Association of a polymorphism of the apolipoprotein E gene with chronic kidney disease in Japanese individuals with metabolic syndrome

The purpose of the present study was to identify genetic variants that confer susceptibility to chronic kidney disease (CKD) in Japanese individuals with metabolic syndrome. The study population comprised 2150 Japanese individuals with metabolic syndrome, including 411 subjects with CKD [estimated glomerular filtration rate (eGFR) < 50 mL/min/1.73m²] and 1739 controls (eGFR ≥ 60 mL/min/1.73m²). The genotypes for 100 polymorphisms of 80 candidate genes were determined. The chi-square test, multivariable logistic regression analysis with adjustment for covariates, as well as a stepwise forward selection procedure revealed that nine polymorphisms of APOE, ABCA1, PTGS1, TNF, CPB2, AGTR1, OR13G1, and GNB3 were associated (P < 0.05) with the prevalence of CKD. Among these polymorphisms, the ? 219G → T polymorphism of APOE (rs405509) was most significantly associated with CKD in Japanese individuals with metabolic syndrome.     

ADAMTSL-6 Is a Novel Extracellular Matrix Protein That Binds to Fibrillin-1 and Promotes Fibrillin-1 Fibril Formation

ADAMTS (A disintegrin and metalloproteinase with thrombospondin motifs)-like (ADAMTSL) proteins, a subgroup of the ADAMTS superfamily, share several domains with ADAMTS proteinases, including thrombospondin type I repeats, a cysteine-rich domain, and an ADAMTS spacer, but lack a catalytic domain. We identified two new members of ADAMTSL proteins, ADAMTSL-6α and -6β, that differ in their N-terminal amino acid sequences but have common C-terminal regions. When transfected into MG63 osteosarcoma cells, both isoforms were secreted and deposited into pericellular matrices, although ADAMTSL-6α, in contrast to -6β, was barely detectable in the conditioned medium. Immunolabeling at the light and electron microscopic levels showed their close association with fibrillin-1-rich microfibrils in elastic connective tissues. Surface plasmon resonance analyses demonstrated that ADAMTSL-6β binds to the N-terminal half of fibrillin-1 with a dissociation constant of ∼80 nm. When MG63 cells were transfected or exogenously supplemented with ADAMTSL-6, fibrillin-1 matrix assembly was promoted in the early but not the late stage of the assembly process. Furthermore, ADAMTSL-6 transgenic mice exhibited excessive fibrillin-1 fibril formation in tissues where ADAMTSL-6 was overexpressed. All together, these results indicated that ADAMTSL-6 is a novel microfibril-associated protein that binds directly to fibrillin-1 and promotes fibrillin-1 matrix assembly.     

Site-specific sampling of taurine from rat brain followed by on-line sample pre-concentration, throughout in-capillary derivatization and capillary electrophoresis

A method of pinpoint-sampling followed by on-line pre-concentration of the sample, throughout in-capillary derivatization and capillary electrophoretic separation was evaluated by demonstrating the detection of taurine, 2-aminoethanesulfonic acid at a specific location of a rat brain. The direct sampling of taurine from the rat brain was accomplished by using voltage injection associated with two kinds of driving forces, electrophoretic flow and electroosmotic flow (EOF). The capillary tube (75 microm of inner diameter x 375 microm of outer diameter) of the capillary electrophoresis (CE) apparatus was already filled with a CE run buffer, viz., 40 mM phosphate-borate buffer (pH 10) containing 2mM o-phthalaldehyde (OPA)/N-acetylcysteine (NAC) as the derivatization reagent. One end of a platinum wire (0.5mm o.d.), used as the anode, and the inlet end of capillary tube (from which a 1.0 cm long polyimide coating was removed), were pricked down onto the surface of either the cerebrum or cerebellum of a rat brain at a location of very small dimension. When a low voltage (5 kV, 30s) was applied, taurine began to move from the rat brain into the capillary tube, and, simultaneously, electric focusing of taurine occurred by the action of "the pH-junction effect" at the inlet end of the capillary tube. After completing the injection, both the platinum wire and capillary tube were detached from the brain and dipped into the run buffer in an anode reservoir filed with the same solution as that in the capillary tube for the CE apparatus. Then, by applying a high voltage (20 kV) between the ends of the capillary tube, taurine was automatically derivatized to yield the fluorescent derivative, separated and detected with fluorescence (E(x)=340 nm, E(m)=455 nm) during migration throughout the capillary tube. The migration profiles obtained from cerebrum and cerebellum appeared to be different, but the peak corresponding to taurine was identified on both electropherograms. The efficacy of the present method including sample on-line pre-concentration prior to throughout in-capillary derivatization CE was first verified with several preliminary experiments by using samples of taurine in water, saline and a piece of 1.5% agar-gel block, as an alternate standard for the rat brain used in this study.     

Non-genomic modulation of dopamine release by bisphenol-A in PC12 cells

An endocrine disruptor chemical, bisphenol-A (BPA), is reported to have several short-term actions in various tissues and/or cells; however, the mechanisms of these actions have not been fully elucidated. We investigated short-term actions evoked by BPA in pheochromocytoma PC12 cells. BPA elicited dopamine release in PC12 cells in a dose-dependent manner. A selective N-type calcium channel antagonist (omega-conotoxin GVIA) and a ryanodine receptor blocker (ryanodine) inhibited the BPA-induced dopamine release. The expression of ryanodine receptor mRNA was detected by RT-PCR in PC12 cells. Subsequently, in order to prove whether membrane receptors participate in BPA-evoked dopamine release, a guanine nucleotide-binding protein inhibitor [guanosine 5'-(beta-thio) diphosphate], cyclic AMP antagonist (Rp-cAMPS) or protein kinase A inhibitor (H7 or H89) was added to PC12 cells prior to BPA-treatment. All of these agents suppressed BPA-evoked dopamine release, indicating that multiple signaling pathways may be involved in BPA-evoked dopamine release in PC12 cells. In conclusion, we demonstrated that BPA induced dopamine release in a non-genomic manner through guanine nucleotide-binding protein and N-type calcium channels. These findings illustrate a novel function of BPA and suggest that exposure to BPA influences the function of dopaminergic neurons.     

Lipid metabolism in the cockroach, Periplaneta americana, is activated by the hypertrehalosemic peptide, HTH-I

Phosphatidylcholine and phosphatidylethanolamine are the major constituents of the phospholipid pool in cockroach (Periplaneta americana) fat body and hemolymph. Both species of phospholipid are significantly decreased 6h after injecting hypertrehalosemic hormone I (HTH-I) into the hemocoel. Loss of phospholipid is accompanied by an accumulation of the phospholipid degradation products glycerophosphorylcholine and glycerol. HTH-I also increases phospholipase activity in the hemolymph and this is thought to be responsible for the depletion of hemolymph phospholipid. Phospholipase activity peaks approximately 2h after injection of HTH-I and returns to normal at 6h. In vitro, total phospholipid in the fat body is decreased by HTH-I whereas the concentration of diacylglycerol displays a corresponding increase. HTH-I elevates free fatty acid levels but has no effect on triacylglycerol. These effects of HTH-I are blocked by the phospholipase inhibitor mepacrine.     

Imidocarb,a potent anti-protozoan drug,up-regulates interleukin-10 production by murine macrophages

Interleukin-10 (IL-10), a potent antiinflammatory and immunosuppressive cytokine, plays an important role in the regulation of immune responses. To discover small molecules that stimulate IL-10 production, a cell-based screening assay was performed using a murine macrophage cell line, RAW264.7. Imidocarb, (3,3'-bis-2-imidazolin-2-yl)-carbanilide, which has been used as an anti-protozoan drug for the prevention and treatment of babesiosis in cattle, was thus identified. Imidocarb markedly enhanced LPS-induced IL-10 production not only by RAW264.7 cells but also by murine peritoneal macrophages in a concentration-dependent manner. It also augmented IL-10 production in the presence of zymosan, a yeast cell wall component. In contrast, imidocarb inhibited LPS-induced tumor necrosis factor (TNF)-alpha production by peritoneal macrophages. In mice, intraperitoneal administration of imidocarb significantly increased serum IL-10 levels and lowered TNF-alpha levels. Our results suggest that a novel anti-inflammatory property of imidocarb could lead to new therapeutic approaches in inflammatory conditions.     

Augmented lipopolysaccharide-induction of the histamine-forming enzyme in streptozotocin-induced diabetic mice

Disorders of the microcirculation and reduced resistance to infection are major complications in diabetes. Histamine enhances capillary permeability, and may also reduce cellular immunity. Here we demonstrate that streptozotocin (STZ)-induced diabetes in mice not only enhances the activity of the histamine-forming enzyme, histidine decarboxylase (HDC), but also augments the lipopolysaccharide (LPS)-induced elevation of HDC activity in various tissues, resulting in a production of histamine. The augmentation of HDC activity occurred as early as 2 days after STZ injection, but was not seen in nondiabetic mice. When given to STZ-treated mice, nicotinamide, an inhibitor of poly(ADP-ribose) synthetase, reduced both the elevation of blood glucose and the elevations of HDC activity and histamine production. These results suggest that hyperglycemia may initiate a sequence of events leading not only to an enhancement of basal HDC activity, but also to a sensitization of mice to the HDC-inducing action of LPS. We hypothesize that bacterial infections and diabetic complications may mutually exacerbate one another because both involved an induction of HDC.     

Intraspecific variability of the tandem repeats in Nicotiana putrescine N-methyltransferases

Plant Molecular Biology -     

In vitro fertilization rate of horse oocytes with partially removed zonae

Frozen-thawed ejaculated stallion spermatozoa were preincubated for 3 h in BO medium containing 5 mM caffeine and then treated with 0.1 mu M calcium ionophore A23187 for 60 sec. Aliquots of the sperm suspension (final concentration 1-2 x 10(7)/ml) were added to the oocytes which had been matured in vitro for 32 h. In Experiment 1, there were 3 groups of oocytes; cumulus intact, denuded zona-intact, and zona-free. Cumulus cells were removed with 0.5% hyaluronidase and the zona pellucida with 0.1% protease. The oocytes were fixed 20 h after insemination with acetic acid:ethanol (1:3) and stained with 1% orcein. The sperm penetration rate of zona-free oocytes was 83%, whereas the sperm penetration rate was very low (1 to 3%) in the cumulus-enclosed or zona-intact oocytes. In Experiment 2, denuded zona-intact oocytes were placed in PBS supplemented with 10% fetal bovine serum 1 h before the end of in vitro maturation. The zona pellucida was micromanipulated with a metal microblade under x 100 magnification within 20 min of treatment with 0.3 M sucrose. For partial zona dissection, a slit in the zona pellucida was made. For partial zona removal, oocytes were transferred to protein-free PBS to fix the oocytes on the bottom of the Petri-dish and to remove a piece of the zona pellucida. Micromanipulated oocytes were subjected to in vitro fertilization as described above. Zona-intact and zona-free oocytes treated with sucrose solution for 20 min were used as controls. The penetration rates were 4 (2 57 ), 12 (7 58 ), 52 (31 60 ), and 86% (44 51 ) for zona-intact, partially zona dissected, partially zona removed, and zona-free oocytes, respectively. Proportions of oocytes with monospermic penetration were 100 (2 2 ), 57 (4 7 ), 58 (18 31 ), and 34% (15 44 ), respectively. In Experiment 3, sperm penetration and male pronucleus formation in the partially zona removed oocytes were examined at 2.5 to 20.0 h of insemination. Sperm penetration started 2.5 h post-insemination (22%, 11 49 ), and increased to 38% (21 55 ) at 5 h, to 46% (23 50 ) at 10 h, and to 56% (27 48 ) at 20 h. The transformation of sperm heads into male pronuclei was first observed 10 h post insemination. These results indicate that assisted fertilization techniques may be a useful tool for achieving fertilization and embryo production in vitro in horses.