Effect of the N-terminal hydrophobic sequence of hepatitis B virus surface antigen on the folding and assembly of hybrid beta-galactosidase in Escherichia coli

To investigate the mechanism of inclusion body formation and the effect of a hydrophobic sequence on the in vivo polypeptide folding, the aggregation caused by recombinant fusion beta-galactosidase in Escherichia coli was examined. Two plasmids were constructed: pTBG(H-) carried only the preS2 sequence of the hepatitis B virus surface antigen (HBsAg) in front of the beta-galactosidase gene (lacZ) while pTBG(H+) carried an additional sequence encoding the amino-terminal hydrophobic sequence of the S region of HBsAg between preS2 and lacZ. Unlike cells expressing the fusion protein not containing the hydrophobic sequence, E. coli JM109/pTBG(H+) exhibited temperature-sensitive production of beta-galactosidase. As the culture temperature increased the activity decreased dramatically. This decrease in activity was not due to a decrease in fusion polypeptide production, but rather the fusion polypeptides containing the hydrophobic sequence aggregated within the cells at high temperature. However once the fusion polypeptides folded into proper conformation at low temperature, they maintained the activity even at high temperature. The results indicate that aggregation is a consequence of incorrect folding and assembly of the polypeptides, and is not derived from the native structure. The aggregates of the pTBG(H+)-encoded fusion polypeptides did not revert to active form when the culture temperature was lowered.     

Selection and proliferation of rapid growing cell lines from embryo derived cell cultures of yew tree (Taxus cuspidata Sieb. et Zucc)

Calli were induced from 300,000 embryos isolated from immature to mature stage of seeds collected on late September from 14 elite trees. When the embryos were cultured onto plastic Petri-dish containing 20 mL of modified B5 basal medium supplemented with 3% (w/v) sucrose, 500 mg/L casein hydrolysate, 250 mg/L myo-inositol, 0.5% (w/v) polyvinyl polypyrrolidon (PVPP), 2×MS vitamins, 0.5 mg/L gibberellic acid, and 10 mg/L 2,4-D after 2 weeks of culture, yellowish-white calli were immediately formed on the surfaces of embryos, and subcultured for 4 weeks in same culture medium. Because most of calli maintained for more than 3 months were revealed differences in their colors, surface texture, and growth rate, visual selection was made for first round screening. When the size of visually selected calli larger than 19 mm in their diameter were inoculated, persistent proliferation was observed. Among the plating methods tested for the selection of rapid growing cell lines at single cell and/or small cell aggregate level, 2-layer spread plating revealed as the best for single cell cloning. To enhance cell growth and maintain high rate of viability for long-term culture of yew cells in bioreactor, final cell volume less than 50% in SCV seemed to be the best. Time course study revealed that 30% of inoculum density was suitable for fed batch culture. Among the tested conditional media, the rate of 1∶2 (old medium: fresh medium) was recorded at the best for cell growth.     

Modulation of Intracellular Calcium Levels by Calcium Lactate Affects Colon Cancer Cell Motility through Calcium-Dependent Calpain

Cancer cell motility is a key phenomenon regulating invasion and metastasis. Focal adhesion kinase (FAK) plays a major role in cellular adhesion and metastasis of various cancers. The relationship between dietary supplementation of calcium and colon cancer has been extensively investigated. However, the effect of calcium (Ca²⁺) supplementation on calpain-FAK-motility is not clearly understood. We sought to identify the mechanism of FAK cleavage through Ca²⁺ bound lactate (CaLa), its downstream signaling and role in the motility of human colon cancer cells. We found that treating HCT116 and HT-29 cells with CaLa immediately increased the intracellular Ca²⁺ (iCa²⁺) levels for a prolonged period of time. Ca²⁺ influx induced cleavage of FAK into an N-terminal FAK (FERM domain) in a dose-dependent manner. Phosphorylated FAK (p-FAK) was also cleaved in to its p-N-terminal FAK. CaLa increased colon cancer cells motility. Calpeptin, a calpain inhibitor, reversed the effects of CaLa on FAK and pFAK cleavage in both cancer cell lines. The cleaved FAK translocates into the nucleus and modulates p53 stability through MDM2-associated ubiquitination. CaLa-induced Ca²⁺ influx increased the motility of colon cancer cells was mediated by calpain activity through FAK and pFAK protein destabilization. In conclusion, these results suggest that careful consideration may be given in deciding dietary Ca²⁺ supplementation to patient undergoing treatment for metastatic cancer.     

Association between misalignment of circadian rhythm and obesity in Korean men: Sixth Korea National Health and Nutrition Examination Survey

ABSTRACT

Background: The disruption of circadian rhythm has been found to associate with obesity in vivo and in vitro. Sleep duration, eating habits, total feeding time, and nightshift work can also affect circadian rhythms. This study investigated the association between misalignment of circadian rhythm and obesity in Korean men, using a cross-sectional database.

Methods: This study used data from the Korean National Health and Nutrition Examination Survey (KNHANES), whose study population was 3,658 men aged 18 to 60 years. General and abdominal obesity was defined as a body mass index (BMI) ≥ 25 kg/m² and waist circumference ≥ 90 cm, respectively. Circadian rhythm factors were determined with a self-report questionnaire and included breakfast frequency, sleep duration, and work time. Frequency of breakfast was divided into regular breakfast (five to seven times a week) and irregular breakfast (less than five times a week). Sleep duration was divided into less than 7 hours, 7–9 hours, and over 9 hours. Working time was defined as day/evening, night shift, and other type. The adjusted odds ratios (aORs) and 95% confidence intervals (CIs) for general and abdominal obesity were calculated using multivariable logistic regression according to the number of factors that disturb the circadian rhythm.

Results: Participants with 1 (aOR 1.34, 95% Cl 1.10–1.61) and ≥2 (aOR 1.62, 95% Cl 1.29–2.05) factors disturbing circadian rhythms were associated with elevated risk for general obesity. Similarly, those with 1 (aOR 1.33, 95% Cl 1.09–1.63) and ≥2 (aOR 1.70, 95% Cl 1.32–2.20) factors had elevated risk for abdominal obesity.

Conclusions: Factors disturbing the circadian rhythm were associated with general and abdominal obesity. Additional studies are needed, and associations with metabolic diseases should be investigated.     

Enzymatic synthesis of two ginsenoside Re-beta-xylosides.

Two new beta-xylosyl derivatives of ginsenoside Re, 20(S)-protopanaxatriol 6-O-alpha-L-rhamnopyranosyl-(1 --> 2)-[beta-D-xylopyranosyl-(1 --> 4)]-beta-D-glucopyranosyl-20-O-beta-D-glucopyranoside and 20(S)-protopanaxatriol 6-O-alpha-L-rhamnopyranosyl-(1 --> 2)-[beta-D-xylopyranosyl-(1 --> 6)]-beta-D-glucopyranosyl-20-O-beta-D-glucopyranoside, were respectively synthesized from p-nitrophenyl beta-D-xylopyranoside and phenyl beta-D-xylopyranoside as donors and ginsenoside Re as the acceptor in 25% acetone and acetonitrile by a cellulase preparation from Trichoderma viride and a beta-galactosidase preparation from Aspergillus oryzae. The latter enzyme preparation also catalyzed the hydrolysis of ginsenoside Re to the minor saponin, ginsenoside Rg2.     

Proteoglycans and glycosaminoglycans induce gap junction synthesis and function in primary liver cultures

Intercellular communication via gap junctions, as measured by dye and electrical coupling, disappears within 12 h in primary rat hepatocytes cultured in serum-supplemented media or within 24 h in cells in a serum-free, hormonally defined medium (HDM) designed for hepatocytes. Glucagon and linoleic acid/BSA were the primary factors in the HDM responsible for the extended life span of the electrical coupling. After 24 h of culture, no hormone or growth factor tested could restore the expression of gap junctions. After 4-5 d of culture, the incidence of coupling was undetectable in a serum-supplemented medium and was only 4-5% in HDM alone. However, treatment with glycosaminoglycans or proteoglycans of 24-h cultures, having no detectable gap junction protein, resulted in synthesis of gap junction protein and of reexpression of electrical and dye coupling within 48 h. Most glycosaminoglycans were inactive (heparan sulfates, chondroitin-6 sulfates) or only weakly active (dermatan sulfates, chondroitin 4-sulfates, hyaluronates), the weakly active group increasing the incidence of coupling to 10-30% with the addition of 50-100 micrograms/ml of the factor. Treatment of the cells with 50-100 micrograms/ml of heparins derived from lung or intestine resulted in cells with intermediate levels of coupling (30-50%). By contrast, 10-20 micrograms/ml of chondroitin sulfate proteoglycan, dermatan sulfate proteoglycan, or liver-derived heparin resulted in dye coupling in 80-100% of the cells, with numerous cells showing dye spread from a single injected cell. Sulfated polysaccharides of glucose (dextran sulfates) or of galactose (carrageenans) were inactive or only weakly active except for lambda-carrageenan, which induced up to 70% coupling (albeit no multiple coupling in the cultures). The abundance of mRNA (Northern blots) encoding gap junction protein and the amounts of the 27-kD gap junction polypeptide (Western blots) correlated with the degree of electrical and dye coupling indicating that the active glycosaminoglycans and proteoglycans are inducing synthesis and expression of gap junctions. Thus, proteoglycans and glycosaminoglycans, especially those found in abundance in the extracellular matrix of liver cells, are important in the regulation of expression of gap junctions and, thereby, in the regulation of intercellular communication in the liver. The relative potencies of heparins from different tissue sources at inducing gap junction expression are suggestive of functional tissue specificity for these glycosaminoglycans.     

Pregnancy diagnosis in zoo animals by estrogen determination in feces

Estrogen concentration in feces was investigated in five different herbivorous species of zoo animals. Using a nonspecific estrogen radioimmunoassay, in four species (red buffalo, yak, Grevy's zebra, and Nubian ibex) pregnancy was revealed by measuring estrogen concentration in feces. In hippopotamus, the levels of fecal estrogens were not different between pregnant and nonpregnant animals.     

Development of immunoassay methods by use of liposomes

In order to develop liposomes for use in an immunoassay system, the preparation of immune liposomes and their characterization have been investigated. Liposomes have potential use in extremely sensitive analytical immunoassays, in addition to serving as an attractive drug delivery system. This liposome immunoassay system is based on membrane immunochemistry and an enzymatic reaction. An intense yellow color, easily detectable with the naked eye, was produced quite rapidly by the lysis of bovine serum albumin (BSA)-labeled, alkaline phosphatase-entrapped liposomes in the presence of anti-BSA rabbit serum and active complement under alkaline conditions. Sensitive detection is possible because of the antigen-antibody complex reaction, which leads to liposome lysis and an enzymatic reaction. The liposome immunoassay method offers a rapid, simple, and sensitive testing procedure which can quantitatively and qualitatively determine the presence or absence of antigenic materials and antibodies.     

Purified maturation promoting factor phosphorylates pp60c-src at the sites phosphorylated during fibroblast mitosis

We have previously shown that overexpressed chicken pp60c-src has retarded mobility, novel serine/threonine phosphorylation, and enhanced kinase activity during NIH 3T3 cell mitosis. Here we show that novel mitotic phosphorylations occur at Thr 34, Thr 46, and Ser 72. The possibility, previously raised, that Ser 17 is dephosphorylated during mitosis is excluded. The phosphorylated sites lie in consensus sequences for phosphorylation by p34cdc2, the catalytic component of maturation promoting factor (MPF). Furthermore, highly purified MPF from metaphase-arrested Xenopus eggs phosphorylated both wild-type and kinase-defective pp60c-src at these sites. Altered phosphorylation alone is sufficient to account for the large retardation in mitotic pp60c-src electrophoretic mobility: phosphorylation of normal pp60c-src by MPF retarded mobility and dephosphorylation of mitotic pp60c-src restored normal mobility. These results suggest that pp60c-src is one of the targets for MPF action, which may account in part for the pleiotropic changes in protein phosphorylation and cellular architecture that occur during mitosis.