• Journal of Microbiology and Biotechnology
• /
• v.20 no.2
• /
• pp.375-382
• /
• 2010

The available techniques for heterologous protein secretion in Lactobacillus strains are limited. The aim of the present study was to develop an efficient protein-secretion system using recombinant lactobacilli for various applications such as live delivery of biotherapeutics. For the construction of expression vectors, the Lactobacillus brevis slpA promoter, Lactobacillus casei prtP signal sequence, and mouse IL-10 sequences were used as a model system. Interestingly, the slpA promoter exhibited strong activity in L. casei, contrary to previous observations. In order to stabilize replication of the plasmid in E. coli, a removable terminator sequence was built into the promoter region. For the improvement of secretion efficiency, a DTNSD oligopeptide was added to the cleavage site of signal peptidase. The resulting plasmids provided remarkably efficient IL-10 secretion. Accumulation of the protein in the culture supernatant varied widely according to the pH conditions. By analysis of the secreted protein, formation of homodimers, and biological activity, IL-10 was confirmed to be functional. The presently constructed plasmids could be useful tools for heterologous protein secretion in L. casei.

• Journal of Nutrition and Health
• /
• v.27 no.9
• /
• pp.910-923
• /
• 1994

The effects of individual fatty acids, differing in their degree of unsaturation(18:0, 18:1, 18:2 and 18:3) on the biosynthesis and secretion and lipids were investigated in Hep-G2 cells. Synthesis of apolipoprotein was measured by the incorporation of 3H-leucine into apolipoprotein(d<1.21g/ml) and synthesis of lipids was measured by the incorporation of 3H-glycerol and 14C-acetate into various lipid classes. Inclusion of 1.0mM of each fatty acids into the culture medium significantly increased the synthesis of total apolipoprotein and Apo B(p<0.05). However, addition of fatty acid did not affect the synthesis of cellular and medium protein. Among different fatty acids tested, oleic acid had the greatest effect on Apo B synthesis. While stearic, linoleic and linolenic acid, all had similar effects. The secretion of triglyceride into the medium markedly increased in all fatty acid groups being 5-6 times over the albumin control. The triglyceride secretion was the highest int he oleic acid group. The secretion of phospholipid and cholesterol also increased with triglyceride output. A positive relationship existed between the output of lipoprotein-triglyceride and Apo B. Since the synthesis of Apo B was significantly increased when various fatty acids were included into the culture medium, part of the apparently stimulated synthesis of the apolipoprotein may be in response to the increased formation and secretion of lipoprotein lipids.

• Korean Journal of Microbiology
• /
• v.31 no.3
• /
• pp.203-207
• /
• 1993

Five different secretion vectors were constructed by varying the signal sequences and .alpha.-antitrypsin (.alpha.1-AT) a numan secretory protein, was produced from yeast cells. The signal sequences used are those of acid phosphatase (PH05) and .alpha.-factor (M f.alphal1) of Saccharomyces cerevisiae, glucoamylase (STA1) of Saccharomyces diastaticus, and human .alpha.1-AT. Four vectors directed the efficient secretion of .alpha.1-AT ito the culture media. The secretion vector carrying the glucoamylase signal sequence (pGAT11) showed the highest efficiency of secretion. About 70% of .alpha.1-AT produce dwere secreted into the media. The endo H treatment of partially purified .alpha.1-AT indicates that the secreted .alpha.1-AT appeared to be glycosylated. This glycosylation pattern was altered when amino acid substitution mutations were introduced at the three glycosylation sites of .alpha.-AT.

• Toxicological Research
• /
• v.18 no.4
• /
• pp.363-367
• /
• 2002

Reactive of gen species (ROS) contribute to several cellular function and are involved in the regulation of signal transduction, gene expression, and proliferation. In the present study, we investigated the effect of $H_2O_2$ treatment on IgM secretion in LPS-stimulated murine B Iymphoma, CH12.LX. Cells were treated directly With $H_2O_2$ and stimulated with LPS. $H_2O_2$ treatment during 72 h time period inhibited IgM secretion in LPS-stimulated CH12.LX cells in a dose- and time-dependent manners. After treatment with 50 $\mu\textrm{M}$ $H_2O_2$ during 72 h time period, the level of IgM in LPS-stimulated CH12.LX cells was markedly decreased, whereas cell viability was not significantly changed. Addition of $H_2O_2$ concomitantly with LPS, or 12 h post-LPS stimulation, produced a significant inhibition of IgM secretion, Whereas inhibitory effect of $H_2O_2$ on IgM secretion was not observed when added 24 h after LPS stimulation. These findings suggest that $H_2O_2$ can inhibit the secretion of IgM in LPS-stimulated CH15.LX cells, and may alter the events necessary for terminal B cell differentiation.

• Asian-Australasian Journal of Animal Sciences
• /
• v.13 no.8
• /
• pp.1044-1049
• /
• 2000

Three wethers fitted with silastic catheters for collection of pancreatic juice, and cannulas located in the abomasum and the duodenum were used to investigate the effects of different hay and energy intake on pancreatic exocrine secretion. The wethers were fed Italian ryegrass hay or alfalfa hay at maintenance energy requirement and alfalfa hay ad libitum. High energy intake from alfalfa significantly increased abomasal flow of dry matter and both the concentration and daily secretion of ${\alpha}-amylase $. The high energy intake also tended to increase daily secretion of lipase, trypsin and chymotrypsin through the large volume of pancreatic juice. Compared with Italian ryegrass hay, alfalfa hay at the maintenance decreased abomasal dry matter flow, but increased concentration of ${\alpha}-amylase $ in the pancreatic juice, and tended to increase daily secretion of ${\alpha}-amylase $. The secretion of the other enzymes was not different between the two hays at maintenance intake. These results suggest that the kind of hay could change the concentration of ${\alpha}-amylase $ in the pancreatic juice, and that the intake level of alfalfa hay affects the ${\alpha}-amylase $ concentration and the juice volume secreted from the pancreas.

• Journal of Microbiology and Biotechnology
• /
• v.14 no.2
• /
• pp.379-384
• /
• 2004

Fibroblast growth factor-2 (FGF-2) is known to modulate numerous cellular functions in various cell types, including cell proliferation, differentiation, survival, adhesion, migration, and motility, and also in processes such as wound healing, angiogenesis, and vasculogenesis. FGF-2 regulates the expression of several molecules thought to mediate critical steps during angiogenesis. This study examines the mechanisms underlying FGF-2-induced cell migration, using human umbilical vein endothelial cells (HUVECs). FGF-2 induced the nondirectional and directional migration of endothelial cells, which are inhibited by MMPs and plasmin inhibitors, and induced the secretion of matrix metalloproteinase-3 (MMP3) and MMP-9, but not MMP-l and MMP-2. FGF-2 also induced the secretion of the tissue inhibitor of metalloproteinase-l (TIMP-I), but not of TIMP- 2. Also, the pan-PKC inhibitor inhibited FGF-2-induced MMP-9 secretion. It is, therefore, suggested that FGF-2 induces the migration of cultured endothelial cells by means of increased MMPs and plasmin secretion. Furthermore, FGF-2 may increase MMP-9 secretion by activating the PKC pathway.

• Molecules and Cells
• /
• v.26 no.3
• /
• pp.291-298
• /
• 2008

Human karyopherin ${\beta}3$, highly homologous to a yeast protein secretion enhancer (PSE1), has often been reported to be associated with a mediator of a nucleocytoplasmic transport pathway. Previously, we showed that karyopherin ${\beta}3$ complemented the PSE1 and KAP123 double mutant. Our research suggested that karyopherin beta has an evolutionary function similar to that of yeast PSE1 and/or KAP 123. In this study, we performed yeast two-hybrid screening to find a protein which would interact with karyopherin ${\beta}3$ and identified apolipoprotein A-I (apo A-I), a secretion protein with a primary function in cholesterol transport. By using in vitro binding assay, co-immunoprecipitation, and colocalization studies, we defined an interaction between karyopherin ${\beta}3$ and apo A-I. In addition, overexpression of karyopherin ${\beta}3$ significantly increased apo A-I secretion. These results suggest that karyopherin ${\beta}3$ plays a crucial role in apo A-I secretion. These findings may be relevant to the study of a novel function of karyopherin ${\beta}3$ and coronary artery diseases associated with apo A-I.

• Food Science and Biotechnology
• /
• v.18 no.5
• /
• pp.1284-1287
• /
• 2009

Tumor necrosis factor (TNF)-${\alpha}$ is chronically elevated in adipose tissues of obese rodents and humans. Increased levels of TNF-${\alpha}$ have been implicated in both the induction of atherogenic adipokines, such as plasminogen activator inhibitor (PAI)-1, and the inhibition of the anti-atherogenic adipokine, adiponectin. In this study, we investigated the effects of trans-stilbene, piceatannol, rhaponticin, and piceid on the TNF-${\alpha}$-induced atherogenic changes of adipokines in 3T3-L1 cells. Exposure to TNF-${\alpha}$ for 24 hr increased PAI-1 secretion and decreased adiponectin secretion. Among stilbenoids, piceatannol significantly inhibited the increased secretion of PAI-1 induced by TNF-${\alpha}$. Adiponectin secretion decreased by TNF-${\alpha}$ was recovered after trans-stilbene and rhaponticin treatments. Our results showed that stilbenoids exerted different effects on TNF-${\alpha}$-induced changes in adipokines secretion in 3T3-L1 adipocytes according to their structural characteristics.

• Food Science and Biotechnology
• /
• v.15 no.4
• /
• pp.485-493
• /
• 2006

Lactococcus species are noninvasive and nonpathogenic microorganisms that are widely used in industrial food fermentation and as well-known probiotics. They have been modified by traditional methods and genetic engineering to produce useful food-grade materials. The application of genetically modified lactococci in the food industry requires their genetic elements to be safe and stable from integration with endogenous food microorganisms. In addition, selection for antibiotic-resistance genes should be avoided. Several expression and secretion signals have been developed for the production and secretion of useful proteins in lactococci. Food-grade systems composed of genetic elements from lactic acid bacteria have been developed. Recent developments in this area have focused on food-grade selection markers, stabilization, and integration strategies, as well as approaches for controlled gene expression and secretion of foreign proteins. This paper reviews the expression and secretion signals available in lactococci and the development of food-grade markers, food-grade cloning vectors, and integrative food-grade systems.

• Natural Product Sciences
• /
• v.25 no.2
• /
• pp.165-171
• /
• 2019

Although the functions of a standardized extract of Gingko biloba leaves (EGb $761^{(R)}$) has been reported with regard to neurobiological properties, no attention has been paid to the impact of EGb $761^{(R)}$ on the neuronal regulation of energy homeostasis. To evaluate the hypothesis that EGb $761^{(R)}$ affect the secretion of peptide tyrosine tyrosine (PYY) and the activation of free fatty acid receptor 4 (FFA4), which are involved in the neuronal circuitries that control energy homeostasis by inducing the transfer of information about the influx of energy to the brain, we examined whether EGb $761^{(R)}$ can stimulate PYY secretion in the enteroendocrine NCI-H716 cells and if EGb $761^{(R)}$ can activate FFA4 in FFA4-expressing cells. In NCI-H716 cells, EGb $761^{(R)}$ stimulated PYY secretion and the EGb $761^{(R)}$-induced PYY secretion was involved in the increase in intracellular $Ca^{2+}$ concentration and the activation of FFA4. Furthermore, in FFA4-expressing cells, EGb $761^{(R)}$ activated FFA4. These results suggest that EGb $761^{(R)}$ may affect the control of energy homeostasis via the regulation of PYY secretion and FFA4 activation.