Yukinori Eguchi

Selenium regulates transcription factor NF-κB activation during the acute phase reaction

BACKGROUND We reported a reciprocal relationship between reduced serum selenium (Se) and elevated serum C-reactive protein (CRP) in various pathological conditions in comparison with the levels in 141 healthy subjects. To clarify the implications of these observations, the effect of Se on nuclear factor (NF)-kappaB, which upregulates the CRP synthesis in the liver, was examined. METHODS Human hepatoma cell line HuH-7 was cultured in medium with 2% fetal calf serum (FCS) for 3 days for the Se deprivation, followed by another 3 days in the same medium containing sodium selenite prior to stimulation of the cells with either monocyte-conditioned medium (MoCM) or tumor necrosis factor-alpha (TNF-alpha). NF-kappaB activation and the synthesis of CRP in hepatocytes were examined by a non-radioisotope (non-RI) gel shift assay for the nuclear extract from the cells and by a highly sensitive ELISA for the cellular extract, respectively. RESULTS The NF-kappaB activation induced by MoCM and TNF-alpha were inhibited by Se at the physiological levels. The maximum activation of NF-kappaB was induced by TNF-alpha or MoCM at a Se concentration (0.5 approximately 1 micromol/l) which was half the level of the serum Se in healthy subjects and was equivalent to level in subjects with pathological conditions together with high serum CRP values. Under the same conditions, the hepatocytes synthesized maximal amounts of CRP. CONCLUSIONS Selenium at physiological levels mediates inhibition of the activation of the transcription factor NF-kappaB which regulates genes that encode inflammatory cytokines, and that conversely, the reduction of selenium induces the synthesis of CRP by hepatocytes during the acute phase response.

Alterations of protein kinase C, 8-hydroxydeoxyguanosine, and K-ras oncogene in rat lungs exposed to passive smoking

To investigate the effects of exposure to sidestream cigarette smoke (CS) on the initiation and promotion of lung cancer, two groups of 8 or 10 rats were exposed to CS for a 1 h period twice a day for 8, 12, or 20 weeks. The protein kinase C (PKC) activity of the lung exhibited significant changes of 120, 86 and 81% in the CS groups, compared with the respective control group values in the three exposure periods. The in vitro activation of PKC by the active oxygens was efficiently eliminated by hydroxyl radical scavengers, indicating that hydroxyl radicals are responsible for the PKC activation. For the alterations in the lung nucleus caused by passive smoking, the 12- and 20-week exposure CS groups showed significant increases in the accumulation of 8-hydroxydeoxyguanosine. One rat with K-ras activation by G:C transversion (GGT-->GCT) at codon 12 was found among 26 rats of the CS groups in the three exposure periods. These results show that active oxygens introduced by passive smoking may contribute to K-ras activation as an initiator of a tumor model, possibly through the oxygen-induced DNA damage, and may also contribute to an initial activation and the subsequent down-regulation of PKC as a promoter.

Effects of calcium sources and soluble silicate on bone metabolism and the related gene expression in mice

OBJECTIVE The effects of five calcium (Ca) sources were compared for bone biochemical and mechanical properties and the related gene expression using mice, from the viewpoint of their soluble silicon (Si) content. METHODS Weanling male mice were fed diets containing 1% Ca supplemented with CaCO(3) as the control (CT), coral sand (CS), fossil stony coral (FSC), fish bone (FC) and eggshell (EC) powders, and 50 ppm of Si in the CT diet for 6 mo. The mRNA expressions related to bone remodeling were quantified by real-time polymerase chain reaction. RESULTS Soluble Si content was 9.83, 7.17, 2.48, 0.29, and 0.20 ppm for the CS, FC, FSC, EC, and Ca-deficient basal diets, respectively. Si, CS, and FSC, in order, significantly increased dry and ash weights, Ca and hydroxyproline contents, and alkaline phosphatase and decreased tartrate-resistant acid phosphatase and urinary excretion of hydroxyproline compared with the CT group. Si significantly increased and FC decreased femoral strength and stiffness. In the mRNA expression related to osteoblastogenesis, Si and CS significantly increased runt-related transcription factor 2. Si, CS, and FSC, in order, significantly decreased and FC and EC increased peroxisome proliferator-activated receptor-gamma. In the mRNA expression related to osteoclastogenesis, Si and CS significantly increased and FC and EC decreased the osteoprotegerin/receptor activator of nuclear factor-kappaB ligand ratio, whereas Si and CS decreased transforming growth factor-beta. CONCLUSION The results indicated that soluble silicate and CS, with the highest Si content among Ca sources, improved bone biochemical and mechanical properties through stimulation of gene expression related to osteoblastogenesis and suppression of that related to osteoclastogenesis.

Effects of soluble silicon compound and deep-sea water on biochemical and mechanical properties of bone and the related gene expression in mice

Silicon has been known as an essential element for bone formation. The silicon contents of sea water increase with increasing of depth: 1.8 ppm Si in deep-sea water (DW) at 612 m in depth versus 0.06 ppm in surface sea water (SW). The effects of soluble silicon (Si) and DW from which NaCl was eliminated were studied in comparison with tap water (TW) and SW in cell cultures and in animal experiments using the control strain of senescence accelerated mouse, SAMR1. Si at 10 ppm as sodium metasilicate or 10% DW in the α-MEM medium stimulated cellular viability, marker enzymes of osteoblast and osteoclast cell lines, and the 45CaCl2 uptake in those cells in comparison with the medium control. After weanling SAMR1 were maintained for 6 months on a diet containing 200 ppm Si and 39% of DW and SW, DW and Si improved bone biochemical indices such as femoral weight, mineral and collagen content, and marker enzymes of bone formation and resorption as well as mechanical properties as compared to TW. In the femoral bone marrow of SAMR1, the mRNA expression of bone morphogenetic protein-2 (BMP-2), interleukin-11 (IL-11), and runt-related transcription factor 2 (Runx 2), which stimulate osteoblast development as well as type I procollagen (COL1A1) mRNA, were significantly increased in both DW and Si groups. The expressions of both osteoprotegerin (OPG) and receptor activator of NF-κB ligand (RANKL) were also elevated, resulting in distinct increases of the OPG/RANKL ratio in both DW and Si groups. The results indicated that a soluble silicate and deep-sea water as its natural material stimulated cell growth in both osteoblasts and osteoclasts in cell culture and promoted bone metabolic turnover in favor of bone formation through stimulation of the related mRNA expression in animal experiments.

Soluble silica and coral sand suppress high blood pressure and improve the related aortic gene expressions in spontaneously hypertensive rats

Silicon is rich in the normal human aorta but decreases with age and the development of atherosclerosis. We hypothesized that soluble silica (Si) and coral sand (CS), as a natural Si-containing material, would suppress high blood pressure (BP) in spontaneously hypertensive rats (SHRs), and clarify the observed antihypertensive mechanism by cell cultures by quantifying messenger RNA expressions in the aorta. In SHR fed diets containing 1% Ca supplemented with CaCO(3) as the control (CT) and CS in a Ca-deficient diet and containing 50 mg/kg Si in the CT diet for 8 weeks, systolic BP was significantly (P < .05) lowered by 18 mm Hg for the Si group and 16 mm Hg for the CS group compared with the control CT group with 207 mm Hg. Magnesium (Mg) uptake by rat aortic smooth muscle cells significantly increased (177%, P < .005) in cells cultured with a physiologic Mg level plus Si compared with those with no Si addition. Furthermore, the increase of systolic BP by the CT diet was significantly suppressed by 17 mm Hg (P < .001) in SHR fed the diet containing Mg along with Si, but not by the Mg-deficient diet with or without Si. Soluble silica and CS treatments suppressed the aortic gene expressions of angiotensinogen and growth factors related to vascular remodeling, whereas, Si stimulated the expression of peroxisome proliferator-activated receptor-γ, the activation of which has anti-inflammatory and antihypertensive effects on vascular cells. These findings suggest that Si reduces hypertension in SHR by stimulating the intracellular Mg uptake and related gene expression in the aorta.

High yield DNA extraction from the snake cast-off skin or bird feathers using collagenase

A simple method to extract DNA in high yield from the snake cast-off skin or bird feathers was developed. The molecular weight of the extracted DNA was higher by this than the conventional method and the yield of DNA was increased by more than one hundred fold. The DNA extracted by this method could be used for PCR and other analyses. This method could be applied to various samples, for instance, extracting DNA from bird feather in general.

Sex- and age-related variations in the in vitro heparin-releasable lipoprotein lipase from mononuclear leukocytes in blood.

An in vitro heparin release of lipoprotein lipase (LPL) from whole blood, mainly from monocytes, was demonstrated by (1) the time-course of lipolytic activity with the presence of 10 U/ml heparin at 37 degrees C, (2) the distribution of LPL activity in monocyte and lymphocyte fractions, (3) an immuno-inactivation with anti-LPL immunoglobulin (IgG) and (4) responses to various compounds such as NaCl, protamine sulfate, heparin, and serum activator. The in vitro heparin-releasable LPL activity from blood correlated well with the LPL activity of postheparin plasma obtained from both normolipidemic and hyperlipidemic rabbits. Studies in humans revealed sex- and age-related variations in the in vitro heparin-releasable LPL from monocytes in the blood of 134 normal subjects and 24 hypertriglyceridemic subjects: The mean LPL activity was significantly higher in normal females over the age of 30, than in the corresponding males. In the hypertriglyceridemic group, the LPL activity was also higher in females than in males, but it was not significant. The in vitro heparin-releasable LPL activity from monocytes in blood was comparable to the LPL activity derived from adipose tissue and postheparin plasma, and thus it reflects lipoprotein metabolism.

Anti-diabetic effects including diabetic nephropathy of anti-osteoporotic trace minerals on diabetic mice

OBJECTIVE In our previous study to evaluate the effects of soluble silicon (Si) on bone metabolism, Si and coral sand (CS) as a natural Si-containing material suppressed peroxisome proliferator-activated receptor γ (PPARγ), which regulates both glucose and bone metabolism and increases adipogenesis at the expense of osteogenesis, leading to bone loss. In this study, we investigated the anti-diabetic effects of bone-seeking elements, Si and stable strontium (Sr), and CS as a natural material containing these elements using obese diabetic KKAy mice. METHODS Weanling male mice were fed diets containing 1% Ca supplemented with CaCO(3) as the control and CS, and diets supplemented with 50 ppm Si or 750 ppm Sr to control diet for 56 d. The mRNA expressions related to energy expenditure in the pancreas and kidney were quantified by real-time polymerase chain reaction. RESULTS At the end of feeding, plasma glucose, insulin, leptin, and adiponectin levels decreased significantly in three test groups, while pancreatic PPARγ and adiponectin mRNA expression levels increased significantly toward the normal level, improving the glucose sensitivity of β-cells and inducing a significant decrease in insulin expression. The renal PPARγ, PPARα, and adiponectin expression levels, histologic indices of diabetic glomerulopathy, and plasma indices of renal function were also improved significantly in the test groups. CONCLUSION Taken together, anti-osteoporotic trace minerals, Si and Sr, and CS containing them showed novel anti-diabetic effects of lowering blood glucose level, improving the tolerance to insulin, leptin, and adiponectin, and reducing the risk of glomerulopathy through modulation of related gene expression in the pancreas and kidney.

Habutobin recognizes Thr7 in the sequence of fibrinopeptide A of rabbit fibrinogen.

Habutobin, a thrombin-like enzyme from Trimeresurus flavoviridis venom, cleaves only the Arg(16)-Gly(17) bond in the rabbit Aalpha chain and releases fibrinopeptide A (FPA). To investigate the role of amino acid residues in the rabbit FPA sequence upon habutobin action, we examined the inhibitory effects of FPA and peptides containing partial sequences of FPA on the habutobin action. Fibrinopeptides from rabbit, human, bovine and dog were isolated and rabbit FPA was fragmented using dilute HCl. Rabbit FPA inhibited the action of habutobin although FPA from human, bovine and dog did not. Among the fragments of rabbit FPA, a heptapeptide Aalpha 3-9, the N-terminal region of rabbit FPA, competitively inhibited the release of FPA by habutobin, whereas the C-terminal hexapeptide of FPA (Aalpha 11-16) exerted no effect on the habutobin action. Synthetic tripeptides Ser-Thr-Phe corresponding to Aalpha 6-8 and Ala-Thr-Phe also inhibited the habutobin action, but Ser-Asp-Phe and Ala-Thr-Gly did not. It is concluded that habutobin would recognize the region around Thr(7)-Phe(8) in the sequence of rabbit FPA (Aalpha 1-16) prior to the cleavage of the Arg(16)-Gly(17) bond.

Amino Acid Sequence of the α- and β-Globin Chains of the Erabu Sea Snake (Laticaudia semifasciata)

We determined the complete amino acid sequences of the Erabu sea snake (Laticaudia semifasciata) hemoglobin by analyzing the intact globin chains, enzymatically digested fragments, and chemical cleavage fragments to clarify the molecular evolution and phylogenetic classification of the sea snake. The Erabu sea snake has two types of hemoglobin components, Hb-I and Hb-II, which contain different α- and β-chains. This is the second report of the complete primary structure for hemoglobin of snakes. The sequences were compared with those of other reptilian hemoglobins. Amino acids at positions critical for the structure and physiological functions of hemoglobin were loosely conserved. The requirements for binding of ATP and of diphosphoglycerate as allosteric effectors of β-globins seemed to be fulfilled.