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Endothelial fenestrae are the transcellular pores existing on the capillary walls which are organized in clusters referred to as sieve plates. They are also divided by a diaphragm consisting of plasmalemma vesicle-associated protein (PLVAP). In this study, we examined the involvement of fibronectin signaling in the formation of fenestra and diaphragm in endothelial cells. Results showed that Itga5 and Itgb1 were expressed in PECAM1-positive endothelial cells isolated from the anterior lobe (AL) of the rat pituitary, and integrin α5 was localized at the fenestrated capillaries of the rat pituitary and cultured PECAM1-positive endothelial cells isolated from AL (CECAL). Inhibition of both integrin α5β1 and FAK, a key molecule for integrin-microtubule signaling, respectively, by ATN-161 and FAK inhibitor 14, caused the delocalization of PLVAP at the sieve plates and depolymerization of microtubules in CECAL. Paclitaxel prevented the delocalization of PLVAP by the inhibition of integrin α5β1. Microtubule depolymerization induced by colcemid also caused the delocalization of PLVAP. Treatment of CECAL with ATN-161 and colcemid caused PLVAP localization at the Golgi apparatus. The localization of PLVAP at the sieve plates was inhibited by BFA treatment in a time-dependent manner and spread diffusely to the cytoplasm. These results indicate that a constant supply of PLVAP proteins by the endomembrane system via the Golgi apparatus is essential for the localization of PLVAP at sieve plates. In conclusion, the endomembrane transport pathway from the Golgi apparatus to sieve plates requires microtubule cytoskeletons, which are regulated by fibronectin-integrin α5β1 signaling.

Endothelial fenestrae are transcellular pores that pierce the capillary walls in endocrine glands such as the pituitary. The fenestrae are covered with a thin fibrous diaphragm consisting of the plasmalemma vesicle-associated protein (PLVAP) that clusters to form sieve plates. The basal surface of the vascular wall is lined by basement membrane (BM) composed of various extracellular matrices (ECMs). However, the relationship between the ECMs and the endothelial fenestrae is still unknown. In this study, we isolated fenestrated endothelial cells from the anterior lobe of the rat pituitary, using a dynabeads-labeled antibody against platelet endothelial cell adhesion molecule 1 (PECAM1). We then analyzed the gene expression levels of several endothelial marker genes and genes for integrin α subunits, which function as the receptors for ECMs, by real-time polymerase chain reaction (PCR). The results showed that the genes for the integrin α subunit, which binds to collagen IV, fibronectin, laminin-411, or laminin-511, were highly expressed. When the PECAM1-positive cells were cultured for 7 days on collagen IV-, fibronectin-, laminins-411-, or laminins-511-coated coverslips, the sieve plate structures equipped with probably functional fenestrae were maintained only when the cells were cultured on fibronectin. Additionally, real-time PCR analysis showed that the fibronectin coating was effective in maintaining the expression pattern of several endothelial marker genes that were preferentially expressed in the endothelial cells of the fenestrated capillaries. These results indicate that fibronectin functions as the principal factor in the maintenance of the sieve plate structures in the endothelial cells of the fenestrated capillary.

The production and secretion of adrenocorticotropin, a proopiomelanocortin (POMC)-derived hormone, by corticotrophs in the anterior pituitary, is regulated by corticotrophin-releasing hormone (CRH) and glucocorticoids. We have previously demonstrated that adrenalectomy induces α-tubulin N-acetyltransferase 1 (ATAT1) expression and α-tubulin acetylation in corticotrophs. However, the regulatory mechanism of ATAT1 expression and the function of acetylated microtubules in corticotrophs are unclear. Here, we analyze the effect of CRH or dexamethasone on Atat1 expression in the mouse corticotroph AtT20 cell line. The expression of Atat1 was increased by CRH and decreased by dexamethasone in AtT20 cells. We examined the effect of Atat1 knockdown on the expression of POMC-associated genes and the dexamethasone-induced nuclear translocation of glucocorticoid receptor (GR) by real-time polymerase chain reaction and Western blot analysis, respectively. Atat1 knockdown resulted in a significant increase in the expression of ACTH-producing genes and decreased the dexamethasone-induced nuclear translocation of GR accompanied with a reduction in α-tubulin acetylation. Atat1 overexpression resulted in a significant increase in α-tubulin acetylation and the dexamethasone-induced nuclear translocation of GR. These results suggest that the acetylated microtubules function as the rail-line for the transportation of GR into the nucleus. We conclude that ATAT1 finely tunes the cellular responses of corticotrophs to hormonal stimulation through an intracellular feedback circuit.

Microtubules play an important role in the intracellular transport of secretory granules in endocrine cells and in mitosis and the maintenance of cell morphology and are composed of heterodimers of α- and β-tubulin. α-Tubulin N-acetyltransferase 1 (ATAT1), which acetylates the lysine residue at position 40 of α-tubulin, functions not only in stabilizing microtubule structures and forming the primary cilium assembly but also in vesicular trafficking in neurons. However, the localization of ATAT1 and the role of α-tubulin acetylation in endocrine cells in the pituitary are still poorly understood. Corticotrophs in the anterior lobe of the pituitary produce and secrete adrenocorticotropin (ACTH). Although removal of the adrenal gland, a target organ of ACTH, is reported to promote the synthesis and secretion of ACTH in corticotrophs and to induce structural alterations in their organelles, uncertainty remains as to whether the acetylation of α-tubulin is involved in such intracellular events of corticotrophs. We investigate the expression and localization of ATAT1 and the acetylation of α-tubulin in the pituitary of normal and adrenalectomized rats. We find that ATAT1 is localized to the Golgi apparatus of endocrine cells in the anterior lobe of normal pituitary and that the expression levels of ATAT1 and acetylation levels of α-tubulin increase following adrenalectomy. These results agree with the hypothesis that the acetylation of α-tubulin by ATAT1 regulates the intracellular transport of secretory granules in corticotrophs.

Background: Previous studies reported that exposure to dioxins was associated with an increased risk of various diseases in general populations., Objectives: The aim of this study was to examine the association between levels of dioxins in blood and allergic and other diseases., Methods: We conducted a cross-sectional study on 1,063 men and 1,201 women (aged 15-76 years), who were living throughout Japan and not occupationally exposed to dioxins, during 2002-2010. In fasting blood samples, polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and dioxin-like PCBs (DL-PCBs) were analyzed by isotope dilution high-resolution gas chromatography/mass spectrometry. We obtained information on life style and self-reported history of diseases using a questionnaire. Blood pressure, blood levels of hemoglobin A1c, and serum lipids were also measured. Multiple logistic regression models were used to analyze the association between dioxin levels in blood and various diseases., Results: Toxic equivalents of PCDDs/PCDFs and total dioxins showed significant inverse dose-response relationships with atopic dermatitis, after adjustments for potential confounders. The highest quartile for total dioxins had an adjusted odds ratio of 0.26 (95 % confidence interval 0.08-0.70) compared to the reference group (first quartile). The odds ratios for hypertension, diabetes mellitus, hyperlipidemia, gout in men, and gynecologic diseases in women significantly increased with increasing toxic equivalents of PCDDs/PCDFs, DL-PCBs, and total dioxins in blood., Conclusions: The present findings suggest that background exposure to dioxins was associated with reduced risk of atopic dermatitis. The results also support the idea that low-level exposure to dioxins is associated with an increased risk of diabetes, hypertension, and hyperlipidemia.

Purpose: The purpose of the present study was to investigate the factors associated with blood levels of each congener of polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and dioxin-like polychlorinated biphenyls (DL-PCBs) in the Japanese population., Methods: A cross-sectional study was performed on 1,656 subjects (755 men and 901 women) aged 15-73 years, who were living in 90 different areas of 30 prefectures in Japan. Blood levels of 29 PCDD, PCDF, and DL-PCB congeners were determined by high-resolution gas chromatography/mass spectrometry. In addition, a questionnaire survey on life style, including dietary habit, was carried out., Results: The median total toxicity equivalent (TEQ) was 17 pgTEQ/g lipid. After adjustment for age, sex, body mass index, smoking habit, and consumption of other food groups, six PCDDs/PCDFs with 4-6 substituted chlorine atoms and 10 DL-PCBs, but not HeptaCDD/F or OctaCDD, showed significant positive correlations with the frequency of intake of fish and shellfish. Furthermore, significant positive relationships were also found between plasma levels of docosahexaenoic acid (DHA), a biomarker of fish intake, and 10 PCDDs/PCDFs with 4-6 chlorine atoms and 10 DL-PCBs. The partial correlation coefficients with plasma DHA were significantly higher for DL-PCBs than for PCDDs/PCDFs, and partial correlation coefficients for PCDDs/PCDFs significantly decreased with increasing number of chlorine atoms (Spearman r = -0.80, P = 0.001)., Conclusions: Blood levels of PCDDs/PCDFs with 4-6 chlorine atoms and DL-PCBs were positively associated with fish intake in the Japanese population. These results may be explained by the higher degree of bioaccumulation of these congeners in fish and shellfish in the ecosystem, and the high consumption of fish among the Japanese population.

Despite many previous studies, the question has not been settled as to whether some human rotavirus strains are more virulent than others. Since disease severity is most clearly reflected by the hospitalization status of the infected children, we examined whether there was any difference in the distribution of dominant strains between inpatient and outpatient groups. The study population comprised 763 children with acute diarrhea who were treated at a general hospital in Honjo City, Akita, Japan, during 1986-1997. Rotaviruses from stool specimens were classified into 77 electropherotypes using polyacrylamide gel electrophoresis. A single dominant strain or two co-dominant strains circulated simultaneously with some infrequent strains in most rotavirus seasons. Over the 11 rotavirus seasons, there was no significant difference in the relative frequencies of 15 rotavirus strains between the inpatient and the outpatient groups when strains of rotavirus were defined by their electropherotypes in polyacrylamide gel electrophoresis. However, infection with one G1 strain that co-dominated with a G4 strain carrying an identical electropherotype except the VP7 gene resulted in a statistically significantly reduced risk of hospitalization. There was no significant difference in the relative frequencies of four major G-serotypes or long/short RNA pattern. We conclude that the virulence or disease-causing potential of human rotavirus is not substantially different in the majority of strains.

Objectives: To evaluate background exposure levels and determinants of the individual variations in the exposure to dioxins in Japan., Methods: A cross-sectional study was performed on 131 men and 122 women (aged 20-76 years), who resided in five prefectures of Japan and had no occupational exposure to dioxins. Seven polychlorinated dibenzo-p-dioxins (PCDDs), ten polychlorinated dibenzo-furans (PCDFs) and 12 polychlorinated biphenyls (PCBs), which are assigned a toxicity equivalent factor, were determined in fasting blood. Biochemical analysis of plasma and a questionnaire survey on life-style, including dietary habit, were also performed. Factors associated with the levels of dioxin-related compounds in blood were evaluated by multiple linear regression., Results: The median of total toxicity equivalents (TEQs) in men and women was 17 and 16 pg TEQ/g lipid, respectively, with no gender difference. After adjustment for age and other covariates, plasma concentrations of eicosapentaenoic acid, a biomarker of fish intake, were found to be positively associated with blood levels of total dioxin, PCDDs, PCDFs and PCBs, all of which were expressed on a TEQ basis (P<0.01). The frequency of intake of coastal fish, such as horse mackerel, mackerel and sardine, was also associated with TEQ-based concentrations of PCDFs (P=0.03) and PCBs (P=0.08). The intake of raw fish was positively related to total dioxins (P=0.06) and PCBs (P=0.03)., Conclusions: The level of intake of marine fish, especially raw fish and coastal varieties, may be associated with increased blood levels of dioxin-related compounds among the population. Despite high fish consumption in Japan, the body burden of dioxins in the population was not found to be higher than that in western countries.

Objectives: This paper evaluates the associations of previous exposure to environmental cadmium (Cd) and renal function with total mortality and cancer incidence., Methods: The study population comprised 275 residents (aged 40-92 years at baseline) in a Cd-polluted area located on Tsushima Island, Nagasaki, Japan. In the study area, the dietary intake of Cd decreased because the soil of the Cd-polluted rice fields was replaced with new soil between 1980 and 1983. The mortality rate from 1982 to 1997 and cancer incidence from 1985 to 1996 were investigated. Standardized mortality and incidence ratios (SMR and SIR) were calculated by using regional reference rates. The associations of renal function and urinary Cd levels with total mortality and cancer incidence were evaluated with Cox regression models., Results: The SMR for all subjects, and those with a urinary beta2-microglobulin (U-beta2M) concentration > or = 1,000 microg/g creatinine (Cr) and < 1,000 microg/g Cr was estimated at 90 [95% confidence interval (CI) 73-109], 138 (95% CI 101-183) and 66 (95% CI 49-87), respectively. After adjustment for age and other potential confounders, in men, serum beta2M (S-beta2M) (> or = 2.3 mg/l) and in women, serum Cr (> or = 21.2 mg/ 100 ml), relative clearance of beta2M (> or = 21%) and U-beta2M (> or = 1,000 microg/g Cr), were associated with a significantly increased risk of mortality, with hazard ratios exceeding 2.0. After further adjustment for log(U-beta2M), the rate ratio of deaths associated with, in men, increased S-beta2M was 2.53 (95% CI 0.97-6.65) and, in women, increased serum Cr (S-Cr) concentrations was 2.75 (95% CI 1.24-6.14). Urinary Cd concentrations (> or = 10 microg/g Cr) were not significantly associated with mortality. The overall SIR of all malignant neoplasms was 71 (95% CI 44-107)., Conclusions: These findings suggest that renal tubule dysfunction and a reduced glomerular filtration rate are predictors of mortality among persons previously exposed to environmental Cd. However, the results also suggest that overall mortality rates in Cd-polluted areas are not necessarily increased, because of the low mortality among those with no, or only slight, signs of low-molecular weight proteinuria. Overall cancer incidence may not be increased among residents in Cd-polluted areas.