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Glial-cell-line-derived neurotrophic factor (GDNF) is a most potent survival factor for dopaminergic neurons. In addition, GDNF was also found to promote neurite outgrowth in dopaminergic neurons. However, despite the potential clinical and physiological importance of GDNF, its mechanism of action is unclear. Therefore, we employed a state-of-the-art proteomic technique, DIGE (Difference in two-dimensional gel electrophoresis), to quantitatively compare profiles of phosphoproteins of PC12-GFRalpha1-RET cells (that stably overexpress GDNF receptor alpha1 and RET) 0.5 and 10 h after GDNF challenge with control. A total of 92 differentially expressed proteins were successfully identified by mass spectrometry. Among them, the relative levels of phosphorylated Hsp27 increased significantly both in 0.5 and 10 h GDNF-treated PC12-GFRalpha1-RET cells. Confocal microscopy and Western blot results showed that the phosphorylation of Hsp27 after GDNF treatment was accompanied by its nuclear translocation. After the mRNA of Hsp27 was interfered, neurite outgrowth of PC12-GFRalpha1-RET cells induced by GDNF was significantly blocked. Furthermore, the percentage of neurite outgrowth induced by GDNF was also reduced by the expression of dominant-negative mutants of Hsp27, in which specific serine phosphorylation residues (Ser15, Ser78 and Ser82) were substituted with alanine. Our data also revealed that p38 MAPK and ERK are the upstream regulators of Hsp27 phosphorylation. Hence, in addition to the numerous novel proteins that are potentially important in GDNF mediated differentiation of dopaminergic cells revealed by our study, our data has indicated that Hsp27 is a novel signaling molecule involved in GDNF-induced neurite outgrowth of dopaminergic neurons.

To find a rapid single nucleotide polymorphism (SNP) loci typing method in the mitochondrial DNA (mtDNA) coding regions, we genotyped 16 SNP loci in the mitochondrial DNA coding region in Han, Miao and Tujia populations by the multiplex-amplified product-length polymorphism (mAPLP) technique. This method generates allele-specific fragments that are different in length through PCR amplification using allele-specific forward (or reverse) primers different in size and a common reverse (or forward) primer. Results showed that both of the allelic frequency of 3970T in Han and Tujia populations were 17%, which were significantly different from that of in the Miao population (P0.01). The allelic frequency of 8020A in Han population was 6% , which was different from that of in the Miao and Tujia populations (P0.05) . In all of 300 samples, a total of 45 different haplotypes were identified, 12 of which were found in all the three populations, 10 were shared by two populations and 23 haplotypes existed only in one of the populations. Among these 23 haplotypes, 8, 6 and 12 haplotypes were exclusively observed in the Han, Miao and Tujia populations, respectively.

Pituitary adenylate cyclase activating polypeptide (PACAP) exhibits a protective effect against neural injury in vitro and in vivo. However, it has not been reported whether peripheral intravenous administration of PACAP could confer benefits in animal models of Parkinson's disease (PD). Furthermore, the underlying molecular mechanisms responsible for these effects are poorly understood. In the present experiments, we determined the effects and mechanism of action of intravenously administered PACAP27 in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice. Our results indicate that intravenous injection of PACAP27 offers neuroprotective effects in the MPTP-induced PD mouse model which may not be directly associated with the expression levels of the monoamine transporters. However, this effect may be correlated with its ability to selectively regulate not only K(ATP) subunits, but D2 receptors in the striatum. Our findings suggest that the benefit of PACAP may accompany with changes not only in dopaminergic neurotransmission, but also in cholinergic neurotransmission that are relatively associated with the K(ATP) subunits and D2 receptors in the striatum.

Fruit aroma of Guoqing 1, Miyagawa Wase, and Owari from three different Satsuma mandarin varieties were investigated by headspace solid phase microextraction (HS-SPME) combined with GC-MS. The results showed that there were 73, 71, and 66 aroma components in the three varieties, and the total contents were 584.67, 505.29, and 494.63 μg g^(-1), respectively. Total 29 constituents were common in the three varieties. It was also found that Guoqing 1, Miyagawa Wase, and Owari had 12, 5, and 2 unique components, respectively. The key aroma components were limonene, linalool, γ-terpinene, β-myrcene, α-pinene, and octanal in the three fruits. Guoqing 1 contained more key aroma compounds than Miyagawa Wase and Owari.

Biomineralization involves proteins and/or other macromolecules directly in controlling the mineral crystal nucleation/induction, growth, and maturation. To identify these proteins in bone, bovine bone EDTA/NaCl extract was passed through a hydroxyapatite column followed by washing with 0.5 M NaCl and the bound proteins were collected and analyzed by mass spectrometry. More than 30 proteins were identified. While as described previously, albumin, alpha2-HS glycoprotein, decorin, biglycan, osteoadherin, osteonectin, etc. were included, collagen alpha2 (I), matrix extracellular phosphoglycoprotein, secreted phosphoprotein 24, chondroadherin, lumican, perlecan, thrombospondin 1, nucleobindin, etc. were for the first time shown to directly interact with calcium phosphate mineral.

Loss-of-function mutations in the parkin gene have recently been shown to be responsible for autosomal recessive juvenile Parkinsonism. However, the exact mechanism of pathogenesis remains unclear. This study explores the effect of Parkin downregulation on dopaminergic cells in Parkinson's disease. We generated small interfering RNA plasmids that target the parkin gene and transfected them into PC12 cells to mimic in vivo loss-of-function. We found that these small plasmids were able to effectively inhibit endogenous Parkin expression in PC12 cells. Downregulation of Parkin decreased the amount of glutathione and superoxide dismutase activity without affecting the amount of malondialdehyde. Moreover, Parkin knockdown rendered PC12 cells more susceptible to cell death induced by the proteasome inhibitor lactacystin. These results indicate that downregulation of Parkin may damage the antioxidation defenses of dopaminergic cells and increase their susceptibility to proteasome inhibitor-induced toxicity.

It is well known that there is a close connection between tame kernels and ideal class groups of number fields. However, the latter is a very difficult subject in number theory. In this paper, we prove some results connecting the pn-rank of the tame kernel of a cyclic cubic field F with the pn-rank of the coinvariants of \( \mu _{{p^{n} }} \otimes Cl{\left( {{\fancyscript O}_{{E,T}} } \right)} \)under the action of the Galois group, where \( E = F{\left( {\varsigma _{{p^{n} }} } \right)} \) and T is the finite set of primes of E consisting of the infinite primes and the finite primes dividing p. In particular, if F is a cyclic cubic field with only one ramified prime and p = 3, n = 2, we apply the results of the tame kernels to prove some results of the ideal class groups of E, the maximal real subfield of E and F(ζ3).

A novel HPLC-UV method with pre-column derivatization by using 2-mercaptoethanol was established for determination of 1,2-[bis(1,2-benzisoselenazolone-3(2H)-ketone)]-ethane (BBSKE) in dog plasma. The derivatives were identified by mass spectrometry. The method had a good linear range of 0.05-2 microg/ml (r(2)=0.9995). The lower limit of quantification (LOQ) was 0.05 microg/ml. The precision and accuracy were less than 7%. After dosing of BBSKE (30 mg/kg, p.o. and 0.79 mg/kg, i.v.) in dogs, AUC(0-t) were 5.72+/-2.42 and 1.35+/-0.41 microg h/ml; t(1/2) were 4.6+/-2.1 and 1.7+/-0.6h, respectively. The method was successfully applied to the pharmacokinetic study in dogs.

Background Cell culture plays a pivotal role in cancer research. However, culture-induced changes in biological properties of tumor cells profoundly affect research reproducibility and translational potential. Establishing culture conditions tailored to the cancer cell of origin could resolve this problem. For glioma research, it has been previously shown that replacing serum with defined growth factors for neural stem cells (NSCs) greatly improved the retention of gene expression profile and tumorigenicity. However, among all molecular subtypes of glioma, our laboratory and others have previously shown that the oligodendrocyte precursor cell (OPC) rather than the NSC serves as the cell of origin for the proneural subtype, raising questions regarding the suitability of NSC-tailored media for culturing proneural glioma cells. Methods OPC-originated mouse glioma cells were cultured in conditions for normal OPCs or NSCs, respectively, for multiple passages. Gene expression profiles, morphologies, tumorigenicity, and drug responsiveness of cultured cells were examined in comparison with freshly isolated tumor cells. Results OPC media-cultured glioma cells maintained tumorigenicity, gene expression profiles, and morphologies similar to freshly isolated tumor cells. In contrast, NSC-media cultured glioma cells gradually lost their OPC features and most tumor-initiating ability and acquired heightened sensitivity to temozolomide. Conclusions To improve experimental reproducibility and translational potential of glioma research, it is important to identify the cell of origin, and subsequently apply this knowledge to establish culture conditions that allow the retention of native properties of tumor cells.

Ataxia telangiectasia is an autosomal recessive multisystem disorder characterized by progressive cerebellar ataxia with onset in childhood, oculocutaneous telangiectasia, increased serum alpha-fetoprotein, immunodeficiency, chromosomal instability, and radiation hypersensitivity. Ataxia-telangiectasia mutated gene (ATM) is one of the known genes to be associated with ataxia telangiectasia. We reported the clinical and genetic findings of three early-onset Chinese patients who demonstrated ataxia, oculomotor apraxia, choreoathetosis, myoclonus and telangiectasia of eyes. Sequence analysis of ATM revealed two known nonsense mutations c.8287C>T and c.9139C>T in the siblings. Though the siblings carried the same mutations, they showed different clinical features involving strephenopodia, exotropia, torsion dystonia, myoclonus and extrapyramidal impairments. The other patient was compound heterozygotes for ATM: c.8911C>T and c.7141_7151delAATGGAAAAAT, both of which were not reported previously and not found in 200 control chromosomes. This study widens the spectrum of mutations and phenotypes in ataxia telangiectasia.

The aim of this study was to better understand the effect of amyloid-β plaques on magnetic resonance T2 relaxation time. We investigated these changes associated with age in an APP/PS1 mouse model of AD at 7 Tesla, combined with histology. Ten double-transgenic AD and ten wild type (WT) female mice (aged 12-20 months) were used in a cross-sectional study. Mean T2 values and standard deviations were calculated for each Regions of interest (ROIs) on T2 map. Immunohistochemistry for amyloid plaques and fluorescence staining with thioflavine S were performed of brain sections after imaging. The results showed that mean T2 values of the hippocampus, cortex, corpus callosum, and thalamus of older mice were significantly lower than of the younger. Compared to WT mice, the T2 values of the hippocampus, corpus callosum, and thalamus in younger AD mice were significantly greater, while the T2 values of the hippocampus and cortex in older AD mice were significantly less. Aβ-40 immunohistochemistry and thioflavine S stainging were positive in the matched region both for younger and older AD mice, while neither Aβ-40 nor thioflavine S were observed in WT mice. These findings suggest that regional T2 values of AD mice may decrease with age, and changes in T2 values in AD mice may be influenced by many factors besides amyloid-β plaque accumulation. Furthermore, they support that the standard deviation of the mean T2 value should be considered as well as the mean.

Bone sialoprotein (BSP) is one of the major non-collagenous glycosylated phosphoproteins of the extracellular matrix in bone. In vitro studies suggest that BSP may play important roles in the initiation and/or growth of calcium-phosphate crystals. To investigate the potential role of BSP in more complex in vivo environments, we implanted purified bovine BSP with type-I collagen as a carrier into surgically created rat calvarial defects and thoracic subcutaneous pouches. The responses to the implants were assessed by histochemistry, immunohistochemistry, in situ hybridization, quantitative real-time PCR, and biochemical analyses. BSP-collagen, but not collagen alone, elicited mineral deposition in the matrix of proliferating cells near the dura at days 4-5 followed by osteoblast differentiation and synthesis of new bone in the mid-portion of the calvarial defects. In contrast, implantation of BSP-collagen into subcutaneous pouches did not induce calcification or osteogenesis over the same experimental period. We explored the underlying mechanisms for the site-specific responses to BSP-collagen implants and found that higher levels of calcium content and alkaline phosphatase activity at the cranial site at days 2-5 were associated with the BSP-mediated calcification. We also found that BSP stimulated osteoblast differentiation through up-regulation of cbfa1 and osterix, key transcription factors of osteoblast differentiation, which occurred in the calvarial defects but not in the subcutaneous tissue. These results demonstrate that BSP stimulates calcification and osteogenesis in a site-specific manner, and that local environment and the specificities of responding cells may play critical roles in the function of BSP in vivo.

Loss-of-function mutations in the parkin gene are known to result in autosomal recessive juvenile parkinsonism, which causes selective degeneration of nigrostriatal dopaminergic neurons in the absence of Lewy bodies. Here, we show that overexpression of parkin protects PC12 cells from neurotoxin of the proteasome inhibitor lactacystin and increases the accumulation of ubiquitin-protein conjugates and the formation of ubiquitin-positive inclusions induced by lactacystin. However, the protective effect of parkin against lactacystin-induced apoptosis is not associated with its ability to promote the formation of ubiquitinated inclusions. It is likely that Lewy body formation may be only a compensatory mechanism of dopaminergic neurons attempting to counteract toxicity, and not the ultimate cause of neuronal death.

Background Red blood cell distribution width (RDW) has been shown to predict mortality in critically ill patients. To our knowledge, whether or not RDW is associated with clinical outcomes of obstetric patients requiring critical care has not been evaluated. Methods This was a single centre, retrospective, observational study of obstetric patients admitted to the intensive care unit (ICU). Patients were excluded from the analysis if they had known haematological diseases or recently underwent blood transfusion. Patients who died or were discharged from the ICU within 24 hours of admission were also excluded. Patient clinical characteristics at ICU admission were retrieved from the medical charts. Multiple logistic regression was used to estimate OR and 95% CI for inhospital mortality associated with RDW. The receiver operating characteristic curve was used to examine the performance of RDW, alone or in combination with the Acute Physiology and Chronic Health Evaluation II score (APACHE II), in predicting inhospital mortality. Results A total of 376 patients were included in the study. The hospital mortality rate was 5.32%. A significant association was found between baseline RDW levels and hospital mortality (OR per per cent increase in RDW, 1.31; 95% CI 1.15 to 1.49). Further adjustment for haematocrit and other potential confounders did not appreciably alter the result (p

Objectives—Intracranial pressure usually increases after severe brain injury. However, a method for noninvasive evaluation of intracranial pressure is still lacking. The purpose of this study was to explore the potential role of transcranial color Doppler sonography in assessing intracranial pressure by observing the middle cerebral artery blood flow parameters in patients with increased intracranial pressure of varying etiology. Methods—The hemodynamic changes in the middle cerebral artery in patients with varying degrees of increased intracranial pressure were investigated by transcranial color Doppler sonography in 93 patients who had emergency surgery for brain injury. Results—Middle cerebral artery Doppler flow spectra changed regularly as intracranial pressure increased. The pulsatility index (PI) and resistive index (RI) had a significantly positive correlation with intracranial pressure (r = 0.90 and 0.89, respectively; P < .001), whereas the middle cerebral artery diastolic velocity showed a significant negative correlation with intracranial pressure (r = –0.52; P < .01). A receiver operating characteristic curve showed that the RI and PI cutoff values were 0.705 and 1.335, respectively, for predicting increased intracranial pressure, with sensitivity of 0.885 and specificity of 0.970. Conclusions—In addition to the PI and RI, middle cerebral artery diastolic flow velocity measurement by transcranial color Doppler sonography may also be a useful variable for evaluating intracranial pressure in patients with acute brain injury.

Levosimendan is a calcium-sensitizing agent shown to prevent myocardical contractile depression in various heart diseases. In this study, we investigated the effect of levosimendan on cardiac dysfunction and apoptosis in hypothermic preservation rat hearts. Isolated rat hearts were preserved in Celsior solution with or without levosimendan. The left ventricular developed pressure (LVDP) recovery rate of isolated rat heart significantly decreased, and the apoptosis index increased after 9 hours of hypothermic preservation. Supplement Celsior solution with levosimendan (10 and 10 mole/L) enhanced the LVDP recovery rate and reduced apoptosis. Levosimendan inhibited the hypothermic preservation-induced calpain activation and cleavage of Bid. Levosimendam induced increased myocardial inducible nitric oxide synthase but not endothelial nitric oxide synthase expression. A selective inducible nitric oxide synthase inhibitor 1400W, and a mitochondrial ATP-sensitive potassium (KATP) channel blocker 5-hydroxydecanoate but not a sarcolemmal KATP channel blocker HMR-1098 prevented improvement effect of levosimendam on LVDP recovery rate, abolished the inhibitory effect of levosimendan on hypothermic preservation-induced activation of calpain, cleavage of Bid, and apoptosis. These data suggested that Celsior solution supplement with levosimendan improved cardiac function recovery and reduced myocyte apoptosis in hypothermic preservation rat hearts.

ATP13A2 (PARK9) mutations are related to Kufor-Rakeb syndrome (KRS). We performed genetic analysis of the Ala746Thr variant in an independent cohort of the patients with PD and healthy controls from mainland China. The Ala746Thr variant was present in 1/532 (0.19%) of PD compared with 1/480 (0.21%) of healthy controls (odds ratio=0.90, 95% CI 0.06, 14.39, P=1.00). The two subjects carried the heterozygous genotype. Subset analysis in the group/=50 years of age revealed a prevalence of 0.7% in PD compared with 0% in healthy controls and in the group50 years of age showed 0% in PD versus 0.3% in healthy controls. We did not observe a significant association between Ala746Thr and Parkinson's disease in Han Chinese population, even after stratification by age at onset. The results suggested that Ala746Thr variant was not a major susceptible factor for PD in Han Chinese people.

A new dicoumarin, named as dimeresculetin (1), together with another dicoumarin, euphorbetin (2) and esculetin (3) were isolated from the ethyl acetate extract of the dried whole plants of Viola yedoensis Makino. The structure of 1 was elucidated as 7-hydroxy-6-[(6,7-dihydroxy-2-oxo-2H-1-benzopyran-5-yl)oxy]-2H-1-benzopyran-2-one on the basis of extensive NMR, as well as the other spectral analysis. Compounds 1-3 exhibited anticoagulant activities with respect to activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT).

• We identified one de novo novel mutation in KCNJ2 gene in a Chinese patient with Andersen–Tawil syndrome.

An 80 kDa protein was purified from calf bone by HCl-demineralization followed by 0.5 M EDTA/1.0 M NaCl extraction and sequential chromatography on DE-52, hydroxyapatite, and TSK-gel G3000SW HPLC columns. From the DE-52 column the protein was eluted at three different fractions, of which one further separated into two fractions on the hydroxyapatite column, indicating that the protein is present in four different molecular forms designated as 80 k-I-1, k-I-2, k-II, k-III. The N-terminal sequence analysis of all four forms gave the same sequence, SEQYNQEPNNV. Several tryptic internal peptides were also generated, purified and sequenced, leading to the identification of several repeat sequences, IFLGXXEI. Homology searching of the N-terminal and internal sequences indicates that this is a novel protein. Both 80 k-I-2 and k-III had similar amino acid composition with high contents of Asx, Glx and Leu and contained 7 and 16 phosphoserines per 1000 total amino acids, respectively. The 80 k-I-1 and 80 k-II forms were stained with Rhodamine B specific for phosphoproteins. The four forms contained different contents of neutral sugars ranging from 5.5 to 26% (w/w protein) and ~ 1.7% sialic acid. These data indicated that the 80 kDa protein exists in four isomeric forms, at least based on the different post-translational modifications. The evaluation of the 80 kDa glycosylated phosphoprotein under alkylating, reducing and non-reducing conditions indicated that this protein undergoes polymerization through intermolecular disulphide bonds. Furthermore, the 80 kDa protein and osteonectin (ON), both of which are cysteine-rich proteins, can cross-link with each other via disulphide bonds, and this process can be induced to take place in vitro under experimental conditions. The occurrence of such a phenomenon in vivo was confirmed from the presence of similar high Mr components containing both 80 kDa and ON in the same SDS/PAGE bands, detected by the respective antibody reactions in crude bone extracts which were extracted in the presence of alkylating agent.

To investigate the effect of Resveratrol on Hydrogen peroxide-treated human umbilical vein endothelial cells (HUVEC).Oxidative stress in HUVEC was induced by hydrogen peroxide (H2O2). The cells were divided in to 5 groups: A. Control group; B. H2O2 group; C. Res + H2O2 group; D. Res + H2O2 + LY294002 group; E. H2O2 + LY294002 group. The cell viability were measured by cck-8 assay; Malondialdehyde (MDA) and Superoxide dismutase (SOD) were determined by using commercially available kits; Reactive oxygen species (ROS)were detect by flow cytometer; the expression of Nuclear factor-E2-related factor(Nrf2), Protein kinase B(Akt), Mammalian target of rapamycin (mTOR) were determined by Western blot.Compared with control, resveratrol increased the cells viability (36.14 ± 2.20) to (72.05 ± 2.46) (P = 0.004), decreased MDA formation (299.38 ± 12.42) to (159.17 ± 7.30)(P = 0.006), elevated SOD activity(35.61 ± 3.26) to (81.37 ± 4.55) (P = 0.007), inhibited ROS production (177.86 ± 8.98) to (133.96 ± 4.17) (P = 0.003). These changes were accompany with the activation of p-Nrf2 (198.57 ± 6.89) to (371.10 ± 8.41) (P = 0.005), p-Akt (102.93 ± 3.29) to (221.62 ± 6.51) (P = 0.004), p-mTOR (93.26 ± 4.12) to (172.14 ± 5.74) (P = 0.008). However these protective effect of resveratrol were abolished by LY294002.Resvertrol could protects HUVEC cells from H2O2-induced oxidative stress, in which the PI3K-Akt-mTOR pathway may be involved.

This paper studied the community characteristics of two sand-fixing plants (Artemisia ordosica and Caragana korshinskii) under different configuration modes (1 m x 1 m and 2 m x 2 m) in the Shapotou region of Northwest China as well as the water relation, gas exchange, and their adjustment mechanisms of the plants under natural and artificial precipitation conditions. With the variation of soil water content, the physiological water consumption and growth characteristics of A. ordosica differed from those of C. korshinskii. A. ordosica presented obvious fluctuation in the stomatal conductance, water potential, transpiration rate, photosynthetic rate, and rapid growth, and had higher water consumption than C. korshinskii. However, the variations of the above-mentioned indices of C. korshinskii were relatively slow and more constant. The C. korshinskii had a lower photosynthetic rate but a very high accumulated biomass over years than A. ordosica. The response procedures and adjustment mechanisms of the two plants under water stress differed, with a water-conserving mechanism for A. ordosica and a water-saving mechanism for C. korshinskii. In extremely drought years, the C. korshinskii had stronger capabilities of water-saving and stress tolerance than A. ordosica. It was suggested that the selection of sand-fixing plants should have a view to the benefits in water saving and sand fixation, and also, to the stability of sand-fixing forest.

In this study, we explored the rationality of processing methods and mechanism of Aconiti Lateralis Radix (Fuzi) through comparing the chemical contents of diester alkaloids (DAs) and monoester alkaloids (MAs) in the raw material of Fuzi and its processed products. The results showed that the toxicity potency of MAs is at least lower than 1/64 to 1/180 of the toxicity potency of DAs. The contents of DAs in processed Fuzi decreased to 1/76.5 to 1/38.3 of the value of raw Fuzi. The contents of MAs in processed Fuzi significantly increased by 4.6 to 5.2 fold or basically the same as that of the raw Fuzi. The values of MAs/DAs of processed Fuzi were enhanced by 30 to 390 fold of the raw Fuzi. It was found that the contents of DAs were insignificantly different between "Wu dan fu pian" (steaming or stir-frying without Danba) and "Dan fu pian" (steaming or stir-frying with Danba). The result suggested that the abilities of "eliminating toxicity" of different processing methods were equivalent at all. In contrast, the contents of MAs contained in "Wu dan fu pian" were of 5.3 to 8.7 fold higher than the values in "Dan fu pian". This result suggested the processing method by steaming or stir-frying without Danba might have better effect for "conserving property" than the method processed with Danba stipulated by China Pharmacopoeia. We believe that the new processing method without Danba can be recommended in further application due to it offers a simple procedure and it will not introduce inorganic impurities in the products.

A new oxidation system of Fenton-like system (ZVI/EDDS/Air) has been developed to degrade 2,4-chlorophenols (2,4-DCP) in aqueous solution. The influences of initial conditions, i. e., EDDS concentration, iron dosage, aeration rate, 2,4-DCP concentration and pH as well as reaction temperature on the degradation of 2,4-DCP were studied. The results demonstrated that this ZVI/EDDS/Air system was able to effectively degrade 2,4-DCP in aqueous solution, and the degradation of 2,4-DCP conforms to the pseudo-first-order reaction kinetics equation. Removal of above 99% 2,4-DCP was achieved in ZVI/EDDS/Air system at room temperature and pressure after 1 h reaction when the initial conditions were 2,4-DCP 100 mg x L(-1), EDDS 0.80 mmo x L(-1), ZVI 20 g x L(-1), aeration rate 2 L x (min x L)(-1). Compared with ZVI/EDTA/Air system, ZVI/EDDS/Air system showed higher efficiency in the degradation of 2,4-DCP at ambient circumstance and was more environmentally benign.

Bone sialoprotein (BSP) containing an Arg-Gly-Asp cell-binding sequence was purified from bovine bone 4 M guanidine-HCl extract after HCl demineralization by a series of chromatographic procedures. When this protein was coated on culture dishes in the presence of type I collagen, it increased both DNA content and alkaline phosphatase (ALP) activity in osteoblast-like MC3T3-E1 cells, and stimulated calcification in the cells, whereas fibronectin, another cell-binding protein, showed a marked increase in the DNA content but had little effect on the ALP activity. These findings suggest that BSP is mitogenic for preosteoblasts and differentiating the cells into osteoblasts, thereby stimulating bone calcification.

Parkinson's disease (PD) is pathologically characterized by the presence of α-synuclein positive intracytoplasmic inclusions. The missense mutation, A53T α-synuclein is closely related to hereditary, early-onset PD. Accumulating evidences suggest that pathological accumulation of A53T α-synuclein protein will perturb itself to be efficiently and normally degraded through its usual degradation pathway, macroautophagy-lysosome pathway, therefore toxic effects on the neuron will be exacerbated. Based on the above fact, we demonstrated in this study that A53T α-synuclein overexpression impairs macroautophagy in SH-SY5Y cells and upregulates mammalian target of rapamycin (mTOR)/p70 ribosomal protein S6 kinase (p70S6K) signaling, the classical suppressive pathway of autophagy. We further found that curcumin, a natural compound derived from the curry spice turmeric and with low toxicity in normal cells, could efficiently reduce the accumulation of A53T α-synuclein through downregulation of the mTOR/p70S6K signaling and recovery of macroautophagy which was suppressed. These findings suggested that the regulation of mTOR/p70S6K signaling may be a participant of the accumulation of A53T α-synuclein protein-linked Parkinsonism. Meanwhile curcumin could be a candidate neuroprotective agent by inducing macroautophagy, and needs to be further investigated by clinical application in patients suffering Parkinson's disease.

Recently, many studies have attempted to illustrate the mechanism of autophagy in protection against oxidative stress to the heart induced by H2O2. However, whether resveratrol-induced autophagy involves the p38 mitogen-activated protein kinase (MAPK) pathway is still unknown. This study aimed to investigate whether treating H9c2 cells with resveratrol increases autophagy and attenuates the cell death and apoptosis induced by oxidative stress via the p38 MAPK pathway. Resveratrol with or without SB202190, an inhibitor of the p38 MAPK pathway, was added 30 min before H2O2. After H2O2 treatment, the cells were incubated under 5% CO2 at 37 °C for 24 h to assess cell survival and death or incubated for 20 min for Western blot and transmission electron microscopy. Flow cytometry was used to detect apoptosis after 6 h of H2O2 treatment. Resveratrol at 20 µmol/L protected H9c2 cells treated with 100 µmol/L H2O2 from oxidative damage. It increased cell survival and markedly decrease lactate dehydrogenase release. In addition, resveratrol increased autophagy and decreased H2O2-induced apoptosis. Furthermore, the protective effects of resveratrol were inhibited by 10 µmol/L SB202190. Thus, resveratrol protected H2O2-treated H9c2 cells by upregulating autophagy via the p38 MAPK pathway.

Acute fatty liver of pregnancy (AFLP) is a rare disease of progressive hepatic insufficiency and secondary systemic complications that induce significant maternal risk. The application of combining plasma exchange (PE) and continuous hemodiafiltration (CHDF) is a novel concept for patients with AFLP. Since 2002, we have utilized the combination of PE with CHDF as adjunctive medical therapy for 11 AFLP patients with multiple organ dysfunction. Before PE and CHDF initiation, four patients had signs and symptoms of encephalopathy, four required ventilatory support, and all 11 were developing liver failure, significant renal compromise, and coagulopathy. PE combined with CHDF for patients was initiated a mean of 2 days postpartum (range, days 0-3). Daily or every other day PE combined with CHDF was undertaken on two to eight occasions for each of the 11 patients. Ten patients responded with composite clinical and laboratory improvement and were discharged to the ward, then cured and discharged from hospital; one patient died of septic shock. Average duration of hospitalization was 17 days (range, days 9-38) from time of admission to discharge; the average duration of intensive care unit was 10 days (range, days 4-23). No significant PE- and CHDF-related complications occurred. These results indicate that combing PE and CHDF in a series-parallel circuit is an effective and safe treatment for patients with severe AFLP. This finding may have important implications for the development of an effective treatment for patients with AFLP suffering multiple organ dysfunction.

The aim of this research was to determine acute toxicity of the essential oil of Lonicera japonica Thunb. (Caprifoliaceae) flower buds against the booklouse (Liposcelis bostrychophila Badonnel) and the maize weevils (Sitophilus zeamais Motschulsky). Essential oil of L. japonica flower buds was obtained by hydrodistillation and analyzed by gas chromatography-mass spectrometry (GC-MS). A total of 25 components of the essential oil were identified. The principal compounds in the essential oil were estragole (80.17%) and linalool (6.05%). The essential oil exhibited strong contact toxicity against S. zeamais and L. bostrychophila with LD50 values of 21.54 µg/adult and 64.04 µg/cm2, respectively. The constituent compounds, estragole (LD50 = 49.95 µg/cm2) and linalool (LD50 = 172.54 µg/cm2) also possessed contact toxicity against L. bostrychophila. L. japonica essential oil and its constituent compounds (estragole and linalool) exhibited fumigant toxicity against S. zeamaiswith LC50 values of 13.36, 14.10 and 10.46 mg/L, respectively. The essential oil of L. japonica(LC50 = 0.20 mg/L) and its constituent compounds, estragole (LC50 = 0.16 mg/L) and linalool (LC50 = 0.41 mg/L) possessed fumigant toxicity against L. bostrychophila. The results indicated that the essential oil of L. japonica and its constituent compounds showed potential in terms of contact and fumigant toxicity against grain storage insects. Key words: Lonicera japonica, Liposcelis bostrychophila, Sitophilus zeamais, contact toxicity, fumigant, essential oil composition, estragole, linalool.

This paper researches the management system of Windows network privacy safety. The component, policies and application of P3P protocol are studied. The VC++ programming methods are proposed to send and receive cookie selectively in order to implement the IE privacy system. The methods mainly include two algorithms. One called rejection algorithm which IE only store acceptable Cookie. The other called Cookie selection algorithm which IE selects certain Cookie to add the Cookie header. It is a effective method to protect network privacy for users.

Sevoflurane postconditioning is a potential clinical measure to protect myocardial. This experiment was designed to investigate the efficacy of sevoflurane postconditioning against ischemia-reperfusion injury. A total of 132 Japanese White Rabbits were enrolled into this study. They were underwent 15-, 30-, or 60-min left anterior descending coronary (LAD) artery occlusion, respectively. At the end of LAD artery occlusion, they randomly received a 5-min inhalation of air (control group), 1% sevoflurane (1% sev group), 2% sevoflurane (2% sev group), 4% sevoflurane (4% sev group) or an IV bolus injection of 5 mg/kg of NIM811 [a specific inhibitor of mitochondrial permeability transition pores (mPTP)]. Infarct size was determined after 2 h of reperfusion (triphenyltetrazolium chloride straining, percentage of risk area). The infarct sizes were significantly (P 0.05) reduced after 15 min ischemia (5.5 ± 3.3%, 5.8 ± 3.6% vs. 20.3 ± 6.9% for 2% sev, 4% sev vs. control, respectively) and 30 min ischemia (23.5 ± 5.0%, 20.7 ± 5.9% vs. 50.9 ± 10.2%, for 2% sev, 4% sev vs. control, respectively; P 0.05). However, it had no effect on infarct size after 60 min ischemia (64.1 ± 5.9%, 62.3 ± 7.6% vs. 72.7 ± 9.2% for 2% sev, 4% sev vs. control, respectively, P 0.05).The efficacy of sevoflurane postconditioning gradually weakened with increasing ischemia duration and disappears after 60 min ischemia in rabbits in vivo.

The aim of the present study was to investigate the effects of glucocorticoids (GCs) on appetite and gene expression of the hypothalamic appetite regulatory peptides, neuropeptide Y (NPY), agouti-related protein (AGRP) and cocaine and amphetamine-regulated transcript (CART), in non-obese and obese rats. Both non-obese and obese rats were randomly assigned to three groups: normal saline, low- and high-dose GC groups (NSG, LDG and HDG, respectively), which received an intraperitoneal injection with normal saline (0.2 ml/100 g) or hydrocortisone sodium succinate at 5 and 15 mg/kg, respectively, for 20 days. The expression levels of NPY, AGRP and CART mRNA in the hypothalamus were measured by real-time quantitative PCR. Non-obese and obese rats were found to undergo weight loss after GC injection, and a higher degree of weight loss was observed in the HDG rats. The average and cumulative food intakes in the obese and non-obese rats injected with high-dose GC were lower compared to that in the NSG (p

Matrices of dentin and bone were compared with respect to the content of cell-attachment sialoproteins. The levels of two sialoproteins, osteopontin (OPN) and bone sialoprotein (BSP), were determined in dentin and bone by immunochemical procedures. Polyclonal antibodies against bovine BSP and an antibody against the amino-terminal decapeptide of rat OPN were used. The relative levels of OPN and BSP in dentin were less than one-tenth of the levels in bone. The differences between dentin and bone levels of OPN and BSP were thus larger than those for osteonectin or bone Gla protein in the two tissues. The scarcity of the cell-attachment proteins in dentin may reflect the metabolic inactivity of dentin.

This study is to elucidate the metabolic pathway of 1,2-[bis (1,2-benzisoselenazolone-3 (2H)-ketone)]-ethane (BBSKE) in rats. Rats were administrated with a single dose of BBSKE 200 mg x kg(-1). The metabolites in rat urine, feces, bile and plasma were identified by LC-MSn analysis. The characterization of fragment ions from LC-MSn chromatography and mass spectrometry was applied to the investigation of structures of metabolites. Three phase I metabolites were detected in rat urine and feces. Two of them were also found in plasma and one existed in bile. These products were derived from oxidized, methylated and S-methylated BBSKE, separately. One phase II glucuronide of BBSKE was also found in bile. Therefore, it is possible that BBSKE was metabolized by oxidization, methylation and glucuronidation.

To observe the effect of moxibustion of "Shenshu" (BL 23) and "Zusanli"(ST 36) on synovial nuclear factor (NF)-kappaB p65 expression, and plasma adrenocorticotropic hormone (ACTH) and serum cortisol (CS) contents in rheumatic arthritis (RA) rats with adrenalectomy (ADX) so as to study the underlying mechanism in ameliorating RA.Fifty male SD rats were randomly divided into control (n=10), model (n=10), moxibustion (n=10), ADX (n=10) and false ADX (n=10) groups according to SPSS-aided random digits table. RA model was established by injecting 0.1 mL Freund's complete adjuvant into the right paw. ADX operation was performed on the sixth day after successful establishment of RA model. Moxibustion was given to "Shenshu" (BL 23) and "Zusanli" (ST 36) from the 7th day on, five moxa cones each session, and once daily for 18 days. Then plasma ACTH and serum CS contents, for which the blood was collected during night, were detected by ELISA, and NF-kappaB p 65 immunoactivity in synovial tissue was detected by immunohistochemistry.In comparison with control group, the foot volumes (swelling degree) of model, moxibustion, ADX, and false ADX groups increased significantly (P0.01), while compared with model group, the swelling degrees of moxibustion and false ADX groups were decreased remarkably after the treatment (P0.01). No significant difference was found between ADX and model groups in the foot volume (P0.05). Compared with control group, plasma ACTH content in RA model group decreased obviously (P0.05), while serum CS level and NF-kappaB p 65 immunoactivity increased apparently in model group (P0.01). Compared with model group, serum CS contents in moxibustion, ADX and false ADX groups and synovial NF-kappaB p 65 immunoactivity in moxibustion and false ADX groups reduced considerably (P0.01, P0.05). No significant difference was found between ADX and model groups in synovial NF-kappaB p 65 immunoactivity (P0.05).Moxibustion treatment can reduce inflammation reactions in RA rats, which is closely associated with its effects in upregulating plasma ACTH, downregulaing serum CS level and synovial NF-kappaB p 65 immunoactivity, and the intact hypothalamus-pituitary-adrenal axis (HPAA).

To study the accumulation rules of dry substance and active components in the Radix Aconiti Lateralis Praeparata for providing the basic evidences to collection time.The Roots were collected periodically and their fresh weight and dry weight were measured. The contents of total alkaloids and three diester aconitum alkaloids aconitine, hypaconitine and mesaconitine were determined by titration and HPLC respectively.During the stage of root inflation, the content of total alkaloids was at a certain level, while the content of diester aconitum alkaloids were increased gradually. The total amount of alkaloid and diester aconitom alkaloids were increased rapidly from the last third of April to the middle third of June, and then maintained at relative constant levels and were decreased from the first third of July.It is more preferable to collect the Radix Aconiti Lateralis Praeparata during the period from the last third of June to first third of July.

Past studies of bone extracellular matrix phosphoproteins such as osteopontin and bone sialoprotein have yielded important biological information regarding their role in calcification and the regulation of cellular activity. Most of these studies have been limited to proteins extracted from mammalian and avian vertebrates and nonvertebrates. The present work describes the isolation and purification of two major highly glycosylated and phosphorylated extracellular matrix proteins of 70 and 22 kDa from herring fish bones. The 70-kDa phosphoprotein has some characteristics of osteopontin with respect to amino acid composition and susceptibility to thrombin cleavage. Unlike osteopontin, however, it was found to contain high levels of sialic acid similar to bone sialoprotein. The 22-kDa protein has very different properties such as very high content of phosphoserine (∼270 Ser(P) residues/1000 amino acid residues), Ala, and Asx residues. The N-terminal amino acid sequence analysis of both the 70-kDa (NPIMA(M)ETTS(M)DSKVNPLL) and the 22-kDa (NQDMAMEASSDPEAA) fish phosphoproteins indicate that these unique amino acid sequences are unlike any published in protein databases. An enzyme-linked immunosorbent assay revealed that the 70-kDa phosphoprotein was present principally in bone and in calcified scales, whereas the 22-kDa phosphoprotein was detected only in bone. Immunohistological analysis revealed diffusely positive immunostaining for both the 70- and 22-kDa phosphoproteins throughout the matrix of the bone. Overall, this work adds additional support to the concept that the mechanism of biological calcification has common evolutionary and fundamental bases throughout vertebrate species.

CD200-CD200R signaling holds microglia in a quiescent state. Parkinson disease (PD) neurodegeneration may be associated with impairment of CD200-CD200R-mediated microglia silencing in the substantia nigra (SN). In this study, an anti-CD200R blocking antibody (ACDR) selectively and significantly enhanced the susceptibility of dopaminergic neurons to neurotoxicity induced by rotenone (Rot) and iron (Ir) in mesencephalic neuron/glia cultures. Microglia were shown to mediate dopaminergic neurotoxicity induced by ACDR/Rot (combination of ACDR and Rot) and ACDR/Ir (combination of ACDR and Ir). ACDR significantly enhanced the microglial activation induced by Rot and Ir in neuron/glia cultures. NADPH oxidase-mediated superoxide generation was a key contributor to dopaminergic neurotoxicity induced by ACDR/Rot and ACDR/Ir. p38 MAPK contributed to NADPH oxidase activation induced by ACDR/Rot and ACDR/Ir. Interestingly, there were a decrease in CD200 expression (mRNA and protein) and an enhancement of microglial response (MHCII mRNA and ICAM-1 protein) in the rat SN with aging. ICAM-1 expression was significantly inversely correlated with CD200 expression. These results strongly indicate the participation of SN CD200-CD200R dysfunction in the etiopathogenesis of PD and provide a new insight into the molecular mechanisms underlying the involvement of aging in PD and help to elucidate the mechanisms of the combined involvement of immune/inflammatory factors, environmental substances, and aging in PD.

Objective:Ubiquitin proteasome system dysfunction is believed to play an important role in the development of Parkinson's disease (PD), and almost all studies till now have mainly focused on the susceptibility of dopaminergic neurons to proteasome inhibition. However, in fact, there are many other types of neurons such as cholinergic ones involved in PD. In our present study, we attempt to figure out what effect the failure of ubiquitin proteasome function would execute on cholinergic cells in culture.Methods:We treated cholinergic cells in culture with various doses of lactacystin. Then MTT assay was used to evaluate the cellular viability and the Annexin V-PI method was used to detect apoptosis. Both cellular soluble and insoluble polyubiquitinated proteins were detected by western blot. Furthermore, the mitochondrial membrane potential was analyzed using JC-1 and the intracellular production of reactive oxygen species (ROS) was determined using the fluorescent probe CM-H2DCFDA.Results:We found that low doses of lactacystin were enough to induce significant apoptotic cell death, disturb the mitochondrial membrane potential, and cause oxidative stress. We also found that the amounts of polyubiquitinated proteins dramatically increased with high doses, although the loss of cells did not increase accordingly.Conclusions:Our results suggest that cholinergic cells are sensitive to ubiquitin proteasome system dysfunction, which exerts its toxic effect by causing mitochondrial dysfunction and subsequent oxidative stress, not through polyubiquitinated proteins accumulation.

In this work, we engineered the α/β fold of mannanase Man23 based on its molecular structure analysis to obtain more stable variants. By introducing 31 single-site mutations in the α/β fold and shuffling them, the incorporation of four mutations (K178R, K207R, N340R, and S354R) displayed a good balance between high activity and stability at higher temperature and broader pH. This quartet variant was characterized by an almost threefold increased activity and a sevenfold increased stability compared to native mannanase Man23. Our results suggest that such work is safe to increase our target protein stability with no loss of activity.

After analyzing many typical association rule mining algorithms, a new algorithm, named as BOFP-V, is proposed for frequent item set mining. FP-V vectors are introduced in order to convert that of frequent item set mining to the course of the vectors operating. The existing Apriori algorithm produces a lot of candidacy sets and needs scanning database many times, and BOM algorithm entails and operation of k vertors with times. Overcoming these drawbacks, BOFP-V algorithm needs scanning database only once. Therefore, the proposed algorithm is obviously superior to Apriori and BOM algorithm in efficiency.

Background: There is much variation in the response of both individuals and different ethnic populations to opioids, with genetic differences being responsible for interindividual variation. The m-opioid receptor single nucleotide polymorphism (rs number 1799971) at nucleotide position 118 (OPRM1 118A>G) affects the analgesic response to opioids. Objective: This study aimed to investigate the association between the OPRM1 118A>G polymorphism and the effects of fentanyl-induced analgesia, respiratory depression, and anesthetic recovery responses in a population of Han Chinese patients. Study Design: The study was a case series in a hospital setting, with 1 year of study and 1 year of follow-up. A total of 189 patients (92 males and 97 females; American Society of Anesthesiologists Physical Status I or II, Glasgow Coma Scale = 15) who were scheduled for laparoscopic abdominal surgery received intravenous midazolam (Versed®) 0.08-0.01 mg/kg and fentanyl (Duragesic®) 5.0 μg/kg. The main outcome measure was the degree of postoperative pain, as assessed using the Visual Analog Scale (VAS). VAS scores were recorded 5, 15, 30, 45, and 60 minutes after a fentanyl bolus injection in the post-anesthesia care unit (PACU). The minute expiratory volume, end-tidal carbon dioxide concentration (EtCO2) and respiratory rate (RR) were measured continuously. The incidence of fentanyl-induced respiratory depression (RR 45 mmHg) was recorded at its appearance and treated with respiratory assistance. Blood gas analysis was done 15, 30, 45, and 60 minutes after extubation. These parameters were correlated with genotyping results of genomic DNA extracted from whole blood. Results: Patients with the OPRM1 118 AG or GG genotypes had significantly higher VAS pain scores 15 and 30 minutes after a fentanyl bolus injection in the PACU than AA genotype patients (p G polymorphism lessens the analgesic response to fentanyl and the time to awakening and extubation but has no clinically significant effect on the incidence of respiratory depression.

At present, the most effective treatment for pulmonary alveolar proteinosis (PAP) remains whole-lung lavage in spite of the usually accompanying severe hypoxemia, which is expected to be prevented by hyperoxygenated solution improving oxygen supply during lavage. In this study, the efficacy and safety of the effect of hyperoxygenated solution were evaluated.Five patients underwent whole-lung lavage over a 28-month period. Each lung was lavaged with hyperoxygenated (HO) and normal saline solution (plain lactated Ringer's solution, NO) randomly and alternatively until the reclaimed fluid was clear. Random number was generated by computer before every cycle of lavage. If the number was odd, the patient was assigned to receive a lavage cycle with hyperoxygenated solution (HO group, n = 109); if the number was even, normal saline solution was used (NO group, n = 115). Data of saturation of peripheral oxygen (SPO(2)), mean arterial pressure (MAP), central venous pressure (CVP), heart rate (HR) and end-tidal carbon dioxide tension (P(ET)CO(2)) were taken down at 0, 30, 60, 90, 120, 150, 180, 210 and 240 seconds from the beginning of the instillation of solution, and frequency and volume of unilateral lung lavage were also recorded. Time interval between the left and the right lung lavage was 1 week.No patient was withdrawn from the study due to low SPO(2) or leakage. Oxygen pressure was (730.21 +/- 7.43) mmHg in the hyperoxygenated solution against (175.73 +/- 5.92) mmHg in the normal saline solution (P0.01). Compared with baseline, SPO(2) increased significantly as the instillation of solution began (P0.01), leveled for about 30 seconds (P0.05), and then decreased significantly to the lowest at the time of drainage (compared with 120 seconds or peak, P0.01). SPO2 was higher in HO group than in NO group (P0.01). There were no significant differences in MAP, HR, CVP and P(ET)CO(2) between HO group and NO group (P0.05) and also among different time points (P0.05).During the lung lavage for pulmonary alveolar proteinosis, hyperoxygenated solution could significantly improve oxygen supply in comparison with normal saline solution without obvious side effects.

1. In the present study, we compared the elastin and collagen content of thoracic aortic medial and adventitial layers from Wistar-kyoto (WKY) and spontaneously hypertensive rats (SHR). In addition, the effects of losartan, an angiotensin II receptor antagonist, and spironolactone, a mineralocorticoid receptor antagonist, on collagen and elastin content were determined. 2. Prehypertensive (4-week-old) and hypertensive (16-week-old) SHR were randomly divided into three groups treated with either 0.9% NaCl, losartan (20 mg/kg per day) or spironolactone (200 mg/kg per day). Prehypertensive and hypertensive SHR were treated for 12 and 16 weeks, respectively. Age-matched WKY rats were not treated with NaCl, losartan or spironolactone and served as the control group. 3. The medial and adventitial layers of the thoracic aorta were composed mainly of elastin and collagen, respectively, in both SHR and WKY rats. Compared with WKY rats, SHR exhibited greater collagen and elastin content in the media, but decreased collagen and elastin content in the adventitial layer. Both medial and adventitial collagen and elastin content increased significantly with age in both strains and was greater in 32-week-old rats compared with 16-week-old rats. Spironolactone treatment decreased collagen content in the media of thoracic aortas from prehypertensive SHR, whereas losartan decreased collagen content in the media of aortas from hypertensive SHR. In contrast, neither spironolactone nor losartan had any effect on adventitial collagen content in prehypertensive and hypertensive SHR. Medial collagen and elastin were positively related to pulse pressure (PP), but there was no correlation between adventitial mass or collagen content and PP or mean arterial pressure in untreated and treated SHR and WKY rats. 4. In conclusion, the composition of the medial and adventitial layers of the thoracic aorta differs and treatment of SHR with losartan and spironolactone decreases collagen content when delivered at the hypertensive or prehypertensive stage, respectively. However, neither drug has any effect on adventitial collagen content in SHR.

We have determined transmembrane protein tyrosine phosphorylation (outside-in signaling) in cultured osteoclasts and macrophages in response to added native purified bone sialoprotein (nBSP) and its dephosphorylated form (dBSP). There were selective/differential and potent inhibitory effects by dBSP and minimal effect by nBSP on intracellular tyrosine phosphorylation in macrophages and osteoclasts. Further studies on the downstream gene expression effects led to identification of a large number of differentially expressed genes in response to nBSP relative to dBSP in both macrophages and osteoclasts. These studies were extended to a bone resorption model using live mouse neonatal calvarial bone organ cultures stimulated by parathyroid hormone (PTH) to undergo bone resorption. Inclusion of nBSP in such cultures showed no effect on type I collagen telopeptide fragment release, hence overall bone resorption, whereas addition of dBSP abolished the PTH-induced bone resorption. The inhibition of bone resorption by dBSP was shown to be unique since in complementary experiments use of integrin receptor binding ligand, GRGDS peptide, offered only partial reduction on overall bone resorption. Quantitative RANKL analysis indicated that mechanistically the PTH-induced bone resorption was inhibited by dBSP via down-regulation of the osteoblastic RANKL production. This conclusion was supported by the RANKL analysis in cultured MC3T3-E1 osteoblast cells. Overall, these studies provided direct evidence for the involvement of covalently bound phosphates on BSP in receptor mediated "outside-in" signaling via transmembrane tyrosine phosphorylation with concurrent effects on downstream gene expressions. The use of a live bone organ culture system augmented these results with further evidence that links the observed in vivo variable state of phosphorylation with bone remodeling.

A rapid and sensitive liquid chromatography/electrospray ionization tandem mass spectrometric (LC/ESI-MS/MS) method has been developed to determine 1, 2-[bis(1,2-benzisoselenazolone-3(2H)-ketone)]-ethane (BBSKE), a novel antineoplastic agent, in rat plasma. The analytes were separated on a C18 column with a mobile phase of methanol-water (75:25, v/v) and detected using a triple-quadrupole mass spectrometer in positive mode with the selective reaction monitoring. The characteristic ion dissociation transitions were m/z 603.0 --> 448.9 for derivatized BBSKE and m/z 631.0 --> 476.8 for derivatized internal standard. The assay was linear over a range of 1-1000 ng/mL with a lower limit of quantification of 1 ng/mL. Intra- and inter-day precisions were less than 9.6 and 5.0%, respectively, and the accuracy ranged from -5.2 to 4.0%. The validated method was successfully applied to the characterization of pharmacokinetic profile of BBSKE after oral administration in rats. Cop