• Journal of Microbiology and Biotechnology
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• v.14 no.3
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• pp.490-497
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• 2004

Although the E. coli heat-labile enterotoxin B subunit (LTB) is known to be a potent mucosal adjuvant towards co-administrated unrelated antigens and immunoregulator in T-helper 1-type-mediated autoimmune diseases, a more efficient and useful LTB is still required for prospective vaccine adjuvants. To determine whether a novel chimeric LTB subunit would produce an enhanced mucosal adjuvant activity and immune response, a number of LTB subunits were genetically fused with chimeric proteins using the epitope genes of the envelope glycoprotein E2 (gp51-54) from the classical swine fever virus (CSFV). It was found that the total serum immunoglobulin (Ig) levels of BALB/c mice orally immunized with chimeric proteins containing an N-terminal linked LTB subunit (LE1, LE2, and LE3) were higher than those of mice immunized with LTB, E2 epitope, and chimeric proteins that contained a C-terminal linked LTB subunit. In particular, immunization with LE1 markedly increased both the total serum Ig and fecal IgA level compared to immunization with LTB or the E2 epitope. Accordingly, the current results demonstrated that the LTB subunit in a chimeric protein exhibited a strong mucosal adjuvant effect as a carrier molecule, while the chimeric protein containing the LTB subunit stimulated the mucosal immune system by mediating the induction of antigen-specific serum Ig and mucosal IgA. Consequently, an LE1-mediated mucosal response may contribute to the development of effective antidiarrhea vaccine adjuvants.

• Journal of Microbiology and Biotechnology
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• v.17 no.6
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• pp.897-904
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• 2007

The aim of this paper is to discuss the methodology of our investigation of the dynamics of protein degradation and the total in situ protealytic activity in meso/eutrophic, eutrophic, and hypereutrophic freshwater environments. Analysis of the kinetics and rates of enzymatic release of amino acids in water samples preserved with sodium azide allows determination of the concentrations of labile proteins $(C_{LAB})$, and their half-life time $(T_{1/2})$. Moreover, it gives more realistic information on resultant activity in situ $(V_{T1/2})$ of ecto- and extracellular proteases that are responsible for the biological degradation of these compounds. Although the results provided by the proposed method are general y well correlated with those obtained by classical procedures, they better characterize the dynamics of protein degradation processes, especially in eutrophic or hypereutrophic lakes. In these environments, processes of protein decomposition occur mainly on the particles and depend primarily on a metabolic activity of seston-attached bacteria. The method was tested in three lakes. The different degree of eutrophication of these lakes was clearly demonstrated by the measured real proteolytic pattern and confirmed by conventional trophic state determinants.

• Asian-Australasian Journal of Animal Sciences
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• v.21 no.5
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• pp.754-768
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• 2008

The past decades have seen enormous advances in our understanding of how GH acts. GH is a pituitary-derived polypeptide hormone that has diverse physiological effects including the regulation of bone growth, carbohydrate and lipid metabolism. The effects of GH are mediated directly and indirectly through IGF-I. In addition, GH stimulates the hepatic production of ALS. In postnatal life, IGF-I and -II circulate as 150 kDa ternary complexes consisting of one molecule each of IGFBP-3 or IGFBP-5, IGF-I or IGF-II and ALS. It is now known that ALS increases significantly the half-lives of the IGFs, IGFBP-3 and -5, and therefore is responsible for maintaining a circulating reservoir for each of these proteins.

• Journal of Periodontal and Implant Science
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• v.39 no.sup2
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• pp.231-237
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• 2009

Purpose: Cytolethal distending toxin (CDT) is a family of heat-labile cytotoxins produced by several gram-negative mucosa-associated pathogens, including Aggregatibacter actinomycetemcomitans. CDT is well known to be capable of inducing growth arrest, morphological alterations, and eventually death in various cells. CDT belongs to a tripartite $AB_2$ toxin (CdtB: the enzymatic A subunit; CdtA and CdtC: the heterodimeric B subunit). Previous studies proposed that CdtA and CdtC together bind to a cell surface receptor and glycolipids act as a receptor for A. actinomycetemcomitans CDT (AaCDT). In this study, recombinant CdtA and CdtC proteins of AaCDT were co-expressed in a bacterial expression system and tested for their affinity for $GM_1$ ganglioside. Methods: The genes for CdtA and CdtC from A. actinomycetemcomitans Y4 were utilized to construct the expression vectors, pRSET-cdtA and pET28a-cdtC. Both CdtA and CdtC proteins were expressed in Escherichia coli BL21(DE3) and then purified using hexahistidine (His6) tag. The identity of purified protein was confirmed by anti-His6 antibody and monoclonal anti-CdtA antibody. Furthermore, the affinity of recombinant protein to $GM_1$ ganglioside was checked through ELISA. Results: Recombinant CdtA and CdtC proteins were expressed as soluble proteins and reacted to anti-His6 and monoclonal anti-CdtA antibodies. ELISA revealed that purified soluble CdtA-CdtC protein bound to $GM_1$ ganglioside, while CdtA alone did not. Conclusions: Co-expression of CdtA and CdtC proteins enhanced the solubility of the proteins in E. coli, leading to convenient preparation of active CdtA-CdtC, a critical material for the study of AaCDT pathogenesis.

• Journal of the Korean Society of Food Science and Nutrition
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• v.30 no.4
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• pp.574-578
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• 2001

Myofibrillar proteins were prepared from red muscle and white muscle of fresh water fish and sea water fish, and their thermostabilities and effect of temperature on the myofibrillar ATPase activities were compared. Differences in temperature dependency of myofibrillar ATPase activities were found between two species. Thermodynamic data for inactivation of myofibrillar proteins, such as D value, Z value, $\Delta$ $H^{{\neq}}$, $\Delta$ $G^{{\neq}}$ and $\Delta$ $S^{\neq}$ revealed that thermostabilities of myofibrillar proteins from fresh water fish were higher than those from sea water fish, and that myofibrillar proteins from red muscle were more heat labile than those from white muscle.

• Archives of Pharmacal Research
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• v.27 no.2
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• pp.206-216
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• 2004

The functional role of protein carboxylmethylation (PCM) has not yet been clearly elucidated in the tissue level. The biochemical feature of PCM in porcine spleen was therefore studied by investigating the methyl accepting capacity (MAC) of natural endogenous substrate proteins for protein carboxyl O-methyltransferase (PCMT) in various conditions. Strong acidic and alkaline-conditioned (at pH 11.0) analyses of the MAC indicated that approximately 65％ of total protein methylation seemed to be mediated by spleen PCMT. The hydrolytic kinetics of the PCM products, such as carboxylmethylesters (CMEs), under mild alkaline conditions revealed that there may be three different kinds of CMEs [displaying half-times (T$_{1}$2/) of 1.1 min (82.7％ of total CMEs), 13.9 min (4.6％), and 478.0 min (12.7％)], assuming that the majority of CME is base-labile and may be catalyzed by class I PCMT. In agreement with these results, several natural endogenous substrate proteins (14, 31 and 86 kDa) were identified strikingly by acidic-conditioned electrophoresis, and their MAC was lost upon alkaline conditions. On the other hand, other proteins (23 and 62 kDa) weakly appeared under alkaline conditions, indicating that PCM mediated by class II or III PCMT may be a minor reaction. The MAC of an isolated endogenous substrate protein (23-kDa) was also detected upon acidic-conditioned electrophoresis. Therefore, our date suggest that most spleen PCM may be catalyzed by class I PCMT, which participates in repairing aged proteins.

• YAKHAK HOEJI
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• v.35 no.5
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• pp.444-455
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• 1991

Two kinds of new lectin fractions (LOA-I, LOA-II) were obtained from loach (Misgurnus spp.) meat by 0.15 M NaCl extraction, salt fractionation, ion exchange and hydroxyapatite column chromatographies. On polyacrylamide gel electrophoresis, LOA-I exhibited one major and a few minor bands, but LOA-II exhibited three minor bands. The partially purified loach lectins agglutinated not only erythrocytes of human B and AB type, rabbit, dog, but also murine splenic lymphocytes. Agglutinability was relatively labile at various pH and stable at increasing temperature, but was not affected by tested several metal ions. By the sugar specificity test, D-glucosamine and metyl-$\beta$-galactopyranose inhibited agglutinating activity at a final concentration of 3 mM. The lectins contained relatively high amounts of aspartic acid, valine and leucine, but sulfur containing amino acids, cystein, methionine and isoleucine were not determined. LOA-I, LOA-II lectins were nonmitogenic toward murine lymphocytes.

• Fisheries and Aquatic Sciences
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• v.22 no.9
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• pp.19.1-19.10
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• 2019

We investigated the insulin-like growth factor 1 (IGF-1)/AKT signaling pathway involved in muscle formation, growth, and movement in the adductor muscle of triploid Pacific oyster, Crassostrea gigas. Large and small triploid oysters (LTs and STs) cultured under identical conditions were screened, and the signaling pathways of individuals with superior growth were compared and analyzed. mRNA and protein expression levels of actin, troponin, tropomyosin, and myosin, proteins important in muscle formation, were higher in LTs compared with STs. Expression levels of IGF-1, IGF binding protein (IGFBP), and IGFBP complex acid-labile subunit were also higher in LTs compared with STs. Phosphorylation of the IGF receptor as well as that of AKT was high in LTs. In addition, the expression of phosphomammalian target of rapamycin and phospho-glycogen synthase kinase $3{\beta}$ was increased and the expression of Forkhead box O3 was decreased in LTs. Therefore, we suggested that the IGF-1/AKT signaling pathway affects the formation, growth, and movement of the adductor muscle in triploid oysters.

• Journal of Microbiology and Biotechnology
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• v.20 no.3
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• pp.550-557
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• 2010

Escherichia coli (E. coli) heat-labile enterotoxin B subunit (LTB) was regarded as one of the most powerful mucosal immunoadjuvants eliciting strong immunoresponse to coadministered antigens. In the research, the high-level secretory expression of functional LTB was achieved in P. pastoris through high-density fermentation in a 5-1 fermentor. Meanwhile, the protein was expressed in E. coli by the way of inclusion body, although the gene was cloned from E. coli. Some positive yeast and E. coli transformants were obtained respectively by a series of screenings and identifications. Fusion proteins LTB-6$\times$His could be secreted into the supernatant of the medium after the recombinant P. pastoris was induced by 0.5% (v/v) methanol at $30^{\circ}C$, whereas E. coli transformants expressed target protein in inclusion body after being induced by 1 mM IPTG at $37^{\circ}C$. The expression level increased dramatically to 250-300 mg/l supernatant of fermentation in the former and 80-100 mg/l in the latter. The LTB-6$\times$His were purified to 95% purity by affinity chromatography and characterized by SDS-PAGE and Western blot. Adjuvant activity of target protein was analyzed by binding ability with GMI gangliosides. The MW of LTB-6$\times$His expressed in P. pastoris was greater than that in E. coli, which was equal to the expected 11 kDa, possibly resulted from glycosylation by P. pastoris that would enhance the immunogenicity of co-administered antigens. These data demonstrated that P. pastoris producing heterologous LTB has significant advantages in higher expression level and in adjuvant activity compared with the homologous E. coli system.

• Journal of Microbiology and Biotechnology
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• v.19 no.5
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• pp.502-510
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• 2009

Although the Escherichia coli heat-labile enterotoxin B subunit (LTB) has already been expressed in several different systems, including prokaryotic and eukaryotic organisms, studies regarding the synthesis of LTB into oligomeric structures of pentameric size in the budding yeast Saccharomyces cerevisiae have been limited. Therefore, this study used a functional signal peptide of the amylase 1A protein from rice to direct the yeast-expressed LTB towards the endoplasmci reticulum to oligomerize with the expected pentameric size. The expression and assembly of the recombinant LTB were confirmed in both the cell-free extract and culture media of the recombinant strain using a Western blot analysis. The binding of the LTB pentamers to intestinal epithelial cell membrane glycolipid receptors was further verified using a GM1-ganglioside enzyme-linked inmmunosorbent assay (GM1-ELISA). On the basis of the GM1-ELISA results, pentameric LTB proteins comprised approximately 0.5-2.0% of the total soluble proteins, and the maximum quantity of secreted LTB was estimated to be 3 mg/l after a 3-day cultivation period. Consequently, the synthesis of LTB monomers and their assembly into biologically active aligomers in a recombinant S. cerevisiae strain demonstrated the feasibility of using a GRAS microorganism-based adjuvant, as well as the development of carriers against mucosal disease.