Qing-Xi Chen

Identification of candidate prostate cancer biomarkers in prostate needle biopsy specimens using proteomic analysis.

Although serum prostate specific antigen (PSA) is a well‐established diagnostic tool for prostate cancer (PCa) detection, the definitive diagnosis of PCa is based on the information contained in prostate needle biopsy (PNBX) specimens. To define the proteomic features of PNBX specimens to identify candidate biomarkers for PCa, PNBX specimens from patients with PCa or benign prostatic hyperplasia (BPH) were subjected to comparative proteomic analysis. 2‐DE revealed that 52 protein spots exhibited statistically significantly changes among PCa and BPH groups. Interesting spots were identified by MALDI‐TOF‐MS/MS. The 2 most notable groups of proteins identified included latent androgen receptor coregulators [FLNA(7–15) and FKBP4] and enzymes involved in mitochondrial fatty acid β‐oxidation (DCI and ECHS1). An imbalance in the expression of peroxiredoxin subtypes was noted in PCa specimens. Furthermore, different post‐translationally modified isoforms of HSP27 and HSP70.1 were identified. Importantly, changes in FLNA(7–15), FKBP4, and PRDX4 expression were confirmed by immunoblot analyses. Our results suggest that a proteomics‐based approach is useful for developing a more complete picture of the protein profile of PNBX specimen. The proteins identified by this approach may be useful molecular targets for PCa diagnostics and therapeutics.

Inhibition Kinetics of Chlorobenzaldehyde Thiosemicarbazones on Mushroom Tyrosinase

2-Chlorobenzaldehyde thiosemicarbazone (2-Cl-BT) and 4-chlorobenzaldehyde thiosemicarbazone (4-Cl-BT) were synthesized, and their inhibitory kinetics on the activity of mushroom tyrosinase were investigated. Results showed that these compounds exhibited significant inhibitory potency on both monophenolase activity and diphenolase activity of tyrosinase. For the monophenolase activity, both compounds could decrease the steady-state activity of the enzyme sharply, without any influence on the lag period. The IC50 values of them were estimated to be 15.4 μM and 6.7 μM, respectively. For the diphenolase activity, both compounds belonged to reversible inhibitors, but their mechanisms were different: 2-Cl-BT was a noncompetitive type inhibitor, while 4-Cl-BT was a mixed-type inhibitor. Their inhibition constants were determined and compared.

Purification and Properties of Endoglucanase from a Sugar Cane Bagasse Hydrolyzing Strain, Aspergillus glaucus XC9

An endoglucanase (EG) from Aspergillus glaucus XC9 grown on 0.3% sugar cane bagasse as a carbon source was purified from the culture filtrate using ammonium sulfate, an anion exchange DEAE Sepharose fast flow column, and a Sephadex G-100 column, with a purification fold of 21.5 and a recovery of 22.3%. The ideal time for EG production is on the fourth day at 30 degrees C using bagasse as a substrate. Results obtained indicate that the enzyme was a monomer protein, and the molecular weight was determined to be 31 kDa. The optimum pH and temperature of EG for the hydrolysis of carboxymethylcellulose sodium (CMC-Na) were pH 4.0 and 50 degrees C, respectively. EG was stable over the pH range from 3.5 to 7.5 and at temperatures below 55 degrees C. Kinetic behavior of EG in the hydrolysis of CMC-Na followed Michaelis-Menten kinetics with constant K(m) of 5.0 mg/mL at pH 4.0 and 50 degrees C. The enzyme activity was stimulated by Fe(2+) and Mn(2+) but inhibited by Cd(2+), Pb(2+), and Cu(2+). The EDC chemical modification suggested that at least one carboxyl group probably acted as a proton donor in the enzyme active site.

Synthesis and Antityrosinase Mechanism of Benzaldehyde Thiosemicarbazones: Novel Tyrosinase Inhibitors

p-Hydroxybenzaldehyde thiosemicarbazone (HBT) and p-methoxybenzaldehyde thiosemicarbazone (MBT) were synthesized and established by (1)H NMR and mass spectra. Both compounds were evaluated for their inhibition activities on mushroom tyrosinase and free-cell tyrosinase and melanoma production from B(16) mouse melanoma cells. Results showed that both compounds exhibited significant inhibitory effects on the enzyme activities. HBT and MBT decreased the steady state of the monophenolase activity sharply, and the IC(50) values were estimated as 0.76 and 7.0 μM, respectively. MBT lengthened the lag time, but HBT could not. HBT and MBT inhibited diphenolase activity dose-dependently, and their IC(50) values were estimated as 3.80 and 2.62 μM, respectively. Kinetic analyses showed that inhibition type by both compounds was reversible and their mechanisms were mixed-type. Their inhibition constants were also determined and compared. The research may supply the basis for the development of new food preservatives and cosmetic additives.

NMR, HPLC-ESI-MS, and MALDI-TOF MS analysis of condensed tannins from Delonix regia (Bojer ex Hook) Raf and their bioactivities

The structures of the condensed tannins isolated from leaf, fruit, and stem bark of Delonix regia (Bojer ex Hook.) Raf. have been investigated with (13)C nuclear magnetic resonance ((13)C NMR) and high performance liquid chromatography electrospray ionization mass spectrometry (HPLC-ESI-MS) coupled with thiolysis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analyses. The results showed that these condensed tannins from D. regia possessed structural heterogeneity in monomer units and degree of polymerization. Propelargonidin (PP) and procyanidin (PC) were found in the leaf, fruit, and stem bark of D. regia, while prodelphinidin (PD) was found only in the leaves. The polymer chain lengths of condensed tannins from leaf and fruit organs were detected to be trimers to hexadecamers but from trimers to tridecamers for stem bark. B-type linkages were present in all these compounds. Condensed tannins from different parts of D. regia can be explored as tyrosinase inhibitors and food antioxidants because of their potent antityrosinase and antioxidant activities. The inhibitor concentration leading to 50% enzyme activity (IC(50)) was estimated to be 38 ± 1, 73 ± 2, and 54 ± 1.5 μg/mL for the condensed tannins of leaf, fruit, and stem bark. Condensed tannins extracted from stem bark exhibited the highest antioxidant activity; the DPPH scavenging activity (IC(50)) and the FRAP values were 90 ± 2 μg/mL and 5.42 ± 0.09 mmol AAE/g, respectively.

Inactivation Kinetics of Mushroom Tyrosinase in the Dimethyl Sulfoxide Solution

Mushroom tyrosinase (EC 1.14.18.1) is a kind of copper-containing oxidase that catalyzes both the hydroxylation of tyrosine into o-diphenols and the oxidation of o-diphenols into o-quinones and then forms brown or black pigments. In the present paper, the effects of dimethyl sulfoxide on the enzyme activity for the oxidation of L-3,4-dihydroxyphenylalanine (L-DOPA) have been studied. The results show that low concentrations of dimethyl sulfoxide (DMSO) can lead to reversible inactivation of the enzyme, and the IC50 is estimated to be 2.45 M. Inactivation of the enzyme by DMSO is classified as mixed type. The kinetics of inactivation of mushroom tyrosinase at low concentrations of DMSO solution has been studied using the kinetic method of the substrate reaction. The rate constants of inactivation have been determined. The results show the free enzyme molecule is more fragile than the enzyme–substrate complex in the DMSO solution. It is suggested that the presence of the substrate offers marked protection of this enzyme against inactivation by DMSO.

Inhibitory effects of cupferron on the monophenolase and diphenolase activity of mushroom tyrosinase

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

Inhibitory Effects of Propyl Gallate on Tyrosinase and Its Application in Controlling Pericarp Browning of Harvested Longan Fruits

Tyrosinase (EC 1.14.18.1), also known as polyphenol oxidase (PPO), is a key enzyme in pigment biosynthesis of organisms. The inhibitory effects of propyl gallate on the activity of mushroom tyrosinase and effects of propyl gallate on pericarp browning of harvested longan fruits in relation to phenolic metabolism were investigated. The results showed that propyl gallate could potently inhibit diphenolase activity of tyrosinase. The inhibitor concentration leading to 50% activity lost (IC50) was determined to be 0.685 mM. Kinetic analyses showed that propyl gallate was a reversible and mixed type inhibitor on this enzyme. The inhibition constants (K(IS) and K(I)) were determined to be 2.135 and 0.661 mM, respectively. Furthermore, the results also showed that propyl gallate treatment inhibited activities of PPO and POD in pericarp of harvested longan fruits, and maintained higher contents of total phenol and flavonoid of longan pericarp. Moreover, propyl gallate treatment also delayed the increases of browning index and browning degree in pericarp of harvested longan fruits. Therefore, application of propyl gallate may be a promising method for inhibiting tyrosinase activity, controlling pericarp browning, and extending shelf life of harvested longan fruits.

Cloning and tissue expressions of seven chitinase family genes in Litopenaeus vannamei

GH18 chitinase is a multi-gene family. The family plays important physiological roles in Crustacea, e.g. ecdysis and defense against pathogen. However, data about GH18 family are rather limited in Crustacea. In the study, different cloning strategies were adopted to clone chitinase genes of Litopenaeus vannamei, which is the most widely cultured shrimp. Seven chitinase family members were identified. Analysis of domain architectures showed the repeated CBM18 modules and catalytic domain of enzymatically inactive chitolectin in Crustacea for the first time. Comparing to the three known groups of crustacean chitinase, four of the seven members are located on new evolutionary clades thus enriched the chitinase family of Crustacea. Tissue expression profiles were investigated in eight tissues. Expression of CHT5 and CHID1 were both detected in the hemocyte by which the innate immunity activity was carried out. The domain architectures, evolutionary relationships and tissue expression patterns all provide reasonable explanation for the existence of multiple genes in crustacean chitinase family.

Inhibitory effects of anticancer peptide from Mercenaria on the BGC-823 cells and several enzymes

An anti‐cancer peptide was purified from the Mercenaria (Meretrix meretrix Linnaeus) by the method of chromatography on Sephadex G‐25 and FPLC, and its molecular weight was determined to be 3147 Da by the way of MALDI‐TOF mass spectrum. The effects of this peptide on human gastric gland carcinoma cells (BGC‐823) and their cytoskeletal morphology were investigated. The results showed that the peptide could inhibit the proliferation of BGC‐823 cells and obviously destroy the skeletal structures of the cells. When the concentration of the peptide reached 4.0 μg/ml, the inhibition percentage of the cell growth was about 60%. The effects of this anticancer peptide on the activities of superoxide dismutase (SOD), alkaline phosphatase (ALP) and tyrosinase were studied. The results showed that the peptide activated ALP and SOD, but inhibit the tyrosinase activity. When the concentration of the peptide reached to 0.5 μg/ml, the relative activities of SOD, ALP and tyrosinase were determined to be 188.5%, 122.0% and 27.5%, respectively.