Castalagin and vescalagin purified from leaves of Syzygium samarangense (Blume) Merrill & L.M. Perry: Dual inhibitory activity against PARP1 and DNA topoisomerase II.

Inhibition of poly(ADP-ribose) polymerase 1 (PARP1) is one of the most promising strategies for cancer chemotherapy, and a number of inhibitors possessing nicotinamide-like structures are being developed. To discover new types of PARP1 inhibitors, we screened a large number of substances of plant origin and isolated two inhibitory substances from the leaves of Syzygium samarangense (Blume) Merrill & L.M. Perry. The inhibitory substances were identified as vescalagin and its epimer castalagin by analyses using nuclear magnetic resonance and mass spectrometry. The IC50 of purified vescalagin and castalagin for PARP1 inhibition were 2.67 and 0.86 µM, respectively. Unlike most of synthetic PARP1 inhibitors, castalagin showed a mixed type inhibition, of which Ki was 1.64 µM. When SH-SY5Y cells were treated with these ellagitannins at concentrations of less than 5 µM, cellular poly(ADP-ribosyl)ation was obviously attenuated. Castalagin and vescalagin also possessed inhibitory activity against DNA topoisomerase II, implying that they function as dual inhibitors in cells.

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.

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 ß-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.

15-Deoxy-Delta(12,14)-prostaglandin J(2) impairs the functions of histone acetyltransferases through their insolubilization in cells.

The cyclopentenonic prostaglandin 15-deoxy-Delta(12,14)-PG J(2) (15d-PGJ(2)) is a metabolite derived from PGD(2). Although 15d-PGJ(2) has been demonstrated to be a potent ligand for peroxisome proliferator activated receptor gamma (PPARgamma), the functions are not fully understood. In order to examine the effect of 15d-PGJ(2) on histone acetyltransferases (HATs), several lines of cell including mouse embryonic fibroblast (MEF) cells were exposed to 15d-PGJ(2). Three types of HAT, p300, CREB-binding protein (CBP), and p300/CBP-associated factor (PCAF), selectively disappeared from the soluble fraction in time- and dose-dependent manners. Inversely, HATs in the insoluble fraction increased, suggesting their conformational changes. The decrease in the soluble form of HATs resulted in the attenuation of NF-kappaB-, p53-, and heat shock factor-dependent reporter gene expressions, implying that the insoluble HATs are inactive. The resultant insoluble PCAF and p300 seemed to be digested by proteasome, because proteasome inhibitors caused the accumulation of insoluble HATs. Taken together, these results indicate that 15d-PGJ(2) attenuates some gene expressions that require HATs. This inhibitory action of 15d-PGJ(2) on the function of HATs was independent of PPARgamma, because PPARgamma agonists could not mimick 15d-PGJ(2) and PPARgamma antagonists did not inhibit 15d-PGJ(2).

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 alpha-MEM medium stimulated cellular viability, marker enzymes of osteoblast and osteoclast cell lines, and the (45)CaCl(2) 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-kappaB 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.

Recombinant human fibrinogen expressed in the yeast Pichia pastoris was assembled and biologically active.

Fibrinogen is a large plasma glycoprotein with a molecular mass of 340kDa that plays a critical role in the final stage of blood coagulation. Human plasma fibrinogen is a dimeric molecule comprising two sets of three different polypeptides (Aalpha, 66kDa; Bbeta, 55kDa; gamma, 48kDa). To express recombinant human fibrinogen in the methylotrophic yeast Pichia pastoris, we constructed an expression vector containing three individual fibrinogen chain cDNAs under the control of the mutated AOX2 (mAOX2) promoter. First, P. pastoris GTS115 was transformed with the vector, but the expressed recombinant fibrinogen suffered severe degradation by yeast-derived proteases under conventional nutrient culture conditions. Fibrinogen degradation was prevented by using the protease A-deficient strain SMD1168 as a host strain and regulating the pH of the culture to between 5.5 and 7.0. Western blot analysis revealed that the Aalpha, Bbeta and gamma chains of recombinant fibrinogen were assembled and secreted as a complete molecule. The Bbeta chain of the recombinant fibrinogen was N-glycosylated but the Aalpha chain, as in plasma fibrinogen, was not. The gamma chains however were heterologous, one being N-glycosylated and the other not. The recombinant fibrinogen was capable of forming a thrombin-induced clot in the presence of factor XIIIa and both the glycosylated and the non-glycosylated gamma chains were involved in the formation of cross-linking fibrin. The present study indicates that the recombinant fibrinogen expressed in P. pastoris, although different from plasma fibrinogen in post-translational modification, is correctly assembled and biologically active.

Selenium regulates transcription factor NF-kappaB 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 serum selenium concentrations in the acute phase of pathological conditions.

BACKGROUND: Selenium (Se), an essential trace element, is known to be a cofactor of antioxidative selenoenzymes such as glutathione peroxidase and thioredoxin reductase. METHODS: We assessed the pathophysiological significance of selenium (Se) by comparing the concentrations of serum Se and C-reactive protein (CRP) in healthy subjects (141; M=71, F=70) vs. patients with various pathological conditions. RESULTS: In normal males in their 40s, peak serum Se concentrations were observed (2.03+/-0.30 microg/g of serum protein, 128%, P<0.001) vs. males in their 20s (1.59+/-0.20), whereas a peak was observed in females in their 30s (1.87+/-0.31, 119%, P<0.025) vs. those in their 20s (1.57+/-0.22). The serum Se concentrations in the high CRP value group (n=40, 1.07+/-0.29 microg/g, 64.1%), the rheumatoid arthritis (RA) test positive group (n=24, 1.37+/-0.29, 82.0%), the lung cancer group (n=16, 1.38+/-0.30, 82.6%), and the adult T-cell leukemia (ATL) group (n=22, 1.26+/-0.35, 75.4%) were significantly lower (P<0.001) than those in the healthy subjects (1.67+/-0.29 microg/g). This finding was confirmed by inducing acute phase response (APR) in rats by injection of lipopolysaccharide (LPS), which produced a significant decrease of Se in plasma and liver (69.5% and 81.6% vs. untreated rats, P<0.05). In contrast, the Se content in muscle, kidney, lung, spleen, heart, and thymus showed increases of <10%. Se mobilized from liver after LPS-challenge appeared to be translocated to muscle, and Se concentrations recovered by 80 h after APR to the control concentrations in parallel with the subsidence of APR. CONCLUSIONS: The reduction of Se in the liver and plasma during APR may be associated with the increased CRP synthesis in the liver.