Tung-Kung Wu

Antioxidant properties of a new antioxidative peptide from algae protein waste hydrolysate in different oxidation systems

Microalgae have been a popular edible food, but there are no known reports on the antioxidative peptides derived from microalgae. The algae protein waste, which is normally discarded as animal feed, is a by-product during production of algae essence from microalgae, Chlorella vulgaris. Algae protein waste was hydrolyzed using pepsin, and a potent antioxidative peptide of VECYGPNRPQF was separated and isolated. The peptide could efficiently quench a variety of free radicals, including hydroxyl radical, superoxide radical, peroxyl radical, DPPH radical and ABTS radicals, and performed more efficiently than that observed for BHT, Trolox and peptides from marine protein sources in most cases. The purified peptide also has significant protective effects on DNA and prevents cellular damage caused by hydroxyl radicals. In addition, the peptide has gastrointestinal enzyme-resistance and no cytotoxicity observed in human lung fibroblasts cell lines (WI-38) in vitro. These results demonstrate that inexpensive algae protein waste could be a new alternative to produce antioxidative peptides.

Anticancer and Antioxidant Activities of the Peptide Fraction from Algae Protein Waste

Algae protein waste is a byproduct during production of algae essence from Chlorella vulgaris. There is no known report on the anticancer peptides derived from the microalgae protein waste. In this paper, the peptide fraction isolated from pepsin hydrolysate of algae protein waste had strong dose-dependent antiproliferation and induced a post-G1 cell cycle arrest in AGS cells; however, no cytotoxicity was observed in WI-38 lung fibroblasts cells in vitro. The peptide fraction also revealed much better antioxidant activity toward peroxyl radicals and LDL than those of Trolox. Among these peptides, a potent antiproliferative, antioxidant, and NO-production-inhibiting hendecapeptide was isolated, and its amino acid sequence was VECYGPNRPQF. These results demonstrate that inexpensive algae protein waste could be a new alternative to produce anticancer peptides.

Saccharomyces cerevisiae Oxidosqualene- Lanosterol Cyclase: A Chemistry-Biology Interdisciplinary Study of the Protein's Structure-Function-Reaction Mechanism Relationships

The oxidosqualene cyclases (EC 5.4.99-) constitute a family of enzymes that catalyze diverse cyclization/rearrangement reactions of (3S)-2,3-oxidosqualene into a distinct array of sterols and triterpenes. The relationship between the cyclization mechanism and the enzymatic structure is extremely complex and compelling. This review covers the historical achievements of biomimetic studies and current progress in structural biology, molecular genetics, and bioinformatics studies to elucidate the mechanistic and structure-function relationships of the Saccharomyces cerevisiae oxidosqualene-lanosterol cyclase-catalyzed cyclization/rearrangement reaction.

Crystal Structure and Inhibition Studies of Transglutaminase from Streptomyces mobaraense

The crystal structure of the microbial transglutaminase (MTGase) zymogen from Streptomyces mobaraense has been determined at 1.9-Å resolution using the molecular replacement method based on the crystal structure of the mature MTGase. The overall structure of this zymogen is similar to that of the mature form, consisting of a single disk-like domain with a deep active cleft at the edge of the molecule. A major portion of the prosequence (45 additional amino acid residues at the N terminus of the mature transglutaminase) folds into an L-shaped structure, consisting of an extended N-terminal segment linked with a one-turn short helix and a long α-helix. Two key residues in the short helix of the prosequence, Tyr-12 and Tyr-16, are located on top of the catalytic triad (Cys-110, Asp-301, and His-320) to block access of the substrate acyl donors and acceptors. Biochemical characterization of the mature MTGase, using N-α-benzyloxycarbonyl-l-glutaminylglycine as a substrate, revealed apparent Km and kcat/Km values of 52.66 mm and 40.42 mm−1 min−1, respectively. Inhibition studies using the partial prosequence SYAETYR and homologous sequence SQAETYR showed a noncompetitive inhibition mechanism with IC50 values of 0.75 and 0.65 mm, respectively, but no cross-linking product formation. Nevertheless, the prosequence homologous oligopeptide SQAETQR, with Tyr-12 and Tyr-16 each replaced with Gln, exhibited inhibitory activity with the formation of the SQAETQR-monodansylcadaverine fluorophore cross-linking product (SQAETQR-C-DNS). MALDI-TOF tandem MS analysis of SQAETQR-C-DNS revealed molecular masses corresponding to those of NSQAETQC-C-DNS and C-DNS-NQRC sequences, suggesting the incorporation of C-DNS onto the C-terminal Gln residue of the prosequence homologous oligopeptide. These results support the putative functional roles of both Tyr residues in substrate binding and inhibition.

Molecular genetic analysis reveals that a nonribosomal peptide synthetase-like (NRPS-like) gene in Aspergillus nidulans is responsible for microperfuranone biosynthesis

Genome sequencing of Aspergillus species including Aspergillus nidulans has revealed that there are far more secondary metabolite biosynthetic gene clusters than secondary metabolites isolated from these organisms. This implies that these organisms can produce additional secondary metabolites, which have not yet been elucidated. The A. nidulans genome contains 12 nonribosomal peptide synthetase (NRPS), one hybrid polyketide synthase/NRPS, and 14 NRPS-like genes. The only NRPS-like gene in A. nidulans with a known product is tdiA, which is involved in terrequinone A biosynthesis. To attempt to identify the products of these NRPS-like genes, we replaced the native promoters of the NRPS-like genes with the inducible alcohol dehydrogenase (alcA) promoter. Our results demonstrated that induction of the single NRPS-like gene AN3396.4 led to the enhanced production of microperfuranone. Furthermore, heterologous expression of AN3396.4 in Aspergillus niger confirmed that only one NRPS-like gene, AN3396.4, is necessary for the production of microperfuranone.

Post-resuscitative clinical features in the first hour after achieving sustained ROSC predict the duration of survival in children with non-traumatic out-of-hospital cardiac arrest

AIM OF THE STUDY Although sustained return of spontaneous circulation (ROSC) can be initially established after resuscitation from non-traumatic out-of-hospital cardiac arrest (OHCA) in some children, many of the children lose spontaneous circulation during hospital stay and do not survive to discharge. The aim of this study was to determine the clinical features during the first hour after ROSC that may predict survival to hospital discharge. METHODS We retrospectively evaluated the medical records of 228 children who presented to the emergency department without spontaneous circulation following non-traumatic OHCA during the period January 1996 to December 2008. Among these children, 80 achieved sustained ROSC for at least 20 min. The post-resuscitative clinical features during the first hour after achieving sustained ROSC that correlated with survival, median duration of survival, and death were analyzed. RESULTS Among the 80 children who achieved sustained ROSC for at least 20 min, 28 survived to hospital discharge and 6 had good neurologic outcomes (PCPC scale=1 or 2). Post-resuscitative clinical features associated with survival included sinus cardiac rhythm (p=0.012), normal heart rate (p=0.008), normal blood pressure (p<0.001), urine output>1 ml/kg/h (p=0.002), normal skin color (p=0.016), lack of cardiopulmonary resuscitation (CPR)-induced rib fracture (p=0.044), initial Glasgow Coma Scale score>7 (p<0.001), and duration of in-hospital CPR<or=10 min (p<0.001). Furthermore, these variables were also significantly associated with the duration of survival (all p<0.05). CONCLUSIONS The most important predictors of survival to hospital discharge in children with OHCA who achieve sustained ROSC are a normal heart rate, normal blood pressure, and an initial urine output>1 ml/kg/h.

Enzymatic formation of multiple triterpenes by mutation of tyrosine 510 of the oxidosqualene-lanosterol cyclase from saccharomyces cerevisiae

Triterpene cyclases constitute a family of enzymes that catalyze diverse and complex carbocationic cyclization/rearrangement reactions of squalene and (3S)-2,3-oxidosqualene (OS) to generate a distinct array of sterols and triterpenes. A major determinant for the triterpenoid diversity is believed to be the precise control of conformation between substrate and enzyme, as well as the position of the carbocation intermediate formation. For example, both oxidosqualene-lanosterol cyclase (ERG7, EC 5.4.99.7) and oxidosqualene-cycloartenol synthase (CAS, EC 5.4.99.8) bind oxidosqualene in a chair–boat– chair conformation, initiate and propagate cyclization to form a protosteryl cation, and then promote 1,2-shifts of hydride and methyl groups to the lanosteryl C8 cation. The lanosterol formation is accomplished through the final deprotonation, abstracting a proton originally at C9 or after a hydride shift from C9 to C8. Cycloartenol is formed after a hydride shift from C9 to C8, followed by 9b,19-cyclopropane ring closure. Elegant molecular-genetic and bioorganic investigations have recently identified several amino acid residues that are critical in probing putative active sites and determining prod-

Importance of Saccharomyces cerevisiae Oxidosqualene-Lanosterol Cyclase Tyrosine 707 Residue for Chair-Boat Bicyclic Ring Formation and Deprotonation Reactions

A contact mapping strategy was applied to identify putative amino acid residues that influence the oxidosqualene-lanosterol B-ring cyclization reaction. A bicyclic intermediate with two altered deprotonation products, in conjunction with lanosterol, were isolated from the ERG7(Y707X) mutants, indicating that the Tyr707 residue may play a functional role in stabilizing the chair-boat bicyclic C-8 cation and the lanosteryl C-8/C-9 cation intermediates.

CMOS chip as luminescent sensor for biochemical reactions

We describe a novel biochemical sensing method and its potential new biosensing applications. A light-sensitive complementary metal oxide semiconductor (CMOS) chip prepared through a standard 0.5-/spl mu/m CMOS process was developed for measuring biochemical reactions. A light producing enzymatic reaction catalyzed by horseradish peroxidase (HRP) was designed as a platform reaction to determine the concentration of hydrogen peroxide (H/sub 2/O/sub 2/) by the CMOS chip with a standard semiconductor parameter analyzer (HP4145). The kinetics of enzymatic reaction were determined and compared with a standard and sophisticated fluorometer (Hitachi F-4500) in a biochemical laboratory. Similar results were obtained by both instruments. Using glucose oxidase as an example, we further demonstrated that the HRP platform can be used to determine other H/sub 2/O/sub 2/ producing reactions with the CMOS system. The result points to an important application of the CMOS chip in biological measurements and in diagnosis of various health factors.

Identification of a Vibrio furnissii Oligopeptide Permease and Characterization of Its In Vitro Hemolytic Activity

We describe purification and characterization of an oligopeptide permease protein (Hly-OppA) from Vibrio furnissii that has multifaceted functions in solute binding, in in vitro hemolysis, in antibiotic resistance, and as a virulence factor in bacterial pathogenesis. The solute-binding function was revealed by N-terminal and internal peptide sequences of the purified protein and was confirmed by discernible effects on oligopeptide binding, by accumulation of fluorescent substrates, and by fluorescent substrate-antibiotic competition assay experiments. The purified protein exhibited host-specific in vitro hemolytic activity against various mammalian erythrocytes and apparent cytotoxicity in CHO-K1 cells. Recombinant Hly-OppA protein and an anti-Hly-OppA monoclonal antibody exhibited and neutralized the in vitro hemolytic activity, respectively, which further confirmed the hemolytic activity of the gene product. In addition, a V. furnissii hly-oppA knockout mutant caused less mortality than the wild-type strain when it was inoculated into BALB/c mice, indicating the virulence function of this protein. Finally, the in vitro hemolytic activity was also confirmed with homologous ATP-binding cassette-type transporter proteins from other Vibrio species.