Ling-Jing Zhang

Purification and characterization of a collagenolytic serine proteinase from the skeletal muscle of red sea bream (Pagrus major).

A collagenolytic serine proteinase (CSP) was purified from red sea bream (Pagrus major) skeletal muscle to homogeneity by ammonium sulfate fractionation and chromatographies including DEAE-Sephacel, Phenyl Sepharose and Hydroxyapatite. The molecular mass of CSP was approximately 85 kDa as estimated by SDS-PAGE and gel filtration. Optimum temperature and pH of CSP were 40 degrees C and 8.0, respectively. CSP was specifically inhibited by serine proteinase inhibitors, while inhibitors to other type proteinases did not show much inhibitory effects. The K(m) and k(cat) values of CSP for Boc-Leu-Lys-Arg-MCA were 3.58 microM and 0.13 s(-1) at 37 degrees C, respectively. Furthermore, CSP hydrolyzed gelatin and native type I collagen effectively though its degradation on myosin heavy chain (MHC) was not significant, suggesting its involvement in the texture tenderization of fish muscle during the post-mortem stage.

Purification, characterization, and analysis of the allergenic properties of myosin light chain in procambarus clarkii

Myosin light chain (MLC) plays a vital role in cell and muscle functions and has been identified as an allergen in shrimp. In this study, MLC with a molecular mass of 18 kDa was purified from crayfish (Procambarus clarkii) muscle. Its physicochemical characterization showed that the purified MLC is a glycoprotein with 4.3% carbohydrate, highly stable to heat, acid-alkali, and digestion, and weakly retains IgE-binding activity when its secondary structure was altered. Serological assays suggested that conformational epitopes predominate over linear epitopes in the purified MLC. Two isoforms of the MLC gene (MLC1 and MLC2) were cloned, and the purified MLC was identified as MLC1. Analysis of the secondary and tertiary structures of the MLCs indicated that MLC1 has four conformational epitopes and three linear epitopes, whereas MLC2 had a major conformational epitope and three linear epitopes. These results are significant for understanding hypersensitization of humans to crayfish.

Suppression of Th2 immune responses by the sulfated polysaccharide from Porphyra haitanensis in tropomyosin-sensitized mice.

The sulfated polysaccharide from Porphyra was hypothesized to exhibit immunoregulatory, anti-tumor and anti-inflammatory activity, but its anti-allergic activity is not fully understood. Therefore, the aim of this study was to isolate sulfated polysaccharide from Porphyra haitanensis (PHPS) and investigate its anti-allergic potential using a tropomyosin (TM)-induced mouse allergy model. Intraperitoneal injection of PHPS suppressed the allergic reaction by modulating serum IgE, IgG1 and IgG2a levels in mice. In particular, when PHPS was injected prior to the first immunization with TM, the IgE level decreased by 34.2% compared with the control (PBS) group. Oral therapeutic administration of PHPS to TM-sensitized mice decreased histamine release and repaired the pathology in the jejunum of the small intestine. In vitro, the mRNA expressions of the TM-induced Th2 cytokines (interleukin-4 (IL-4), IL-5 and IL-13) in splenic lymphocytes were reduced by PHPS; however, the expression of Th1 and regulatory cytokines (interferon gamma (IFN-γ) and IL-10) were up-regulated in PHPS-treated splenic lymphocytes. In the splenic lymphocyte supernatant, the IL-4, IL-13 and IFN-γ levels were also regulated by PHPS. Moreover, PHPS induced IFN-γ secretion via the Jun N-terminal kinase (JNK) and Janus kinase 2 (JAK2) signaling pathways. Therefore, these results suggest that PHPS suppresses the TM-induced allergic reaction, possibly by modulating the imbalance of the Th1/Th2 immune response.

Purification and Characterization of Protamine, the Allergen from the Milt of Large Yellow Croaker (Pseudosciaena crocea), and Its Components.

The protamine in fish milt can cause anaphylaxis in humans. To determine the allergen in the milt of large yellow croaker (Pseudosciaena crocea), crude extracts were incubated with sera from allergic patients. The results showed that a 12 kDa multicomponent protein was the major allergen in the milt of large yellow croaker. The multicomponent protein was purified, and physicochemical characterization showed that it was a glycoprotein, highly stable in acid-alkali conditions, and weakly retained immunoglobulin E (IgE)-binding activity at high temperatures. Separation and immunoreactivity analysis of the components of the multicomponent protein showed that it had six components, and component 5 had the strongest IgE-binding activity with patient sera. N-terminal sequencing confirmed the multicomponent protein was protamine. Following analysis of protamine from different fish by reversed-phase liquid chromatography and circular dichroism spectra, the protamines from different fish were found to have a similar secondary structure, although their components were different.

Identification and inhibition of histamine-forming bacteria in blue scad (Decapterus maruadsi) and chub mackerel (Scomber japonicus).

In this study, we investigated the differences in histamine accumulation between blue scad and chub mackerel and methods of inhibiting histamine-forming bacteria and controlling histamine accumulation in fish. The free histidine contents in blue scad and chub mackerel were 1.45 and 2.75 mg/g, respectively. The histamine-forming bacteria isolated from them were identified as Citrobacter freundii, Citrobacter braakii, and Enterobacter aerogenes using 16S rDNA sequence analysis, the VITEK 2 Compact system, and MALDI-TOF MS. The histamine-producing capacities of C. freundii, C. braakii, and E. aerogenes were 470, 1,057, and 4,213 mg/liter, respectively, after culture at 37°C for 48 h. Among the different antimicrobials and preservatives tested, potassium sorbate and sodium diacetate effectively inhibited the histamine-forming bacteria and their histamine production. After chub mackerel was dipped into 0.5% potassium sorbate or sodium diacetate, its histamine content increased more slowly at room temperature. Therefore, a potassium sorbate or sodium diacetate dipping treatment could effectively control histamine accumulation in fish.

Involvement of clip-domain serine protease in the anti-Vibrio immune response of abalone (Haliotis discus hannai)–Molecular cloning, characterization and functional analysis

ABSTRACT Vibrio parahemolyticus (V. parahemolyticus) is a major pathogen for abalone, an important economical shellfish in coastal area of China. There is little known about the abalone innate immune system against pathogen infection. Clip‐domain serine proteases (cSPs) are increasingly recognized to play important roles in host immune defense in invertebrates. In this study, we cloned a cSP (Hdh‐cSP) from abalone (Haliotis discus hannai). We found out that Hdh‐cSP was widely expressed in multiple tissues of abalone, with highest level in the immune‐like organ, hepatopancreas. V. parahemolyticus infection induced significantly elevated expression of Hdh‐cSP in addition to better‐characterized innate immune component genes including Rel/NF‐&kgr;B, allograft inflammatory factor (ALInFa), macrophage expressed protein (MEP) and caspase‐8. Importantly, the silencing of Hdh‐cSP reduced the expression of these genes, suggesting that Hdh‐cSP was an upstream regulatory factor in V. parahemolyticus infection. Further analysis showed that apoptosis of hemocytes was inhibited when the transcription of Hdh‐cSP was knocked down, suggesting that Hdh‐cSP participated in cell apoptosis by regulation of caspase 8 expression in V. parahemolyticus infection. Therefore, our study established an important role of cSP in the innate immunity against V. parahemolyticus infection in abalone. HighlightsA clip‐domain serine protease (Hdh‐cSP) was identified in Haliotis discus hannai.Induced expression of Hdh‐cSP was observed after Vibrio parahemolyticus infection.Hdh‐cSP regulated the expression of some well‐known immune factors.Hdh‐cSP was involved in the regulation of hemocytes apoptosis in abalone.