Use of NaCl prevents aggregation of recombinant COMP–Angiopoietin-1 in Chinese hamster ovary cells

To investigate the effect of hyperosmotic medium on production and aggregation of the variant of Angiopoietin-1 (Ang1), cartilage oligomeric matrix protein (COMP)–Ang1, in recombinant Chinese hamster ovary (CHO) cells, CHO cells were cultivated in shaking flasks. NaCl and/or sorbitol were used to raise medium osmolality in the range of 300–450 mOsm/kg. The specific productivity of COMP–Ang1, q_COMP–Ang1, increased as medium osmolality increased. At NaCl-450 mOsm/kg, the q_COMP–Ang1 was 7.7-fold higher than that at NaCl-300 mOsm/kg, while, at sorbitol-450 mOsm/kg, it was 2.9-fold higher than that at sorbitol-300 mOsm/kg. This can be attributed to the increased relative mRNA level of COMP–Ang1 at NaCl-450 mOsm/kg which was approximately 2.4-fold higher than that at sorbitol-450 mOsm/kg. Western blot analysis showed that COMP–Ang1 aggregates started to occur in the late-exponential phase of cell growth. When sorbitol was used to raise the medium osmolality, a severe aggregation of COMP–Ang1 was observed. On the other hand, when NaCl was used, the aggregation of COMP–Ang1 was drastically reduced at NaCl-400 mOsm/kg. At NaCl-450 mOsm/kg, the aggregation of COMP–Ang1 was hardly observed. This suggests that environmental conditions are critical for the aggregation of COMP–Ang1. Taken together, the use of NaCl-induced hyperosmotic medium to cell culture process turns out to be an efficient strategy for enhancing COMP–Ang1 production and reducing COMP–Ang1 aggregation.     

Development of a Saccharomyces cerevisiae strain for the production of 1,2-propanediol by gene manipulation

The main goal of this research was to achieve a more efficient production of 1,2-propanediol (1,2-PD) using mutated Saccharomyces cerevisiae. 1,2-PD cannot be produced by wild type S. cerevisiae. To develop a S. cerevisiae mutant that could produce 1,2-PD, the mgs gene of E. coli-K12 MG1655 and the dhaD gene of Citrobacter freundii were inserted into yeast expression vectors such as pESC-URA and pESC-TRP and transformed into the wild type of S. cerevisiae. As a result, the batch fermentation of S. cerevisiae YPH500, harboring an mgs gene inserted pJES27 vector, resulted in a yield of 0.17 g/L. On the other hand, the methylglyoxal synthase of the recombinant S. cerevisiae YPH500, harboring a dhaD gene inserted pJES29 vector, was inactive and produced no detectable amount of 1,2-PD. Therefore, in order to achieve a maximum yield of 1,2-PD, we selected the pESC-TRP vector that is able to co-express the dhaD gene with the pJES27 vector. By inserting the dhaD gene into the pESC-TRP vector, the pJES30 vector was constructed. The maximal yield of 1,2-PD achieved in a 1% galactose batch fermentation by pJES27 and pJES30 harboring S. cerevisiae was 0.45 g/L.     

In vitro anti-inflammatory activity of lignans isolated from Magnolia fargesii

The overproduction of nitric oxide (NO) and prostaglandin E₂ (PGE₂) causes neurodegenerative diseases, such as Alzheimer’s disease and Parkinson’s disease. Four lignans, (+)-eudesmin (1), (+)-magnolin (2), (+)-yangambin (3) and a new structure named as epimagnolin B (4) were isolated from Magnolia fargesii (Magnoliaceae) as the inhibitors of NO production in LPS-activated microglia. The most potent compound 4 inhibited the production of NO and PGE₂ and the expression of respective enzyme iNOS and COX-2 through the suppression of I-κB-α degradation and nuclear translocation of p65 subunit of NF-κB.     

Synthesis of phenylisothiourea derivatives as inhibitors of NO production in LPS activated macrophages

A series of phenylisothioureas were synthesized as inhibitors of NO production in lipopolysaccharide-activated macrophages. We investigated the effect of lipophilic moiety and N- or S-substituents of the phenylisothioureas on the activity. Inhibitory activities of carbazole-linked phenylisothioureas were superior to the corresponding simple phenylisothiourea derivatives. Among these compounds, 12b having N-ethyl and S-isopropyl groups on phenylisothiourea moiety was the most potent in the inhibition of NO production. They inhibited NO production through the suppression of the LPS-induced translocation of p65 subunit of NF-κB and the followed suppression of the iNOS protein and mRNA expression.     

Insulin-sensitizing activities of tanshinones,diterpene compounds of the root of Salvia miltiorrhiza Bunge

In this study, the effects of the extract and four tanshinone compounds from the dried root of Salvia miltiorrhiza Bunge (Labiatae) on the tyrosine phosphorylation of the insulin receptor (IR) β-subunit and the downstream signaling were examined in Chinese-hamster ovary cells expressing human insulin receptors (CHO/IR cells) as well as in 3T3-L1 adipocytes. In addition the translocation of the glucose transporter 4 was investigated in 3T3-L1 adipocytes. Total extract of Danshen (1–10 μg/ml) and the four tanshinones (10 μM) did not show any activity, but the total extract and the tanshinone I, IIA and 15, 16-dihydrotanshinone I except cryptotanshinone enhanced the activity of insulin (1 nM) on the tyrosine phosphorylation of the IR as well as the activation of the downstream kinases Akt, ERK1/2, and GSK3β. In the adipocytes the same IR-downstream signaling and the translocation of glucose transporter 4 were demonstrated by the three tanshinones in the presence of insulin. These insulin-sensitizing activities of tanshinones may be useful for developing a new class of specific IR activators as anti-diabetic agents.     

COMP-Ang1 ameliorates leukocyte adhesion and reinforces endothelial tight junctions during endotoxemia

Endotoxemia is characterized by multiple dysfunctions of the micro-vascular endothelium. Of these, vascular leakage is an initial event that aggravates vascular dysfunction can lead to systemic vascular collapse and organ failure. Thus, prevention of vascular leakage may ameliorate endotoxin-induced dysfunctions of blood vessels. Here we examine the effect of an angiopoietin-1 variant, COMP-Ang1, on endotoxin-induced vascular leakage in mice. COMP-Ang1 significantly reduced endotoxin-induced vascular leakage in the lung, heart, and kidney, but not in liver or intestine. Interestingly, COMP-Ang1 attenuated endotoxin-induced lung damage, presumably due to reduced infiltration of macrophages. Moreover, COMP-Ang1 restored the level of PECAM-1 expression, which is significantly reduced by endotoxin challenge. This study suggests that COMP-Ang1 reduces endotoxin-induced vascular leakage by restoration of cellular junctions and subsequent attenuation of leukocyte infiltration.     

Hypoxia protects articular chondrocytes from thapsigargin-induced apoptosis

The present study investigates the effect of low oxygen concentrations on thapsigargin-induced apoptosis and reactive oxygen species (ROS)-related signaling in articular chondrocytes. Chondrocytes were obtained from normal canine knee cartilage and were treated with different concentrations of thapsigargin for 24 h under normoxic (21% oxygen tension) or hypoxic (1% oxygen tension) conditions. The cells treated with thapsigargin under normoxic conditions showed a dose-dependent induction of apoptosis. However, the cellular changes and apoptotic events that occurred following thapsigargin treatment, were completely inhibited by hypoxia, including loss of mitochondrial transmembrane potential (MTP), ROS generation and JNK phosphorylation. Moreover, the cells exposed to hypoxic conditions showed increased expression of the anti-apoptotic proteins xIAP-2 and Bcl-2. We demonstrate that hypoxia inhibited thapsigargin-induced apoptosis in chondrocytes by regulating ROS-related signaling and the expression of anti-apoptotic proteins. We propose that maintaining hypoxic conditions in articular cartilage may be required for the prevention of chondrocyte and cartilage diseases such as arthritis.     

Structural Snapshots of Heparin Depolymerization by Heparin Lyase I

Heparin lyase I (heparinase I) specifically depolymerizes heparin, cleaving the glycosidic linkage next to iduronic acid. Here, we show the crystal structures of heparinase I from Bacteroides thetaiotaomicron at various stages of the reaction with heparin oligosaccharides before and just after cleavage and product disaccharide. The heparinase I structure is comprised of a β-jellyroll domain harboring a long and deep substrate binding groove and an unusual thumb-resembling extension. This thumb, decorated with many basic residues, is of particular importance in activity especially on short heparin oligosaccharides. Unexpected structural similarity of the active site to that of heparinase II with an (α/α)₆ fold is observed. Mutational studies and kinetic analysis of this enzyme provide insights into the catalytic mechanism, the substrate recognition, and processivity.     

Lactic acid fermentation of Chinese yam (Dioscorea batatas Decne) flour and its pharmacological effect on gastrointestinal function in rat model

To develop a health-aid preparation of Chinese yam (Dioscorea batatas Decne), lactic acid fermentation was attempted using a mixed starter comprising ofLactobacillus acidophilus, Streptococcus thermophilus, andBifidobacterium bifidus. The anaerobic fermentation of a 5% Chinese yam flour suspension gave a uniform suspension of pH 4.35, containing 7.76×10⁶ CFU/mL lactic acid bacteria (LAB), and which was found to be acceptable to the panel from a sensory assessment. During the administration of the lactic acid fermented (LAF) Chinese yam to Sprague Dawley rats for 6 weeks, a smaller body weight gain, but greater excretion of feces were observed, implying the creation of a healthy gastrointestine on the administration of LAF Chinese yam, which was also confirmed by the gastrointestinal motility of the feed in rats fed on LAF Chinese yam. The constipation induced by loperamide was further suppressed in a rat group fed on a LAF Chinese yam diet, which was qualified from healthy gastrointestinal flora established by LAB. A serochemical analysis revealed a slight improvement in the blood glucose, neutral lipid and total cholesterol concentrations on administration of LAF Chinese yam, suggesting LAF Chinese yam could be served as a healthy-aid preparation, even for hyperglycemia or hyperlipidemia patients.