ALK(R1275Q) perturbs extracellular matrix, enhances cell invasion and leads to the development of neuroblastoma in cooperation with MYCN.

Overexpression of MYCN is a hallmark of neuroblastoma (NB). ALK(R1275Q), an activating mutation of ALK (anaplastic lymphoma kinase), has been found in sporadic and familial NB patients. In this report, we demonstrated that ALK(R1275Q) knock-in, MYCN transgenic compound mice developed NB with complete penetrance. Transcriptome analysis revealed that ALK(R1275Q) globally downregulated the expression of extracellular matrix (ECM)- and basement membrane (BM)-associated genes in both primary neuronal cells and NB tumors. Accordingly, ALK(R1275Q)/MYCN tumors exhibited reduced expression of ECM/BM-related proteins as compared with MYCN tumors. In addition, on MYCN transduction, ALK(R1275Q)-expressing neuronal cells exhibited increased migratory and invasive activities. Consistently, enhanced invasion and metastasis were demonstrated in ALK(R1275Q)/MYCN mice. These results collectively indicate that ALK(R1275Q) confers a malignant potential on neuronal cells that overexpress MYCN by impairing normal ECM/BM integrity and enhancing tumor growth and dissemination. Moreover, we found that crizotinib, an ALK inhibitor, almost completely inhibited the growth of ALK(R1275Q)/MYCN tumors in an allograft model. Our findings provided insights into the cooperative mechanism of the mutated ALK and overexpressed MYCN in the pathogenesis of NB and demonstrated the effectiveness of crizotinib on ALK(R1275Q)-positive tumors.

Respiratory complications after haematopoietic stem cell transplantation in a patient with chronic granulomatous disease.

Chronic granulomatous disease (CGD) is an inherited immunodeficiency disorder caused by defects in NADPH oxidase and characterized by recurrent life-threatening bacterial and fungal infections. Although CGD has been considered to be a target for gene therapy, bone marrow transplantation (BMT) is now selected as the radical treatment in most cases. We performed BMT in a patient with CGD with severe infections and experienced respiratory complications of diffuse alveolar haemorrhage and/or infection-associated alveolar haemorrhage. We suggest that attention be paid to signs of onset of alveolar haemorrhage during BMT in CGD patients.

Identification and production of a bacteriocin from Enterococcus mundtii QU 2 isolated from soybean.

AIMS: Identification of the bacteriocin produced by Enterococcus mundtii QU 2 newly isolated from soybean and fermentative production of the bacteriocin. METHODS AND RESULTS: The bacteriocin produced by Ent. mundtii QU 2 inhibited the growth of various indicator strains, including Enterococcus, Lactobacillus, Leuconostoc, Pediococcus and Listeria. The bacteriocin activity was stable at wide pH range and against heat treatment, but completely abolished by proteolytic enzymes. The bacteriocin was purified from the culture supernatant by the three-step chromatographic procedure. Mass spectrometry, amino acid sequencing and DNA sequencing revealed that the bacteriocin was similar to class IIa bacteriocins produced by other Ent. mundtii strains. The bacteriocin production decreased in the absence of glucose, nitrogen sources, or Tween 80 in MRS medium. Additionally, it was strongly suppressed by addition of Ca(2+) (CaCO(3) or CaCl(2)). In pH-controlled fermentations, the highest bacteriocin production was achieved at pH 6.0, whereas the highest cell growth was obtained at pH 7.0. CONCLUSIONS: Ent. mundtii QU 2 produced a class IIa bacteriocin. Some growth factors (e.g. Ca(2+) and pH) influenced the bacteriocin production. SIGNIFICANCE AND IMPACT OF THE STUDY: A new soybean isolate, Ent. mundtii QU 2 was found to be a class IIa bacteriocin producer. Factors influencing the bacteriocin production described herein are valuable for applications of the bacteriocins from Ent. mundtii strains.

Enhanced insulin gene expression by reduced intracellular glutathione level in insulin secreting cells MIN6.

Glutathione (GSH) participates in deoxidization and elimination of hydrogen peroxide and other reactive oxygen species, and plays an important part in the antioxidant system. To investigate the effect of GSH content on insulin gene expression, we utilized a stable transfectant, designated as ribo-MIN6 cells, which were stably transfected with the ribozyme of gamma-glutamylcysteine synthetase (gamma-GCS), exhibiting approximately 50% reduction of intracellular GSH content. We transiently transfected a luciferase expression vector driven by human preproinsulin gene promoter spanning from -1998 to +237 (pINS-1998/luc) and several deletion constructs into ribo-MIN6. Furthermore, transient transfection with ribozyme vector and pINS-1998/luc into wild-type MIN6 cells was also carried out. Luciferase activity was about 9-fold higher in ribo-MIN6 cells as compared to wild-type MIN6 cells. In the transient transfection of pINS-1998/luc with gamma-GCS ribozyme vector into wild-type MIN6 cells, the luciferase activity was increased in proportion to the added amounts of ribozyme vector. In transfection with deletion constructs, two major sites were found to be critical for insulin promoter activity. For the wild-type MIN6 cells, regions important for the promoter activity were also located at regions similar to those of ribo-MIN6 cells. Our results suggest that the suppression of intracellular GSH level might, in part, regulate the insulin gene expression.

Decrease in the insulin receptor protein level by anti-insulin receptor antibodies: roles of tyrosine kinase activity and receptor internalization.

To investigate the mechanism of severe impairment of insulin action in type B insulin resistance, we extracted IgG from the serum of a patient with type B insulin resistance (B-IgG) and analyzed the inhibiting effect of B-IgG not only on insulin signaling but also on IGF-I signaling in Chinese hamster ovary (CHO) cells expressing human insulin receptor or human IGF-I receptor. Preincubation with 1 mg/ml B-IgG prevented insulin-induced phosphorylation of insulin receptor and insulin receptor substrate-1 (IRS-1) but did not alter the IGF-I-induced phosphorylation of the IGF-I receptor and IRS-1. (125)I-insulin binding was inhibited by 93% after preincubation with B-IgG at 37 degrees C and was recovered up to 50% of the control value by acid washing. However, when cells were preincubated with B-IgG at 4 degrees C, the insulin binding completely recovered the control value by acid washing. (125)I-IGF-I binding was not altered by B-IgG preincubation. Immunoblot study revealed that the protein level of the insulin receptor was strongly decreased by preincubation with 1 mg/ml B-IgG at 37 degrees C, but never at 4 degrees C. The IRS-1 protein level did not change by B-IgG preincubation. In order to know the role of the insulin receptor internalization in the inhibiting effect of B-IgG, we employed CHO cells expressing mutant insulin receptors which do not undergo internalization (CHO-K1018R). B-IgG incubation of CHO-K1018R at 37 degrees C failed to decrease the protein level of the insulin receptor. The present data indicate that IgG from the diabetic patient with type B insulin resistance decreased insulin receptor protein level, probably due to the enhanced degradation rate of the insulin receptor, in which insulin receptor tyrosine kinase activity and internalization are required for this process. This effect of B-IgG was specific for the insulin receptor with no effect on either IGF-I receptor or IRS-1, as reflected by the IGF-I effectiveness on glycemic control in this patient.

Comparison of serum concentrations of soluble adhesion molecules in diabetic microangiopathy and macroangiopathy.

AIMS: To clarify the correlation between serum concentrations of soluble adhesion molecules and diabetic microangiopathy or macroangiopathy in patients with Type 2 diabetes. METHODS: Patients with diabetic retinopathy and intima-media thickness of common carotid artery (CCA-IMT) < 1.1 mm were classified as the microangiopathy group (n = 62). Patients with CCA-IMT > or = 1.1 mm and without retinopathy were classified as the macroangiopathy group (n = 95). Patients with CCA-IMT < 0.9 mm and without retinopathy were assigned to the no complications group (n = 139). Clinical characteristics and soluble intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin levels were compared between the groups. RESULTS: Patients with microangiopathy had a significantly longer duration of diabetes, were hypertensive and more likely to have a positive family history of diabetes than the control group. Patients with macroangiopathy were more likely to be smokers, hypertensive, and have a family history of hypertension. Soluble ICAM-1, VCAM-1, and E-selectin levels were significantly higher in the microangiopathy group than in the control group. Soluble VCAM-1 and E-selectin levels, but not ICAM-1 levels, were significantly elevated in the macroangiopathy group. These results were unchanged after adjustment for age, sex, duration of diabetes, blood pressure, HbA1c, HDL-cholesterol, and smoking status. CONCLUSIONS: Our results suggest that soluble adhesion molecules are related to both diabetic micro- and macroangiopathy. The relative contributions of adhesion molecules may be greater in the former than latter patients with Type 2 diabetes.

Antibodies to GAD in Japanese patients classified as Type 2 diabetes at diagnosis. High titre of GAD Ab is a predictive marker for early insulin treatment--report of west Japan (Kyushu, Yamaguchi, Osaka) study for GAD Ab(+) diabetes.

AIM: We evaluated the prevalence of GAD Ab in Japanese Type 2 diabetic patients treated with oral hypoglycaemic agents (OHA) and/or diet and followed GAD Ab(+) patients to assess the usefulness of GAD Ab as a marker for future insulin treatment prospectively. METHODS: A total of 2658 Japanese Type 2 diabetic patients treated by OHA and/or diet were randomly selected between April 1996 and December 1998. The clinical characteristics at entry were assessed and patients were followed for 1-3 years. RESULTS: The overall prevalence of GAD Ab among Type 2 diabetic patients was 2.0%. Forty-five had a history of diabetes of < or = 5 years (short history) while those with duration > 5 years (long history) totalled nine. Among them, 47% of patients with a short history did not require insulin in the follow-up period. However, none of those with a long history required insulin treatment within 2 years. Comparison of patients based on GAD titre in those with short history showed that 33% of patients in the high-titre group (> or = 20 U) required no insulin treatment in the first year of follow-up. In contrast, this proportion was 80% in the first and 67% in the second year in the low-titre group (< 20 U). CONCLUSIONS: The prevalence of GAD Ab in Japanese patients with a short and long history of diabetes was 2.8% and 0.9%, respectively. The presence of GAD Ab in Japanese Type 2 diabetic patients with a short history of diabetes is a marker for early insulin treatment.

Sweeping on a microchip: concentration profiles of the focused zone in micellar electrokinetic chromatography.

On-line sample concentration by sweeping was investigated in microchip micellar electrokinetic chromatography (MEKC), By changing the distance between the injection cross and the detection points, the profile of the concentration process and the diffusion process in sweeping was elucidated. Rhodamine B injected for 4 s was best concentrated by sweeping at 9.4 mm from the injection cross and the enhancement factor was 450. At the longer distance from this point the peak of Rhodamine B was broadened and diluted by diffusion. The diffusion constant of Rhodamine B calculated from the experiment was 5.7 x 10(-6) cm2s(-1). The mixture of rhodamine B, sulforhodamine B, and cresyl fast violet was concentrated by sweeping and separated by MEKC at the same time.

Differential splenic migration of dendritic cells after immunologic unresponsiveness in rat hepatic allografts induced by pretransplant donor-specific transfusion.

BACKGROUND: Donor dendritic cells migrate into the recipient spleen after hepatic transplantation. We previously reported that immunologic unresponsiveness to rat hepatic allografts can be induced by prior donor-specific blood transfusion (DST). We investigated the phenotype and splenic distribution of donor dendritic cells after allografting and DST. METHODS: Donor dendritic cells were identified with anti-rat dendritic cell (OX-62) and anti-donor class II MHC (RT1B(a)) (OX-76) antibodies. The phenotype of dendritic cells was determined with antibodies to CD45RC, CD62L, and the maturation markers CD80 (B7-1) and CD86 (B7-2). The cytokine profile of sorted CD45RC(+) OX-62(+) and CD45RC(-) OX-62(+) dendritic cells was analyzed by reverse transcription polymerase chain reaction (RT-PCR). RESULTS: Pretransplant DST significantly prolonged rat hepatic allograft survival. Immunostaining revealed OX76(+)/OX-62(+) cells in the splenic red pulp of animals receiving pretransplant DST and in the white pulp of untreated animals after transplantation. The ratio of splenic CD45RC(-) OX-62(+) cells to CD45RC(+) OX-62(+) cells was significantly higher in DST recipients than in untreated animals. CD62L, CD80, and CD86 were lower on CD45RC(-) OX-62(+) than CD45RC(+) OX-62(+) cells. RT-PCR revealed that sorted CD45RC(-) OX-62(+) cells expressed interleukin (IL)-4 and IL-10. In contrast, sorted CD45RC(+) OX-62(+) cells expressed only IL-2 and interferon gamma (IFN-gamma). CONCLUSION: Differential splenic migration of CD45RC(-) dendritic cells is associated with immunologic unresponsiveness to rat hepatic allografts.

CTLA4 gene polymorphism contributes to the mode of onset of diabetes with antiglutamic acid decarboxylase antibody in Japanese patients: genetic analysis of diabetic patients with antiglutamic acid decarboxylase antibody.

AIM: The mode of onset is occasionally similar in Type 1 and Type 2 diabetes mellitus, and some patients with Type 2 diabetes are positive for antiglutamic acid decarboxylase antibody (GAD Ab). We investigated the contribution of Type 1 diabetes susceptibility genes to the progression of the insulin-deficient state and mode of onset of Type 2 diabetes in GAD Ab-positive (GAD-Ab+) patients. We examined the variable number of tandem repeats in the promoter region of the insulin gene (INS-VNTR, insulin-dependent diabetes mellitus (IDDM) 2) and cytotoxic T lymphocyte antigen 4 (CTLA4, IDDM12) as representative of Type 1 diabetes susceptibility genes. METHODS: Patients with Type 2 diabetes who were GAD-Ab+ (n = 51) were selected for this study. In INS-VNTR, the class I allele was classified according to length (1S, 25-38 repeat units; 1M, 39-41 repeat units; 1L, 42-44 repeat units) and the exact class I allele length was analysed by specific polymerase chain reaction (PCR) amplifications. Analyses of classes II and III were performed by Southern blot. CTLA4 gene polymorphism (exon 1 position 49, G/A) was analysed by PCR-restriction fragment length polymorphism. RESULTS: The distribution of INS-VNTR was no different between Type 1 diabetes and Type 2 diabetes with GAD Ab. The allele frequencies of CTLA4 gene polymorphism G and A in Type 2 diabetes/GAD-Ab+ were significantly different from those of Type 1 diabetes/GAD-Ab+ (G: 53%, A: 47% vs. G: 84%, A: 16%; P < 0.0001). CONCLUSIONS: Our data showed that GAD-Ab+ Japanese patients presenting with Type 2 diabetes have shifted A allele while patients with abrupt onset have shifted G allele of CTLA4 gene polymorphism. Our results suggest that immunological function and polymorphism of the CTLA4 gene may contribute to the pathogenesis and progression of Type 1 diabetes.

Novel compound heterozygous mutations for lipoprotein lipase deficiency. A G-to-T transversion at the first position of exon 5 causing G154V missense mutation and a 5' splice site mutation of intron 8.

We systematically investigated the molecular defects causing a primary LPL deficiency in a Japanese male infant (patient DI) with fasting hyperchylomicronemia (type I hyperlipoproteinemia) and in his parents. Patient DI had neither LPL activity nor immunoreactive LPL mass in the pre- and post-heparin plasma. The patient was a compound heterozygote for novel mutations consisting of a G-to-T transversion at the first nucleotide of exon 5 [+1 position of 3' acceptor splice site (3'-ass) of intron 4] and a T-to-C transition in the invariant GT at position +2 of the 5' donor splice site (5'-dss) of intron 8 (Int8/5'-dss/t(+2)c). The G-to-T transversion, although affecting the 11 nucleotide of the 3'-consensus acceptor splice site, resulted in a substitution of Gly(154) to Val (G154V; GG(716)C(-->)GTC). The mutant G154V LPL expressed in COS-1 cells was catalytically inactive and hardly released from the cells by heparin. The Int8/5'-dss/t(+2)c mutation inactivated the authentic 5' splice site of intron 8 and led to the utilization of a cryptic 5'-dss in exon 8 as an alternative splice site 133 basepairs upstream from the authentic splice site, thereby causing joining of a part of exon 8 to exon 9 with skipping of a 134-bp fragment of exon 8 and intron 8. These additional mutations in the consensus sequences of the 3' and 5' splice sites might be useful for better understanding the factors that are involved in splice site selection in vivo.

Clinical and biologic characteristics for recurring neuroblastoma at mass screening cases in Japan.

BACKGROUND: It is said that most cases detected by neuroblastoma mass screening at 6 months of age tend to have a favorable clinical course after a surgical resection either with or without mild chemotherapy. However, a few cases have an unfavorable outcome. In the current study, the authors analyzed the clinical and biologic characteristics for recurring neuroblastoma in mass screening cases. METHODS: In 245 cases detected through mass screening in the Kyushu area in Japan, the clinical data and biologic features (N-myc status, DNA ploidy, Shimada histology, neuron-specific enolase (NSE), ferritin) were investigated, whereas, in particular, the data for recurring cases also were analyzed. RESULTS: Of 245 cases, 28 tumors had one or more biologically unfavorable prognostic factors, and 6 patients experienced recurrence. Three of the six patients with recurring disease underwent a complete resection of the primary tumor, whereas three cases had undergone an incomplete resection of the tumor. Regarding the initial chemotherapy, three cases received mild chemotherapy, two cases received no chemotherapy, and one case had high-dose multidrug chemotherapy. Regarding biologic prognostic factors, four of six cases with recurring disease had one or more unfavorable factors, whereas two cases had no unfavorable factors. Regarding the outcome after recurrence, four cases are CR, one case has a stable residual tumor, and one case died of disease with N-myc amplification. CONCLUSIONS: Most neuroblastomas detected by mass screening at 6 months of age have biologically favorable factors. However, approximately 10% of the cases had one or more unfavorable factors and thus might have a higher risk of recurrence than the patients with no unfavorable factors. Conversely, some cases with recurring disease had no unfavorable factors; however, the reason for this is still unclear. A long-term follow-up for mass screening cases is important, and it also might be necessary to research the established biologic factors and identify other new prognostic factors.

Hepatocyte nuclear factor-1alpha inhibits insulin promoter factor 1-dependent transactivation of the human insulin gene.

To investigate the regulational interaction of hepatocyte nuclear factor-1alpha (HNF-1alpha) and insulin promoter factor 1 (IPF1) on insulin gene expression, either or both of the expression vectors carrying each transcription factor were transiently transfected into HeLa cells, RINm5F cells and MIN6 cells together with the luciferase reporter construct driven by a human preproinsulin gene promoter (-1998 to +237) designated as, pINS-1998/luc. IPF1-transfection into HeLa cells strongly stimulated the luciferase activity to 725 fold that of the basal level. In contrast, HNF-1alpha-transfection resulted in only a 6.7 fold increase. In co-transfection experiments, increasing the amount of HNF-1alpha resulted in an 84.5% and 74.4% decrease in IPF1-stimulated luciferase activity in HeLa and RINm5F cells, respectively. Deletion constructs designated as pINS-248/luc, pINS-213/luc and pINS-185/luc were transfected into RINm5F cells to determine the role of the A3 element and its 5' flanking sequence in the inhibitory effect of HNF-1alpha. The results showed that the inhibiting effects of HNF-1alpha with pINS-213/luc and pINS-185/luc were significantly smaller than those with both pINS-1998/luc and pINS-248/luc. Transfection into MN6 cells with pINS-1998/luc in the absence of IPF1 resulted in constitutional transactivation of the insulin gene, and this transactivation was abolished by the co-transfection with HNF-1alpha. The present data indicate that IPF1 rather than HNF-1alpha predominantly transactivates the insulin gene, and that HNF-1alpha inhibits IPF1-dependent insulin gene transactivation mediated through the 5' flanking sequence of the A3 element. It is suggested that HNF-1alpha may be involved in insulin gene expression as a negative regulator.

Asbestos-related lung cancer and mesothelioma in Japan.

In Japan, crocidolite had been used for asbestos cement pipe and spraying, and amosite had been used for building board and spraying. These two types of asbestos had stopped to use in Japan in the late 1970s. An extreme increase in imported asbestos (all 3 commercial types) was observed between 1960 and 1974. In 1960, 77,000 tons of asbestos were imported, and reached the peak as 352,316 tons in 1974. This extreme rise of asbestos imports corresponds with the recent rapid increase in mortality of malignant pleural mesothelioma. Between 1995 and 1999, an estimated mean annual death from pleural mesothelioma was about 500. The annual number of compensated occupational respiratory cancers due to asbestos exposure has also been increasing. Up to the end of March 2000, 162 cases with malignant mesothelioma and 197 cases with lung cancer were compensated. As for lung cancer, epidemiological studies are scanty in Japan. Limited environmental data of the working places in asbestos textile factories suggests that heavy asbestos exposure in the past made deaths from respiratory diseases. Less asbestos exposure will enable exposed workers to survive enough to reach cancer age. Even now smoking rate among males in Japan are over 50%. So lung cancer deaths caused by the interaction between smoking and asbestos exposure will be continuing.

Isolation and evaluation of nonsiderophore cyclic peptides from marine sponges.

The world's oceans are iron-deficient environments and there is little knowledge available regarding iron uptake by marine sponges. To understand iron-related biofunctions in marine organisms, iron-binding natural compounds from marine sponges were investigated. Here we reported a natural compound haliclonamide A and its analogue haliclonamide B were isolated from the marine sponge Haliclona sp. and their structures were investigated by spectroscopic analysis. The structure of haliclonamide A was determined to consist of novel cyclic peptides containing oxazole and methyloxazoline rings. Mass spectra revealed that these two compounds formed a 1:1 stable complex with trivalent iron but not with divalent iron. EPR analysis showed that these compounds will bind with Fe(III) and Cr(III) specifically, but will not bind to other cation ions such as Cu2+, Zn2+, Co2+, Ni2+, Al3+, and Ti3+. The binding constant of compound-iron complex was 10(19) which is lower than the binding constant of siderophores. The Fe(III) concentration in this sponge tissue was shown to be 10 and 100 times higher than the other sponge tissues and seawater. This indicated the sponge Haliclona sp. may possibly uptake iron through nonsiderophore metal-binding peptides haliclonamides A and B. It also suggests that iron uptake activity of marine organisms may occur through nonsiderophore metal-binding peptides in natural ocean.

Insulin resistance and coronary risk factors in Japanese type 2 diabetic patients with definite coronary artery disease.

Insulin resistance is known as an important risk factor for coronary artery disease (CAD). However, CAD-related mortality in Japanese type 2 diabetics is lower than in Caucasians. To investigate whether insulin resistance is related to CAD in Japanese type 2 diabetics, we measured insulin sensitivity and several coronary risk factors in Japanese patients with type 2 diabetes with and without CAD. Thirty-three patients with definite CAD and 33 age- and sex-matched patients without CAD (control) were studied. Insulin sensitivity was assessed by the K index of insulin tolerance test (KITT). Clinical characteristics, classical risk factors, lipoprotein (a), and insulin sensitivity were compared between the two groups. Patients with CAD had a significantly longer duration of diabetes (9.0 +/- 1.4 vs. 5.5 +/- 0.9 years, P < 0.05, respectively), were mostly hypertensive (69.7 vs. 39.4%, P < 0.05), and more likely to be treated with insulin (45.5 vs. 18.2%, P < 0.05) compared with the control. Concerning the metabolic parameters, patients with CAD had a significantly higher insulin resistance than control (2.40 +/- 0.15 vs. 3.23 +/- 0.17%/min, P < 0.01, respectively), higher triglyceride (1.39 +/- 0.10 vs. 1.05 +/- 0.05 mmol/l, P < 0.05), lower HDL cholesterol (1.05 +/- 0.05 vs. 1.28 +/- 0.06 mmol/l, P < 0.05), and higher lipoprotein (a) (27.5 +/- 4.3 vs. 17.4 +/- 2.0 mg/dl, P < 0.05). Multiple logistic regression analysis indicated that hypertension, insulin resistance, high lipoprotein (a) and triglyceride, and low HDL cholesterol were independently related to CAD. Our results suggest that insulin resistance per se is a significant risk factor for CAD in Japanese patients with type 2 diabetes.

Association between IA-2 autoantibody epitope specificities and age of onset in Japanese patients with autoimmune diabetes.

The IA-2 is a major autoantigen of type 1 diabetes belonging to the protein tyrosine phosphatase family. We report on the humoral autoimmunity to an alternatively-spliced variant of IA-2 (IA-2 variant) and autoimmune-mediated diabetes age of onset association with IA-2 autoantibody epitope specificities, in 144 recent-onset patients with type 1 diabetes and 54 GAD autoantibody-positive patients with type 2 diabetes. The cytoplasmic domain of IA-2 (IA-2ic) detected a somewhat greater proportion of patients expressing autoantibodies than IA-2 variant (56%vs. 52% of patients with type 1 diabetes and 17%vs. 9% of GAD autoantibody-positive patients with type 2 diabetes). Conversely, only 1% of IA-2 variant autoantibody-positive patients failed to react to IA-2ic construct. Among 80 patients with type 1 diabetes who were positive for autoantibodies to IA-2ic, 8% recognized the juxtamembrane region (JM, representing amino acids 601-629) only, 64% bound the protein tyrosine phosphatase (PTP)-like domain of IA-2 only, and 29% bound both JM and PTP epitopes. Autoantibodies to the PTP-like domain were prevalent in children and adolescents with type 1 diabetes. The age of disease onset in patients with IA-2JM autoantibodies only, was significantly higher than those in patients reacted with the PTP-like domain of IA-2 (P< 0.02). Among GAD autoantibody-positive patients with type 2 diabetes reacted with IA-2ic, 44% bound the JM region only, and 33% bound epitopes in the PTP-like domain only; 22% had autoantibodies to both regions. The frequency of GAD autoantibody-positive patients with type 2 diabetes positive for autoantibodies to the JM region only, was significantly higher than that in patients with type 1 diabetes (P< 0.01). IA-2PTP autoantibodies were significantly associated with HLA-DR4, while the additional reactivity to IA-2JM was associated with HLA-DR9 allele. These results suggest that autoantibody recognition of IA-2 epitopes in autoimmune diabetes is associated with age of disease onset, which may reflect the intensity of the beta-cell destruction process.

Improved survival for patients with advanced neuroblastoma after high-dose combined chemotherapy based in part on N-myc amplification.

BACKGROUND/PURPOSE: In spite of many different kinds of chemotherapy for neuroblastoma, the prognosis for advanced neuroblastoma remains unsatisfactory. In particular, the outcome of advanced neuroblastoma with high copies of the N-myc gene tend to be poor. Therefore, the new high-dosage combined chemotherapy regimens for advanced neuroblastoma based in part on the N-myc amplification status has been utilized in the Kyushu area of Japan since 1991. This study aims to investigate whether these new regimens based in part on N-myc amplification have improved the survival rate of stage III and stage IV patients in comparison with the old regimens. METHODS: Between 1983 and 1995, 77 patients over 1 year of age and with stage III or IV neuroblastoma were registered in the Kyushu Area. Between 1983 and 1990, 49 patients received 1 of 2 combined chemotherapy regimens consisting of cyclophosphamide, cisplatin plus VM-26, and Adriamycin plus DTIC. Since 1991, two new regimens (New A1 and A3) have been administered based on the N-myc amplification status in a total of 28 patients. The New A1 regimen, which consists of cyclophosphamide, cisplatin, Adriamycin, and VP-16 has been administered in cases of less than 10 copies of N-myc, whereas the A3 regimen, consisting of a higher dose of cyclophosphamide, cisplatin, Adriamycin, and VP-16, has been administered in cases of more than 10 copies of N-myc. The survival rate was then compared between the old regimens and the new regimens. RESULTS: The 3-year survival rate (61.5%) for patients treated by the new regimens was significantly higher than that (32.7%) for patients treated by the old regimens (P <.01). Regarding the 24 cases of more than 10 copies of N-myc, the 3-year survival rate (35.9%) of the 13 patients treated by the A3 regimen was higher than that (0%) of the 11 patients treated by the old regimens (P <.05). However, in the 19 stage IV patients treated by the new regimens, the 3-year survival rate (11.1%) of the 9 cases of more than 10 copies was significantly lower than that (77.8%) of the 10 cases of less than 10 copies of N-myc (P <.01). CONCLUSIONS: These results suggest that high-dose combined chemotherapy based in part on the N-myc amplification status significantly improved the prognosis of patients with advanced neuroblastoma. However, stage IV patients with N-myc amplification still require a more effective treatment modality.

Attenuation of expression of gamma-glutamylcysteine synthetase by ribozyme transfection enhance insulin secretion by pancreatic beta cell line, MIN6.

Low levels of intracellular antioxidant enzyme activities as well as glutathione (GSH) concentrations have been described in pancreatic beta cells. We examined the effects of intracellular GSH depletion on insulin secretion and the role of intracellular GSH in signal transduction in beta cell line, MIN6 cells. Anti-gamma-glutamylcysteine synthetase (gamma-GCS) heavy subunit ribozyme was stably transfected to MIN6 cells to reduce intracellular GSH concentration. In the presence of 10 mM glucose, ribozyme-transfected cells (RTC) increased insulin secretion from 0.58 microg/10(6) cells/h in control cells (CC) to 1.48 microg/10(6) cells/h. This was associated with increased intracellular Ca(2+) concentration in RTC, detected by fluo-3 staining. Our results demonstrated that intracellular GSH concentration might influence insulin secretion by MIN6 cells, and suggest that enhanced insulin secretion by beta cells conditioned by chronic depletion of GSH is mediated by increased intracellular Ca(2+) concentration.