FUT2 non-secretor status is associated with Type 1 diabetes susceptibility in Japanese children.

AIM: To examine the contribution of the FUT2 gene and ABO blood type to the development of Type 1 diabetes in Japanese children. METHODS: We analysed FUT2 variants and ABO genotypes in a total of 531 Japanese children diagnosed with Type 1 diabetes and 448 control subjects. The possible association of FUT2 variants and ABO genotypes with the onset of Type 1 diabetes was statistically examined. RESULTS: The se2 genotype (c.385A>T) of the FUT2 gene was found to confer susceptibility to Type 1A diabetes in a recessive effects model [odds ratio for se2/se2, 1.68 (95% CI 1.20-2.35); corrected P value = 0.0075]. CONCLUSIONS: The FUT2 gene contributed to the development of Type 1 diabetes in the present cohort of Japanese children.

Variants associated with autoimmune Type 1 diabetes in Japanese children: implications for age-specific effects of cis-regulatory haplotypes at 17q12-q21.

AIMS: The aim of this study was to clarify the significance of previously reported susceptibility variants in the development of autoimmune Type 1 diabetes in non-white children. Tested variants included rs2290400, which has been linked to Type 1 diabetes only in one study on white people. Haplotypes at 17q12-q21 encompassing rs2290400 are known to determine the susceptibility of early-onset asthma by affecting the expression of flanking genes. METHODS: We genotyped 63 variants in 428 Japanese people with childhood-onset autoimmune Type 1 diabetes and 457 individuals without diabetes. Possible association between variants and age at diabetes onset was examined using age-specific quantitative trait locus analysis and ordered-subset regression analysis. RESULTS: Ten variants, including rs2290400 in GSDMB, were more frequent among the people with Type 1 diabetes than those without diabetes. Of these, rs689 in INS and rs231775 in CTLA4 yielded particularly high odds ratios of 5.58 (corrected P value 0.001; 95% CI 2.15-14.47) and 1.64 (corrected P value 5.3 × 10-5 ; 95% CI 1.34-2.01), respectively. Age-specific effects on diabetes susceptibility were suggested for rs2290400; heterozygosity of the risk alleles was associated with relatively early onset of diabetes, and the allele was linked to the phenotype exclusively in the subgroup of age at onset ≤ 5.0 years. CONCLUSIONS: The results indicate that rs2290400 in GSDMB and polymorphisms in INS and CTLA4 are associated with the risk of Type 1 diabetes in Japanese children. Importantly, cis-regulatory haplotypes at 17q12-q21 encompassing rs2290400 probably determine the risk of autoimmune Type 1 diabetes predominantly in early childhood.

Ultrastructure of placental hyperplasia in mice: comparison of placental phenotypes with three different etiologies.

Hyperplastic placentas have been reported in several experimental mouse models, including animals produced by somatic cell nuclear transfer, by inter(sub)species hybridization, and by somatic cytoplasm introduction to oocytes followed by intracytoplasmic sperm injection. Of great interest are the gross and histological features common to these placental phenotypes--despite their quite different etiologies--such as the enlargement of the spongiotrophoblast layers. To find morphological clues to the pathways leading to these similar placental phenotypes, we analyzed the ultrastructure of the three different types of hyperplastic placenta. Most cells affected were of trophoblast origin and their subcellular ultrastructural lesions were common to the three groups, e.g., a heavy accumulation of cytoplasmic vacuoles in the trophoblastic cells composing the labyrinthine wall and an increased volume of spongiotrophoblastic cells with extraordinarily dilatated rough endoplasmic reticulum. Although the numbers of trophoblastic glycogen cells were greatly increased, they maintained their normal ultrastructural morphology, including a heavy glycogen deposition throughout the cytoplasm. The fetal endothelium and small vessels were nearly intact. Our ultrastructural study suggests that these three types of placental hyperplasias, with different etiologies, may have common pathological pathways, which probably exclusively affect the development of certain cell types of the trophoblastic lineage during mouse placentation.

Estimation of reduced and increasing health risks by installation of urban wastewater systems.

This paper aims to propose a methodological framework for quantifying the reduction and increase of health risks associated with urban wastewater systems. A risk assessment model was used to quantify reduction in disease burden while a life cycle assessment approach was used to quantify increasing risks by environmental loading. Disability adjusted life years (DALYs) was used to quantify health risks. This framework was applied to a hypothetical watershed to evaluate health risks by installation of wastewater treatment systems. In this hypothetical case, 55 DALYs per year of health risk would be reduced for 200,000 people in the downstream community by constructing wastewater systems for 200,000 people, while a range from 1.9 to 22 DALYs per year of health risks would be generated by materials and energy consumption for construction and operation of wastewater systems. However, this result would significantly change, depending on the parameters used in this analysis. The proposed methodology should be improved to obtain more precise results, but it will suggest useful information to discuss the overall effects due to the installation of various types of urban wastewater systems.

Evaluation of nitrate removal by continuous culturing of an aerobic denitrifying bacterium, Paracoccus pantotrophus.

Nitrate removal under aerobic conditions was investigated using pure cultures of Paracoccus pantotrophus, which is a well-known aerobic-denitrifying (AD) bacterium. When a high concentration of cultures with a high carbon/nitrogen (C/N) ratio was preserved at the beginning of batch experiments, subsequently added nitrate was completely removed. When continuous culturing was perpetuated, a high nitrate removal rate (66.5%) was observed on day 4 post-culture, although gradual decreases in AD ability with time were observed. The attenuation in AD ability was probably caused by carbon limitation, because when carbon concentration of inflow water was doubled, nitrate removal efficiency improved from 18.1% to 59.6%. Bacterial community analysis using the polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) method showed that P. pantotrophus disappeared in the suspended medium on day 8 post-culture, whereas other bacterial communities dominated by Acidovorax sp. appeared. Interestingly, this replaced bacterial community also showed AD ability. As P. pantotrophus was detected as attached colonies around the membrane and bottom of the reactor, this bacterium can therefore be introduced in a fixed form for treatment of wastewater containing nitrate with a high C/N ratio.

Mouse macrophage metalloelastase gene transfer into a murine melanoma suppresses primary tumor growth by halting angiogenesis.

Mouse macrophage metalloelastase (MME) has been associated with the generation of angiostatin, an internal fragment of plasminogen, which inhibits angiogenesis. To clarify whether tumor cells that consistently generate MME can suppress angiogenesis and, therefore, inhibit the growth of primary tumors in vivo, we transfected a cDNA coding for MME into murine B16-BL6 melanoma cells that grow rapidly and are MME deficient. The generation of active MME in MME-transfected clones was confirmed by immunoprecipitation followed by in vitro cleavage of plasminogen. Subcutaneous implantation of these stable clones in C57BL/6 mice inhibited primary tumor growth by an average of 73% (P = 0.00002), which directly correlated with a significant reduction of blood vessel formation (approximately 76%) in such tumors. Microangiography revealed massive angiogenesis in control tumors (mock and vector); however, in MME-transfected primary tumors it demonstrated a decreased and disrupted vascular network. Western blot analysis using a specific anti-mouse angiostatin antibody demonstrated a strong 38-kDa immunoreactive band in MME-transfected tumors and in the serum of mice bearing those tumor cells. These results show that placing MME gene directly into B16-BL6 melanoma cells is an effective approach to suppress primary tumor growth in vivo because it halts angiogenesis. Our data provide a feasible and promising strategy for gene therapy of cancer by targeting tumor vasculature.

Leptin before and after insulin therapy in children with new-onset type 1 diabetes.

Serum leptin levels reflect the amount of body fat. However, several reports suggest that insulin may also regulate serum leptin levels. This study was aimed at testing whether leptin levels are low in newly diagnosed patients with type 1 diabetes and increase after institution of insulin therapy. Nineteen children with new-onset type 1 diabetes were studied. Serum leptin levels were measured at presentation before insulin therapy was initiated (day 0), 1 day after insulin therapy (day 1), 3-5 days after insulin therapy (day 3-5), and at 3 months of follow-up (3 months). The control group consisted of 19 healthy children matched for age and body mass index. On day 0 leptin levels were lower in the patients compared with those in controls (3.3 +/- 0.2 vs. 6.2 +/- 0.9 ng/mL; P < 0.005). After insulin therapy, leptin levels increased significantly by day 1 without significant weight change and became comparable to control values by days 3-5. Before insulin therapy, leptin did not correlate with weight, body mass index, or hemoglobin A1c. After insulin therapy, leptin levels on days 3-5 correlated with insulin dose (r = 0.43; P = 0.03). The results of this study demonstrate that children with new-onset type 1 diabetes have low leptin levels before insulin therapy. Leptin levels increase within 24 h of insulin therapy and become comparable to nondiabetic levels by 3-5 days. This rapid increase in leptin after 24 h of insulinization is independent of changes in body weight and is postulated to be due to a stimulatory effect of insulin on leptin production, nutritional replenishment, or both factors together.

Protection by vascular endothelial growth factor against sinusoidal endothelial damage and apoptosis induced by cold preservation.

BACKGROUND: It is well known that sinusoidal endothelial cell (SEC) damage during cold preservation of liver tissue is closely involved in early graft failure. The objective of this study was to investigate the involvement of apoptosis in the SEC damage induced by cold preservation and to demonstrate the protective effect of vascular endothelial growth factor (VEGF) on SEC injury, including apoptotic changes. METHODS: Isolated SECs and liver tissue of Wistar rats were cold-preserved in University of Wisconsin (UW) solution, and the protective effect of VEGF was then investigated. Isolated SECs were cultured for 24 hr, and divided into the following 3 groups: Group A, in which the cells were cultured for an additional 27 hr, Group B, in which the cells were cold-preserved in UW solution for 3 hr, and then recultured for 24 hr, and Group C, in which 20 ng/ml of VEGF was added to both the culture medium and the UW solution of cells cultured according to the Group B protocol. Each group of SECs was morphologically examined using the phase contrast microscopic method and the transmission electron microscopic method (TEM), and quantitatively analyzed using the WST-1 assay. Rat livers were cold-preserved in UW solution and divided into the VEGF(+) group and the VEGF(-) group, depending on whether VEGF was added or not. Each group of livers were analyzed by scanning electron microscopic method (SEM) after 24 hr of preservation. The hyaluronic acid uptake rate (HUR) was also determined after 6 hr of preservation. After 24 hr of preservation and 6 hr of reperfusion, tissues were examined by TEM and by the terminal deoxynucleotidyl transferase d-uridine triphosphate nick end labeling (TUNEL) assay. RESULTS: The phase contrast microscopic method and the WST-1 assay showed a protective effect of VEGF against the injury to isolated SECs during cold preservation and subsequent reculturing. Apoptosis was detected immediately by TEM after isolation of SECs, and the number of apoptotic cells increased with the incubation time. This increase was accelerated after cold preservation. The scanning electron microscopic method and the hyaluronic acid uptake rate showed a protective effect of VEGF against SEC damage in the cold-preserved livers. In the liver tissue, the TEM and the TUNEL assay detected apoptosis of SECs only after cold preservation and subsequent reperfusion. VEGF suppressed the apoptosis of SECs induced by cold preservation in both isolated cells and liver tissue. CONCLUSIONS: We demonstrated that SEC damage in the cold preservation of liver tissue was caused mainly by apoptosis, which required subsequent reperfusion. Moreover, isolated SECs showed spontaneous occurrence of apoptotic changes during culture, and these changes were accelerated by the preceding cold preservation. This is the first report to demonstrate the apoptotic changes of SECs seen here were inhibited by VEGF.

Morphologic alteration of hepatocytes and sinusoidal endothelial cells in rat fatty liver during cold preservation and the protective effect of hepatocyte growth factor.

BACKGROUND: Fatty liver grafts are considered to be one of the main factors of primary nonfunctioning graft in transplantation. We investigated here, the hepatic damage during cold preservation in a rat fatty liver model by ultrastructural observation, and examined the effect of human recombinant hepatocyte growth factor (hrHGF) on amelioration of the cold-preserved graft condition. METHODS: Wistar rats were fed a choline-deficient diet (CDD) for 7 days. Livers were stored in cold University of Wisconsin (UW) solution for 0, 4, and 24 hr. We evaluated the ultrastructural alteration of the hepatocytes, sinusoidal architecture, and endothelial cells (SECs) by scanning and transmission electron microscopy. Ex vivo, we measured alanine aminotransferase (ALT) in first effluent as an index of hepatocyte injury and the hyaluronic uptake rate (HUR) as that of SEC damage. We injected hrHGF into rats fed CDD for 7 days through the portal vein and also added it to the UW solution to determine whether or not the agent ameliorated the hepatic damage in cold-preserved fatty livers. RESULTS: In rats fed CDD for 7 days, the lesion occupied by fat deposits appeared to enlarge with the duration of cold preservation leading to the disarrangement of sinusoidal architecture. Furthermore, sinusoidal endothelial damage, in which gaps, blebs, microvilli, and sinusoid denudation were detected, appeared to be more severe in these livers than in the corresponding control livers. ALT significantly increased in the 4-hr cold-preserved livers of rats fed CDD for 7 days. HUR decreased with 4-hr cold preservation and/or with CDD feeding. Administration of hrHGF prevented the expansion of fatty droplets and reduced SEC injury as detected by morphological observations. Increase of ALT in first effluent was inhibited to about one fourth the level observed in the 4-hr cold-preserved livers of rats fed CDD. Moreover, HUR significantly increased with the pretreatment of hrHGF. CONCLUSION: The hepatic injury in both hepatocytes and SECs in cold-preserved fatty liver graft developed more rapidly and severely than in the corresponding controls and demonstrated a protective effect of hrHGF.

Brachydactyly type A-4 (Temtamy type) with short stature in a Japanese girl and her mother.

We report on a Japanese girl and her mother with brachydactyly. Their 2nd and 5th middle phalanges were short and the latter was fused with the distal phalanx in one of the patients. Length and shape of proximal and distal phalanges as well as metacarpals seemed normal. These findings are consistent with brachydactyly type 4-A, which is a rare subtype and has not been adequately documented. Short stature was reported only in some cases of brachydactyly type A-1, A-6, C, and E, but not in the other subtypes. Our patients with type A-4 brachydactyly also had short stature.

Assessment of subcutaneous fat using ultrasonography in the Ullrich-Turner syndrome.

Subcutaneous fat was measured using an ultrasonic method (SF-US) in girls with Ullrich-Turner syndrome (UTS) and in height and weight-matched controls. The method is not influenced by changes in cutaneous compressibility, which is frequently abnormal in UTS especially at a younger age. SF-US in the arms was significantly less in UTS patients (ulnar SF-US: P < 0.05; triceps SF-US: P < 0.005) than in the control girls. Results in suprailiac SF-US measurement was also similar (P < 0.05). Though the mean SF-US in the subscapular region was less in UTS patients than the control girls, there was no statistically significant difference. It is demonstrated that subcutaneous fat in UTS patients is less in their arms than in girls without UTS and that trunkal fatness is not increased but rather decreased in the UTS. The ratio of lean body mass to weight seems higher in UTS patients than in girls with similar weight and height.

Fat distribution in overweight patients with Ullrich-Turner syndrome.

Overweight patients with Ullrich-Turner syndrome (UTS) and control children with similar weight/height and indices of overweight were studied to clarify the unique fat distribution in the syndrome. Triceps and ulnar skin-fold thickness (SFT) in UTS patients was significantly less than that of obese children without the syndrome. The means of SFT at the subscapular and paraumbilical regions were also less in the patients than control girls, though significance was not documented. Thus, increased body weight in UTS children seems mainly to be due to excess of adipose tissue, not in the limbs but on the trunk, and/or due to the increment of lean body mass.

Ullrich-Turner syndrome and anorexia nervosa.

We report on a 6-year-old girl with Ullrich-Turner syndrome and anorexia nervosa. The diagnosis was made at 6 years and she became anorectic at 14 years. She had been treated with low doses of estrogen just before the onset of anorexia. In spite of remarkable decrease in food intake, her body weight was in the normal range compared to standard weight. Rohrer indices were also normal, probably due to abnormal habitus in individuals with the syndrome. The pathogenetic relationship between this disorder and the hormone treatment in the onset of anorexia nervosa is discussed.

Application of the hybridization AT-tailing method for detection of human immunodeficiency virus RNA in cells and simian immunodeficiency virus RNA in formalin-fixed and paraffin-embedded tissues.

An in situ hybridization (ISH) AT-tailing method (HybrAT) was developed for the detection of viral genomes in infected cells and tissues. The method consists of hybridization with oligonucleotide probe which has a 3' end oligo d(A-T) tag, followed by elongation of the oligo d(A-T) by deltaTth DNA polymerase in the presence of the labeled nucleotide. The in situ HybrAT detected human immunodeficiency virus type 1 (HIV-1) in cells and simian immunodeficiency virus (SIV) in formalin-fixed and paraffin embedded sections with a sensitivity comparable to RNA ISH. The advantage of this method over other methods is discussed.

Nitrous oxide production in high-loading biological nitrogen removal process under low COD/N ratio condition.

Effects of influent COD/N ratio on N2O emission from a biological nitrogen removal process with intermittent aeration, supplied with high-strength wastewater, were investigated with laboratory-scale bioreactors. Furthermore, the mechanism of N2O production in the bioreactor supplied with low COD/N ratio wastewater was studied using 15N tracer method, measuring of reduction rates in denitrification pathway, and conducting batch experiments under denitrifying condition. In steady-state operation, 20-30% of influent nitrogen was emitted as N2O in the bioreactors with influent COD/N ratio less than 3.5. A 15N tracer study showed that this N2O originated from denitrification in anoxic phase. However, N2O reduction capacity of denitrifiers was always larger than NO3(-)-N or NO2(-)-N reduction capacity. It was suggested that a high N2O emission rate under low COD/N ratio operations was mainly due to endogenous denitrification with NO2(-)-N in the later part of anoxic phase. This NO2(-)-N build-up was attributed to the difference between NO3(-)-N and NO2(-)-N reduction capacities, which was the feature observed only in low COD/N ratio operations.

Inhibitory effects of modified oligonucleotides complementary to the leader RNA on the multiplication of mouse hepatitis virus.

Phosphorothioate oligonucleotides (PS-oligo) and PS-oligos with cholesterol conjugates (ChPS-oligo) complementary to the leader RNA of strain JHM of mouse hepatitis virus (JHMV) were more effective inhibitors of viral multiplication than natural oligodeoxynucleotides (PO-oligo) in JHMV-infected DBT cells. PS- and ChPS-oligos were 1,000 times more potent than unmodified PO-oligo. No significant difference was observed in the inhibitory efficiency between PS-oligo and ChPS-oligo. Sequence-dependent inhibition of viral multiplication was shown at low concentrations (0.001-0.1 M) of antisense PS-oligo and ChPS-oligo. Phosphorothioate oligodeoxycytidine, PS-(dC)20, and PS-(dC)20 with cholesterol conjugates, and PS- and ChPS-oligo which have no significant homology to the JHMV sequences, showed inhibitory effects on JHMV multiplication at concentrations higher than 0.5 M. These results showed that PS-oligo and ChPS-oligo were more potent than PO-oligo in the inhibition of JHMV multiplication, and that PS-oligo and ChPS-oligo may inhibit JHMV multiplication by two different mechanisms, that is by sequence-dependent and sequence-independent manners.

Inhibition of mouse hepatitis virus multiplication by phosphorothioate analogues of oligodeoxynucleotides complementary to the leader RNA.

Phosphorothioate oligodeoxynucleotides (PS-oligo) complementary to a leader RNA of mouse hepatitis virus (MHV) were more effective inhibitors of MHV multiplication than natural oligodeoxynucleotides (PO-oligo). Sequence-dependent inhibition of viral multiplication was shown at low concentrations (0.001-0.1 microM) of antisense PS-oligo. Phosphorothioate oligodeoxycytidine, PS-(dC)20 and PS-oligo, which has no significant homology to the MHV sequence, showed inhibitory effects on MHV multiplication at concentrations higher than 0.5 microM. These results showed that PS-oligo was more potent than PO-oligo in inhibition of MHV multiplication and that PS-oligo may inhibit MHV multiplication by two different mechanisms, that is, in sequence-dependent and -independent manners.

Comparison of aerobic denitrifying activity among three cultural species with various carbon sources.

Abilities of three aerobic denitrifiers such as Alcaligenes faecalis, Microvirgula aerodenitrificans and Paracoccus pantotrophus were compared from the viewpoints of nitrate removal efficiency and organic matter utilization. First, the effect of carbon source was investigated. Although nitrate reduction was observed in all strains under aerobic conditions, a change of carbon source considerably affected the denitrification ability. In the case of P. pantotrophus, nitrate and nitrite were completely removed in three days under sodium acetate or leucine as a carbon source. In the case of A. faecalis, sufficient nitrate removal was observed only when sodium acetate or ethanol was added. P. pantotrophus and A. faecalis showed a higher ability of nitrate removal than that of M. aerodenitrificans. Therefore, P. pantotrophus was selected in order to investigate the effects of concentration and repetitive addition of carbon. Sodium acetate was used as a sole carbon source. Nitrate was not reduced when the carbon concentration was below 500 mgC/L. However, when carbon source was added repeatedly, nitrate was reduced under 100 mgC/L after the optical density of the bacterium reached above 1.0. This result indicated that a high enough level of bacterial density was necessary to express aerobic denitrification activity.

Nitrate removal with low N2O emission by application of sulfur denitrification in actual agricultural field.

Sulfur denitrification was applied to the agricultural field and the characteristics of the treatment were evaluated from the viewpoints of nitrate removal efficiency and nitrous oxide (N2O) emission. Two actual sites where sulfur denitrification was performed were surveyed. One is a valley bottom field, where groundwater contaminated with nitrate is coming up as spring water. The nitrate concentration in influent was about 45 mgN/L. The other was wastewater from a plastic greenhouse. The nitrate concentration in inflow water was about 200 mgN/L. Nitrate was almost removed by the containers packed with sulfur (S0)-CaCO3 blocks in both sites. Increase of sulfate indicated that nitrate was removed by sulfur denitrification. This was also estimated stoichiometrically from the relationships between the removed nitrate and produced sulfate. The N2O was supersaturated in water at most sampling points and the highest concentration of dissolved N2O reached 900 microgN/L in Saitama in March. It seemed that insufficient nitrate removal caused accumulation of intermediates during denitrification, such as nitrite and N2O, in this month. However, the emission ratio of N2O to the removed nitrate during these processes was kept low, ranging from 0.01 to 0.19%, at both two sites throughout all surveys.

Evaluation of appropriate system for reclaimed wastewater reuse in each area of Tokyo using GIS-based water balance model.

The appropriate type of reclaimed wastewater reuse system in each area of Tokyo was evaluated from the aspect of economic efficiency, using a GIS-based water balances model. The following four reclaimed wastewater reuse systems and conventional waterworks and sewerage system were evaluated; "Rain water storage and use system", "Onsite wastewater treatment and reuse system", "Sewage treatment and reuse at an intermediate point on the sewer pipe" and "Treated water supply system in sewage treatment plant". In the case that we install them to office and residential buildings, the supplied volume by reclaimed wastewater reuse systems is 693 thousands m3/d, this corresponds to 15% of total water demand in the area. Furthermore, the effects of the following scenarios brought about by technological innovation in water treatment were investigated; the case that flush water in toilet and wastewater from kitchen are also available as source in a "onsite wastewater treatment and reuse system" and the case that reclaimed water is used for laundering in residential buildings. When reclaimed water is used for laundering in residential buildings, the supplied volume by these systems increases to 814 thousand m3/d in the case that these systems are installed to office and residential buildings.