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Determining absolute ages of orogenic faults is critical to understanding the deformation process in the upper crust, but obtaining age remains a problem due to the lack of readily available techniques. Carbonates occur as veins in faults in a range of geological settings, and thus it is a suitable mineral for U-Pb geochronology. Here, we apply the new approach of U-Pb dating on syn-tectonic dolomite veins from the Gelouang gold deposit in the western Qinling Orogen to unravel the absolute timing of the fault formation shedding new light on the regional upper crustal deformation archive. In situ LA-ICP-MS U-Pb dating of dolomite yielding a successful age of 115–112 Ma demonstrates that the dolomite precipitated coeval with tectonic events ascribed to the post-orogenic deformation phase in the Qinling Orogen. This event is possibly correlated with broader intracontinental processes and might be an inevitable response to the extensional deformation of the Qinling Orogen. The presented LA-ICP-MS dolomite U-Pb age successfully represents the age of a specific structure that encompasses the intracontinental process in the Qinling Orogen. Moreover, it demonstrates the utility of the method to decipher a response to complex deformation histories on a regional scale.

Fibulin-1 (FBLN1) is involved in the progression of some types of cancer. However, the role of FBLN1 in cutaneous melanoma (CM) has not been examined. The purpose of this study was to understand the molecular mechanisms and clinical significance of FBLN1 inactivation in CM. The expression of FBLN1 mRNA in CM tissues and adjacent normal skin tissues was analyzed by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). Methylation-specific polymerase chain reaction was performed to examine the methylation status of the FBLN1 gene promoter. Furthermore, the methylation status of FBLN1 was analyzed with the clinicopathological characteristics and overall survival. qRT-PCR showed FBLN1 mRNA levels in cancerous tissues to be significantly decreased compared with that in adjacent normal skin tissues. The rate of FBLN1 promoter methylation was significantly higher in CM tissues than in adjacent normal skin tissues (P

The advanced glycation end products (AGEs) are associated with increased cardiac endothelial injury. However, no causative link has been established between increased AGEs and enhanced endothelial injury after ischemia/reperfusion. More importantly, the molecular mechanisms by which AGEs may increase endothelial injury remain unknown. Adult rat cardiac microvascular endothelial cells (CMECs) were isolated and incubated with AGE-modified bovine serum albumin (BSA) or BSA. After AGE-BSA or BSA preculture, CMECs were subjected to simulated ischemia (SI)/reperfusion (R). AGE-BSA increased SI/R injury as evidenced by enhanced lactate dehydrogenase release and caspase-3 activity. Moreover, AGE-BSA significantly increased SI/R-induced oxidative/nitrative stress in CMECs (as measured by increased inducible nitric oxide synthase expression, total nitric oxide production, superoxide generation, and peroxynitrite formation) and increased SI/R-induced nitrative inactivation of thioredoxin-1 (Trx-1), an essential cytoprotective molecule. Supplementation of EUK134 (peroxynitrite decomposition catalyst), human Trx-1, or soluble receptor of advanced end product (sRAGE) (a RAGE decoy) in AGE-BSA precultured cells attenuated SI/R-induced oxidative/nitrative stress, reduced SI/R-induced Trx-1 nitration, preserved Trx-1 activity, and reduced SI/R injury. Our results demonstrated that AGEs may increase SI/R-induced endothelial injury by increasing oxidative/nitrative injury and subsequent nitrative inactivation of Trx-1. Interventions blocking RAGE signaling or restoring Trx activity may be novel therapies to mitigate endothelial ischemia/reperfusion injury in the diabetic population.

To examine the in vivo effects of atorvastatin (AT) on arterial calcification in rats, arterial calcification was established by subcutaneous injection of vitamin D3 and Warfarin. Intragastric administration of AT began 4 days before establishment of arterial calcification in the AT group (n = 6). Blood samples were taken and abdominal aortas were collected and stained. After induction of calcification, plasma Ca2+ levels in the CA and AT groups were significantly higher than those before treatment and in the untreated controls. Plasma Ca2+ levels in the AT group were significantly lower than in the CA group. The relative calcification area in aortic specimens from the AT group was significantly smaller than in the CA group. Rat aortic vascular smooth muscle cells (VMSC) were isolated from abdominal aortic segments and pre-treated with AT (1, 5, or 10 μM) for 24 hr. Cells in the calcification (CA) group and the AT group were cultured with β-glycerophosphate, insulin and vitamin C for 14 days to induce cell calcification. Calcium deposition and alkaline phosphatase activity were significantly increased in the CA group compared to untreated controls (p

The present study defines the expression of Toll-like Receptor 2 (TLR2), and the modulatory role of Glycogen synthase kinase (GSK)-3beta inhibitor on TLR2/Nuclear Factor-kappa B (NF-kappaB) signaling following myocardial ischemia-reperfusion (MI-R) injury in rats.Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC) were used to analyze the presence and quantity of TLR2 mRNA and protein. Tumor necrosis factor (TNF)-alpha mRNA and interleukin-6 (IL-6) mRNA were analyzed by RT-PCR. The activation of NF-kappaB was detected by Western Blot and the myocardial infarct size by Evans blue-TTC staining.Following 30 min of myocardial ischemia, a significant up-regulation of TLR2 mRNA was revealed by RT-PCR from 1 to 24 h post reperfusion. IHC demonstrated high protein expression levels of TLR2. Administration of the GSK-3beta inhibitor 4-benzyl-2-methyl-1, 2, 4-thiadiazolidine-3, 5-dione (TDZD-8) 5 min prior to reperfusion following 1 h reperfusion down-regulated mRNA levels of TLR2 and downstream proinflammatory cytokines (P0.05 vs. MI-R), decreased the activity of NF-kappaB and the size of the myocardial infarct (P0.05 vs. MI-R).Our results demonstrate that TLR2 and its signaling components are activated by MI-R. TDZD-8 administration attenuates TLR2/NF-kappaB signaling, suggesting a possible mechanism whereby GSK-3beta inhibition improves the outcome of MI-R.

Objective To investigate the effects of static magnetic field(SMF) on the viability, adhesion molecule expression of human umbilical vessel endothelial cell. Methods Magnetic flux intensity was 0.1 mT, 1 mT, 10 mT, Cell viability and proliferation were measured with 3 H-TdR and MTT methods; and apoptosis of human umbilical vein endothelial cell (HUVEC) was studied by flow cytometry and transmission electric microscopy. ELISA was used to measure the expression of ICAM-1 and VCAM-1 on endothelium. Results 0.1 mT SMF had no effects on the growth of HUVEC, however, SMF of 1 mT, 10 mT attenuated growth of HUVEC. 10 mT static magnetic field could induce apoptosis and necrosis of HUVEC. 10 mT SMF enhanced the expression of ICAM-1 and VCAM-1 on endothelium. Conclusion The effect of SMF depends on the intensity of SMF. 10 mT SMF has adverse effects on human umbilical vessel endothelial cell.

To elucidate the mechanism of nacre biomineralization, the mantle of Pinctada fucata (P. fucata) from the South China Sea was used. Using the mantle cDNA library and the ESTs we have cloned through suppression subtractive hybridization (SSH), ten novel genes including PFMG1 were obtained through nested PCR. Bioinformative results showed that PFMG1 had a high homology (40%) with Onchocerca volvulus calcium-binding protein CBP-1 and had two EF-hand calcium-binding domains from the 81st to the 93rd amino acid and from the 98th to the 133rd amino acid in the deduced amino acid sequence. The results of multitissue RT-PCR and in situ hybridization demonstrated the high expression of PFMG1 in the mantle of P. fucata and confirmed the SSH method. The results of GST-PFMG1 on CaCO3 crystallization showed significant effects on nucleation and precipitation of CaCO3. PFMG1 was cloned into the pcDNA.3.1/myc-HisA vector and was subsequently transfected into MC3T3-E1 cells. RT-PCR revealed upregulation of the marker genes related to cell growth, differentiation, and mineralization, and BMP-2, osterix, and osteopontin were upregulated as a result. This research work suggests that PFMG1 plays an important role in the nacre biomineralization, and the SSH method can pave the way for the bulk cloning and characterization of new genes involved in biomineralization in P. fucata and may accelerate research on the mechanism of pearl formation.

Mushroom tyrosinase (EC 1.14.18.1) is a copper containing oxidase that catalyzes both the hydroxylation of tyrosine into o-diphenols and the oxidation of o-diphenols into o-quinones. In the present study, the kinetic assay was performed in air-saturated solutions and the kinetic behavior of this enzyme in the oxidation of L-tyrosine and L-DOPA has been studied. The effects of cupferron on the monophenolase and diphenolase activity of mushroom tyrosinase have been studied. The results show that cupferron can inhibit both monophenolase and diphenolase activity of mushroom tyrosinase. The lag phase of tyrosine oxidation catalyzed by the enzyme was obviously lengthened and the steady-state activity of the enzyme decreased sharply. Cupferron can lead to reversible inhibition of the enzyme, possibly by chelating copper at the active site of the enzyme. The IC(50) value was estimated as 0.52 microM for monophenolase and 0.84 microM for diphenolase. A kinetic analysis shows that the cupferron is a competitive inhibitor for both monophenolase and diphenolase. The apparent inhibition constant for cupferron binding with free enzyme has been determined to be 0.20 microM for monophenolase and 0.48 microM for diphenolase.

Genistein and daidzein are two major isoflavonoids in dietary soybean that have inhibition effect on the cell growth of different tumor cell lines. We previously reported the anti-tumor activities of genistein and daidzein in human co1on tumor (HCT) cells and their different ability to enhance the activation of murine lymphocytes. In the present study, the effect of genistein and daidzein on the cell growth, cell cycle progression, and differentiation of murine K1735M2 and human WM451 cel1s was investigated. It was found that genistein could inhibit the cell growth of two metastatic melanoma cell lines, murine Kl735M2 and human WM45l in a dose-dependent manner. Flow cytometry showed that genistein could cause arrest of both Kl735M2 and WM45l at G2/M phase, while daidzein increased the cell numbers at S phase, decreased the cell numbers at G1 phase. Detection of melanin and morphological observation showed that genistein can induce Kl735M2 and WM45l to produce dendrite-like structure and produce more melanin by 80%. In contrast, daidzein only retarded the growth of K1735M2 and did not induce differentiation in either K1735M2 or WM451. These results suggest that genistein and daidzein in soybean can inhibit certain malignant phenotype of melanoma via different mechanisms and be potential medical candidates for melanoma cancer therapy.

To investigate the effects of liver X receptor (LXR) agonist on adipose-derived mesenchymal stem cells (AD-MSCs) implantation into infarcted hearts of mice.AD-MSC(Fluc+) which stably expressed firefly luciferase (Fluc) were isolated from β-actin-Fluc transgenic mice and characterized by flow cytometry. Male FVB mice were randomly allocated into the following four groups (n = 10 each): (1) sham group; (2) MI + PBS group; (3) MI + AD-MSC(Fluc+) group; (4) MI + AD-MSC(Fluc+) + LXR agonist (T0901317) group. AD-MSC(Fluc+) or PBS were injected intramyocardial into peri-infarcted region of mice heart after permanent left anterior descending (LAD) artery ligation. Bioluminescence imaging (BLI) was performed for quantification of injected cells retention and survival. Cardiac function was evaluated by echocardiography.The AD-MSC(Fluc+) were positive for CD44 and CD90 by flow cytometry. BLI evidenced the firefly luciferase expression of AD-MSC(Fluc+) which was positively correlated with cell numbers (r(2) = 0.98). The results of BLI in vivo revealed that LXR agonist could improve the survival of AD-MSC(Fluc+) at day 7, 14 and 21 after transplantation compared with AD-MSC(Fluc+) alone group. Cardiac function was further improved in combination therapy group compared with AD-MSC(Fluc+) alone group (P0.05).LXR agonist T0901317 can improve the retention and survival of intramyocardial injected AD-MSC(Fluc+) post-MI, and the combination therapy of T0901317 and AD-MSC(Fluc+) has a synergetic effect on improving cardiac function in this model.

The nacreous layer of molluskan shells, which consists of highly oriented aragonitic crystals and an organic matrix (including chitin and proteins), is a product of biomineralization. This paper briefly introduces the recent research advances on nacre biomineralization of shells from bivalves and gastropods, which mainly focus on analysis of the micro- and nano-structure and components of shell nacreous layers, and investigations of the characteristics and functions of matrix proteins from nacre. Matrix proteins not only participate in construction of the organic nacre framework, but also control the nucleation and growth of aragonitic crystals, as well as determine the polymorph specificity of calcium carbonate in nacre. Moreover, the inorganic aragonite phase also plays an active role in organizing nacre microstructure. Based on these studies, several models to illustrate the formation mechanism related to lamellar nacre in bivalves, and columnar nacre in gastropods are introduced.

To examine the in vivo effects of atorvastatin (AT) on arterial calcification in rats, arterial calcification was established by subcutaneous injection of vitamin D3 and Warfarin. Intragastric administration of AT began 4 days before establishment of arterial calcification in the AT group (n=6). Blood samples were taken and abdominal aortas were collected and stained. After induction of calcification, plasma Ca(2+) levels in the CA and AT groups were significantly higher than those before treatment and in the untreated controls. Plasma Ca(2+) levels in the AT group were significantly lower than in the CA group. The relative calcification area in aortic specimens from the AT group was significantly smaller than in the CA group. Rat aortic vascular smooth muscle cells (VMSC) were isolated from abdominal aortic segments and pre-treated with AT (1, 5, or 10 μM) for 24 hr. Cells in the calcification (CA) group and the AT group were cultured with β-glycerophosphate, insulin and vitamin C for 14 days to induce cell calcification. Calcium deposition and alkaline phosphatase activity were significantly increased in the CA group compared to untreated controls (p0.01). This effect was ameliorated by AT (all p0.01). In vivo administration of AT reduced arterial calcification and plasma Ca(2+) concentration. In vitro, AT reduced calcification markers in rat aortic vascular smooth muscle cells.

To shorten operation time and improve survival rate of rats with myocardial ischemia or myocardial infarction, we use a novel device comprised of a face mask and a head/neck retainer in this study. We report the basic design of the novel respiratory face mask (RFM) and evaluate its performance in a rat model of myocardial ischemia. The device is cost-effective and easier to handle than other devices, such as tracheal intubation. Compared with conventional tracheal intubation, we found that RFM shortens operation time significantly while keeping blood indices normal; the mean operation time for rats in the mask group was (32+/-3) min, and that for the intubation group was (45+/-7) min (P0.05). Moreover, the size and shape of the RFM can be changed according to the body weight of rats. In conclusion, RFM is an appropriate device for the establishment of myocardial infarction or ischemia-reperfusion in rats.

To investigate the antagonism of LY333531 on the increased permeability of cardiac microvascular endothelial cells (CMECs) induced by high glucose.The cultured CMECs from rats were randomly divided into four groups: normal group, high glucose group (25 mmol/L), high glucose+LY333531 (10 micromol/L) group and high glucose+saline group. The permeability of cell monolayer was detected using in vitro vascular permeability assay kit. Cell apoptosis was determined by TUNEL and the expression of PKCbeta II was analyzed by immunofluorescence and Western blot in each group.Compared with normal group, the permeability (400.0+/-20.00 vs 223.3+/-25.17; P0.01) of cell monolayer cultured in high glucose medium was increased at a higher apoptosis rate (55.00%+/-5.000% vs 2.333%+/-1.155%; P0.01) and PKCbeta II expression (0.4767+/-0.0751 vs 0.1733+/-0.0208; P0.01). However, the high glucose+LY333531 group showed noticeable attenuation on both permeability (360+/-17.32 vs 400.0+/-20.00; P0.05) and apoptosis (25.00%+/-5.000% vs 55.00%+/-5.000%; P0.01) with reduced PKCbeta II expression (0.2800+/-0.0700 vs 0.4767+/-0.0751; P0.01). No significant effects of saline on the cell permeability, apoptosis and PKCbeta II expression were observed.The antagonism of LY333531 has shown obvious effects on the impairment of high glucose to the permeability of CMECs.

To investigate the effects of constant magnetic field (CMF) on proliferation and migration of bone marrow-derived endothelial progenitor cells (EPCs) under rapamycin intervention.EPCs were isolated from rat bone marrow by density gradient centrifugation and cultured on fibronectin-coated dishes. Six days later the attached cells were divided into 5 groups: control group, rapamycin (1 ng/ml) group, and 3 rapamycin + CMF groups (treated with CMF of the doses 0.1 mT, 0.5 mT, and 1.0 mT respectively). Samples were collected 24 hours after incubation. Cell proliferation was measured by MTT chromatometre. EPC migration was detected with modified Boyden chamber assay.The EPC proliferation ability of the rapamycin group, expressed by absorbance, was (0.252 +/- 0.006), significantly lower than that of the control group [(0.328 +/- 0.025), P0.05]. The number of migrating EPC was (31 +/- 3) cells, significantly lower than that of the control group [(48 +/- 5), P0.05]. The EPC proliferation ability of the rapamycin + CMF 0.5 mT and 1.0 mT groups, expressed by absorbance, were (0.278 +/- 0.008) and (0.280 +/- 0.010) respectively, both significantly higher than that of the control group (both P0.05). The migrating EPC number of the rapamycin + CMF 0.5 mT and 1.0 mT groups were (37 +/- 3) and (38 +/- 4) respectively, both significantly higher than that of the control group (both P0.05).CMF of the doses of 0.5 mT and 1.0 mT antagonizes the effects of rapamycin on EPCs, increasing the proliferation and migration of EPCs.

To prepare an inclusion complex of daidzein and hydropropyl-beta-cyclodextrin to enhance the solubility of daidzein.The inclusion complex of daidzein was prepared by the solution stirring method. The binary system of daidzein and HP-beta-CD was confirmed by differential thermal, thermogravimetry analysis, infrared spectroscopy and X-ray diffractometry.The drug content in the inclusion complex was 6. 76% and the solubility was 13.68 mg x mL(-1). The identification results showed that the inclusion complex was formed.The preparation method of the inclusion complex of daidzein and hydropropyl-beta-cyclodextrin is simple and available, with a increased solubility of daidzein.

The effect of daidzein, an important isoflavone, on the differentiation and mineralization in MC3T3-E1 cells, a mouse calvaria osteoblast-like cell line, was investigated. The MTT assay, the alizarin red S and von Kossa staining, the measurement of calcium (Ca) and phosphorus (P) concentrations by inductively coupled plasma-atomic emission spectrometry and the nitrophenol liberation method were used to determine the cell proliferation, mineralization and intracellular alkaline phosphatase (ALP) activity, respectively. Daidzein enhanced the cell proliferation after the culture for 2 days and the effect reached maximum on day 6. ALP activity and cellular Ca and P contents were increased time- and dose-dependently when the cells were treated with daidzein in the presence of disodium beta-glycerophosphate and L-ascorbic acid. Differentiation of the cells to the mature osteoblasts was prompted under incubation in the presence of daidzein for 21 days, by the time the mineralized nodules formed. The calcium depositions of the cells by alizarin red S staining were increased significantly after the culture with daidzein as long as 28 days. It has been demonstrated that daidzein may be able to enhance the bone differentiation and mineralization and prompt the bone formation in the early growing stage and the late growing stage of osteoblasts.

To study the effect of alendronate on artery calcification in rats.(1) 4-week SD male rats were randomly divided into 3 groups: alendronate group (AL, n = 6), calcification group (CA, n = 6) and normal group (N, n = 6). In AL and CA group, artery calcification of rat was established by subcutaneous injection of vitamin D3 (300,000 U x kg(-1) x d(-1) for 3 days) and Warfarin (15 mg x 100 g(-1) x 12 h(-1) for 4 days); In AL group, at 4 days before establishment of artery calcification, alendronate (1 mg x kg(-1) x 24 h(-1)) was administered with subcutaneous injection and continued to be given to the end of the study. Abdominal aortae were collected for paraffin section and stained with von Kossa staining to observe the area of calcification. (2) Rat aortic vascular smooth muscle cells (VSMC) were cultured in vitro with tissue explant. All cells were divided into 5 groups: normal group, calcification group (control group), and alendronate 10(-9), 10(-7) and 10(-5) mol/L group. Before inducing calcification, alendronate 10(-9), 10(-7) and 10(-5) mol/L group were individually pre-treated with final concentrations of 10(-9), 10(-7) and 10(-5) mol/L alendronate for 24 hours. Beta-glycerophosphate were then added in the calcification group and in all the alendronate groups to induce VSMC calcification. All cells were cultured for 14 days. Cell crawling slice was applied to Alizarin red S staining to observe VSMC calcification. Colorimetric method was applied to measure the contents of Ca2+, cell proteins, and ALP activity. The ratio of contents of Ca2+ and cell proteins was cell calcium deposits. Cell proliferation was measured with tetrazolium salt (MTT) method.(1) With von Kossa staining the black deeply stained structure was found to be decreased in AL group. (2) As compared with the control group, in all the alendronate groups, showed that the number of calcium nodules [(6.8 +/- 2.7, 6.2 +/- 4.2, 5.3 +/- 2.4) % vs (7.4 +/- 3.8)%], and cell calcium depositions [(5.2 +/- 1.2, 4.8 +/- 1.7, 3.5 +/- 1.8)% vs (5.6 +/- 1.6)%], cell ALP activity and cell proliferation decreased significantly and dose-dependently.Alendronate can inhibit the artery calcification in rats.

To evaluate the possibility and efficiency of nanoparticle as a new vector in vascular endothelial growth factor (VEGF) gene transference, and investigate the efficacy of direct gene transfer of nanoparticle with VEGF(165) gene into ischemic myocardium.Nanoparticle-VEGF (Np/VEGF) complex was prepared with poly (D, L-lactide-co-glycolide) (PLGA) loading VEGF(165) gene and the envelopment efficiency and size of the complex were determined. The Np/VEGF was transfected into the cultured myocardial cells, RT-PCR and ELISA were used to evaluate the transfection of VEGF. Suspension of Np/VEGF was injected into the myocardial tissue of 4 rabbits. 96 hours after operation myocardial tissue was obtained, made into sections, and observed with electron microscope. New Zealand White rabbits underwent thoracotomy followed by ligation of left anterior descending coronary artery to establish ischemic models. The New Zealand White rabbits were divided into 3 groups: Np/VEGF group (n = 12, nanoparticle with VEGF(165) were injected into the cordial myocardium), blank plasmid group (n = 12, injected with blank VEGF(165) plasmid), and control group (n = 8, injected with normal saline). Ultrasonography and immunohistochemistry with factor VIII related antigen were conducted to evaluate the cardiac function and the collateral circulation of the occluded artery. One month later the rabbits were killed to observe the vascularization of capillaries in the ischemic myocardium.The envelopment efficiency of the Np/VEGF complex thus prepared, 50 - 300 nm in size, were 1.87% y. RT-PCR and ELISA showed that VEGF gene had been successfully transfected into myocardial cells by the nanoparticles. A great number of nanoparticles were observed in the myocardial cytoplasm and nuclei. One month after operation, the ventricular wall motor amplitude of the Np/VEGF group was 1.87 mm +/- 0.32 mm, significantly larger than those of the blank plasmid group (1.59 mm +/- 0.24 mm, P0.05) and control group (0.93 mm +/- 0.40 mm, P0.05); and the left ventricular ejection fraction of the Np/VEGF group was 60% +/- 10%, significantly higher than those of the blank plasmid group (50% +/- 6%, P0.05) and control group (40% +/- 8%, P0.05). The capillary density at low power field (x 100) of the Np/VEGF group was 57 +/- 12, significantly higher than those of the VEGF group (41 +/- 14) and control group (24 +/- 8).Nanoparticle can act as a vector to transfect specific gene in vitro and in vivo. Direct gene transfer of nanoparticle with DNA encoding VEGF into the ischemic rabbit myocardium can increase capillary number; therefore it may be a novel therapeutic approach for myocardial ischemia.

Mushroom tyrosinase (EC 1.14.18.1) is a copper containing oxidase that catalyzes both the hydroxylation of tyrosine into o-diphenols and the oxidation of o-diphenols into o-quinones, and then forms brown or black pigments. In the present study, the effects of some flavonoids on the oxidation of L-3,4-dihydroxyphenylalanine (L-DOPA) have been studied. The results show that flavonoids can lead to reversible inhibition of the enzyme. A kinetic analysis showed that the flavonols are competitive inhibitors, whereas luteolin is an uncompetitive inhibitor. The rank order of inhibition was: quercetingalanginmorin; fisetin3,7,4;-trihydroxyflavone; luteolinapigeninchrysin.

Genistein and daidzein are two major isoflavonoids in dietary soybean that have inhibition effect on the cell growth of different tumor cell lines. We previously reported the anti-tumor activities of genistein and daidzein in human co1on tumor (HCT) cells and their different ability to enhance the activation of murine lymphocytes. In the present study, the effect of genistein and daidzein on the cell growth, cell cycle progression, and differentiation of murine K1735M2 and human WM451 cel1s was investigated. It was found that genistein could inhibit the cell growth of two metastatic melanoma cell lines, murine Kl735M2 and human WM45l in a dose-dependent manner. Flow cytometry showed that genistein could cause arrest of both Kl735M2 and WM45l at G(2)/M phase, while daidzein increased the cell numbers at S phase, decreased the cell numbers at G(1) phase. Detection of melanin and morphological observation showed that genistein can induce Kl735M2 and WM45l to produce dendrite-like structure and produce more melanin by 80%. In contrast, daidzein only retarded the growth of K1735M2 and did not induce differentiation in either K1735M2 or WM451. These results suggest that genistein and daidzein in soybean can inhibit certain malignant phenotype of melanoma via different mechanisms and be potential medical candidates for melanoma cancer therapy.

Gonadotropin-releasing hormone (GnRH) has been reported to exist in many non-hypothalamic tissues, such as the placenta, gonads, and mammary glands, while there still have been no reports concerning the existence and expression of GnRH in the mammalian digestive system. Immunocytochemistry and in situ hybridization results show that GnRH molecule and GnRH mRNA are both exclusively distributed in exocrine pancreas, and RT-PCR result further proves that GnRH transcription units do exist in the pancreas, which possess the same sequence as the hypothalamus GnRH mRNA. Quantitative analysis indicates that mRNA levels in rat pancreas remain at a low level (less than 10% of that in hypothalamus) without sexual or developmental difference. This is the first report suggesting the existence and gene expression of GnRH in rat pancreas.

Green crab (Scylla serrata) alkaline phosphatase (EC 3.1.3.1) is a metalloenzyme which catalyzes the nonspecific hydrolysis of phosphate monoesters. Some pollutants in seawater affect the enzyme activity causing loss of the biological function of the enzyme, which affects the exuviating crab-shell and threatens the survival of the animal. The present paper studies the effects of thiohydroxyal compounds on the activity of green crab alkaline phosphatase. The results show that thiohydroxyal compounds can lead to reversible inhibition. The equilibrium constants have been determined for dithiothreitol (DTT) and mercaptoethanol (ME) binding with the enzyme and/or the enzyme-substrate complexes. The results show that both DTT and ME are non-competitive inhibitors. The kinetics of enzyme inactivation by ME at low concentrations has been studied using the kinetic method of the substrate reaction. The results suggest that at pH 10.0, the action of ME on green crab ALP is first quick equilibrium binding and then slow inactivation. The microscopic rate constants were determined for inactivation and reactivation. The rate constant of the forward inactivation (k+0) is much larger than that of the reverse reactivation (k−0). Therefore, when the ME concentration is sufficiently large, the enzyme is completely inactivated.

The effect of ethanol on the activity of Penaeus penicillatus acid phosphatase has been studied. The results show that ethanol significantly inhibits enzyme activity as a non-competitive inhibitor, with Ki 8.75%. The conformational changes of the enzyme molecule induced by ethanol were followed using fluorescence emission, ultraviolet difference and circular dichroism (CD) spectra. Increasing the ethanol concentration caused the fluorescence emission intensity of the enzyme to increase. The ultraviolet difference spectra of the enzyme denatured with ethanol had two negative peaks at 220 and 278 nm, and a positive peak at 240 nm. Increasing the ethanol concentration produced a small shoulder peak at 287 nm in addition to the increases in the negative magnitudes of the 220 and 278 nm peaks. The changes of the fluorescence and ultraviolet difference spectra reflected the changes of the microenvironments of the tryptophan and tyrosine residues of the enzyme. The CD spectrum changes of the enzyme show that the secondary structure of the enzyme also changed. The results suggest that ethanol is a non-competitive inhibitor and the conformational integrity of the enzyme is essential for its activity.