Beiwei Zhu

Characterization of an i-type lysozyme gene from the sea cucumber Stichopus japonicus, and enzymatic and nonenzymatic antimicrobial activities of its recombinant protein

Because sea cucumbers lack a well-developed immune system and can ingest pathogenic bacteria together with food, some form of active antibacterial substances must be present in the body for defense. In this study, the cDNA of an i-type lysozyme from the sea cucumber Stichopus japonicus (designated SjLys) was cloned by RT-PCR and RACE PCR techniques. The full length cDNA of SjLys was 713 bp with an open reading frame of 438 bp coding for 145 amino acids. Two catalytic residues (Glu34 and Asp47), conserved in i-type lysozymes, and a highly conserved region near the active site, MDVGSLSCG(P\Y)(Y\F)QIK, were detected in SjLys. In addition, the domain structure analysis of SjLys showed that it is highly similar to the medicinal leech destabilase, which belongs to a new phylogenetic family of invertebrate lysozymes possessing both glycosidase and isopeptidase activities. To gain insight into the in vitro antimicrobial activities of SjLys, the mature peptide coding region was heterologously expressed in Escherichia coli. The recombinant SjLys protein displayed an inhibitive effect on the growth of the tested Gram-positive and Gram-negative bacteria. A remarkable finding is that the recombinant SjLys exhibited more potent activities against all tested bacterial strains after heat-treating at 100 degrees C for 50 min. These results indicated that the S. japonicus lysozyme is an enzyme with combined enzymatic (glycosidase) and nonenzymatic antibacterial action.

Docosahexaenoic acid induces ERK1/2 activation and neuritogenesis via intracellular reactive oxygen species production in human neuroblastoma SH-SY5Y cells

Docosahexaenoic acid (22: 6n-3; DHA) is a long chain polyunsaturated fatty acid that exists highly enriched in fish oil, and it is one of the low molecular weight food chemicals which can pass a blood brain barrier. A preliminary survey of several fatty acids for expression of growth-associated protein-43 (GAP-43), a marker of axonal growth, identified DHA as one of the most potent inducers. The human neuroblastoma SH-SY5Y cells exposed to DHA showed significant and dose-dependent increases in the percentage of cells with longer neurites. To elucidate signaling mechanisms involved in DHA-enhanced basal neuritogenesis, we examined the role of extracellular signal-regulated kinase (ERK)1/2 and intracellular reactive oxygen species (ROS) production using SH-SY5Y cells. From immunoblotting experiments, we observed that DHA induced the ROS production, protein tyrosine phosphatase inhibition, mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) phosphorylation, and sequentially ERK1/2 phosphorylation, the last of which was significantly reduced by MEK inhibitor U0126. Both antioxidants and MEK inhibitor affected DHA-induced GAP-43 expression, whereas the specific PI3K inhibitor LY294002 did not. We found that total protein tyrosine phosphatase activity was also downregulated by DHA treatment, which was counteracted by antioxidant pretreatment. These results suggest that the ROS-dependent ERK pathway, rather than PI3K, plays an important role during DHA-enhanced neurite outgrowth.

Purification and characterization of a cathepsin L-like enzyme from the body wall of the sea cucumber Stichopus japonicus

Cathepsin L-like enzyme was purified from the body wall of the sea cucumber Stichopus japonicus by an integral method involving ammonium sulfate precipitation and a series of column chromatographies on DEAE Sepharose CL-6B, Sephadex G-75, and TSK-GEL. The molecular mass of the purified enzyme was estimated to be 63 kDa by SDS–PAGE. The enzyme cleaved N-carbobenzoxy-phenylalanine-arginine 7-amido-4-methylcoumarin with K m (69.92 μM) and k cat (12.80/S) hardly hydrolyzed N-carbobenzoxy-arginine-arginine 7-amido-4-methylcoumarin and L-arginine 7-amido-4-methylcoumarin. The optimum pH and temperature for the purified enzyme were found to be 5.0 and 50 °C. It showed thermal stability below 40 °C. The activity was inhibited by sulfhydryl reagents and activated by reducing agents. These results suggest that the purified enzyme was a cathepsin L-like enzyme and that it existed in the form of its enzyme-inhibitor complex or precursor.

Purification and bioactivity of a sulphated polysaccharide conjugate from viscera of abalone Haliotis discus hannai Ino

Abstract A sulphated polysaccharide conjugate ACP I was purified from the viscera of abalone Haliotis discus hannai Ino, and its immunostimulatory and anti-tumour activities were studied in vivo. Effect of ACP I on immune function was investigated in normal and cyclophosphamide-induced immunosuppressive mice. The anti-tumour effect was investigated by growth inhibition of tumour. Dose-dependent increase in spleen and thymus relative weight, lymphocyte proliferation, phagocytosis of macrophage, natural killer cell activity and antibody production was observed in all the tested mice administered with 10, 20 and 40 mg kg−1 body weight of ACP I. Significant increase of the above function was observed especially in immunosuppressive mice at dose of 40 mg kg−1. A significant inhibition of tumour growth was observed in 10, 20 and 40 mg kg−1 ACP I groups, with inhibitory rates of 40.24, 41.91 and 46.43%, respectively. Results suggested that ACP I might have immunomodulating and anti-tumour potential deserving application in functional food industry.

Autophagy plays a potential role in the process of sea cucumber body wall “melting” induced by UV irradiation

The changes of tissue appearances and structures in the process of UV-induced “melting” for sea cucumber (Stichopus japonicus) body wall were studied. And the localization and determination of acid phosphatase (ACP), Cathepsin B and Cathepsin L activities were also investigated. The results show that the connective tissue was damaged with many hollows emerging and the regular collagen bundles were broken apart into irregular fragments. Margination of condensed chromatin at the nuclear membrane was observed. Both Golgi’s body and endoplasmic reticulum swelled, curled, and eventually double-or multi-lamellar vesicles were formed. A number of autophagic vesicles distributed in all through the whole cytoplasm. ACP becomes more active after UV irradiation. The activities of cathepsin B and cathepsin L increased in UV-treated sea cucumbers and both achieved their maximum under certain conditions. It indicates that autophagy plays a potential role in the “melting” process for sea cucumber body wall induced by UV irradiation.

(−)-Epigallocatechin-3-gallate induces up-regulation of Th1 and Th2 cytokine genes in Jurkat T cells

In the present study, we found that (-)-epigallocatechin-3-gallate (EGCG) significantly up-regulated the mRNA expression of the Th1/Th2 cytokines including IL-2, IFN-gamma, IL-5 and IL-13 in Jurkat T cells. The EGCG-induced mRNA up-regulation of IL-2 and IL-5 was predominantly affected by the extracellular signal-regulated protein kinase (ERK) signalling, whereas IL-13 gene expression, the most responsive to the EGCG treatment, was dependent on neither ERK nor c-jun NH(2)-terminal kinase (JNK) signalling. IFN-gamma gene expression was partially mitigated by both inhibitors of the ERK and JNK pathways. Furthermore, catalase significantly attenuated the intracellular peroxide production, phosphorylation of ERK and JNK, and all cytokine gene expressions induced by EGCG. In addition, physiologically relevant concentrations of both EGCG and H(2)O(2)-induced up-regulation of IL-5 gene expression. Our findings provide biological evidence that EGCG induces Th1/Th2 cytokine mRNA expression via H(2)O(2) production followed by activation of ERK or JNK in Jurkat T cells.

Proteolysis of noncollagenous proteins in sea cucumber, Stichopus japonicus, body wall: characterisation and the effects of cysteine protease inhibitors.

Proteolysis of noncollagenous proteins in sea cucumber, Stichopus Japonicus, body wall (sjBW) was investigated. The proteins removed from sjBW by SDS and urea extraction were mainly noncollagenous proteins with molecular weights about 200kDa (Band I) and 44kDa (Band II), respectively. Band I and Band II were identified as major yolk protein (MYP) and actin, respectively, from holothurian species by liquid chromatography-mass spectrometry (LC-MS/MS) with significant scores. Based on TCA-soluble oligopeptide assay, the optimum proteolysis condition of noncollagenous proteins was at 46.3°C and pH 6.1, by response surface methodology. The proteolysis of MYP, and actin, was partially inhibited by cysteine protease inhibitors, including Trans-epoxysuccinyl-l-leucyl-amido (4-guanidino) butane (E-64), iodoacetic acid, antipain and whey protein concentrate. These results suggest that cysteine proteases are partially involved in the proteolysis of noncollagenous proteins in body wall of sea cucumber, S. japonicus.

Effect of Dimethyl Sulfides on the Induction of Apoptosis in Human Leukemia Jurkat Cells and HL-60 Cells

Organosulfur compounds have been established to possess anticancer effects. To provide a better understanding of the biological function of dimethyl sulfides, dimethyl monosulfide (Me2S), dimethyl disulfide (Me2S2), dimethyl trisulfide (Me2S3) and dimethyl tetrasulfide (Me2S4) were used as experimental materials to investigate their effects on apoptosis induction in human leukemia Jurkat cells and HL-60 cells. Treatment with 20 μM dimethyl sulfides for 24 h decreased the viability of both cells. The cell viability-reducing effect of these sulfides was in the following order: Me2S4 ≈ Me2S3 > Me2S2 ≈ Me2S for Jurkat cells and Me2S4 > Me2S3 > Me2S2 ≈ Me2S for HL-60 cells. Me2S3 and Me2S4 significantly induced DNA fragmentation and caspase-3 activation. The addition of GSH or NAC completely suppressed the sulfide-induced apoptosis. Our results indicate that dimethyl sulfides with a larger number of sulfur atoms more strongly induced apoptosis in both human leukemia cells via ROS production and caspase-3 activation.

Characterization of glycerophospholipid molecular species in six species of edible clams by high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry

The molecular species of glycerophosphocholine (GPCho), glycerophosphoethanolamine (GPEtn), glycerophosphoserine (GPSer), lysoglycerophosphocholine (LGPCho) and lysoglycerophosphoethanolamine (LGPEtn) from six species of edible clams were characterized by using high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry. At least 435, 453, 468, 443, 427 and 444 glycerophospholipid (GP) molecular species were characterized, respectively, from Cyclina sinensis, Mactra chinensis Philippi, Mactra veneriformis Reeve, Meretrix meretrix, Ruditapes philippinarum and Saxidomus purpurata. Most of the predominant GP molecular species in clam contained polyunsaturated fatty acids (PUFA), mainly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), indicating that clam is a potential resource of GP enriched PUFA. According to the amount of the major molecular species containing EPA and DHA, Cyclina sinensis was the best fit species for GPCho, Mactra veneriformis Reeve was the best fit species for GPEtn, Mactra chinensis Philippi was the best fit species for GPSer and LGPEtn, and Saxidomus purpurata was the best fit species for LGPCho.

Comparison of polysaccharides of Haliotis discus hannai and Volutharpa ampullacea perryi by PMP-HPLC-MSn analysis upon acid hydrolysis

Haliotis discus hannai Ino (Haliotis) is a highly valued marine shellfish, and it is sometimes replaced by another cheaper Gastropoda mollusk, Volutharpa ampullacea perryi (Volutharpa). Polysaccharides from pleopods, viscera and gonads of these two gastropods were compared by analyzing the mono- and di-saccharides in their acid hydrolysates using high performance liquid chromatography-mass spectrometry (HPLC-MS(n)) after 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatization. Disaccharide analysis revealed the distribution of uronic acid-containing polysaccharides (UACPs) in the biological samples. GlcA-(1 → 2)-Man, GlcA-(1 → 3)-GalN, and another disaccharide consisting of a hexuronic acid linked to a hexose were found in the hydrolysates, which indicated the existence of AGSP (abalone gonad sulfated polysaccharide) with the backbone composed of → 2)-α-Man(1 → 4)-β-GlcA(1 → repeating unit, AAP (abalone glycosaminoglycan-like polysaccharide) with the backbone of → 3)-GalNAc-(1 → 2)-GlcA-(1 → 3)-GalNAc-(1 → 4)-GlcA-(1 → repeating unit, and unidentified DS1P containing a hexuronic acid linked to a hexose unit, respectively. As shown by extracted ion chromatograms (XICs), AAP was the only UACP found in pleopods of the two gastropods; gonads and viscera of Haliotis contained DS1P and AGSP, while those of Volutharpa contained DS1P, AGSP as well as AAP. Monosaccharides in the acid hydrolysates were demonstrated in XICs by extracting their corresponding PMP derivative quasi-molecular ions one by one, and the results indicated the similar conclusion to the disaccharide analysis. Therefore, it could be concluded that polysaccharides from pleopods of the two gastropods are very similar, while those from their viscera and gonads differ greatly.