Mutation of residue arginine 330 of arginine kinase results in the generation of the oxidized form more susceptible.

Arginine kinase (AK), a crucial enzyme for the energy metabolism of invertebrates, catalyzes the reversible phosphorylation of arginine by Mg(2+)ATP to form phosphoarginine and Mg(2+)ADP. Arginine 330 (R330), not involved in the catalysis of phosphoryl transfer, is a residue highly conserved in the phosphagen kinase family. In order to investigate the role of R330 in AK, it was replaced by lysine (R330K). Non-reduced SDS-PAGE analysis suggested that wild type AK (Wt-AK) and R330K existed in two forms, the reduced form (R-AK or R-R330K) and the oxidized form (O-AK or O-R330K), whereas O-R330K was more susceptible to generate than O-AK. Subsequently, an intramolecular disulfide bond in O-R330K was demonstrated to be formed between Cys201 and Cys271 by site-directed mutagenesis. Biochemical analysis revealed that conformational changes of R330K were concomitant with the sharp decline of catalytic activity. These results were further confirmed by structure modeling of AK and R330K. Therefore, it can be concluded that R330 residue plays an important role in the structural stability and activity of AK.

Alantolactone induces activation of apoptosis in human hepatoma cells.

Alantolactone, a sesquiterpene lactone, possesses anti-inflammatory property. In this study, we provide evidence that it could be developed as a novel agent against human liver cancer. We observed that alantolactone treatment to HepG2, Bel-7402 and SMMC-7721 cells, human liver cancer cell lines resulted in a dose-dependent inhibition of cell growth. We selected HepG2 cell line as a test model system. Alantolactone treatment of HepG2 cells resulted in a dose-dependent induction of apoptosis and arrest of cells in G2-M phase. This induction of apoptosis seems to be mediated via modulating the protein levels of Bcl-2 family and activation of caspases. Moreover, caspase-8 and Bid activation, loss of mitochondrial transmembrane potential and cytochrome c release suggest the existence of a cross-talk between the death receptor and the mitochondrial pathways. We also observed that alantolactone treatment of cells resulted in a dose-dependent decrease in NF- κB/p65. In addition, a significant and progressive increase in the level of p53 protein in alantolactone-treated cells was observed. Taken together, our data suggest that alantolactone could be developed as an agent against human liver cancer.

Activation of apoptosis by ethyl acetate fraction of ethanol extract of Dianthus superbus in HepG2 cell line.

Dianthus superbus L. is commonly used as a traditional Chinese medicine. We recently showed that ethyl acetate fraction (EE-DS) from ethanol extract of D. superbus exhibited the strongest antioxidant and cytotoxic activities. In this study, we examined apoptosis of HepG2 cells induced by EE-DS, and the mechanism underlying apoptosis was also investigated. Treatment of HepG2 cells with EE-DS (20-80 µg/ml) for 48 h led to a significant dose-dependent increase in the percentage of cells in sub-G1 phase by analysis of the content of DNA in cells, and a large number of apoptotic bodies containing nuclear fragments were observed in cells treated with 80 µg/ml of EE-DS for 24 h by using Hoechst 33258 staining. These data show that EE-DS can induce apoptosis of HepG2 cells. Immunoblot analysis showed that EE-DS significantly suppressed the expressions of Bcl-2 and NF-κB. Treatment of cells with EE-DS (80 µg/ml) for 48 h resulted in significant increase of cytochrome c in the cytosol, which indicated cytochrome c release from mitochondria. Activation of caspase-9 and -3 were also determined when the cells treated with EE-DS. The results suggest that apoptosis of HepG2 cells induced by EE-DS could be through the mitochondrial intrinsic pathway. High performance liquid chromatography (HPLC) data showed that the composition of EE-DS is complicated. Further studies are needed to find the effective constituents of EE-DS.

Evidences of monomer, dimer and trimer of recombinant human cyclophilin A.

Cyclophilin A (CyPA) is a cytosolic receptor of immunosuppressive drug cyclosporin A (CsA) which possesses peptidyl-prodyl cis/trans isomerase (PPIase) activity. The recombinant human CyPA (rhCyPA) gene has been expressed in E. coli M15. Purification was performed using salting-out, as well as Sephacryl S-100 and DEAE-Sepharose CL-6B column chromatography. The molecular weight is about 18 kDa, confirmed by SDS-PAGE and mass spectrum. The results of Native-PAGE and immunoblotting showed the existence of three bands, which agreed well with the gel filtration results. The molecular mass of the three bands determined via CTAB gel electrophoresis and SDS-PAGE (rhCyPA cross-linked with glutaraldehyde) was 18 kDa, 36 kDa and 54 kDa respectively. Further more, the native rhCyPA and the cross-linked rhCyPA had the similar chromatographic behavior in gel filtration. All of the evidences above suggest that rhCyPA exists in forms of monomer, dimer and trimer. Moreover, we observed that even at low protein concentrations CyPA largely occurs as a dimer in solution, and enzyme kinetic parameters showed that activity of dimer was much higher than monomer or trimer, which probably have some biological significances.

Anticancer, antioxidant and antimicrobial activities of the essential oil of Lycopus lucidus Turcz. var. hirtus Regel.

This study was designed to examine the anticancer, antioxidant and antimicrobial activities of the essential oil from Lycopus lucidus Turcz. var. hirtus Regel. The essential oil treatment to six human cancer cell lines resulted in a dose-dependent inhibition of cell growth. The cytotoxicity of the essential oil on liver carcinoma and breast cancer cell lines was significantly stronger than on other cell lines. The essential oil can induce apoptosis of the liver carcinoma cell line Bel-7402 and decrease the intracellular GSH level. The antioxidant effect of the essential oil was evaluated by using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and hydroxyl radical (OH) scavenging assays. The essential oil exhibited moderate antioxidant activity. The antimicrobial activity of the essential oil was evaluated against eight microorganisms using the disc diffusion and broth microdilution methods. The essential oil also showed moderate antimicrobial activity. These suggest that the essential oil could hold a good potential for use in the pharmaceutical industry.

A theoretical study on the catalytic mechanism of Mus musculus adenosine deaminase.

The catalytic mechanism of Mus musculus adenosine deaminase (ADA) has been studied by quantum mechanics and two-layered ONIOM calculations. Our calculations show that the previously proposed mechanism, involving His238 as the general base to activate the Zn-bound water, has a high activation barrier of about 28 kcal/mol at the proposed rate-determining nucleophilic addition step, and the corresponding calculated kinetic isotope effects are significantly different from the recent experimental observations. We propose a revised mechanism based on calculations, in which Glu217 serves as the general base to abstract the proton of the Zn-bound water, and the protonated Glu217 then activates the substrate for the subsequent nucleophilic addition. The rate-determining step is the proton transfer from Zn-OH to 6-NH(2) of the tetrahedral intermediate, in which His238 serves as a proton shuttle for the proton transfer. The calculated kinetic isotope effects agree well with the experimental data, and calculated activation energy is also consistent with the experimental reaction rate.

Cloning, expression, characterization and phylogenetic analysis of arginine kinase from greasyback shrimp (Metapenaeus ensis).

Arginine kinase (AK) plays an important role in cellular energy metabolism in invertebrate. The encoding AK gene from Shrimp Metapenaeus ensis (M. ensis) was cloned in prokaryotic expression plasmid pET-28a, and it was then expressed in Escherichia coil in dissoluble form. The recombinant protein was purified by following three chromatography steps in turn: CM-Cellulose cation-exchange, Sephacryl S-100HR gel filtrate and DEAE-Sepharose anion-exchange. The purified AK's apparent K(m) was 2.33+/-0.1 and 1.59+/-0.2 mM for ATP and l-arginine, respectively, while its optimum pH and temperature was 8.5 and 30 degrees C in the process of forward reaction, respectively. Phylogenetic analysis of cDNA-derived amino acid sequences for the AKs indicated a close affinity of M. ensis and another shrimp (Litopenaeus vannamei).

A novel extracellular protease with fibrinolytic activity from the culture supernatant of Cordyceps sinensis: purification and characterization.

A novel serine protease with fibrinolytic activity named CSP was purified from the culture supernatant of the fungus Cordyceps sinensis, a kind of Chinese herbal medicine. Analysis of the purified enzyme by SDS-PAGE indicated that CSP was a single polypeptide chain with an apparent molecular weight of 31 kDa, and N-terminal sequencing revealed that the first ten amino acid residues of the enzyme were Ala-Leu-Ala-Thr-Gln-His-Gly-Ala-Pro-Trp-. When casein was used as a substrate, the proteolytic activity of CSP reached its maximum at pH 7.0 and 40 degrees C. The effect of chemical agents on the enzyme activity indicated that CSP is a serine protease with a free cysteine residue near the active site. It hydrolysed fibrinogen, fibrin and casein with a high efficiency, while hydrolysing bovine serum albumin (BSA) and human serum albumin (HSA) to a lesser extent. CSP was found to be a plasmin-like protease, but not a plasminogen activator, and it preferentially cleaved the A alpha chain of fibrinogen and the alpha-chain of fibrin. Therefore, the extracellular protein CSP may represent a potential new therapeutic agent for the treatment of thrombosis.

Changes in mitochondrial membrane potential and reactive oxygen species during wogonin-induced cell death in human hepatoma cells.

Flavonoids exist extensively in plants, and several biological effects of them have been demonstrated. Wogonin is an important flavonoid compound. In this study, wogonin showed obvious growth inhibition on Bel-7402 cells. The major mechanisms of inhibition included cell apoptosis and cytotoxic effects. Wogonin-induced cell death showed characteristics of apoptosis including DNA fragmentation, chromatin condensation, appearance of apoptotic bodies, and an increase in hypodiploid cells. However, the percentage of necrosis cells also increased with the increase of wogonin concentration. Furthermore, treatment with wogonin caused changes of reactive oxygen species (ROS) and mitochondrial membrane potentials (DeltaPsim, MMP), the decrease of the ratio of reduced and oxidized glutathione (GSH/GSSG), cytochrome c release and activation of caspase-9.

Composition, antimicrobial activity and cytotoxicity of essential oils from Aristolochia mollissima.

The compositions of the essential oils from the rhizome and the aerial part of Aristolochia mollissima were analysed by GC-MS, 68 constituents (88.2% of the total oil) and 74 constituents (89.4% of the total oil) were identified, respectively. 2,2,7,7-Tetramethyltricyclo[6.2.1.0(1,6)]undec-4-en-3-one was the most abundant constituent among all in the ratios of 15.9% and 13.5% from the rhizome and the aerial part of A. mollissima, respectively. Among other main compounds, (E)-ß-santalol acetate (10.3%) and camphene (6.7%) were detected in the rhizome oil, spathulenol (6.8%) was detected in the oil from the aerial par of A. mollissima. The antimicrobial activity of the essential oils from the rhizome and the aerial part of A. mollissima was evaluated against 20 microorganisms using disc diffusion and broth microdilution methods. The gram-positive bacteria were more sensitive to both oils than gram-negative bacteria and yeasts. The rhizome oil showed the strongest bactericidal activity against Staphylococcus saprophyticus, whereas the oil from the aerial part of A. mollissima exerted the strongest bactericidal activity against methicillin-resistant and methicillin-senstive Staphylococcus aureus. The in vitro cytotoxicity of both oils on six human cancer cell lines were also examined. The cytotoxicity of the rhizome oil on four cancer cell lines (ACHN, Bel-7402, Hep G2 and HeLa) was significantly stronger than that of the oil from the aerial part of A. mollissima.

A novel fluorescent method for determination of peroxynitrite using folic acid as a probe.

A novel method for the determination of peroxynitrite using folic acid as a fluorescent probe is described. The method is based on the oxidation of the reduced, low-fluorescent folic acid by peroxynitrite to produce a high-fluorescent emission product. The fluorescence increase is linearly related to the concentration of peroxynitrite in the range of 3x10(-8) to 5.0x10(-6)molL(-1) with a correlation coefficient of 0.998, and the detection limit is 1x10(-8)molL(-1). Interferences from some metal ions normally seen in biological samples, and also some anions structurally similar to peroxynitrite were studied. The optimal conditions for the detection of peroxynitrite were evaluated.

Direct electrochemistry and electrochemical catalysis of immobilized hemoglobin in an ethanol-water mixture.

Hemoglobin (Hb) was immobilized on a glassy carbon electrode (GCE) surface by konjac glucomannan (KGM). KGM hydrogel films on GCE have relatively high stabilities in aqueous-ethanol mixtures. The entrapped hemoglobin undergoes fast direct electron transfer reactions in aqueous-organic solvent mixtures. The peak current is bigger, the peak-to-peak separation smaller and the formal potential observed in the cyclic voltammogram is more negative for Hb-KGM/GCE in ethanol-PBS compared to Hb-KGM/GCE in PBS. The electrochemical properties of the Hb in aqueous-organic solution are almost unchanged from with those observed for the purely aqueous solution, suggesting that water pools in the KGM hydrogel play an important role in preventing changes in conformation and making proteins unreactive with polar organic solvents. The immobilized Hb was able to catalyze the reduction of nitric oxide, peroxides (hydrogen peroxide, cumene hydroperoxide, t-butyl hydroperoxide, 2-butanone peroxide), and the dehalogenation of haloethanes (hexachloroethane, pentachloroethane, tetrachloroethane, etc.). The stability and reproducibility of the modified electrode meant that it could be used to determine these substances.

Electrochemical investigation of immobilized hemoglobin: redox chemistry and enzymatic catalysis.

Hb entrapped in the Konjak glucomannan (KGM) film could transfer electrons directly to an edge-plane pyrolytic graphite (EPG) electrode, corresponding to the redox couple of Fe(III)/Fe(II). The redox properties of Hb, such as formal potential, electron transfer rate constant, the stability of the redox state of protein and redox Bohr effect, were characterized by cyclic voltammetry and square wave voltammetry. The stable Hb-KGM/EPG gave analytically useful electrochemical catalytic responses to oxygen, hydrogen peroxide and nitrite.

The presence of anti-mitochondrial antibodies in Chinese patients with liver involvement in systemic lupus erythematosus.

Sixty-six hospitalized patients with systemic lupus erythematosus (SLE) were enrolled into this study. The test for anti-mitochondrial antibodies (AMAs) was performed and biochemical parameters were determined. AMAs were detected in 15 of the 66 patients with SLE. Meanwhile, we compared enzymatic levels in AMA-positive and -negative patients and found that serum aminotransferase levels were significantly higher in AMA-positive patients than in AMA-negative individuals. Furthermore, we found a positive correlation between serum AMA titration and serum aminotransferase levels. This study suggests that AMAs might contribute to the elevation of aminotransferases. Although much remains to be learned about the pathogenesis of autoimmune liver disease associated with AMAs, this report might provide greater insight into the metabolic mechanisms of AMAs in AMA-positive patients.

Probing the importance of hydrogen bonds in the active site of the subtilisin nattokinase by site-directed mutagenesis and molecular dynamics simulation.

Hydrogen bonds occurring in the catalytic triad (Asp32, His64 and Ser221) and the oxyanion hole (Asn155) are very important to the catalysis of peptide bond hydrolysis by serine proteases. For the subtilisin NK (nattokinase), a bacterial serine protease, construction and analysis of a three-dimensional structural model suggested that several hydrogen bonds formed by four residues function to stabilize the transition state of the hydrolysis reaction. These four residues are Ser33, Asp60, Ser62 and Thr220. In order to remove the effect of these hydrogen bonds, four mutants (Ser33-->Ala33, Asp60-->Ala60, Ser62-->Ala62, and Thr220-->Ala220) were constructed by site-directed mutagenesis. The results of enzyme kinetics indicated that removal of these hydrogen bonds increases the free-energy of the transition state (DeltaDeltaG(T)). We concluded that these hydrogen bonds are more important for catalysis than for binding the substrate, because removal of these bonds mainly affects the kcat but not the K(m) values. A substrate, SUB1 (succinyl-Ala-Ala-Pro-Phe-p-nitroanilide), was used during enzyme kinetics experiments. In the present study we have also shown the results of FEP (free-energy perturbation) calculations with regard to the binding and catalysis reactions for these mutant subtilisins. The calculated difference in FEP also suggested that these four residues are more important for catalysis than binding of the substrate, and the simulated values compared well with the experimental values from enzyme kinetics. The results of MD (molecular dynamics) simulations further demonstrated that removal of these hydrogen bonds partially releases Asp32, His64 and Asn155 so that the stability of the transition state decreases. Another substrate, SUB2 (H-D-Val-Leu-Lys-p-nitroanilide), was used for FEP calculations and MD simulations.

Increased serum aminotransferases associated with anti-mitochondrial antibodies in systemic lupus erythematosus patients with autoimmune liver disease.

BACKGROUND: Serum aminotransferase activities are increased in many liver diseases, but the causes for the elevation might be difficult to determine. Whether the elevation of aminotransferases correlates with anti-mitochondrial antibodies (AMA) in systemic lupus erythematosus (SLE) patients with autoimmune liver disease deserves further consideration. METHODS: A meticulous review was done in a large SLE cohort searching for laboratory features of the presence of AMA. Forty-eight hospitalized SLE patients with AMA and 60 randomly selected SLE patients without AMA as a matched case control were enrolled into the retrospective study. Laboratory data were collected, analyzed and compared in SLE patients with and without AMA. RESULTS: Serum activities of aminotransferases were significantly increased in the 48 SLE patients with AMA compared with the 60 subjects without AMA. Meanwhile, we found a positive correlation between serum AMA titers and serum aminotransferase activities. CONCLUSION: Although much remains to be learned about the pathogenesis of autoimmune liver disease associated with AMA, it is possible to suggest that AMA might contribute to the elevation of aminotransferases in SLE patients with the progressive disease.

Reactions of peroxynitrite and nitrite with organic molecules and hemoglobin.

In this work, the reactions of nitrite (NO2-) and peroxynitrite (ONOO-) with organic molecules as well as with hemoglobin (Hb) were examined and the potential interference with the detection of hydrogen peroxide and Hb was investigated. ONOO- at low concentrations (35-140 microM) induced a concentration-dependent oxidation of o-phenylenediamine and guaiacol, and this process can be improved by the addition of Hb in a concentration-dependent manner. This enhancing effect of Hb was possibly due to the formation of such highly reactive species as ferrylHb during the reaction of ONOO- and Hb. NO2- also oxidized the aromatic amine o-phenylenediamine, but its efficiency was much lower than that of ONOO-. A 300-fold excess of NO2- over hydrogen peroxide inhibited the oxidation of Pyrogallol Red mediated by hydrogen peroxide and Hb, which was due in part to the reaction of NO2- with Hb ferryl species compound I and compound II and the phenoxyl radical. These data suggest that ONOO- and NO2- can interfere with the detection of hydrogen peroxide. The overestimation or underestimation of the hydrogen peroxide detected is dependent upon the organic molecule utilized for detection and the relative rate of NO2-, superoxide, and ONOO- generation.

Interaction of daunomycin antibiotic with human serum albumin: investigation by resonant mirror biosensor technique, fluorescence spectroscopy and molecular modeling methods.

Daunomycin (DM) is a clinically used antitumor anthracycline antibiotic, which is transported primarily by human serum albumin (HSA) in the blood. Binding characteristics are therefore of interest for both the pharmacokinetics and pharmacodynamics of DM. A new optical biosensor technique based on the resonant mirror was used to characterize interaction of DM with HSA at different temperatures and the affinity constants were obtained. The HSA-DM interaction is exothermic with having favorable enthalpy and entropy followed by the integrated van't Hoff equation analysis. Fluorescence studies showed that DM has an ability to quench the intrinsic fluorescence of HSA through a static quenching procedure according to the Stern-Volmer equation and DM displays a pH-dependent binding affinity to HSA. Molecular modeling calculations showed that the DM binds HSA to a non-classical drug binding site and further analysis of the binding site of DM within the HSA molecule suggested that hydrophobic contacts, hydrogen bond formation and electrostatic interactions account for the binding of DM.

Acute and genetic toxicity of essential oil extracted from Litsea cubeba (Lour.) Pers.

Litsea cubeba oil is an aromatic essential oil extracted from the fresh fruits of Litsea cubeba (Lour.) Pers. It is used as a flavor enhancer in foods, cosmetics, and cigarettes; as a raw material in the manufacture of citral, vitamins A, E, and K, ionone, methyl ionone, and perfumes; and as an antimicrobial and insecticide. Based on the widespread use of L. cubeba oil, its insolubility in water, resulting in its partition in soil sediment, and its volatility when exposed to the atmosphere, risk of injury due to consumption and occupational exposure may be significant. In the present study, we studied the toxicity of L. cubeba oil with a battery of acute and genetic toxicity tests in Institute of Cancer Research mice and Sprague-Dawley rats. The oral, dermal, and inhalation 50% lethal dose and concentration (LD50 and LC50) of L. cubeba oil were determined. Results indicated that the oral LD50, the dermal LD50, and the inhalation LC50 are approximately 4,000 mg/kg of body weight, in excess of 5,000 mg/kg, and approximatively 12,500 ppm, respectively. We therefore conclude that L. cubeba oil is slightly toxic. In addition, the genetic toxicity of L. cubeba oil was assessed with Salmonella Typhimurium, by determination of the induction of micronuclei in bone marrow cells, and also by testing for chromosome aberration in spermatocyte cells of Institute of Cancer Research mice. The results of genetic toxicity testing of L. cubeba oil in vitro and in vivo were negative.

Identification of intermediate and product from methemoglobin-catalyzed oxidation of o-phenylenediamine in two-phase aqueous-organic system.

Methemoglobin (metHb) was used as a mimetic enzyme for peroxidase to catalyze the oxidation reaction of o-phenylenediamine (OPDA) with H2O2 functioning as an oxidant. A reaction intermediate was obtained in two-phase aqueous-organic system and an absorption peak at 710 nm was confirmed to be that of the intermediate in relation to OPDA. The isolated product and intermediate were characterized by UV-Vis and IR spectrophotometry and HPLC-tandem mass spectrometry. The results showed that the product is 2,3-diaminophenazine, the molecular mass of the intermediate is 212 daltons, and a conceivable structure of the intermediate is suggested. Combining the catalyzed reaction mechanism of peroxidase and our experimental results, a conceivable oxidation reaction mechanism of OPDA and H2O2 using metHb as catalyst is proposed.