Antitumor agents 220. Antitumor-promoting effects of cimigenol and related compounds on Epstein-Barr virus activation and two-stage mouse skin carcinogenesis

Cimigenol (1) and 39 related compounds were screened as potential antitumor promoters by examining the ability of the compounds to inhibit Epstein-Barr virus early antigen (EBV-EA) activation (induced by 12-O-tetradecanoylphorbol-13-acetate) in Raji cells. Structure-activity relationship analysis indicated that compound 1 showed the highest activity and also exhibited significant inhibitory effects on mouse skin tumor promotion in an in vivo two-stage carcinogenesis test. These data suggest that 1 and the related compounds might be valuable anti-tumor promoters.     

Signals of seminal vesicle autoantigen suppresses bovine serum albumin-induced capacitation in mouse sperm

Capacitation is the prerequisite process for sperm to gain the ability for successful fertilization. Unregulated capacitation will cause sperm to undergo a spontaneous acrosome reaction and then fail to fertilize an egg. Seminal plasma is thought to have the ability to suppress sperm capacitation. However, the mechanisms by which seminal proteins suppress capacitation have not been well understood. Recently, we demonstrated that a major seminal vesicle secretory protein, seminal vesicle autoantigen (SVA), is able to suppress bovine serum albumin (BSA)-induced mouse sperm capacitation. To further identify the mechanism of SVA action, we determine the molecular events associated with SVA suppression of BSA's activity. In this communication, we demonstrate that SVA suppresses the BSA-induced increase of intracellular calcium concentration ([Ca2+]i), intracellular pH (pH(i)), the cAMP level, PKA activity, protein tyrosine phosphorylation, and capacitation in mouse sperm. Besides, we also found that the suppression ability of SVA against BSA-induced protein tyrosine phosphorylation and capacitation could be reversed by dbcAMP (a cAMP agonist).     

Slingshot cofilin phosphatase localization is regulated by receptor tyrosine kinases and regulates cytoskeletal structure in the developing Drosophila eye

Animal development requires that positional information act on the genome to control cell fate and cell shape. The primary determinant of animal cell shape is the cytoskeleton and thus the mechanisms by which extracellular signals influence the cytoskeleton are crucial for morphogenesis. In the developing Drosophila compound eye, localized polymerization of actin functions to constrict the apical surface of epithelial cells, both at the morphogenetic furrow and later to maintain the coherence of the nascent ommatidia. As elsewhere, actin polymerization in the developing eye is regulated by ADF/cofilin ('Twinstar', or 'Tsr' in Drosophila), which is activated by Slingshot (Ssh), a cofilin phosphatase. Here we show that Ssh does act in the developing eye to limit actin polymerization in the assembling ommatidia, but not in the morphogenetic furrow. While Ssh does control cell shape, surprisingly there are no direct or immediate consequences for cell type. Ssh protein becomes apically concentrated in cells that express elevated levels of the Sevenless (Sev) receptor-tyrosine kinase (RTK), even those which receive no ligand. We interpret this as a non-signal driven, RTK-dependent localization of Ssh to allow for locally increased actin filament turnover. We suggest that there are two modes of actin remodeling in the developing eye: a non-RTK, non-Ssh mediated mechanism in the morphogenetic furrow, and an RTK and Ssh-dependent mode during ommatidial assembly.     

Sex-specific genetic architecture of human fatness in Chinese: the SAPPHIRe Study

To dissect the genetic architecture of sexual dimorphism in obesity-related traits, we evaluated the sex–genotype interaction, sex-specific heritability and genome-wide linkages for seven measurements related to obesity. A total of 1,365 non-diabetic Chinese subjects from the family study of the Stanford Asia–Pacific Program of Hypertension and Insulin Resistance were used to search for quantitative trait loci (QTLs) responsible for the obesity-related traits. Pleiotropy and co-incidence effects from the QTLs were also examined using the bivariate linkage approach. We found that sex-specific differences in heritability and the genotype–sex interaction effects were substantially significant for most of these traits. Several QTLs with strong linkage evidence were identified after incorporating genotype by sex (G × S) interactions into the linkage mapping, including one QTL for hip circumference [maximum LOD score (MLS) = 4.22, empirical p = 0.000033] and two QTLs: for BMI on chromosome 12q with MLS 3.37 (empirical p = 0.0043) and 3.10 (empirical p = 0.0054). Sex-specific analyses demonstrated that these linkage signals all resulted from females rather than males. Most of these QTLs for obesity-related traits replicated the findings in other ethnic groups. Bivariate linkage analyses showed several obesity traits were influenced by a common set of QTLs. All regions with linkage signals were observed in one gender, but not in the whole sample, suggesting the genetic architecture of obesity-related traits does differ by gender. These findings are useful for further identification of the liability genes for these phenotypes through candidate genes or genome-wide association analysis.     

Cell-based semiquantitative assay for sulfated glycosaminoglycans facilitating the identification of chondrogenesis

Glycosaminoglycans (GAGs), in particular chondroitin sulfate, are an accepted marker of chondrogenic cells. In this study, a cell-based sulfated GAG assay for identifying the chondrogenesis of mesenchymal stem cells was developed. Based on fluorescent staining using safranin O and 4’,6-diamidino-2-phenylindole (DAPI), this method was highly sensitive. The results were both qualitative and quantitative. The method is suitable for identifying the chondrogenic process and also for screening compounds. The method may be helpful for discovering novel bioactive compounds for cartilage regeneration.     

Immobilization of rat erythrocytes for aflatoxicol production

Summary A modified procedure for preparing alginate gel was developed and used to entrap rat erythrocytes. The immobilized erythrocytes showed high enzyme activity for the reduction of aflatoxin B₁ to aflatoxicol. The production of aflatoxicol from aflatoxin B₁ by immobilized erythrocytes was studied for over 3 weeks and the half-life of such a preparation was shown to be about 10 days. The immobilized erythrocytes can be repeatedly used at 37° C for the batch-wise mode of aflatoxicol production without substantial loss of enzyme activity. Haemolysis of immobilized erythrocytes was not observed upon prolonged storage at 4° C. As compared with free erythrocytes, the immobilized erythrocytes were more temperature resistant at 40° C incubation.Part of this work was presented on ROC-Japan Seminar on Applied Microbiology and Enzymology held in Taipei, Republic of China, on March 8, 1984     

Fetal and Childhood Exposure to Phthalate Diesters and Cognitive Function in Children Up to 12 Years of Age: Taiwanese Maternal and Infant Cohort Study

Few studies have examined the association between environmental phthalate exposure and children’s neurocognitive development. This longitudinal study examined cognitive function in relation to pre-and postnatal phthalate exposure in children 2–12 years old. We recruited 430 pregnant women in their third trimester in Taichung, Taiwan from 2001–2002. A total of 110, 79, 76, and 73 children were followed up at ages 2, 5, 8, and 11, respectively. We evaluated the children’s cognitive function at four different time points using the Bayley and Wechsler tests for assessing neurocognitive functions and intelligence (IQ). Urine samples were collected from mothers during pregnancy and from children at each follow-up visit. They were analyzed for seven metabolite concentrations of widely used phthalate esters. These esters included monomethyl phthalate, monoethyl phthalate, mono-butyl phthalate, mono-benzyl phthalate, and three metabolites of di(2-ethylhexyl) phthalate, namely, mono-2-ethylhexyl phthalate, mono(2-ethyl-5-hydroxyhexyl) phthalate, and mono(2-ethyl-5-oxohexyl) phthalate. We constructed a linear mixed model to examine the relationships between the phthalate metabolite concentrations and the Bayley and IQ scores. We found significant inverse associations between the children’s levels of urinary mono(2-ethyl-5-oxohexyl) phthalate and the sum of the three metabolites of di(2-ethylhexyl) phthalate and their IQ scores (β = -1.818; 95% CI: -3.061, -0.574, p = 0.004 for mono(2-ethyl-5-oxohexyl) phthalate; β = -1.575; 95% CI: -3.037, -0.113, p = 0.035 for the sum of the three metabolites) after controlling for maternal phthalate levels and potential confounders. We did not observe significant associations between maternal phthalate exposure and the children’s IQ scores. Children’s but not prenatal phthalate exposure was associated with decreased cognitive development in the young children. Large-scale prospective cohort studies are needed to confirm these findings in the future.     

Overcoming challenges in WAVE Bioreactors without feedback controls for pH and dissolved oxygen

The biopharmaceutical industry is increasing its use of the WAVE Bioreactor for culturing cells. Although this disposable bioreactor can be equipped to provide real-time pH and dissolved oxygen (DO) monitoring and control, our goal was to develop a process for culturing CHO cells in this system without relying on pH and DO feedback controls. After identifying challenges in culturing cells without controlling for pH and DO in the WAVE Bioreactor, we characterized O(2) and CO(2) transfer in the system. From these cell-free studies, we identified rock rate and rock angle as key parameters affecting O(2) transfer. We also identified the concentration of CO(2) in the incoming gas and the rate of gas flow into the headspace as key parameters affecting CO(2) transfer--and therefore pH--in the disposable culture chamber. Using a full-factorial design to evaluate the rock rate, rock angle, and gas flow rate defined for this WAVE Bioreactor process, we found comparable cell growth and pH profiles in the ranges tested for these three parameters in two CHO cell lines. This process supported cell growth, and maintained pH and DO within our desired range--pH 6.8-7.2 and DO exceeding 20% of air saturation--for six CHO cell lines, and it also demonstrated comparable cell growth and viability with the stirred-tank bioreactor process with online pH and DO control. By eliminating the use of pH and DO probes, this process provides a simple and more cost-effective method for culturing cells in the WAVE Bioreactor.