cardiac expansion was seen, with a cardiothoracic ratio (CTR) of 69% on chest x-ray. Two-dimensional echocardiograms showed a large volume of pericardial effusion between the side wall of the left ventricle and the epicardium, and the presence of a parenchymatous tumor. An increase in tumor size was detected on chest computer tomography (CT) scan. Using a pump oxygenator, median sternotomy was performed to reach the epicardium. A pale yellow, soft tumor was seen in the left atrium near the left ventricle. Histologically, the patient was diagnosed as having a malignant schwannoma. We have reported a case of primary malignant schwannoma which was surmised to have arisen from the boundary between the atrium and the ventricle.

Epidemiology of childhood burns in the critical care medical center of Kinki University Hospital in Osaka, Japan.

The objective of the present study was to describe the characteristics of pediatric burns in order to prepare a program for the prevention of severe burn injuries in children. We conducted a retrospective study of burn victims aged 15 years or younger who were hospitalized in our Critical Care Medical Center between 1982 and 1997. There were 73 children with burn injuries hospitalized in our center during the study period. The greatest number were children 1 year old. The average % body surface area burned was 21. 5+/-20.5%. The most important causes of pediatric burns were found to be hot bath water and other hot liquids. Hot bath scalds accounted for about half of the pediatric burns occurring in all age groups, and they were often extensive. Non-bath scalds accounted for about one-third of the pediatric burns and were most frequent in children 2 years and younger. All the injuries sustained at home occurred when a family member was in the house. Similar to many reports from overseas, non-bath scalds were one of the most common causes of burns in this study; however, hot bath scalds were the most important cause. These data are being used to develop a prevention program. We also consider it necessary to educate children and their family members about the dangers of burn injuries.

Application of cryoprecipitate as a hematostatic glue.

BACKGROUND: The effectiveness of cryoprecipitate, harvested from a patient's own fresh frozen plasma, for use in cardiac surgery as a hematostatic glue was studied in 32 randomized elective adult cardiac surgery patients from January 1993 to July 1994. MATERIALS AND METHODS: Patients from the Toho Sakura Hospital were randomly allocated to two groups: Group 1 (n=11) received conventional fibrin glue presently available in our institution; while Group 2 (n=21) received autologous cryoprecipitate as a hematostatic glue. Surgical procedures broken down by group were as follows: Group 1: 4 CABG, 5 valvular surgeries and 2 other. Group 2: 11 CABG, 6 valvular surgery, 4 other. We preserved the patient's own blood and stored pure red cell and fresh frozen plasma (FFP). Cryoprecipitate was prepared from the FFP and preserved until required. RESULTS: Cryoprecipitate had a 5-fold increase in fibrinogen activity (1190+/-311 mg/dl vs 238+/-34 mg/dl p<0.001), a 10-fold increase in factor VIII activity (362+/-219% vs 34+/-11%, p=0.001), and 4.5-fold increase in factor XIII activity (538+/-213% vs 119+/-50%, p<0.001), compared to serum. The amount of bleeding postoperatively was slightly lower in the cryoprecipitate glue group compared to the conventional glue group, but this was not significantly different. CONCLUSIONS: We conclude that autologous samples of human cryoprecipitate prepared from a patient's own FFP had a strong hematostatic effect compared to conventional fibrin glue and was a very valuable hematostatic agent during cardiac surgery.

Partial type of common atrioventricular canal defect associated with mitral stenosis.

We report a 63-year-old woman, with a partial type of common atrioventricular canal and mitral stenosis, who was hospitalized because of dyspnea on exertion. Two-dimensional echocardiogram showed an ostium primum atrial septal defect with two well-formed AV valves located at the same level. However, both anterior and posterior mitral leaflets were markedly thickened with a thickened subvalvular apparatus, and the commisures were fused. Echocardiographic measurements demonstrated a mitral valve area of 1.48 cm2 with mild mitral regurgitation. Cardiac catheterization demonstrated mild pulmonary artery hypertension with a large left to right shunt (72%) at the atrial level. The combination of the partial type of common atrioventricular canal and mitral stenosis is rare; only one similar case has been reported previously in the literature.

Dilated cardiomyopathy associated with hyperthyroidism.

We report a case of dilated cardiomyopathy with hyperthyroidism. A 28-year-old man was admitted because of congestive heart failure and atrial fibrillation, and was newly diagnosed as having hyperthyroidism. Despite administration of antithyroid medication, he developed recurrent congestive heart failure. An echocardiogram revealed a moderately dilated left ventricle with diffuse hypokinesis. Though his thyroid function normalized, the patient's cardiac dysfunction did not improve. Beta-blocker therapy was begun with subsequent improvement in clinical symptoms. This suggests that beta-blocker treatment may be effective in patients with atrial fibrillation associated with cardiomyopathy and hyperthyroidism.

[Clinical and pathological analysis of deep mycoses].

Clinical and pathological analysis were performed on 127 cases of deep mycoses diagnosed by autopsy during the 24 years between 1964 and 1987 in Juntendo University Hospital. The following findings were obtained. 1) There has been a tendency for the number of mycoses to increase each year, especially notable for candidiasis and aspergillosis. 2) Underlying diseases were, in order of incidence, various hematologic diseases, solid tumors, inflammatory diseases and collagen diseases; the most common were various types of leukemia. 3) Candidiasis was often observed in patients with gastrointestinal tract cancers. Aspergillosis was often observed in patients with collagen diseases. 4) Regarding the visceral distribution of mycoses, aspergillosis was observed in the lung, candidiasis was observed in the lung, kidney and intestinal tract in decreasing order, and cryptococcosis was also observed in the lung and central nervous system. 5) There was a probability of fungal infections occurring in cases of lymphopenia. 6) A fever was present at the time of hospitalization in many cases of aspergillosis, and the presence of an indwelling catheter was a common feature in cases of candidiasis. 7) Fungemia was frequently observed in candidiasis, but very rarely in cases of aspergillosis. 8) The large amounts of corticosteroid hormones and blood transfusions were suspected as causative factors of fungal infections.