Pentachlorophenol (PCP), a xenobiocide used to preserve lumbers, is a major environmental pollutant in North America. In spite of an expected high resistance to biodegradation, a number of aquatic and soil bacteria can degrade PCP. In this study, we cloned, expressed and purified tetrachlorobenzoquinone reductase (PcpD), the second enzyme in the PCP biodegradation pathway in Sphingobium chlorophenolicum. PcpD, present mainly as a homo-trimer, exhibited low but statistically significant activity in the reduction of tetrachlorobenzoquinone to tetrachlorohydroquinone. The optimal pH for PcpD activity was 7.0. PcpD was stimulated by tetrachlorohydroquinone at low concentrations but inhibited at high concentrations. Because of the constitutive expression and relatively high catalytic efficiency of downstream enzyme tetrachlorohydroquinone reductive dehalogenase, tetrachlorohydroquinone was unlikely to accumulate in high concentrations, suggesting that PcpD would only be stimulated by tetrachlorohydroquinone under in vivo conditions. It was also shown that PcpD was inhibited by PCP in a concentration-dependent manner. Therefore, PcpD was regulated by tetrachlorohydroquinone and PCP using a possible “Yin-Yang” mechanism, which maintained tetrachlorobeanzoquinone at a level that would neither significantly decrease the biodegradation of PCP nor cause cytotoxicity in S. chlorophenolicum cells. Structural model of PcpD showed that the putative tetrachlorobenzoquinone binding site, adjacent to the cofactor flavin mononucleotide and the 2Fe2S cluster, was situated in a deep pit on the surface and slightly positively charged. Full article

Hypericin, extracted from H. perforatum, can induce the generation of reactive oxygen species by visible light irradiation, which may consequently induce the conformational change of hemoglobin. We have not only employed UV-vis spectroscopy to observe the changes of UV-vis spectra of the protein, which reveals the conformational changes of the protein, but also employed electrochemical method to obtain its enhanced peroxidase activity. The photodynamic effect of hypericin on the conformation and catalytic activity of the protein has also been proven to be strongly dependent on the irradiation time, the hypericin concentration and the presence of oxygen. This work is beneficial not only to the fabrication of more sensitive hydrogen peroxide biosensor, but also to the guidance of the usage of this medicinal herb molecule, since the conformational change of the protein and the enhanced peroxidase can be easily obtained only by visible light irradiation on hypericin, the process of which is so common to happen. Full article

Protein-protein interactions are important for biochemical processes in biological systems. The 3D structure of the macromolecular complex resulting from the protein-protein association is a very useful source to understand its specific functions. This work focuses on computational study for protein-protein docking, where the individually crystallized structures of interacting proteins are treated as rigid, and the conformational space generated by the two interacting proteins is explored extensively. The energy function consists of intermolecular electrostatic potential, desolvation free energy represented by empirical contact potential, and simple repulsive energy terms. The conformational space is six dimensional, represented by translational vectors and rotational angles formed between two interacting proteins. The conformational sampling is carried out by the search algorithms such as simulated annealing (SA), conformational space annealing (CSA), and CSA combined with SA simulations (combined CSA/SA). Benchmark tests are performed on a set of 18 protein-protein complexes selected from various protein families to examine feasibility of these search methods coupled with the energy function above for protein docking study. Full article

Inhibitors of human NAD⁺-dependent protein deacetylases possess great value for deciphering the biology of these enzymes and as potential therapeutics for metabolic and agerelated diseases and cancer. In the current study, we have experimentally demonstrated that, the potent inhibition we obtained previously for one of these enzymes (i.e. sirtuin type 1 (SIRT1)) by simply replacing N^ε-thioacetyl-lysine for N^ε-acetyl-lysine in its peptide substrate, represented a general and efficient strategy to develop potent and selective inhibitors of human NAD⁺-dependent protein deacetylase enzymes. Indeed, by using this simple inhibition strategy, potent (low-micromolar) and selective (≤40-fold) SIRT2 and SIRT3 inhibitors, which were either comparable or superior to currently existing inhibitors, have also been quickly identified in the current study. These inhibitors could be used as chemical biological tools or as lead compounds for further focused structure-activity optimization. Full article

Apoptosis or necrosis of neurons in the central nervous system (CNS) is thehallmark of many neurodegenerative diseases and Traumatic Brain Injury (TBI). Theinability to regenerate in CNS offers little hope for naturally repairing the damagedneurons. However, with the rapid development of new technologies, regenerative medicineoffers great promises to patients with these disorders. Among many events for furtheradvancement of regenerative medicine, extracellular matrix (ECM) plays a critical role forcellular migration and differentiation. To develop a biocompatible and electricallyconductive substrate that can be potentially used to promote growth and regeneration ofneurons and to record intracellular and multisite signals from brain as a probe, a polymericprecursor – SPR 220.7 was fabricated by pyrolysis at temperatures higher than 700 oC.Human Neuroblastoma cells - SK-N-MC, SY5Y, mouse teratocarcinoma cells P-19 and ratPC12 cells were found to attach and proliferate on photoresist derived carbon film.Significantly, neuronal differentiation of PC12 cells induced by NGF was demonstrated byobserving cell shape and size, and measuring the length of neurites under SEM. Our resultsindicated that fabricated carbon could potentially be explored in regenerative medicine forpromoting neuronal growth and differentiation in CNS with neurodegeneration. Full article

It has been suggested that natural killer (NK) cell activity and Th1 immunitymay be involved in the pathogenesis of preeclampsia. This study aimed to investigate theimmunophenotypes of NK cells and type 1/type 2 immunity in both decidua and maternalperipheral blood between normal (n=11) and preeclamptic pregnant women (n=20) by flowcytometry. The results showed that no significant difference was observed between patientsand controls by detecting CD56⁺ CD69⁺ and CD56⁺ CD94⁺ NK cells in both peripheralblood and decidua. Moreover, in preeclamptic patients, decreased percentages of Tc2 andTh2 cells and the increased ratios of Tc1/Tc2 were determined in both decidua andmaternal peripheral blood. In addition, the ratio of Th1/Th2 in peripheral blood alsoincreased. There was no significant difference of immunophenotypes of uNK cells betweenpreeclampsia and normal pregnancy. Local decidua and systematic immunity did notcorrelate with each other. These results suggest that the type 1/type 2 immunity shifted totype 1 immunity including Th1 and Tc1 cells may contribute to the patho-genesis ofpreeclampsia. Full article

Cecropin X is a short cationic peptide with a broad antibacterial and antitumorspectrum. Here, we report the production of a tumor necrosis factor (TNFα)-cecropin Xfusion protein under the control of a temperature-inducible PR promoter in the bacterialexpression vector pRC. During fermentation, we studied and optimized essentialparameters including the type of host cells, medium, timing of induction, post-inductiontime and dissolved oxygen level. Using the suitable conditions in the fermentation, up to20 % ~ 23 % of the total cellular proteins is produced as the fusion protein, mostly in theform of inclusion bodies. After washing, on average about 5.27 g dried inclusion bodiescould be collected from 1 L broth and the purity of inclusion bodies reached 80 %.Cecropin X obtained by cleaving the fusion protein with cyanogen bromide showedremarkable tumorcidal activity against mouse Lewis lung carcinoma 3LL in vivo. Full article

The glycine-N-acyltransferase (GLYAT) is well known to be involved in thedetoxification of endogenous and exogenous xenobiotic acyl-CoA's in mammals.Unfortunately, the knowledge about the gene encoding GLYAT is very limited. Here wereport a novel gene encoding a GLYAT member, designated as GLYATL1, which was1546 base pairs in length and contained an open reading frame (ORF) encoding apolypeptide of 302 amino acids. GLYATL1 was a split gene that was consisted of 7 exonsand 6 introns and mapped to chromosome 11q12.1. The expression of GLYATL1 could befound in liver, kidney, pancreas, testis, ovary and stomach among 18 human tissues by RT-PCR analysis. Subcellular localization of myc-tagged GLYATL1 fusion protein revealedthat GLYATL1 was distributed primarily in the cytoplasm of COS-7 cells. Furthermore,through the pathway profiling assay, the GLYATL1 protein was found to activate HSEsignaling pathway in a dose-dependent manner when overexpressed in HEK293T cells. Full article

The aim of this study was to investigate the characteristic polynomials resulting from the molecular graphs used as molecular descriptors in the characterization of the properties of chemical compounds. A formal calculus method is proposed in order to identify the value of the characteristic polynomial parameters for which the extremum values of the squared correlation coefficient are obtained in univariate regression models. The developed calculation algorithm was applied to a sample of nonane isomers. The obtained results revealed that the proposed method produced an accurate and unique solution for the best relationship between the characteristic polynomial as molecular descriptor and the property of interest. Full article

Loss of imprinting (LOI) of insulin-like growth factor II gene (IGF2) is anepigenetic abnormality associated with human diseases. However, little is known about thecharacteristics of IGF2 imprinting in newborn cord blood cells. METHODS: A total of 923cord blood samples from term singletons and related clinical data were collected; IGF2imprinting status in 273 specimens were successfully analyzed using RT-PCR andrestriction fragment length polymorphism. RESULTS: LOI of IGF2 was detected in 20.9%of informative samples. The mean birth weights (BW) in the LOI and the normal imprintinggroups were 3462.7 ± 460.2 g and 3363.7 ± 427.7 g, respectively. The abdominal perimetersin the LOI group tended to be larger than that in the normal imprinting group. Pregnancycomplications, delivery modes, newborn diseases, occurrences of malignant tumors ingrandparents, and other maternal factors were not associated with LOI of IGF2. 22.2% ofthe infants with IGF2 LOI also showed LOI in their father’s lymphocytes while 21.4% intheir mother’s lymphocytes. CONCLUSIONS: About 20% of Han Chinese newbornsindicated LOI of IGF2 in their cord blood lymphocytes that may represent the epigeneticcharacteristics in this ethnic group. While IGF2 LOI tends to be weakly inherited between parents and offspring, abnormal imprinting seems to be statistically unrelated with phenotypes of newborns, although it might have an association with later phenotypes of infants. Full article

A homeodomain-containing transcription factor Csx/Nkx2.5 is an importantregulator of cardiogensis in mammals. There has been considerable interest in understandingdeterminants of the diverse cardiac phenotypes associated with Csx/Nkx2.5 mutationssituated within or around the homeodomain in patients. To make clear of the functionalproperties of the regions out of homeodomain, we found that mutants locate outside of thehomeodomain retained intact nuclear localization and have nearly normal or increasedtranscriptional activity but impaired DNA binding capability, the C-terminus region exhibitsan inhibitory function on transcriptional activity of wild type Csx/Nkx2.5, and the NK2-Specific Domain is likely to facilitate both DNA binding and protein-protein interaction. Inthe current study, deletion mutant in homeodomain displayed extremely different biologicalappearance from the mutants with deletion outside of the homeodomain, these may explainthe clinical observation that patients with missense situated outside the homeodomain werenot associated with atrioventricular conduction disturbance. Full article

This study was made to explore the effects of acetazolamide on tumor growth,metastasis and the possible mechanisms. The mice bearing with Lewis lung carcinomaswere taken as the animal model. The effects of acetazolamide were compared with thecombination treatment of NaHCO₃ on tumor growth, metastasis and carbonic anhydraseactivity in lung and tumor tissues using imidazole-Tris technique. And also the possible roleof AQP1 in tumor tissues was investigated by Western blot and immuno-histochemicalanalysis. Results showed that acetazolamide alone could sharply reduce the number of lungmetastasis and primary tumor growth, and appeared in a dose-dependent manner.Acetazolamide significantly inhibited carbonic anhydrase activity in tumor tissue. After theaddition of NaHCO₃, the suppression of acetazolamide on tumor growth, number ofmetastasis and carbonic anhydrase activity in primary tumor tissue could not be alteredsignificantly, but the inhibitory rate of metastasis in lung and carbonic anhydrase activity inlung tissue appeared to show a reversal trend in the dose dependency from the acetazolamidetreatment alone. The exactly mechanisms need to be clarified in future. Western blot andimmunohistochemical analysis demonstrated that AQP1 expression in the tumor tissue washigher than both tissue of normal and treated with acetazolamide treatment alone.Combination with NaHCO₃ could not synergistically inhibit the expression of AQP1 withacetazolamide. The results suggested that the mechanism of acetazolamide on anti-tumor especially on its anti-metastasis actions might partly involve either inhibiting the carbonic anhydrase activity or reducing AQP1 water channel protein expression, whatever if treated with or without NaHCO₃. Full article

The expression of mitochondrial voltage-dependent anion channels (VDAC) mayunderlie the protective effects of Limonium sinense (Girard) Ktze root extracts (LSE) againstcarbon tetrachloride-induced liver damage. Pretreatment of mice with 100 mg/kg, 200mg/kg or 400 mg/kg LSE significantly blocked the carbon tetrachloride-induced increase inboth serum aspartate aminotransferase (sAST) and serum alanine aminotransferase (sALT)levels. Ultrastructural observations by electron microscope confirmed hepatoprotection,showing decreased nuclear condensation, ameliorated mitochondrial fragmentation of thecristae and less lipid deposition. Pretreatment with LSE prevented the decrease of thedisruption of mitochondrial membrane potential (15.3%) observed in the liver of the carbontetrachloride-insulted mice, further demonstrating the mitochondrial protection. In addition,LSE treatment (100-400 mg/kg) significantly increased both transcription and translation ofVDAC. The above data suggests that LSE mitigates the damage to liver mitochondriainduced by carbon tetrachloride, possibly through regulation of mitochondrial VDAC, one ofthe most important proteins in the mitochondrial outer membrane. Full article

The aim of the paper is to present the results obtained by utilization of an originalapproach called Molecular Descriptors Family on Structure-Property (MDF-SPR) andStructure-Activity Relationships (MDF-SAR) applied on classes of chemical compoundsand its usefulness as precursors of models elaboration of new compounds with betterproperties and/or activities and low production costs. The MDF-SPR/MDF-SARmethodology integrates the complex information obtained from compound’s structure inunitary efficient models in order to explain properties/activities. The methodology has beenapplied on a number of thirty sets of chemical compounds. The best subsets of moleculardescriptors family members able to estimate and predict property/activity of interest wereidentified and were statistically and visually analyzed. The MDF-SPR/MDF-SAR modelswere validated through internal and/or external validation methods. The estimation andprediction abilities of the MDF-SPR/MDF-SAR models were compared with previousreported models by applying of correlated correlation analysis, which revealed that theMDF-SPR/MDF-SAR methodology is reliable. The MDF-SPR/MDF-SAR methodologyopens a new pathway in understanding the relationships between compound’s structure andproperty/activity, in property/activity prediction, and in discovery, investigation andcharacterization of new chemical compounds, more competitive as costs andproperty/activity, being a method less expensive comparative with experimental methods. Full article

Nicotine and a variety of other drugs and toxins are metabolized by cytochromeP450 (CYP) 2A6. The aim of the present study was to build a quantitative structure-activityrelationship (QSAR) model to predict the activities of nicotine analogues on CYP2A6.Kernel partial least squares (K-PLS) regression was employed with the electro-topologicaldescriptors to build the computational models. Both the internal and external predictabilitiesof the models were evaluated with test sets to ensure their validity and reliability. As acomparison to K-PLS, a standard PLS algorithm was also applied on the same training andtest sets. Our results show that the K-PLS produced reasonable results that outperformed thePLS model on the datasets. The obtained K-PLS model will be helpful for the design ofnovel nicotine-like selective CYP2A6 inhibitors. Full article

In this work, we reported the investigation on the interaction between DNAstrands self-assembled at gold electrodes and an electron transfer protein, cytochrome c. Weobserved that cytochrome c exhibited well-defined electrochemistry in both double-strandedand single-stranded DNA films. This suggested that the electron transfer reaction ofcytochrome c arose possibly due to the electron hopping along DNA strands rather thanwiring along the double helix. We also compared the heterogeneous electron transfer rate ofcytochrome c with that of a ruthenium complex, which further confirmed this mechanism. Full article

Salmonella enterica serovar Typhi is a human enteroinvasive pathogen that canovercome the stress caused by the high osmolarity of the human small intestine and causesystemic infection. To investigate the global transcriptional regulations of S. entericaserovar Typhi exposed to a hyperosmotic environment, a genomic oligo-DNA microarraycontaining 4474 Salmonella genes was prepared. A wild strain of S. enterica serovar TyphiGIFU10007 was grown in LB medium containing 50 mM NaCl to simulate a low osmoticenvironment. The hyperosmotic stress was simulated by an osmotic up-shift, whichincreased the concentration of NaCl in the LB from 50 mM to 300 mM. Genome-wide geneexpressions of S. enterica serovar Typhi at 15 min, 30 min, 60 min, and 120 min after theosmotic up-shift were investigated by the microarray analysis. Gene expression profiles insomewhat later stage (60 ~120 min) of the stress were quite different from those in the earlystage (0 ~ 30 min) of the stress. At 120 min after the osmotic stress, the expression levels of889 genes were obviously changed. However, expression levels of only 382 genes weresignificantly changed at 15 min after the osmotic stress. The expression levels of most SPI-1genes associated with invasion of the pathogen were increased at 120 min after the osmoticup-shift, but were not obviously changed at 15 min or 30 min after the osmotic stress.Expressions of a central regulatory gene, phoP, and sigma factor genes rpoE, rpoD, andrpoS were also changed with different profiles during the osmotic stress. These resultsindicated that the invasive ability of the pathogen is significantly increased after 2 h of hyperosmotic stress, and regulator PhoP and sigma factors RpoE, RpoD appear to participate in the network regulatory mechanisms that benefit the pathogen to adapt hyperosmotic environmental conditions. The later increased invasive ability of S. enterica serovar Typhi after hyperosmotic stress may be one reason why the pathogen performs invading in the distal ileum of human and not in areas of the upper small intestine. Full article

25 kDa branched polyethylenimine (PEI) has successfully been used for in vitroand in vivo gene delivery approaches, but it is cytotoxic. Smaller PEIs are usually non-cytotoxic but less efficient. In order to enhance the gene delivery efficiency and minimizecytotoxicity of PEI, we explored to synthesize cross-linked PEIs with degradable bonds byreacting amines of small branched 2000 Da PEI with small diacrylate (1,4-butanedioldiacrylate or ethyleneglycol dimethacrylate) for 2-6 hours. The efficiency of the cross-linkedPEIs during in vitro delivering plasmid containing enhanced green fluorescent protein(EGFP) gene reporter and their cytotoxicity were assessed in melanoma B16F10 cell andother cell lines. In vivo gene delivery efficiency was evaluated by direct injection delivery ofthe EGFP plasmid/ cross-linked PEI complexes into mice and by estimating the EGFPexpression in animal muscles. Compared to commercially available 25-kDa branched PEI,the cross-linked PEIs reported here could mediate more efficient expression of reporter genethan the 25-kDa PEI control, 19-fold more efficiently in B16F10 cells, 17-fold in 293T cells, 2.3-fold in 3T3 cells, and they exhibited essentially nontoxic at their optimized condition for gene delivery. Furthermore the transfection activity of polyplexs was preserved in the presence of serum proteins. The muscle transfected with the cross-linked PEI prepared here exhibited normal morphology and excellent gene expression. The cross-linked PEIs reported here were evidently more efficient than the commercial 25-kD PEI control and had less cytotoxicity in gene delivery in vitro and in vivo. Full article

This study was designed to evaluate the inhibitory effect of endostatin onchoroidal neovascularization (CNV) in laser-induced rat model. Choroidalneovascularization was induced in Brown Norway (BN) rats by diode-laserphotocoagulation. Rats were randomly divided into five groups (10 animals in each group):endostatin 20mg/kg group, endostatin 10mg/kg group, laser injury group, normal salinegroup and blank control group. The animals were treated with endostatin, normal saline,laser injury or without treatment on day 0 to day 13 after laser photocoagulation. On day 7and 14 after laser photocoagulation, the CNV formation was assessed by fluoresceinangiography and histopathological analysis. VEGF expression in retina was determined byimmunohistochemical assay. In two endostatin groups, the incidence of CNV formation andthe intensity of fluorescein leakage were reduced compared with the two control groups. Nosignificant difference was found between laser injury group and normal saline group. Theexpression of VEGF reached peak at day 7 and then decreased from day 14 afterphotocoagulation. The expression of VEGF was significantly reduced in the two endostaingroups than laser injury group in a dose-dependent way. Endostatin can inhibit the formationof experimental CNV in the rat. Down-regulation of VEGF expression could be one of themechanisms underlying the inhibition of CNV by endostatin. Full article

Hyperphosphorylation of tau leading to aggregated tau and tangle formation is acommon pathological feature of tauopathies, including Alzheimer's disease. Abnormalphosphorylation of tau by kinases, in particular GSK3β, has been proposed as a pathogenicmechanism in these diseases. In this study we demonstrate that the heat shock protein 90(Hsp90) maintains the stability and function of the GSK3β. By using both rat primarycortical neurons and COS-7 cells, we show that Hsp90 inhibitors lead to a reduction of theprotein level of GSK3β, and that this effect is associated with both a decrease in tauphosphorylation at putative GSK3β sites and an induction in heat shock protein 70 (Hsp70)levels. We further show that Hsp90 associates with the GSK3β regulating its stability andfunction and preventing its degradation by the proteasome. Full article

Camptothecin, an anticancer component from Camptotheca acuminate, mayinteract with human serum albumin (HSA) at the subdomain IIA (site I), and then convert toits inactive form(carboxylate form). In this paper, the detailed electrochemical behaviors ofcamptothecin at a pyrolytic graphite electrode is presented. The interaction betweencamptothecin and HSA is also studied by electrochemical technique. By comparing withbovine serum albumin (BSA), which is highly homologous to HSA, we prove thatcamptothecin can specifically bind to HSA. Meanwhile, the inhibitory influence of sodiumsalicylate to this binding is also discussed. Full article

Nephropathy is one of the most common complications of diabetes mellituswhich remains incompletely understood. We reported the expression of myosin light chainkinase (MLCK) in the kidney of diabetic rats and investigated the correlation betweenMLCK and diabetic nephropathy by observing the expression of MLCK. The diabetic modelrats were induced by an intraperitoneal injection of streptozotocin (STZ) and the insulin-treated rats were subcutaneously injected with protamine zine insulin 3u/d. The kidneyswere excised and immersed in 4% polyoxymethylene after 12 weeks later. The expression ofMLCK was analyzed by immunohistochemical staining and Western blot.Immunohistochemical analysis and Western blot assay indicated that the MLCK expressionwas higher in kidney of diabetic rats than that in control and it was decreased in kidney ofinsulin-treated rats. Our results suggested that the over expression of MLCK may be relatedwith the development of diabetic nephropathy. Full article