Activin A is essential for Feeder‐free culture of human induced pluripotent stem cells

Feeder‐free culture of human induced pluripotent stem (hiPS) cells is necessary for their clinical application to avoid adverse effects of foreign proteins. hiPS cells were cultured with combinations of activin (A), CHIR99021 (C), basic fibroblast growth factor (F), and leukemia inhibitory factor (L) under feeder‐free conditions. Culture was terminated after 12 passages or when the cell morphology changed from pluripotency. Pluripotency was analyzed by alkaline phosphatase (ALP) staining and immunostaining with antibodies to Oct3/4, Nanog, SSEA4, and TRA‐1‐60. SB431542 (SB), an activin inhibitor, was added to the culture, and the morphology of the cells was observed. hiPS cells cultured with A, AC, and ACL after 12 passages were positive for ALP staining. Oct3/4 was positive in hiPS cells cultured with A, AC, and ACL. hiPS cells were positive for Nanog when cultured with A and AC; however, Nanog signal was weaker in cells cultured with ACL. SSEA4 was positive in hiPS cells cultured with A and AC but almost negative in those cultured with ACL. hiPS cells were positive for TRA‐1‐60 when cultured with A, AC, and ACL. hiPS cells lose their undifferentiated morphology at six passages when cultured with A + SB, five passages with AC + SB, and nine passages with ACL. We conclude that feeder‐free culture of hiPS cells requires A or AC to maintain pluripotency. J. Cell. Biochem. 114: 584–588, 2013. © 2012 Wiley Periodicals, Inc.     

A novel tumor metastasis suppressor gene LASS2/TMSG1 interacts with vacuolar ATPase through its homeodomain

LASS2/TMSG1 was a novel tumor metastasis suppressor gene, which was first cloned by our laboratory from non‐metastatic and metastatic cancer cell variants of human prostate carcinoma PC‐3M using mRNA differential display in 1999. LASS2/TMSG1 could interact with the C subunit of vacuolar ATPase (V‐ATPase, ATP6V0C) and regulate V‐ATPase activity. In an attempt to provide molecular mechanism of the interaction between LASS2/TMSG1 and V‐ATPase, we constructed four variant transfectants containing different functional domain of LASS2/TMSG1 and stably transfected the variants to human prostate cancer cell line PC‐3M‐1E8 cell with high metastatic potential. Results showed that there were no obvious differences of V‐ATPase expression among different transfected cells and the control. However, V‐ATPase activity and intracellular pH was significantly higher in the variant transfectants with Homeodomain of LASS2/TMSG1 than that in the control using the pH‐dependent fluorescence probe BECEF/AM. Immunoprecipitation, immunofluorescence and immuno‐electron microscope alone or in combination demonstrated the direct interaction of Homeodomain of LASS2/TMSG1 and ATP6V0C. Loss of Homeodomain markedly enhanced the proliferation ability but weakened the apoptotic effect of LASS2/TMSG1 in PC‐3M‐1E8 cells. These lines of results for the first time contribute to the conclusion that LASS2/TMSG1 could regulate V‐ATPase activity and intracellular pH through the direct interaction of its Homeodomain and the C subunit of V‐ATPase. Their interaction could play important roles in the apoptosis of tumor cells. J. Cell. Biochem. 114: 570–583, 2013. © 2012 Wiley Periodicals, Inc.     

Daily Variations of Homocysteine Concentration May Influence Methylation of DNA in Normal Healthy Individuals

The regulation of genetic expression is tightly controlled and well balanced in the organism by different epigenetic mechanisms such as DNA methylation and histone modifications. DNA methylation occurring after embryogenesis is seen mainly as an irreversible event. Even small changes in genomic DNA methylation might be of biological relevance, and several factors influencing DNA methylation have been identified so far, one being homocysteine. In this study, genomic DNA methylation was analyzed and homocysteine plasma levels were measured over a 24 h period in 30 healthy students (15 males and 15 females) exposed to a standard 24 h regime of daytime activity alternating with nighttime sleep. Plasma homocysteine concentrations were measured using HPLC detection. DNA was extracted from whole EDTA blood, and genomic DNA methylation was assessed by fluorescently labeled cytosine extension assay. Both homocysteine and DNA methylation showed 24 h variation. Homocysteine showed a significant daily rhythm with an evening peak and nocturnal nadir in all subjects (p<0.001). Males showed higher overall homocysteine levels compared to females (p=0.002). Genomic DNA methylation showed a significant rhythm with increased levels at night (p=0.021), which was inverse to plasma homocysteine levels.     

CIRCADIAN RHYTHMS DURING CYCLING EXERCISE AND FINGER-TAPPING TASK

The hypothesis of lunar influence on suicide remains widespread, despite the fact that little scientific evidence to substantiate it. We conducted a population‐based study to assess the influence of the lunar phases on suicides according to age, sex, and chosen method. The study included all suicides in Middle Franconia between 1998 and 2003. From a population‐based sample of 3351 events, the files of 3054 suicides (1949 males and 1105 females) were complete for the study variables. Data were categorized by lunar phase, sex, age, and chosen method—“violent” vs. “non‐violent” acts. No significant relationship was detected between the full, absent, and moon's interphases and suicide incidence. Nevertheless, there was a weak association between the absent moon and choice of a non‐violent suicide method in men aged less than the median of 40.2 yrs. There was no evidence of a relationship between suicide and lunar phase. Some explanations for this phenomenon are discussed.     

Chitooligosaccharide inhibits RANKL-induced osteoclastogenesis and ligation-induced periodontitis by suppressing MAPK/ c-fos/NFATC1 signaling

Considering the high rate of osteoclast-related diseases worldwide, research targeting osteoclast formation/function is crucial. In vitro, we demonstrated that chitooligosaccharide (CS) dramatically inhibited osteoclastogenesis as well as osteoclast function dose-dependently. CS suppressed osteoclast-specific genes expression during osteoclastogenesis. Furthermore, we found that CS attenuated receptor activator of nuclear factor kappa B ligand (RANKL)-mediated mitogen-activated protein kinase (MAPK) pathway involving p38, erk1/2, and jnk, leading to the reduced expression of c-fos and nuclear factor of activated T cells c1 (NFATc1) during osteoclast differentiation. In vivo, we found CS protected rats from periodontitis-induced alveolar bone loss by micro-computerized tomography and histological analysis. Overall, CS inhibited RANKL-induced osteoclastogenesis and ligature-induced rat periodontitis model, probably by suppressing the MAPK/c-fos/NFATc1 signaling pathway. Therefore, CS may be a safe and promising treatment for osteoclast-related diseases.     

Amino acid and vitamin requirements in mammalian cultured cells

Summary The development of the tissue culture technique has enabled us to cultivate mammalian cells in a way which is similar to that in use with bacterial cells. As such, the nutritional requirements of mammalian cells in culture have been studied with simplicity and exactness. According to Eagle's extensive works it is accepted that cultured cells generally require 13 amino acids, 8 or 9 vitamins, glucose and 6 inorganic salts. However, although some cultured cells have a capacity for the biosynthesis of Eagle's essential nutrients and others require non-essential nutrients.In this review we will discuss the amino acid and vitamin requirements of cultured cells, and a cell line (R-Y121B · cho) which propagates continuously in a chemically defined medium containing 11 amino acids, 7 vitamins, glucose and 6 ionic salts. Arginine, glutamine, tyrosine and choline are synthesized in the R-Y121B · cho cells.     

Microsatellite variation in Drosophila melanogaster and Drosophila simulans: a reciprocal test of the ascertainment bias hypothesis

Interspecific comparisons of microsatellite loci have repeatedly shown that the loci are longer and more variable in the species from which they are derived (the focal species) than are homologous loci in other (nonfocal) species. There is debate as to whether this is due to directional evolution or to an ascertainment bias during the cloning and locus selection processes. This study tests these hypotheses by performing a reciprocal study. Eighteen perfect dinucleotide microsatellite loci identified from a Drosophila simulans library screen and 18 previously identified in an identical Drosophila melanogaster library screen were used to survey natural populations of each species. No difference between focal and nonfocal species was observed for mean PCR fragment length. However, heterozygosity and number of alleles were significantly higher in the focal species than in the nonfocal species. The most common allele in the Zimbabwe population of both species was sequenced for 31 of the 36 loci. The length of the longest stretch of perfect repeat units is, on average, longer in the focal species than in the non-focal species. There is a positive correlation between the length of the longest stretch of perfect repeats and heterozygosity. The difference in heterozygosity can thus be explained by a reduction in the length of the longest stretch of perfect repeats in the nonfocal species. Furthermore, flanking-sequence length difference was noted between the two species at 58% of the loci sequenced. These data do not support the predictions of the directional-evolution hypothesis; however, consistent with the ascertainment bias hypothesis, the lower variability in nonfocal species is an artifact of the microsatellite cloning and isolation process. Our results also suggest that the magnitude of ascertainment bias for repeat unit length is a function of the microsatellite size distribution in the genomes of different species.      

siRNA, miRNA and HIV: promises and challenges

INTRODUCTION The recent discovery of small interfering RNA (siRNA) revealed an important role for small RNAs in regulating gene expression. First described in plants, as “post- trancriptional gene silencing” (PTGS) [1], RNA interfer- ence (RNAi) is a nucleic-acid based immune defense against viruses, transgenes and transposons [2]. Triggered by double-stranded RNA (dsRNA), RNAi leads to the se- quence specific degradation of a target mRNA [3]. In eukaryotic cells, long dsRN…     

Early and late passage C-6 glial cell growth: Similarities with primary glial cells in culture

Earlier studies in our laboratory have shown that C-6 glial cells in culture exhibit astrocytic properties with increasing cell passage. In this study, we tested the responsiveness of early and late passage C-6 glial cells to various cultures conditions: culture substrata (collagen, poly-L-lysine, plastic), or supplements for the culture medium, DMEM, [fetal calf, or heat inactivated (HI) serum, or media conditioned from mouse neuroblastoma cells (NBCM) or primary chick embryo cultured neurons (NCM)]. Glutamine synthetase (GS) and cyclic nucleotide phosphohydrolase (CNP), astrocytic and oligodendrocytic glial markers, were used. Cell numer and protein content increased exponentially with days in culture regardless of the type of the substratum or cell passage. Differences in cell morphology among the three types of substratum were also reflected on GS activity, which rose by three-fold on culture day 3 for cells grown on collagen; thereafter, GS profiles were similar for all substrata. This early rise in GS is interpreted to reflect differential cell adhesion processes on the substrata; specifically, cell adhesion on the collagen stimulated differentiation into astrocytic phenotype.Analogous to immature glia cells in primary cultures, early passage C-6 glial cells responded to neuronal factors supplied either from NCM or NBCM by expressing reduced GS activity, the astrocytic marker and enhanced CNP activity, the oligodendrocytic marker. Thus, early passage cells can be induced to express either astrocytic or oligodendrocytic phenotype. In accordance with our previous reports on primary glial cells, late passage C-6 cells exhibit their usual astrocytic behavior, responding to serum factors with GS activity. Moreover, whereas NCM or NBCM alone markedly lowered GS activity, a combination with serum restored activity. The present findings confirm our previous observations and further establish the C-6 glial cells as a reliable model to study immature glia.Special issue dedicated to Dr. Paola S. Timiras.