Essential role of caspase-3 in apoptosis of mouse beta-cells transfected with human Fas.

Several recent studies have indicated that the Fas-Fas ligand system may be critical for pancreatic beta-cell destruction in type 1 diabetes. Although the fundamental roles of caspases in the mammalian apoptotic machinery have been elucidated, it is not known which caspase or caspases play a major role in Fas-mediated apoptosis of beta-cells. In this study, we transfected human Fas cDNA into a mouse beta-cell line (betaTC1) and established a beta-cell clone expressing human Fas. This clone, designated hFas/betaTC1, underwent apoptosis when exposed to anti-Fas, showing hallmarks of apoptosis (chromatin condensation, nucleolar disintegration, internucleosomal DNA fragmentation, and annexin V staining), indicating that the mouse beta-cell line has the intact machinery of Fas-mediated apoptosis. The cross-linking of Fas by anti-Fas resulted in the elevation of caspase-3-like, but not caspase-1-like, protease activity 2-12 h after the addition of the anti-Fas. A caspase-3 inhibitor, Z-Asp-Glu-Val-Asp-fluoromethyl ketone, attenuated the Fas-mediated beta-cell apoptosis, while a caspase-1 inhibitor, acetyl-Tyr-Val-Ala-Asp-chloromethylketone, failed to suppress the apoptosis. Thus the Fas-induced death signal apparently bypassed caspase-1 in the cells. Furthermore, an antisense caspase-3 construct blocked caspase-3 activation and substantially suppressed Fas-triggered apoptosis of hFas/betaTC1 cells. These observations suggest the essential role of caspase-3 in Fas-mediated apoptosis of the beta-cell line.

Polymorphism in the 5'-leader cistron of the beta2-adrenergic receptor gene associated with obesity and type 2 diabetes.

We screened the 5'-untranslated region of the beta2-adrenergic receptor gene from 40 obese subjects by the PCR-direct sequencing technique. Two polymorphic sites were identified; a T-->C substitution at -47 and a T-->C substitution at -20. We further analyzed the association of the polymorphisms with obesity in 574 subjects by PCR and restriction digestion. The substitution at -47 was in tight linkage disequilibrium with that at -20. The polymorphisms were also in linkage disequilibrium with codon 16 and codon 27 polymorphisms. Subjects carrying the -47C/-20C allele had greater body mass index (25.5+/-4.5 vs. 24.4+/-4.1 kg/m2, p=0.007) and higher serum triglyceride levels (166+/-160 vs. 139+/-95 mg/dl, p=0.015) than -47T/-20T homozygotes. The variant allele frequency was significantly higher in obese subjects than in non-obese subjects (0.18 vs. 0.11, p=0.0026). Furthermore, an increased frequency of the variant allele was shown in diabetic patients compared with non-diabetic subjects (0.19 vs. 0.11, p=0.0005). The association may be attributable to the greater proportion of diabetic patients in the obese group. The exchange at -47 may alter the expression level of the beta2-adrenergic receptor gene, because the nucleotide substitution at -47 results in a Cys-->Arg exchange at the C terminal of the leader peptide. The -47C/-20C allele may be associated with genetic predisposition to obesity and obesity-related metabolic disorders.

Vascular cell adhesion molecule-1 as a biochemical marker of left ventricular mass in the patients with hypertension.

Plasma levels of soluble intercellular adhesion molecule-1 (sICAM-1), vascular adhesion molecule-1 (sVCAM-1), and E-selectin were measured in 80 outpatients with uncomplicated essential hypertension. Although the levels of E-selectin and sICAM-1 were similar between the patients with and without left ventricular (LV) hypertrophy, sVCAM-1 level was significantly elevated in the patients with LV hypertrophy (759.7+/-154.6 ng/mL nu 984.4+/-240.6 ng/mL, P < .0001). The LV mass normalized to body surface area or height were significantly correlated with sVCAM-1 (r=0.615, P < .0001 and r=0.571, P < .0001, respectively). These results indicate that a soluble adhesion molecule is correlated with LV mass in uncomplicated essential hypertensive patients.

Analysis of trinucleotide-repeat combination polymorphism at the rad gene in patients with type 2 diabetes mellitus.

A combined (GTT)n (ATT)n trinucleotide-repeat polymorphism designated as RAD1 has been identified at intron 2 of the rad gene on chromosome 16q. An association between the total length of the RAD1 locus and type 2 diabetes has been shown in white American subjects, but not in Finns. We genotyped 115 Japanese patients with type 2 diabetes and 114 nondiabetic control subjects at the RAD1 locus by the direct sequencing method, and found 16 RAD1 alleles composed of various combinations of GTTs and ATTs. Allele 14 consisting of four GTTs and seven ATTs accounted for the majority in both control subjects and diabetic patients, suggesting that RAD1 polymorphism is not a major genetic component for susceptibility to common forms of diabetes in the Japanese. There was no significant association between total repeat length and diabetes. However, the frequency of minor alleles containing five GTTs or three GTTs was significantly higher in diabetic patients versus nondiabetic subjects (4.8% v 0.9%, P = .012). Thus, genetic variability at the rad gene in linkage disequilibrium with RAD1 could be associated with a predisposition to type 2 diabetes in the Japanese population.

An experience of ischemic limb salvage associated with myositis ossificans of the left thigh.

A case of ischemic limb salvage associated with myositis ossificans of the left thigh in a 66-year-old man was reported. The patient had a medical history of cerebral palsy and a cervical spinal cord injury, and had an operative past history of hip arthroplasty for fracture of the left femoral neck 10 years before. He showed ischemic symptoms such as paleness, coldness, and loss of the left dorsal arterial pulsation in the left toe, and had a rapidly growing mass in the left thigh. Roentgenography and computed tomography showed a mass 10 cm by 10 cm by 8 cm in size with severe calcification in the left quariceps muscle. Occlusion of the left common femoral artery was found in the arteriogram. Surgery was carried out in order to establish an accurate diagnosis and to rescue the left lower limb. The arterial pulsation was recovered as the result of completely resecting the left quariceps muscle tumor. The pathohistological diagnosis was of myositis ossificans in the quariceps muscle of the thigh. Etidronate disodium was administered in order to prevent a recurrence postoperatively. The patient has been well for the 13 months since surgery.

A case report of epithelioid leiomyosarcoma of transverse mesocolon: diagnosis and treatment.

A case of epithelioid leiomyosarcoma of the transverse mesocolon in a 45-year-old man was reported. The patient had a rapidly growing mass in the left upper quadrant. Ultrasonography, gastrointestinography, and abdominal computed tomography showed that the mass was separated from the pancreas, the gastrointestinal tract, and the retroperitoneal organs. Preoperatively the primary origin of this tumor was related to the transverse mesocolon. On laparotomy the tumor of 5cm by 6cm by 3cm in size was found in the anterior left of the transverse mesocolon and the mass was resected entirely. The patient is well 18 months after surgical treatment with no evidence of recurrence.

A case report of substernal goiter.

A case of substernal goiter is reported. A 78-year-old female was admitted to our hospital with no symptoms. Chest roentgenography on admission showed that a mass of 3 by 5 cm in size with calcification located in the substernal region. Computed tomography of the chest and aortography revealed that the mass was attached to the trachea, but the connection to the great vessels was not clear. Pathological findings of the incisional biopsy specimen showed thyroid tissue with no evidence of malignancy. Our clinical diagnosis was substernal goiter. Surgery was not carried out in this case, based on the literature. Surgery is indicated in case of malignancy or in cases with severe illness such as respiratory disorder and superior vena cava syndrome.

Relationship between occlusal tooth contact patterns and tightness of proximal tooth contact.

The aim of this study was to investigate the relationship between occlusal tooth contact patterns and the tightness of proximal tooth contact (TPTC) during clenching. Twenty young adult volunteers with healthy dentition participated in the study. TPTC between the left second premolar (P2) and the first molar (M1) was measured during clenching at the 50% maximum voluntary contraction level in the intercuspal position (ICP). A silicone impression material was used to make an interocclusal record at the ICP in each subject, and interocclusal records were analysed using an image-processing system. Subjects were classified according to the presence or absence of each type (A, B or C) of occlusal contact. Statistical analysis was performed using the Mann-Whitney U-test. The results of this study exhibited a relationship between B-type contact and the TPTC of maxillary teeth. The experimental group with a lack of B-type contact on maxillary P2 and/or M1 showed a statistically greater TPTC than the group with B-type contact on both of these teeth (P < 0.01). These results suggest that occlusal tooth contact patterns have an influence on TPTC during clenching.

Characterization of phosphorylcholine- (PC) specific IgE-B cells in CBA/N or (CBA/N x BALB/c)F1 male mice.

Surface immunoglobulin isotype of precursors for PC-specific IgE response in CBA/N or NBF1 male mice was studied. Immunization of CBA/N or NBF1 male mice, which had been lethally irradiated and reconstituted with KLH-primed syngeneic spleen cells, with PC-KLH induced the PC-specific IgE, but not the IgM, response. Depletion of mu-bearing cells from KLH-primed spleen cells completely abolished the induction of the anti-PC IgE response. Depletion of T15 idiotype-bearing cells with anti-T15-coated dishes also abrogated the anti-PC IgE response in NBF1 male mice. Mice that were transferred with delta- or epsilon-bearing cell-depleted cell populations did not show the anti-PC IgE response on day 14, but they showed the anti-PC IgE response on day 21. These results indicated that PC-specific IgE-producing cells in CBA/N mice, which were defective in the anti-PC IgM response, were derived from PC-specific mu-bearing cells, and direct precursors bore mu, delta, and epsilon on their surface.

Demonstration of phosphorylcholine-specific IgE B cells in CBA/N mice.

CBA/N mice, which did not make anti-PC IgM or IgG antibody against PC-conjugated T-dependent or T-independent antigens, produced IgE antibody to PC-determinant when they were immunized with PC-KLH. PC-specificity of IgE antibody produced in CBA/N mice was determined by inhibition of PCA reaction with free PC-hapten or C-polysaccharide or by absorption of reaginic activity in the serum with C-polysaccharide. The presence of T15 idiotype on anti-PC IgE antibody produced in CBA/N x BALB/c F1 males also showed that anti-PC IgE antibody in defective mice was PC-specific. The results suggest that PC-specific B epsilon cells may belong to a subpopulation distinct from PC-specific precursors for IgM and IgG responses.

Evaluation of proximal tooth contact tightness at rest and during clenching.

The aims of this study were to develop a device for measuring the tightness of proximal tooth contact and to evaluate the proximal contact tightness using this device at rest and during clenching. Twenty young adult volunteers with healthy dentition participated in this experiment. The tightness of proximal tooth contact between the second premolar and the first molar of both the maxilla and the mandible was measured by pulling a stainless steel strip between them at rest, and at 20 and 50% clenching levels of maximum voluntary contraction of masseter muscles at intercuspal position. Proximal contact tightness increased as the clenching levels of both the maxilla and the mandible increased. At rest, proximal contact tightness was less in the maxilla than in the mandible, whereas during clenching it was less in the mandible. These results indicate that during clenching, the teeth are displaced and they contact appropriately with adjacent teeth, making it possible to exert sufficient occlusal force while maintaining the integrity of dental arches.

Identification of polymorphic loci in the promoter region of the serotonin 5-HT2C receptor gene and their association with obesity and type II diabetes.

AIMS/HYPOTHESIS: Polymorphisms in the upstream region of the 5-HT2C receptor gene could play a part in the development of obesity. METHODS: We screened the upstream region from 27 men by the single strand conformational polymorphism analysis and PCR-direct sequencing and then genotyped 466 non-obese (body mass index < 28 kg/m2) and 123 obese (> or = 28 kg/m2) men including 138 patients with Type II (non-insulin-dependent) diabetes mellitus. RESULTS: Three loci of single nucleotide substitution (G-->A at -995, C-->T at -759, G-->C at -697) and a (GT)n dinucleotide repeat polymorphism at -1,027 were identified. The frequency of -995/-759 and -697 variants was higher in non-obese subjects and that of -995/-759 variants in non-diabetic subjects. In the dinucleotide repeat locus, five alleles were detected including Z containing 17 repeats. The Z - 6 allele was more common in non-obese subjects and the Z + 2 allele in obese subjects. Haplotype 3 (Z - 6, -995A, -759T, -697C) was associated with leanness (p = 0.02) and the absence of diabetes (p = 0.033) and haplotype 9 (Z + 2, -995G, -759C, -697G) with obesity (p = 0.007). Haplotype 2 (Z - 6, -995G, -759C, -697C) tended to be more common in non-obese subjects. A luciferase reporter assay showed that haplotype 2 and haplotype 3 had 1.44- or 2.58-fold higher promoter activities than the most common haplotype 6 (Z, -995G, -759C, -697G). CONCLUSION/INTERPRETATION: The haplotypes containing the nucleotide substitutions could be associated with higher transcription levels of the gene and thereby with resistance to obesity and Type II diabetes. Promoter polymorphisms of the 5-HT2C receptor gene may play an important part in genetic predisposition to the disorders.

Clinical characterization of polymorphisms in the sulphonylurea receptor 1 gene in Japanese subjects with Type 2 diabetes mellitus.

To assess the association of polymorphisms at the sulphonylurea receptor (SUR1) gene with the development of Type 2 diabetes mellitus, 456 subjects, 236 with Type 2 diabetes and 220 non-diabetic controls, were analysed for variants at exon 7, exon 22 and intron 24 of the SUR1 gene by the polymerase chain reaction and restriction fragment length polymorphism. The T761T substitution in exon 22 of the SUR1 gene was not found in either diabetic patients or non-diabetic controls. Both the exon 7 variant and the intron 24 variant were present in both groups at similar frequencies. No significant association was seen between either variant and obesity. Diabetic patients homozygous for the -3C allele of intron 24 had a higher ratio of positive family history than patients homozygous for the -3T allele (p = 0.03). We conclude that these polymorphisms are not major determinants of diabetes and obesity in the Japanese population.

Association of an (A-C)n dinucleotide repeat polymorphic marker at the 5'-region of the aldose reductase gene with retinopathy but not with nephropathy or neuropathy in Japanese patients with Type 2 diabetes mellitus.

AIMS: Recently an (A-C)n dinucleotide repeat polymorphic marker in the 5'-region of the ALR2 gene encoding aldose reductase was found to be associated with diabetic retinopathy in the Chinese population in Hong Kong, and with nephropathy and neuropathy in the British Caucasian population. The present study assessed the association between the polymorphism and microvascular complications in Japanese patients with Type 2 diabetes mellitus. METHODS: DNA from 87 Japanese patients with Type 2 diabetes mellitus and 90 control subjects with normal glucose tolerance were typed for the polymorphic marker by polymerase chain reaction and direct sequencing. RESULTS: Six alleles, namely Z-12, Z-6, Z-4, Z-2, Z, and Z+2 were identified. There was no significant difference in allele distribution between diabetic patients and controls. The Z-2 allele frequency was significantly higher in subjects with diabetic retinopathy than those without retinopathy (0.35 vs. 0.20, P=0.039), suggesting that aldose reductase is involved in the development of diabetic retinopathy. In contrast, the microsatellite marker was not associated with diabetic nephropathy, peripheral or autonomic neuropathy. The discrepancy may be partly attributable to the low frequency of Z+2 allele in the Japanese subjects. CONCLUSIONS: The (A-C)n dinucleotide repeat polymorphism may be a useful genetic marker to screen for patients at high risk of retinopathy.

Association of polymorphisms in the beta2-adrenergic receptor gene with obesity, hypertriglyceridaemia, and diabetes mellitus.

To assess the role of polymorphisms in the beta2-adrenergic receptor gene in the development of obesity and obesity-related metabolic disorders, we analysed Arg16Gly, Gln27Glu, and Thr164Ile polymorphisms in 400 non-obese subjects (body mass index < 27 kg/m2) and 108 obese subjects (body mass index> or =27 kg/m2). The Gln27Glu substitution was twice as common in obese subjects as in non-obese subjects (0.14 vs 0.07, p = 0.001, odds ratio 2.14, 95 % confidence interval 1.35-3.41). The frequency of the Glu27 allele was also higher in patients with Type II (non-insulin-dependent) diabetes mellitus than nondiabetic subjects (0.14 vs 0.07, p = 0.001, odds ratio 2.13, 95% confidence interval 1.34-3.41). Analysis of variance of multiple variables showed an association between 2-h post-load glucose concentrations and body mass index but not with the Glu27 variant, suggesting that the association with diabetes could be secondary to obesity. Obese subjects carrying the variant allele had higher concentrations of serum triglyceride than obese subjects homozygous for the wild type allele (2.68+/-1.90 vs 1.18+/-1.15 mmol/l, p = 0.02). Conversely, the frequency of Gly16 homozygotes was lower in obese women when compared with non-obese women (11% vs 28%, p = 0.01, odds ratio 0.30, 95% confidence interval 0.12-0.75), although the association was not present in male subjects. Thr164Ile substitution was not detected in the subjects of this study. These observations suggest that the amino-terminal polymorphisms of the beta2-adrenergic receptor gene could be involved in the molecular pathogenesis of obesity and hypertriglyceridaemia, and thereby the development of Type II diabetes mellitus.

The Gln27Glu beta2-adrenergic receptor variant is associated with obesity due to subcutaneous fat accumulation in Japanese men.

The Trp64Arg beta3-adrenergic receptor (AR) variant is associated with visceral obesity probably due to decreased lipolysis in visceral fat (H. Kim-Motoyama et al., Diabetologia 40, 469-472, 1997). Functional alteration of beta2AR may also change fat distribution. We investigated the influence of the Gln27Glu beta2AR variant upon obesity and fat distribution. We screened 278 unrelated Japanese men and detected 249 wild-type Gln27 homozygotes, 28 Gln27/Glu27 heterozygotes, and one mutant Glu27 homozygote. The frequency of mutant Glu27 allele was significantly higher in obese subjects than in nonobese/intermediate subjects (0.11 vs 0.04, P = 0. 004). The Gln27/Glu27 heterozygotes had a significantly higher mean age-adjusted body-mass index (BMI) and mean age-adjusted subcutaneous fat area assessed by CT scan than the wild-type homozygotes but not the mean age-adjusted visceral fat areas. In summary, we have found that in Japanese men the Gln27Glu beta2AR variant is associated with obesity due to subcutaneous fat accumulation.