Enteric microflora contribute to constitutive ICAM-1 expression on vascular endothelial cells

Quantitative estimates of endothelial cell adhesion molecule expression have revealed that some adhesion molecules [e.g., intercellular adhesion molecule-1 (ICAM-1)] are abundantly expressed in different vascular beds under normal conditions. The objective of this study was to determine whether the enteric microflora contribute to the constitutive expression of ICAM-1 and other endothelial cell adhesion molecules in the gastrointestinal tract and other regional vascular beds. The dual radiolabeled monoclonal antibody technique was used to measure endothelial expression of ICAM-1, ICAM-2, vascular cell adhesion molecule-1 (VCAM-1), and E-selectin in conventional, germ-free mice and germ-free mice receiving the cecal contents of conventional mice to reestablish the enteric microflora (total association). Constitutive ICAM-1 expression was significantly lower in the splanchnic organs (pancreas, stomach, small and large intestine, mesentery, and liver), kidneys, skeletal muscle, and skin of germ-free mice compared with their conventional counterparts. These differences were abolished after total association of germ-free mice with the indigenous gastrointestinal flora. The expression of ICAM-2, VCAM-1, and E-selectin in the various tissues studied did not differ between conventional and germ-free mice. These findings indicate that the indigenous gastrointestinal microflora are responsible for a significant proportion of the basal ICAM-1 expression detected in both intestinal and extraintestinal tissues.     

Mechanism of superoxide anion generation in the toxic red tide phytoplankton Chattonella marina: possible involvement of NAD(P)H oxidase

Red tide phytoplankton Chattonella marina is known to produce reactive oxygen species (ROS), such as superoxide anion (O(2)(-)), hydrogen peroxide (H(2)O(2)) and hydroxyl radical (&z.rad;OH), under normal physiological conditions. Although several lines of evidence suggest that ROS are involved in the mortality of fish exposed to C. marina, the mechanism of ROS generation in C. marina remains to be clarified. In this study, we found that the cell-free supernatant prepared from C. marina cells showed NAD(P)H-dependent O(2)(-) generation, and this response was inhibited by diphenyleneiodonium, an inhibitor of mammalian NADPH oxidase. When the cell-free supernatant of C. marina was analyzed by immunoblotting using antibody raised against the human neutrophil cytochrome b558 large subunit (gp91phox), a main band of approximately 110 kDa was detected. The cell surface localization of the epitope recognized with this antibody was also demonstrated in C. marina by indirect immunofluorescence. Furthermore, Southern blot analysis performed on genomic DNA of C. marina with a probe covering the C-terminal region of gp91phox suggested the presence of a single-copy gene coding for gp91phox homologous protein in C. marina. These results provide evidence for the involvement of an enzymatic system analogous to the neutrophil NADPH oxidase as a source of O(2)(-) production in C. marina.     

Chromosomal instability syndrome of total premature chromatid separation with mosaic variegated aneuploidy is defective in mitotic-spindle checkpoint

Skin fibroblast cells from two unrelated male infants with a chromosome-instability disorder were analyzed for their response to colcemid-induced mitotic-spindle checkpoint. The infants both had severe growth and developmental retardation, microcephaly, and Dandy-Walker anomaly; developed Wilms tumor; and one died at age 5 mo, the other at age 3 years. Their metaphases had total premature chromatid separation (total PCS) and mosaic variegated aneuploidy. Mitotic-index analysis of their cells showed the absence of mitotic block after the treatment with colcemid, a mitotic-spindle inhibitor. Bromodeoxyuridine-incorporation measurement and microscopic analysis indicated that cells treated with colcemid entered G1 and S phases without sister-chromatid segregation and cytokinesis. Preparations of short-term colcemid-treated cells contained those cells with chromosomes in total PCS and all or clusters of them encapsulated by nuclear membranes. Cell-cycle studies demonstrated the accumulation of cells with a DNA content of 8C. These findings indicate that the infants' cells were insensitive to the colcemid-induced mitotic-spindle checkpoint.     

Human homologue of S. pombe Rad9 interacts with BCL-2/BCL-xL and promotes apoptosis

DNA damage induces apoptosis through a signalling pathway that can be suppressed by the BCL-2 protein, but the mechanism by which DNA damage does this is unknown. Here, using yeast two-hybrid and co-immunoprecipitation studies, we show that RAD9, a human protein involved in the control of a cell-cycle checkpoint, interacts with the anti-apoptotic Bcl-2-family proteins BCL-2 and BCL-x L, but not with the pro-apoptotic BAX and BAD. When overexpressed in mammalian cells, RAD9 induces apoptosis that can be blocked by BCL-2 or BCL-x L. Conversely, antisense RAD9 RNA suppresses cell death induced by methyl methanesulphonate. These findings indicate that RAD9 may have a new role in regulating apoptosis after DNA damage, in addition to its previously described checkpoint-control and other radioresistance-promoting functions.     

Epidermal growth factor stimulates proliferation of mouse uterine epithelial cells in primary culture

Epidermal growth factor (EGF) is one of growth factors that are thought to mediate the stimulatory effects of estrogen on the proliferation of uterine epithelial cells. The present study was attempted to obtain direct evidence for the mitogenic effects of EGF on uterine epithelial cells, and to prove that EGF and EGF receptors are expressed in these cells. Mouse uterine epithelial cells were isolated from immature female mice and cultured with or without EGF for 5 days. EGF (1 to 100 ng/ml) significantly increased the number of uterine epithelial cells, and the maximal growth (141.9+/- 8.3% of controls) was obtained at a dose of 10 ng/ml. In addition, EGF (0.1 to 100 ng/ml) increased the number of DNA-synthesizing cells immunocytochemically detected by bromodeoxyuridine uptake to the nucleus. Northern blot analysis revealed that the uterine epithelial cells expressed both EGF mRNA (4.7 kb) and EGF receptor mRNAs (10.5, 6.6, and 2.7 kb) These results suggest that the proliferation of uterine epithelial cells is regulated by the paracrine and/or autocrine action of EGF. Our previous study demonstrated the mitogenic effect of IGF-I on uterine epithelial cells. To examine whether the EGF- and IGF-I signaling act at the same level in the regulation of the proliferation of uterine epithelial cells, the cultured cells were simultaneously treated with IGF-I and EGF. IGF-I was found to additively stimulate the mitogenic effects of EGF, suggesting that the EGF-induced growth of uterine epithelial cells is distinct from IGF-I-induced growth.     

Selective attenuation of metabolic branch of insulin receptor down-signaling by high glucose in a hepatoma cell line, HepG2 cells

The effects of a high concentration of glucose on the insulin receptor-down signaling were investigated in human hepatoma (HepG2) cells in vitro to delineate the molecular mechanism of insulin resistance under glucose toxicity. Treatment of the cells with high concentrations of glucose (15-33 mm) caused phosphorylation of serine residues of the insulin receptor substrate 1 (IRS-1), leading to reduced electrophoretic mobility of it. The phosphorylation of IRS-1 with high glucose treatment was blocked only by protein kinase C (PKC) inhibitors. The high glucose treatment attenuated insulin-induced association of IRS-1 and phosphatidylinositol 3-kinase and insulin-stimulated phosphorylation of Akt. A metabolic effect of insulin, stimulation of glycogen synthesis, was also inhibited by the treatment. In contrast, insulin-induced association of Shc and Grb2 was not inhibited. Treatment of the cells with high glucose promoted the translocation of PKCepsilon and PKCdelta from the cytosol to the plasma membrane but not that of other PKC isoforms. Finally, PKCepsilon and PKCdelta directly phosphorylated IRS-1 under cell-free conditions. We conclude that a high concentration of glucose causes phosphorylation of IRS-1, leading to selective attenuation of metabolic signaling of insulin. PKCepsilon and PKCdelta are involved in the down-regulation of insulin signaling, and they may lie in a pathway regulating the phosphorylation of IRS-1.     

Y-24180, an antagonist of platelet-activating factor, suppresses interleukin 5 production in cultured murine th(2)cells and human peripheral blood mononuclear cells

Interleukin (IL)-5 has been shown to play an essential role in the pathogenesis of airway inflammation. We investigated the effect of 4-(2-chlorophenyl)-2-[2-(4-isobutylphenyl)ethyl]-6, 9-dimethyl-6 H -thieno[3,2- f ][1,2,4]triazolo[4,3- a][1,4]diazepine (Y-24180), an antagonist of platelet-activating factor (PAF), on the production of IL-5 in cultured D10.G4.1 cells, a murine Th(2)clone, and human peripheral blood mononuclear cells (PBMC). As a result, Y-24180 was found to suppress both the mRNA expression of IL-5 and the subsequent secretion of this cytokine in antigen-stimulated D10.G4.1 cells. Y-24180 also suppressed the production of IL-4, another Th(2)type cytokine, at the level of mRNA expression, however, it hardly affected the mRNA expression for IL-6 or IL-10, thus indicating it to have a selective action against IL-5 and IL-4. The suppressive effect of Y-24180 on the secretion of IL-5 by human PBMC was more potent than that of WEB2086, which is another PAF-antagonist. These results suggest that Y-24180 suppresses IL-5 production through a common pathway which also affects the production of IL-4, even though the mechanism remains to be elucidated as to whether the PAF-antagonistic actions are involved or not.     

Bmp2 and Bmp4 genetically interact to support multiple aspects of mouse development including functional heart development

Bone morphogenetic proteins (BMPs) have multiple roles during embryogenesis. Current data indicate that the dosage of BMPs is tightly regulated for normal development in mice. Since Bmp2 or Bmp4 homozygous mutant mice show early embryonic lethality, we generated compound heterozygous mice for Bmp2 and Bmp4 to explore the impact of lowered dosage of these BMP ligands. Genotyping pups bred between Bmp2 and Bmp4 heterozygous mice revealed that the ratio of adult compound heterozygous mice for Bmp2 and Bmp4 is much lower than expected. During embryogenesis, the compound heterozygous embryos showed several abnormalities, including defects in eye formation, body wall closure defects, and ventricular septal defects (VSD) in the heart. However, the ratio of the compound heterozygous embryos was the same as expected. Caesarean sections at E18.5 revealed that half of the compound heterozygotes died soon after birth, and the majority of the dead individuals exhibited VSD. Survivors were able to grow to adults, but their body weight was significantly lower than control littermates. They demonstrated progressive abnormalities in the heart, eventually showing a branched leaflet in atrioventricular valves. These results suggest that the dosage of both BMP2 and 4 is critical for functional heart formation during embryogenesis and after birth. genesis 47:374–384, 2009. © 2009 Wiley‐Liss, Inc.     

Longitudinal Study of the Decline in Renal Function in Healthy Subjects

Background

Chronic kidney disease is an important concern in preventive medicine, but the rate of decline in renal function in healthy population is not well defined. The purpose of this study was to determine reference values for the estimated glomerular filtration rate (eGFR) and rate of decline of eGFR in healthy subjects and to evaluate factors associated with this decline using a large cohort in Japan.

Methods

Retrospective cross-sectional and longitudinal studies were performed with healthy subjects aged ≥18 years old who received a medical checkup. Reference values for eGFR were obtained using a nonparametric method and those for decline of eGFR were calculated by mixed model analysis. Relationships of eGFR decline rate with baseline variables were examined using a linear least-squares method.

Results

In the cross-sectional study, reference values for eGFR were obtained by gender and age in 72,521 healthy subjects. The mean (±SD) eGFR was 83.7±14.7ml/min/1.73m². In the longitudinal study, reference values for eGFR decline rate were obtained by gender, age, and renal stage in 45,586 healthy subjects. In the same renal stage, there was little difference in the rate of decline regardless of age. The decline in eGFR depended on the renal stage and was strongly related to baseline eGFR, with a faster decline with a higher baseline eGFR and a slower decline with a lower baseline eGFR. The mean (±SD) eGFR decline rate was ‒1.07±0.42ml/min/1.73m²/year (‒1.29±0.41%/year) in subjects with a mean eGFR of 81.5±11.6ml/min/1.73m².

Conclusions

The present study clarified for the first time the reference values for the rate of eGFR decline stratified by gender, age, and renal stage in healthy subjects. The rate of eGFR decline depended mainly on baseline eGFR, but not on age, with a slower decline with a lower baseline eGFR.