Identification of novel fatty acid glucosides from the tropical fruit Morinda citrifolia L.

Two new fatty acid glucosides, 1,6-di-O-octanoyl-β-D-glucopyranose (1) and 6-O-(β-D-glucopyranosyl)-1-O-decanoyl-β-D-glucopyranose (2), were isolated from a methanol extract of the fruit of Morinda citrifolia L. along with five known saccharide fatty acid esters. The structures of these compounds were determined by combination of spectral and chemical analyses. These fatty acid glucosides exhibited inhibitory effect against copper-induced low-density lipoprotein oxidation. Compound 2 had the strongest effect, which was almost comparable to that of butylated hydroxytoluene.     

Effect of sodium butyrate on the production, heterogeneity and biological activity of human thrombopoietin by recombinant Chinese hamster ovary cells

Human thrombopoietin (hTPO) is a heavily glycosylated protein with 6 and 24 potential N- and O-glycosylation sites, respectively. To determine the effect of sodium butyrate (NaBu) on the production and quality of hTPO in recombinant Chinese hamster ovary (rCHO) cells, NaBu (0-10 mM) was added to the cultures of exponentially growing cells. NaBu addition significantly increased both the specific and volumetric hTPO production, although it decreased the cell viability by apoptosis in a dose-dependent manner. The highest hTPO concentration of 82.2 +/- 5.6 microgml-1 was obtained in the culture with 3 mM NaBu addition. Compared with the culture without NaBu addition, the culture with 3 mM NaBu resulted in a 6.4-fold increase in qTPO and a 3.3-fold increase in the final hTPO concentration on day 7. However, NaBu deteriorated the quality of hTPO, resulting from increased heterogeneity, reduced acidic hTPO isoforms, reduced alpha(2 --> 3) sialylation, and decreased in vivo biological activity. We also found that the biological activity of hTPO in the culture with 3 mM NaBu addition collected on day 7 was 72% of that in the culture without NaBu addition. Taken together, the use of NaBu or its optimal concentration for high-level expression of a heavily glycosylated protein like hTPO should be determined by considering its detrimental effect on the quality of glycoprotein.     

The shooty callus induced by suppression of tobacco CHRK1 receptor-like kinase is a phenocopy of the tobacco genetic tumor

CHRK1 encodes a tobacco receptor-like kinase that contains a chitinase-like sequence in the extracellular domain. In a previous study, CHRK1-suppressed transgenic tobacco plants exhibited pleiotropic developmental abnormalities including spontaneous growth of shooty callus from emerging embryos in the absence of any exogenous hormones. In this study, we show that the CHRK1 shooty callus mimics tobacco genetic tumors in its morphology, physiology, and gene expression profiles. Similar to CHRK1 shooty callus, tobacco genetic tumors exhibit shooty callus morphology and hormone-independent shoot organogenesis. Both the CHRK1 callus and genetic tumors constitutively expressed KNOTTED1-type homeobox genes at the high levels, consistent with their vigorous shoot formation. These two types of calli exhibited cell death phenotypes, accompanied by high H₂O₂ production, increased ion leakage, and callose accumulation. Consistently, both types of calli constitutively expressed high levels of defense genes induced during pathogen-mediated HR cell death. These results, together with previous reports that both the CHRK1 shooty callus and tobacco genetic tumor contained high levels of cytokinin, indicate that CHRK1 shooty callus is a phenocopy of tobacco genetic tumor. CHRK1-mediated signal transduction may play a role in the formation of the genetic tumor in tobacco.     

A novel splice acceptor mutation in the DSPP gene causing dentinogenesis imperfecta type II

The dentin sialophosphoprotein (DSPP) gene (4q21.3) encodes two major noncollagenous dentin matrix proteins: dentin sialoprotein (DSP) and dentin phosphoprotein (DPP). Defects in the human gene encoding DSPP cause inherited dentin defects, and these defects can be associated with bilateral progressive high-frequency sensorineural hearing loss. Clinically, five different patterns of inherited dentin defects are distinguished and are classified as dentinogenesis imperfecta (DGI) types I, II, and III, and dentin dysplasia types I and II. The genetic basis for this clinical heterogeneity is unknown. Among the 11 members recruited from the studied kindred, five were affected with autosomal dominant DGI type II. The mutation (g.1188CG, IVS2-3CG) lay in the third from the last nucleotide of intron 2 and changed its sequence from CAG to GAG. The mutation was correlated with the affection status and was absent in 104 unaffected individuals (208 alleles) with the same ethnic and geological background. The proband was in the primary dentition stage and presented with multiple pulp exposures. The occlusal surface of his dental enamel was generally abraded, and the dentin was heavily worn and uniformly shaded brown. The dental pulp chambers appeared originally to be within normal limits without any sign of obliteration, but over time (by age 4), the pulp chambers became partially or completely obliterated. The oldest affected member (age 59) showed mild hearing loss at high-frequency (8 kHz). Permanent dentition was severely affected in the adults, who had advanced dental attrition, premature loss of teeth, and extensive dental reconstruction.     

Differential effects of gadolinium chloride on Kupffer cells in vivo and in vitro

Gadolinium chloride (GdCl) is commonly used to study the role of Kupffer cells in liver disease in vivo. The in vitro effects of GdCl on cultured Kupffer cells are poorly characterised. The aim of this study was to characterise rat Kupffer cell TNFalpha production, phagocytic function, and ED1 and ED2 antigen expression following the administration of GdCl. For in vivo experiments, rats received 10mg/kg GdCl IV or sterile saline. Lipopolysaccharide 3mg/kg IP (LPS) was administered 4h prior to sacrifice on Days 1-3, 5 or 8 following GdCl injection. Hepatic ED1 and ED2 positive macrophage numbers and TNFalpha mRNA levels were determined. For in vitro experiments, Kupffer cells were cultured in the presence of 0-270 microM GdCl for 24h following which viability, TNFalpha protein production in response to LPS (10 ng/ml), phagocytosis, and ED1 and ED2 staining were evaluated. In vivo, the proportion of ED1 positive cells which were ED2 positive was reduced from 87 to 3% and hepatic TNFalpha mRNA levels following LPS declined by 60% over Days 1-5 after injection of GdCl (P<0.01). In vitro, phagocytosis declined with increasing concentrations of GdCl. GdCl (0-27 microM) did not effect cultured Kupffer cell viability, TNFalpha production, ED1 or ED2 staining. We conclude that GdCl significantly reduces ED2 expression by Kupffer cells in vivo. In vitro, GdCl has a dose dependent effect on phagocytosis but only effects viability and TNFalpha production at high concentrations. ED2 expression of cultured Kupffer cells is not affected by GdCl.     

Constitutive production of human leptin by fed-batch culture of recombinant rpoS- Escherichia coli

High-level production of human leptin by fed-batch culture of recombinant Escherichia coli using constitutive promoter system was investigated. For the constitutive expression of the obese gene encoding human leptin, the strong constitutive HCE promoter cloned from the D-amino acid aminotransferase gene of Geobacillus toebii was used. To develop an optimal host-vector system, several different recombinant E. coli strains were compared for leptin production. In flask cultures, E. coli FMJ123, which is a rpoS mutant strain, showed the highest level of leptin production (41% of total proteins). By comparing the expression levels of leptin in several different rpoS- and rpoS+ strains, it could be concluded that rpoS mutation positively affected constitutive production of leptin. For the large-scale production of human leptin, fed-batch cultures of recombinant E. coli FMJ123 were carried out using three different feeding solutions--chemically defined, yeast extract-containing, and casamino acid-containing feeding solutions. Among these, the use of casamino acid-containing feeding solution allowed production of leptin up to 2.1 g/L, which was 2.1- and 1.8-fold higher than that obtained with chemically defined and yeast extract-contained feeding solutions, respectively. These results suggest that the HCE promoter can be used for the efficient production of leptin, and most likely other recombinant proteins, in a constitutive manner.     

Simple aromatic compounds containing propenone moiety show considerable dual COX/5-LOX inhibitory activities

For the development of safer anti-inflammatory agents, simple aromatic compounds containing propenone moiety were prepared and evaluated for their dual COX/5-LOX inhibitory activities. Among the 17 prepared compounds, most of the compounds exhibited considerable COX/5-LOX inhibitory activities. Especially compound C(15) showed the most significant dual COX/5-LOX inhibitory activity.     

Flow cytometric application of helper adenovirus (HAd) containing GFP gene flanked by two parallel loxP sites to evaluation of 293 cre-complementing cell line and monitoring of HAd in Gutless Ad production

Gutless adenoviruses (GAds), namely, all gene-deleted adenoviruses, were developed to minimize their immune responses and toxic effects for a successful gene delivery tool in gene therapy. The Cre/loxP system has been widely used for GAd production. To produce GAd with a low amount of helper adenovirus (HAd) as byproduct, it is indispensable to use 293Cre cells expressing a high level of Cre for GAd production. In this study, we constructed the HAd containing enhanced green fluorescent protein gene flanked by two parallel loxP sites (HAd/GFP). The use of HAd/GFP with flow cytometry allows one to select 293Cre cells expressing a high level of Cre without using conventional Western blot analysis. Unlike conventional HAd titration methods such as plaque assay and end-point dilution assay, it also allows one to monitor rapidly the HAd as byproduct in earlier stages of GAd amplification. Taken together, the use of HAd/GFP with flow cytometry facilitates bioprocess development for efficient GAd production.