The ACC1 gene,encoding acetyl-CoA carboxylase,is essential for growth in Ustilago maydis

Acetyl-CoA carboxylase [ACCase; acetylCoA: carbon dioxide ligase (ADP forming), EC 6.4.1.2] catalyses the ATP-dependent carboxylation of acetylCoA to form malonyl-CoA. We have amplified a fragment of the biotin carboxylase (BC) domain of the Ustilago maydis acetyl-CoA carboxylase (ACC1) gene from genomic DNA and used this amplified DNA fragment as a probe to recover the complete gene from a λEMBL3 genomic library. The ACC1 gene has a reading frame of 6555 nucleotides, which is interrupted by a single intron of 80 bb in length. The gene encodes a protein containing 2185 amino acids, with a calculated M_r of 242 530; this is in good agreement with the size of ACCases from other sources. Further identification was based on the position of putative binding sites for acetyl-CoA, ATP, biotin and carboxybiotin found in other ACCases. A single ACC1 allele was disrupted in a diploid wild-type strain. After sporulation of diploid disruptants, no haploid progeny containing a disrupted acc1 allele were recovered, even though an exogenous source of fatty acids was provided. The data indicate that, in U. maydis, ACCase is required for essential cellular processes other than de novo fatty acid biosynthesis.     

Effect of low oxygen tension on the biological characteristics of human bone marrow mesenchymal stem cells

Culture of mesenchymal stem cells (MSCs) under ambient conditions does not replicate the low oxygen environment of normal physiological or pathological states and can result in cellular impairment during culture. To overcome these limitations, we explored the effect of hypoxia (1 % O₂) on the biological characteristics of MSCs over the course of different culture periods. The following biological characteristics were examined in human bone marrow-derived MSCs cultured under hypoxia for 8 weeks: proliferation rate, morphology, cell size, senescence, immunophenotypic characteristics, and the expression levels of stemness-associated factors and cytokine and chemokine genes. MSCs cultured under hypoxia for approximately 2 weeks showed increased proliferation and viability. During long-term culture, hypoxia delayed phenotypic changes in MSCs, such as increased cell volume, altered morphology, and the expression of senescence-associated-β-gal, without altering their characteristic immunophenotypic characteristics. Furthermore, hypoxia increased the expression of stemness and chemokine-related genes, including OCT4 and CXCR7, and did not decrease the expression of KLF4, C-MYC, CCL2, CXCL9, CXCL10, and CXCR4 compared with levels in cells cultured under normoxia. In conclusion, low oxygen tension improved the biological characteristics of MSCs during ex vivo expansion. These data suggest that hypoxic culture could be a useful method for increasing the efficacy of MSC cell therapies.     

Hypertrophy in skeletal myotubes induced by junctophilin-2 mutant, Y141H, involves an increase in store-operated Ca2+ entry via Orai1

Junctophilins (JPs) play an important role in the formation of junctional membrane complexes (JMC) in striated muscle by physically linking the transverse-tubule and sarcoplasmic reticulum (SR) membranes. Researchers have found five JP2 mutants in humans with hypertrophic cardiomyopathy. Among these, Y141H and S165F are associated with severely altered Ca(2+) signaling in cardiomyocytes. We previously reported that S165F also induced both hypertrophy and altered intracellular Ca(2+) signaling in mouse skeletal myotubes. In the present study, we attempted to identify the dominant-negative role(s) of Y141H in primary mouse skeletal myotubes. Consistent with S165F, Y141H led to hypertrophy and altered Ca(2+) signaling (a decrease in the gain of excitation-contraction coupling and an increase in the resting level of myoplasmic Ca(2+)). However, unlike S165F, neither ryanodine receptor 1-mediated Ca(2+) release from the SR nor the phosphorylation of the mutated JP2 by protein kinase C was related to the altered Ca(2+) signaling by Y141H. Instead, abnormal JMC and increased SOCE via Orai1 were found, suggesting that the hypertrophy caused by Y141H progressed differently from S165F. Therefore JP2 can be linked to skeletal muscle hypertrophy via various Ca(2+) signaling pathways, and SOCE could be one of the causes of altered Ca(2+) signaling observed in muscle hypertrophy.     

ErbB2 overexpression in p53-inactivated mammary epithelial cells

Functional loss of p53 and ErbB2 overexpression are the frequent genetic alterations in human breast carcinomas. Here, we found that ErbB2 expression was upregulated in primary cultured mammary epithelial cells (MECs) isolated from mice with a defect in exons 5 and 6 of the p53 gene (p53(Delta5,6)). The reporter gene activity in the p53(Delta5,6) MECs transfected with the -756bp flanking region of the hErbB2 gene was higher than the wild type MECs. p53 inactivation selectively increased the level of AP-2alpha, but not AP-2beta and AP-2gamma and a mutation of the two AP-2 binding sites completely inhibited the reporter activity.     

poky/chuk/ikk1 is required for differentiation of the zebrafish embryonic epidermis

An epidermis surrounds all vertebrates, forming a water barrier between the external environment and the internal space of the organism. In the zebrafish, the embryonic epidermis consists of an outer enveloping layer (EVL) and an inner basal layer that have distinct embryonic origins. Differentiation of the EVL requires the maternal effect gene poky/ikk1 in EVL cells prior to establishment of the basal layer. This requirement is transient and maternal Ikk1 is sufficient to allow establishment of the EVL and formation of normal skin in adults. Similar to the requirement for Ikk1 in mouse epidermis, EVL cells in poky mutants fail to exit the cell cycle or express specific markers of differentiation. In spite of the similarity in phenotype, the molecular requirement for Ikk1 is different between mouse and zebrafish. Unlike the mouse, EVL differentiation requires functioning Poky/Ikk1 kinase activity but does not require the HLH domain. Previous work suggested that the EVL was a transient embryonic structure, and that maturation of the epidermis required replacement of the EVL with cells from the basal layer. We show here that the EVL is not lost during embryogenesis but persists to larval stages. Our results show that while the requirement for poky/ikk1 is conserved, the differences in molecular activity indicate that diversification of an epithelial differentiation program has allowed at least two developmental modes of establishing a multilayered epidermis in vertebrates.     

Aberrant allocations of inner cell mass and trophectoderm cells in bovine nuclear transfer blastocysts

Abortions of nuclear transfer (NT) embryos are mainly due to insufficient placentation. We hypothesized that the primary cause might be the aberrant allocations of two different cell lineages of the blastocyst stage embryos, the inner cell mass (ICM) and the trophectoderm (TE) cells. The potential for development of NT embryos to blastocysts was similar to that for in vitro fertilized (IVF) embryos. No difference in the total cell number was detected between NT and IVF blastocysts, but both types of embryos had fewer total cells than did in vivo-derived embryos (P < 0.05). The NT blastocysts showed a higher ratio of ICM:total cells than did IVF or in vivo-derived embryos (P < 0.05). Individual blastocysts were assigned to four subgroups (I: <20%, II: 20-40%, III: 40-60%, IV: >60%) according to the ratio of ICM:total cells. Most NT blastocysts were placed in groups III and IV, whereas most IVF and in vivo-derived blastocysts were distributed in group II. Our findings suggest that placental abnormalities or early fetal losses in the present cloning system may be due to aberrant allocations of NT embryos to the ICM and TE cells during early development.     

Ribosomal S6 kinase 2 inhibition by a potent C-terminal repressor domain is relieved by mitogen-activated protein-extracellular signal-regulated kinase kinase-regulated phosphorylation

Ribosomal S6 kinase 2 (S6K2) is a recently identified serine/threonine protein kinase that phosphorylates the 40 S ribosomal protein S6 in vitro. S6K2 is highly homologous to S6K1 in the core kinase and linker regulatory domains but differs from S6K1 in the N- and C-terminal regions and is differently localized primarily to the nucleus because of a C-terminal nuclear localization signal unique to S6K2. We have recently demonstrated that S6K2 is regulated similarly to S6K1 by the mammalian target of rapamycin pathway and by multiple PI3-K pathway effectors in vivo. However, deletion of the C-terminal domain of S6K2 enhances kinase activity, whereas analogous deletion of S6K1 is inhibitory. Here, we characterize the S6K2 C-terminal motifs that confer this differential regulation. We demonstrate that the inhibitory effects of the S6K2 C-terminal domain are only partly attributable to the nuclear localization signal but that three C-terminal proline-directed potential mitogen-activated protein kinase phosphorylation sites are critical mediators of this inhibitory effect. Site-specific mutation of these sites to alanine completely desensitizes S6K2 to activating inputs, whereas mutation to aspartic acid to mimic phosphorylation results in an activated enzyme which is hypersensitive to activating inputs. Pretreatment of cells with the mitogen-activated protein-extracellular signal-regulated kinase kinase (MEK) inhibitor U0126 inhibited S6K2 activation to a greater extent than S6K1. Furthermore, S6K2 mutants with C-terminal deletion or acidic phosphorylation site mutations displayed greatly reduced U0126 sensitivity. Thus, MEK-dependent inputs to C-terminal phosphorylation sites appear to be essential for relief of S6K2 inhibition but less critical for activation of S6K1. These data suggest a mechanism by which weak PI3-K agonists can regulate S6 phosphorylation and selective translation in the presence of mitogen-activated protein kinase signaling.     

Cell culture density affects the proliferation activity of human adipose tissue stem cells

In this study, we investigated the effect of cell density on the proliferation activity of human mesenchymal stem cells (MSCs) derived from adipose tissue (AT‐MSCs) over time in culture. Passage #4 (P4) and #12 (P12) AT‐MSCs from two donors were plated at a density of 200 (culture condition 1, CC1) or 5000 (culture condition 2, CC2) cells cm^?2. After 7 days of incubation, P4 and P12 AT‐MSCs cultured in CC1 were thin and spindle‐shaped, whereas those cultured in CC2 had extensive cell‐to‐cell contacts and an expanded cell volume. In addition, P4 and P12 AT‐MSCs in CC1 divided more than three times, while those in CC2 divided less than once on average. Flow cytometric analysis using 5(6)‐carboxyfluorescein diacetate N‐succinimidyl ester dye showed that the fluorescence intensity of AT‐MSCs was lower in CC1 than in CC2. Furthermore, expression of proliferation‐associated genes, such as CDC45L, CDC20A and KIF20A, in P4 AT‐MSCs was higher in CC1 than in CC2, and this difference was also observed in P12 AT‐MSCs. These data demonstrated that cell culture density affects the proliferation activity of MSCs, suggesting that it is feasible to design a strategy to prepare suitable MSCs using specific culture conditions. Copyright © 2016 John Wiley & Sons, Ltd.     

In vitro 3-D culture demonstrates incompetence in improving maintenance ability of primary hepatocytes

Primary hepatocytes (PHs) are considered the ‘gold standard’ in drug screening owing to their ability to express many drug-metabolizing enzymes and transporters. Culturing hepatocytes and maintaining their fate in vitro is a major issue since last decade. The main problem with in vitro hepatocytes culture is that they rapidly lose their hepatic morphology and liver-specific functions in culture. Herein, we isolated rat PHs, and cultured them in monolayers (2-D) and spheroids (3-D). The 2-D-cultured PHs exhibited elongated morphology, whereas the 3-D-cultured PHs exhibited spheroid morphology with gradual diameter decrease until 7 days. After 7 days of in vitro culture, PHs were analyzed for the expression of hepatic (Alb, Tf, and Afp) and apoptotic markers (Bax and Bcl2), and co-expression of CYP3A1 and Abumin after 2 and 7 days. Furthermore, in both cultures, PHs were induced with 3-methylcholanthrene (3-MC, Cyp1a-specific inducer) and dexamethasone (Cyp3a-specific inducer) for 48 and 72?h, respectively. The mRNA levels of Cyp1a and Cyp3a were analyzed in induced (3-MC, dexamethasone) and non-induced PHs. After 7 days of in vitro culture, PHs exhibited dramatic downregulation of hepatic marker expression in both cultures. Furthermore, apoptotic marker expression was higher in the 2-D-cultured PHs than 3-D-cultured PHs. The mRNA levels of Cyp1a and Cyp3a indicated higher RNA content in the 2-D-cultured PHs after 48?h of induction. Therefore, we concluded that there was no significant difference between the culture systems, and further studies are required to identify the essential components for in vitro PH culture rather than culture systems.