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The fetal heart is highly sensitive to changes in mechanical load. We have previously demonstrated that increased cardiac load can stimulate cell cycle activity and maturation of immature cardiomyocytes, but the effects of reduced load are not known. Sixteen fetal sheep were given either continuous intravenous infusion of lactated Ringer solution (LR) or enalaprilat, an angiotensin-converting enzyme inhibitor beginning at 127 days gestational age. After 8 days, fetal arterial pressure in the enalaprilat-infused fetuses (23.8 [+ or -] 2.8 mmHg) was lower than that of control fetuses (47.5 [+ or -] 4.7 mmHg) (P < 0.0001). Although the body weights of the two groups of fetuses were similar, the heart weight-to-body weight ratios of the enalaprilat-infused fetuses were less than those of the LR-infused fetuses (5.6 [+ or -] 0.5 g/kg vs. 7.0 [+ or -] 0.6 g/kg, P < 0.0001). Dimensions of ventricular myocytes were not different between control and enalaprilat-infused fetuses. However, there was a significant decrease in cell cycle activity in both the right ventricle (P < 0.005) and the left ventricle (P < 0.002) of the enalaprilat-infused fetuses. Thus, we conclude a sustained reduction in systolic pressure load decreases hyperplastic growth in the fetal heart. blood pressure; enalaprilat; hyperplasia; cardiomyocyte doi: 10.1152/ajpregu.00754.2009.

The mRNA expression of myogenic regulatory factors, including myoD1 (myoblast determination factor) gene paralogs, and their regulation by amino acids and insulin-like growth factors were investigated in primary cell cultures isolated from fast myotomal muscle of Atlantic salmon (Salmo salar). The cell cycle and S phase were determined as 28.1 and 13.3 h, respectively, at 18[degrees]C. Expression of myoD1b and myoD1c peaked at 8 days of culture in the initial proliferation phase and then declined more than sixfold as cells differentiated and was correlated with PCNA (proliferating cell nuclear antigen) expression (R = 0.88, P < 0.0001; R = 0.70, P < 0.0001). In contrast, myoDla transcripts increased from 2 to 8 days and remained at elevated levels as myotubes were formed. mRNA levels of myoD1c were, on average, 3.1- and 5.7-fold higher than myoDla and myoD1b, respectively. Depriving cells of amino acids and serum led to a rapid increase in pax7 and a decrease in myoD1c and PCNA expression, indicating a transition to a quiescent state. In contrast, amino acid replacement in starved cells produced significant increases in myoD1c (at 6 h), PCNA (at 12 h), and myoD1b (at 24 h) and decreases in pax7 expression as cells entered the cell cycle. Our results are consistent with temporally distinct patterns of myoD1c and myoD1b expression at the [G.sub.1] and S/[G.sub.2] phases of the cell cycle. Treatment of starved cells with insulin-like growth factor I or II did not alter expression of the myoD paralogs. It was concluded that, in vitro, amino acids alone are sufficient to stimulate expression of genes regulating myogenesis in myoblasts involving autocrine/ paracrine pathways. The differential responses of myoD paralogs during myotube maturation and amino acid treatments suggest that myoD1b and myoD1c are primarily expressed in proliferating cells and myoD1a in differentiating cells, providing evidence for their subfunctionalization following whole genome and local duplications in the Atlantic salmon lineage. myoblast; myogenesis; cell cycle; amino acid stimulation; genome duplication; subfunctionalization; myoD doi: 10.1152/ajpregu.00114.2010.

The unicellular rhodophyte Cyanidioschyzon merolae, having a single plastid and a single mitochondrion, is suitable for the analysis of the cell cycle involving the division of organelles. In conventional methods of synchronous culture of algae, light/dark cycles have been used as signals for synchronization, and the gene expression promoted by light is not separated from the gene expression related to cell cycle progression. We previously devised a novel synchronous culture system with controlled photosynthesis, which is triggered by 6 h-light/18 h-dark cycles combined with different levels of C[O.sub.2]. The cells do not enter S-phase and consequently do not divide after the minimum light period without C[O.sub.2] supplementation, but do divide after a light period with 1% C[O.sub.2]. In this way, we can compare a dividing cycle and a non-dividing cycle. We examined changes in the expression of 74 genes throughout the cell cycle by quantitative RTPCR. The expression of genes for two cyclins (cyclin C and H) and two CDKs (CDKA and CDKD) as well as metabolic enzymes was promoted by light, whereas the expression of genes for G1/S or G2/M cyclins and CDKs as well as DNA replication enzymes and proteins related to organellar division was promoted only in the dividing cycles. These results suggested that C. merolae has a checkpoint for G1/S progression, which is regulated by nutrients within the 6 h light period. DOI 10.1099/mic.0.037754-0

Two prominent timekeeping systems, the cell cycle, which controls cell division, and the circadian system, which controls 24-h rhythms of physiology and behavior, are found in nearly all living organisms. A distinct feature of circadian rhythms is that they are temperature-compensated such that the period of the rhythm remains constant (~24 h) at different ambient temperatures. Even though the speed of cell division, or growth rate, is highly temperature-dependent, the cell-mitosis rhythm is temperature-compensated. Twenty-four-hour fluctuations in cell division have also been observed in numerous species, suggesting that the circadian system is regulating the timing of cell division. We tested whether the cell-cycle rhythm was coupled to the circadian system in immortalized rat-1 fibroblasts by monitoring cell-cycle gene promoter-driven luciferase activity. We found that there was no consistent phase relationship between the circadian and cell cycles, and that the cell-cycle rhythm was not temperature-compensated in rat-1 fibroblasts. These data suggest that the circadian system does not regulate the cell-mitosis rhythm in rat-1 fibroblasts. These findings are inconsistent with numerous studies that suggest that cell mitosis is regulated by the circadian system in mammalian tissues in vivo. To account for this discrepancy, we propose two possibilities: (i) There is no direct coupling between the circadian rhythm and cell cycle but the timing of cell mitosis is synchronized with the rhythmic host environment, or (ii) coupling between the circadian rhythm and cell cycle exists in normal cells but it is disconnected in immortalized cells. cell cycle | reporter gene | temperature compensation | Cyclin B1 | cancer www.pnas.org/cgi/doi/10.1073/pnas.0914078107

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2009.12.018 Byline: Cheng Huang (a)(d), Shanye Gu (b), Pengchun Yu (b), Fudong Yu (c), Chun Feng (a), Ning Gao (e), Jiulin Du (b) Keywords: Cell cycle; Chromatin remodeling; Development; Schimke Immuno-Osseous Dysplasia; smarcal1; Apoptosis Abstract: Mutations in SMARCAL1 cause Schimke Immuno-Osseous Dysplasia (SIOD), an autosomal recessive multisystem developmental disease characterized by growth retardation, T-cell deficiency, bone marrow failure, anemia and renal failure. SMARCAL1 encodes an ATP-driven annealing helicase. However, the biological function of SMARCAL1 and the molecular basis of SIOD remain largely unclear. In this work, we cloned the zebrafish homologue of the human SMARCAL1 gene and found that smarcal1 regulated cell cycle progression. Morpholino knockdown of smarcal1 in zebrafish recapitulated developmental abnormalities in SIOD patients, including growth retardation, craniofacial abnormality, and haematopoietic and vascular defects. Lack of smarcal1 caused G0/G1 cell cycle arrest and induced cell apoptosis. Furthermore, using Electrophoretic Mobility Shift Assay and reporter assay, we found that SMARCAL1 was transcriptionally inhibited by E2F6, an important cell cycle regulator. Over-expression of E2F6 in zebrafish embryos reduced the expression of smarcal1 mRNA and induced developmental defects similar to those in smarcal1 morphants. These results suggest that SIOD may be caused by defects in cell cycle regulation. Our study provides a model of SIOD and reveals its cellular and molecular bases. Author Affiliation: (a) Institute for Nutritional Sciences and Key Laboratory of Nutrition and Metabolism, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China (b) Institute of Neuroscience and State Key Laboratory of Neuroscience, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China (c) Bioinformatics Center and Key Laboratory of Systems Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China (d) School of Pharmacy, Shanghai University of Traditional Chinese Medicine, 1200 Cailun Road, Shanghai 201203, China (e) Department of Pharmacognosy, School of Pharmacy, Third Military Medical University, 30 Gaotanyan Street, Chongqing 400038, China Article History: Received 24 March 2009; Revised 14 December 2009; Accepted 15 December 2009

The goal of this study was to determine whether caveolin-2 (Cav-2) is capable of controlling endothelial cell (EC) proliferation in vitro. To realize this goal, we have directly compared proliferation rates and cell cycle-associated signaling proteins between lung ECs isolated from wild-type (WT) and Cav-2 knockout (KO) mice. Using three independent proliferation assays, we have determined that Cav-2 KO ECs proliferate by ca. 2-fold faster than their WT counterparts. Cell cycle analysis by flow cytometry of propidium iodide-stained cells showed a relatively higher percentage of Cav-2 KO ECs in S and [G.sub.2]/M and lower percentage in [G.sub.o]/[G.sub.1] phases of cell cycle relative to their WT counterparts. Furthermore, an over 2-fold increase in the percentage of S phase-associated Cav-2 KO relative to WT ECs was independently determined with bromodeoxyuridine incorporation assay. Mechanistically, the increase in proliferation/cell cycle progression of Cav-2 KO ECs correlated well with elevated expression levels of predominantly S phase- and [G.sub.2]/M phase-associated cyclin A and B1, respectively. Further mechanistic analysis of molecular events controlling cell cycle progression revealed increased level of hyperphosphorylated (inactive) form of [G.sub.1] to S phase transition inhibitor, the retinoblastoma protein in hyperproliferating Cav-2 KO ECs. Conversely, the expression level of the two cyclin-dependent kinase inhibitors [p16.sup.INK4] and [p27.sup.Kip1] was reduced in Cav-2 KO ECs. Finally, increased phosphorylation (activation) of proproliferative extracellular signal-regulated kinase 1/2 was observed in hyperproliferating Cav-2 KO ECs. Overall, our data suggest that Cav-2 negatively regulates lung EC proliferation and cell cycle progression. phospho-Rb; phospho-ERK1/2; [pl6.sup.INK4]; [p27.sup.Kip1] doi:10.1152/ajpcell.00401.2009.

Pneumocystis carinii (Pc) causes severe pneumonia in immunocompromised hosts. The binding of Pc trophic forms to alveolar epithelial ceils is a central feature of infection, inducing the expression and activation of Pc-Ste20, a gene participating in mating, proliferation, and pseudohyphal growth. In related fungi, Ste20 proteins are generally activated by immediate upstream small G proteins of the Cdc42-like family. PcCdc42 has not been previously described in Pneumocystis. To address the potential role of such a G protein in Pneumocystis, PcCdc42 was cloned from a Pc cDNA library. Using the full-length 576-bp PcCdc42 cDNA sequence, a CHEF blot of genomic DNA yielded a single band, providing evidence that this gene is present as a single copy within the genome. The total length of PcCdc42 cDNA was 576 bp with an estimated molecular mass of ~38 kDa. BLASTP analysis demonstrated greater than 80% homology with other fungal Cdc42p proteins. Northern analysis indicated equal mRNA expression in both cystic and trophic life forms. Heterologous expression of PcCdc42 in Saccharomyces cerevisiae (Sc) demonstrated that PcCdc42p was able to restore growth in an ScCdc42[DELTA] yeast strain. Additional assays with purified PcCdc42 protein demonstrated GTP binding and intrinsic GTPase activity, which was partially but significantly suppressed by Clostridium difficile toxin B, characteristic of Cdc42 GTPases. Furthermore, PcCdc42 protein was also shown to bind to the downstream PCSte20 kinase partner in the presence (but not the absence) of GTP. These data indicate that Pc possesses a Cdc42 gene expressing an active G protein, which binds the down-stream regulatory kinase PcSte20, important in Pc life cycle regulation. Cdc42; G protein doi: 10.1152/ajplung.00191.2009

Fertilization triggers a rise in intracellular [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]) in the egg that initiates a series of events known as egg activation. These events include cortical granule exocytosis that establishes a block to polyspermy, resumption of meiosis, and recruitment of maternal mRNAs into polysomes for translation. Several calcium-dependent proteins, including calcium/calmodulin-dependent protein kinase II (CaMKII), have been implicated in egg activation. However, the precise role of CaMKII in mediating specific events of egg activation and the identity of the isoform(s) present in mouse eggs have not been unequivocally established. Through targeted deletion of the [gamma] isoform of CaMKII, we find that CaMKII[gamma] is the predominant CaMKII isoform in mouse eggs and that it is essential for egg activation. Although CaMKII[[gamma].sup.-/-] eggs exhibit a normal pattern of [Ca.sup.2+] oscillations after insemination and undergo cortical granule exocytosis, they fail to resume meiosis or to recruit maternal mRNAs. Surprisingly, we find that the recruitment of maternal mRNAs does not directly depend on CaMKII, but requires elevated [[[Ca.sup.2+]].sub.i] and metaphase II exit. We conclude that CaMKII[gamma] specifically controls mouse egg activation by regulating cell cycle resumption. CaMKII | fertilization | metaphase II exit www.pnas.org/cgi/doi/10.1073/pnas.0912658106

Spore formation in Bacillus subtilis is a superb experimental system with which to study some of the most fundamental problems of cellular development and differentiation. Work begun in the 1980s and ongoing today has led to an impressive understanding of the temporal and spatial regulation of sporulation, and the functions of many of the several hundred genes involved. Early in sporulation the cells divide in an unusual asymmetrical manner, to produce a small prespore cell and a much larger mother cell. Aside from developmental biology, this modified division has turned out to be a powerful system for investigation of cell cycle mechanisms, including the components of the division machine, how the machine is correctly positioned in the cell, and how division is coordinated with replication and segregation of the chromosome. Insights into these fundamental mechanisms have provided opportunities for the discovery and development of novel antibiotics. This review summarizes how the bacterial cell cycle field has developed over the last 20 or so years, focusing on opportunities emerging from the B. subtilis system. DOI 10.1099/mic.0.035634-0

Shamhart PE, Luther DJ, Hodson BR, Koshy JC, Ohanyan V, Meszaros JG. Impact of type 1 diabetes on cardiac fibroblast activation: enhanced cell cycle progression and reduced myofibroblast content in diabetic myocardium. Am J Physiol Endocrinol Metab 297:E1147-E1153, 2009. First published August 25, 2009; doi: 10.1152/ajpendo.00327.2009.--Diabetic patients are prone to developing myocardial fibrosis and suffer from decreased wound healing capabilities. The purpose of this study was to determine whether diabetes alters cardiac fibroblast activity in the myocardium in a 6-wk streptozotocin-induced type 1 diabetic model. In vivo echocardiography indicated significant dilation of the left ventricle (LV) in the diabetic animals, while cardiac function was comparable to that in the normal group. We isolated cardiac fibroblasts from diabetic and control hearts and observed increased proliferation of the diabetic fibroblasts. Microarray analysis using mRNA collected from whole LVs revealed downregulation of known inhibitors of proliferation, p53 and p21, in the diabetic group, consistent with our proliferation data. Western blot analysis confirmed a reduction in p53 protein expression in the diabetic hearts compared with control. We explored the potential signaling underlying the downregulation of these cell cycle mediators and determined that activated Akt, a signal that inhibits p53, was elevated in the diabetic group. Surprisingly, the hearts from the diabetic group contained lower levels of the myofibroblast marker [alpha]-smooth muscle actin ([alpha]-SMA) and higher levels of desmin and platelet endothelial cell adhesion molecule (PECAM). The isolated fibroblasts from the diabetic group also contained significantly less a-SMA. These data suggest that early-stage diabetic hearts contain highly proliferative fibroblasts, which predisposes the diabetic myocardium to fibrosis, but have fewer myofibroblasts, which may compromise wound healing. cardiac remodeling; fibrosis; cell growth; hyperglycemia; p53; Akt doi: 10.1152/ajpendo.00327.2009.

Phosphorylated Akt (p-Akt), a phosphoinositide-3-OH-kinase-activated protein kinase, is highly expressed in prostate tumors, p-Akt can indirectly hinder p53-dependent growth suppression and apoptosis by phosphorylating Mdm2. Alternatively, p-Akt can directly phosphorylate p21 and restrict it to the cytoplasm for degradation. Because the prostate is the highest zinc-accumulating tissue before the onset of cancer, the effects of physiological levels of zinc on Akt-Mdm2-p53 and Akt-p21 signaling axes in human normal prostate epithelial cells (PrEC) and malignant prostate LNCaP cells were examined in the present study. Cells were cultured for 6 days in low-zinc growth medium supplemented with 0 [zinc-deficient (ZD)], 4 [zinc-normal (ZN)], 16 [zinc-adequate (ZA)], or 32 [zinc-supplemented (ZS)] [micro]M zinc. Zinc status of both cell types was altered in a dose-dependent manner, with LNCaP cells reaching a plateau at >16 [micro]M zinc. For both cell types, p-Akt was higher in the ZD than in the ZN cells and was normalized to that of the ZN cells by treatment with a PI3K inhibitor, LY-294002. PTEN, an endogenous phosphatase targeting Akt dephosphorylation, was hyperphosphorylated (p-PTEN, inactive form) in ZD PREC. Nuclear p-Mdm2 was raised, whereas nuclear p53 was depressed, by zinc deficiency in PREC. Nuclear p21 and p53 were lowered by zinc deficiency in LNCaP cells. Higher percentages of ZD, ZA, and ZS than ZN LNCaP cells were found at the [G.sub.0]/[G.sub.1] phase of the cell cycle, with proportionally lower precentages at the S and [G.sub.2]/M phases. Hence, the increased p-PTEN in ZD PrEC would result in hyperphosphorylation of p-Akt and p-Mdm2, as well as reduction of nuclear p53 accumulation. For ZD LNCaP cells, Akt hyperphosphorylation was probably mediated through p21 phosphorylation and degradation, thus restricting p21 nuclear entry to induce cell cycle arrest. Thus zinc deficiency differentially modulated the Akt-Mdm2-p53 signaling axis in normal prostate ceils vs. the Akt-p21 signaling axis in malignant prostate cells. zinc status; cell cycle progression; signal transduction doi: 10.1152/ajpcell.00042.2009.

We examined the contribution of S phase in timing cell cycle progression during Drosophila embryogenesis using an approach that deletes S phase rather than arresting its progress. Injection of Drosophila Geminin, an inhibitor of replication licensing, prevented subsequent replication so that the following mitosis occurred with uninemic chromosomes, which failed to align. The effect of S phase deletion on interphase length changed with development. During the maternally regulated syncytial blastoderm cycles, deleting S phase shortened interphase, and deletion of the last of blastoderm S phase (cycle 14) induced an extra synchronous division and temporarily deferred mid-blastula transition (MBT) events. In contrast, deleting S phase after the MBT in cycle 15 did not dramatically affect mitotic timing, which appears to retain its dependence on developmentally programmed zygotic transcription. We conclude that normal S phase and replication checkpoint activities are important timers of the undisturbed cell cycle before, but not after, the MBT.

Numerous proteins, many essential for the DNA replication machinery, interact with proliferating cell nuclear antigen (PCNA) through the PCNA-interacting peptide (PIP) sequence called the PIP box. We have previously shown that the oxidative demethylase human AlkB homologue 2 (hABH2) colocalizes with PCNA in replication foci. In this study, we show that hABH2 interacts with a posttranslationally modified PCNA via a novel PCNA-interacting motif, which we term AlkB homologue 2 PCNA-interacting motif (APIM). We identify APIM in >200 other proteins involved in DNA maintenance, transcription, and cell cycle regulation, and verify a functional APIM in five of these. Expression of an APIM peptide increases the cellular sensitivity to several cytostatic agents not accounted for by perturbing only the hABH2--PCNA interaction. Thus, APIM is likely to mediate PCNA binding in many proteins involved in DNA repair and cell cycle control during genotoxic stress.

Omi/ HtrA2 is a mitochondrial serine protease that has a dual function: while confined in the mitochondria, it promotes cell survival, but when released into the cytoplasm, it participates in caspase-dependent as well as caspase-independent cell death. To investigate the mechanism of Omi/HtrA2's function, we set out to isolate and characterize novel substrates for this protease. We have identified Thanatos-associated protein 5 (THAP5) as a specific interactor and substrate of Omi/HtrA2 in cells undergoing apoptosis. This protein is an uncharacterized member of the THAP family of proteins. THAP5 has a unique pattern of expression and is found predominantly in the human heart, although a very low expression is also seen in the human brain and muscle. THAP5 protein is localized in the nucleus and, when ectopically expressed, induces cell cycle arrest. During apoptosis, THAP5 protein is degraded, and this process can be blocked using a specific Omi/HtrA2 inhibitor, leading to reduced cell death. In patients with coronary artery disease, THAP5 protein levels substantially decrease in the myocardial infarction area, suggesting a potential role of this protein in human heart disease. This work identifies human THAP5 as a cardiac-specific nuclear protein that controls cell cycle progression. Furthermore, during apoptosis, THAP5 is cleaved and removed by the proapoptotic Omi/HtrA2 protease. Taken together, we provide evidence to support that THAP5 and its regulation by Omi/ HtrA2 provide a new link between cell cycle control and apoptosis in cardiomyocytes. Omi/HtrA2; Thanatos-associated protein 5; coronary artery disease; apoptosis

GABA cell dysfunction in both schizophrenia (SZ) and bipolar disorder (BD) involves decreased [GAD.sub.67] expression, although this change involves fundamentally different networks of genes in the 2 disorders. One gene that is common to these 2 networks is cyclin D2, a key component of cell cycle regulation that shows increased expression in SZ, but decreased expression in BD. Because of the importance of cell cycle regulation in maintaining functional differentiation and DNA repair, the current study has examined the genes involved in the [G.sub.1] and [G.sub.2] checkpoints to generate new hypotheses regarding the regulation of the GABA cell phenotype in the hippocampus of SZ and BD. The results have demonstrated significant changes in cell cycle regulation in both SZ and BD and these changes include the transcriptional complex (TC) that controls the expression of E2F/DP-1 target genes critical for progression to [G.sub.2]/M. The methyl-CpG binding domain protein (MBD4) that is pivotal for DNA repair, is significantly up-regulated in the stratum oriens (SO) of CA3/2 and CA1 in SZs and BDs. However, other genes associated with the TC, and the [G.sub.1] and [G.sub.2] checkpoints, show complex changes in expression in the SO of CA3/2 and CA1 of both SZs and BDS. Overall, the patterns of expression observed have suggested that the regulation of functional differentiation and/or genomic integrity of hippocampal GABA cells varies according to diagnosis and their location within the trisynaptic pathway. cyclin D2 | DNA polyrnerase | [G.sub.2] checkpoint | nicotinic receptors | p53

A long-standing question in biology is whether there is an intrinsic mechanism for coordinating growth and the cell cycle in metazoan cells. We examined cell size distributions in populations of lymphoblasts and applied a mathematical analysis to calculate how growth rates vary with both cell size and the cell cycle. Our results show that growth rate is size-dependent throughout the cell cycle. After initial growth suppression, there is a rapid increase in growth rate during the [G.sub.1] phase, followed by a period of constant exponential growth. The probability of cell division varies independently with cell size and cell age. We conclude that proliferating mammalian cells have an intrinsic mechanism that maintains cell size.

Erk1/2 mitogen-activated protein kinases (MAPKs) are often hyperactivated in human cancers, where they affect multiple processes, including proliferation. However, the effects of Erk1/2 loss in normal epithelial tissue, the setting of most extracellular signal-regulated kinase (Erk)--associated neoplasms, are unknown. In epidermis, loss of Erk1 or Erk2 individually has no effect, whereas simultaneous Erk1/2 depletion inhibits cell division, demonstrating that these MAPKs are necessary for normal tissue self-renewal. Growth inhibition caused by Erk1/2 loss is rescued by reintroducing Erk2, but not by activating Erk effectors that promote G1 cell cycle progression. Unlike fibroblasts, in which Erk1/2 loss decreases cyclin D1 expression and induces G1/S arrest, Erk1/2 loss in epithelial cells reduces cyclin B1 and c-Fos expression and induces G2/M arrest while disrupting a gene regulatory network centered on cyclin B1-Cdc2. Thus, the cell cycle stages at which Erkl/2 activity is required vary by cell type, with Erk1/2 functioning in epithelial cells to enable progression through G2/M.

The mechanisms that regulate NK cell trafficking are unclear. Phosphoinositide-3 kinases (PI3K) control cell motility and the p110[gamma], and p110[delta] isoforms are mostly expressed in leukocytes, where they transduce signals downstream of G protein coupled receptors (GPCR) or tyrosine kinase receptors, respectively. Here, we set out to determine the relative contribution of p110[gamma], and p110[delta] to NK cell migration in mice. Using a combination of single-cell imaging analysis of transgenic cells reporting on PI3K activity in real time and small molecule inhibitors of p110[gamma], and p110[delta], we show here that the tyrosine-kinase coupled p110[delta] is linked to GPCR signaling and, depending on the GPCR, may even be preferentially activated over p1107. Using gene-targeted mice, we showed that both isoforms were essential for NK cell chemotaxis to CXCL12 and to CCL3 and, in vivo, for normal NK cell migration during pregnancy and to the inflamed peritoneum. By contrast, only p110[delta] was indispensable for chemotaxis to SIP and CXCL10 and for NK cell distribution throughout lymphoid and nonlymphoid tissues and for extravasation to tumors. These results implicate p110[delta] downstream of GPCRs in NK cells and highlight its nonredundant role as a key regulator of NK cell trafficking in health and disease. cell signaling | cell trafficking | innate immunity

Somatic mammalian cells possess well-established S-phase programs with specific regions of the genome replicated at precise times. The ATR-Chk1 pathway plays a central role in these programs, but the mechanism for how Chk1 regulates origin firing remains unknown. We demonstrate here the essential role of cyclin A2-Cdk1 in the regulation of late origin firing. Activity of cyclin A2-Cdk1 was hardly detected at the onset of S phase, but it was obvious at middle to late S phase under unperturbed condition. Chk1 depletion resulted in increased expression of Cdc25A, subsequent hyperactivation of cyclin A2-Cdk1, and abnormal replication at early S phase. Hence, the ectopic expression of cyclin A2-Cdk1AF (constitutively active mutant) fusion constructs resulted in abnormal origin firing, causing the premature appearance of DNA replication at late origins at early S phase. Intriguingly, inactivation of Cdk1 in temperature-sensitive Cdk1 mutant cell lines (FT210) resulted in a prolonged S phase and inefficient activation of late origin firing even at late S phase. Our results thus suggest that cyclin A2-Cdk1 is a key regulator of S-phase programs. Chk1 | DNA replication | molecular combing | ATR protein | checkpoint

Motivated by a problem encountered in the analysis of cell cycle gene expression data, this article deals with the estimation of parameters subject to order restrictions on a unit circle. A normal eukaryotic cell cycle has four major phases during cell division, and a cell cycle gene has its peak expression (phase angle) during the phase that may correspond to its biological function. Because the phases arc ordered along a circle, the phase angles of cell cycle genes are ordered unknown parameters on a unit circle. The problem of interest is to estimate the phase angles using the information regarding the order among them. We address this problem by developing a circular version of the well-known isotonic regression for Euclidean data. Because of the underlying geometry, the standard pool adjacent violator algorithm (PAVA) cannot be used for deriving the circular isotonic regression estimator (CIRE). However, PAVA can be modified to obtain a computationally efficient algorithm for deriving the CIRE. We illustrate the CIRE by estimating the phase angles of some of well-known cell cycle genes using the unrestricted estimators obtained in the literature. KEY WORDS: Circular isotonic regression; Order restrictions on circle; Phase angles; Pool adjacent violator algorithm: Restricted maximum likelihood estimators; von Mises distribution.

There is significant evidence linking nucleocytoplasmic transport to cell cycle control. The budding yeast, Saccharomyces cerevisiae, serves as an ideal model system for studying transport events critical to cell cycle progression because the nuclear envelope remains intact throughout the cell cycle. Previous studies linked the classical nuclear localization signal (cNLS) receptor, importin-[alpha]/Srp1, to the [G.sub.2]/M transition of the cell cycle. Here, we utilize two engineered mutants of importin-[alpha]/Srp1 with specific molecular defects to explore how protein import affects cell cycle progression. One mutant, Srp1-E402Q, is defective in binding to cNLS cargoes that contain two clusters of basic residues termed a bipartite cNLS. The other mutant, Srp1-55, has defects in release of cNLS cargoes into the nucleus. Consistent with distinct in vivo functional consequences for each of the Srp1 mutants analyzed, we find that overexpression of different nuclear transport factors can suppress the temperature-sensitive growth defects of each mutant. Studies aimed at understanding how each of these mutants affects cell cycle progression reveal a profound defect at the G1 to S phase transition in both srp1-E402Q and srp1-55 mutants as well as a modest [G.sub.1]/S defect in the temperature-sensitive srp1-31 mutant, which was previously implicated in [G.sub.2]/M. We take advantage of the characterized defects in the srp1-E402Q and srp1-55 mutants to predict candidate cargo proteins likely to be affected in these mutants and provide evidence that three of these cargoes, Cdc45, Yox1, and Mcm10, are not efficiently localized to the nucleus in importin-[alpha] mutants. These results reveal that the classical nuclear protein import pathway makes important contributions to the [G.sub.1]/S cell cycle transition.

Cell growth and proliferation require coordinated ribosomal biogenesis and translation. Eukaryotic initiation factors (eIFs) control translation at the rate-limiting step of initiation (1,2). So far, only two eIFs connect extracellular [...]

Our recent studies showed that Src family kinases (SFKs) are important mediators of proliferation in renal proximal tubular cells (RPTC). In this study, we elucidate the signaling mechanisms that mediate SFK regulation of cell proliferation and cycle protein expression, and identify the SFK member responsible for these responses in a mouse RPTC line. Akt, a target of phosphoinositide-3-kinase (PI3K), and ERK1/2 were constitutively phosphorylated in RPTC cultured in the presence of serum. While treatment of cells with PP1, a specific SFK inhibitor, completely blocked phosphorylation of ERK1/2 and Akt, only inhibition of PI3K/Akt resulted in decreased RPTC proliferation. Incubation of cells with PP1 decreased cyclin D1 expression, decreased p27 and p57 phosphorylation, and increased p27 and p57 expression, two cyclin-dependent kinase inhibitors. Inhibition of the PI3K pathway decreased expression of cyclin D1 without altering expression of p27 and p57. In contrast, PP1 and PI3K inhibition had no effect on cyclin E and p21. Although RPTC expressed Src, Fyn, and Lyn, only siRNA-mediated knockdown of Src decreased RPTC proliferation, decreased cyclin D1 expression, and increased p27 and p57 expression. These data reveal that Src is a crucial mediator of RPTC proliferation and Src-mediated proliferation is associated with PI3K-dependent upregulation of cyclin D1 and PI3K-independent downregulation of p27 and p57. signal transduction processes; tissue regeneration

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2008.02.054 Byline: Zhirong Bao, Zhongying Zhao, Thomas J. Boyle, John I. Murray, Robert H. Waterston Keywords: Statistics; Single cell; Fate differentiation; cdc25; Skp1-related Abstract: As a fundamental process of development, cell proliferation must be coordinated with other processes such as fate differentiation. Through statistical analysis of individual cell cycle lengths of the first 8 out of 10 rounds of embryonic cell division in Caenorhabditis elegans, we identified synchronous and invariantly ordered divisions that are tightly associated with fate differentiation. Our results suggest a three-tier model for fate control of cell cycle pace: the primary control of cell cycle pace is established by lineage and the founder cell fate, then fine-tuned by tissue and organ differentiation within each lineage, then further modified by individualization of cells as they acquire unique morphological and physiological roles in the variant body plan. We then set out to identify the pace-setting mechanisms in different fates. Our results suggest that ubiquitin-mediated degradation of CDC-25.1 is a rate-determining step for the E (gut) and P.sub.3 (muscle and germline) lineages but not others, even though CDC-25.1 and its apparent decay have been detected in all lineages. Our results demonstrate the power of C. elegans embryogenesis as a model to dissect the interaction between differentiation and proliferation, and an effective approach combining genetic and statistical analysis at single-cell resolution. Author Affiliation: Department of Genome Sciences, University of Washington, Seattle, WA 98195, USA Article History: Received 17 October 2007; Revised 10 February 2008; Accepted 29 February 2008

CUGBP2, a translation inhibitor, induces colon cancer cells to undergo apoptosis. Mc1-1, an antiapoptotic Bc1-2 family protein, interferes with mitochondrial activation to inhibit apoptosis. Here, we have determined the effect of CUGBP2 on Mc1-1 expression. We developed a HCUG2 cell line by stably expressing CUGBP2 in the HCT-116 colon cancer cells. HCUG2 cells demonstrate decreased levels of proliferation and increased apoptosis, compared with HCT-116 cells. Flow cytometry analysis demonstrated higher levels of cells in the [G.sub.2]-M phase. Western blot analyses demonstrated that there was decreased Bc1-2 and Mc1-1 protein but increased expression of Bax, cyclin B1, and Cdc2. Immunocytochemistry also demonstrated increased levels of cyclin B1 and Cdc2 in the nucleus of HCUG2 cells. However, there was colocalization of phosphorylated histone H3 with transferase-mediated dUTP nick-end labeling (TUNEL). Furthermore, immunostaining for [alpha]-tubulin demonstrated that there was disorganization of microtubules. These data suggest that CUGBP2 expression in HCUG2 cells induces the cells to undergo apoptosis during the [G.sub.2]-M phase of the cell cycle. We next determined the mechanism of CUGBP2mediated reduction in Mc1-1 expression. Mc1-1 protein, but not Mcl-1 mRNA, was lower in HCUG2 cells, suggesting translation inhibition. CUGBP2 binds to Mc1-1 3'-untranslated region (3'-UTR) both in vitro and in HCUG2 cells. Furthermore, CUGBP2 increased the stability of both endogenous Mc1-1 and luciferase mRNA containing the Mc1-1 3'-UTR. However, luciferase protein expression from the luciferase-Mc1-1 3'-UTR mRNA was suppressed. Taken together, these data demonstrate that CUGBP2 inhibits Mc1-1 expression by inhibiting Mc1-1 mRNA translation, resulting in driving the cells to apoptosis during the [G.sub.2] phase of the cell cycle. Bc1-2 family member; [G.sub.2]-M arrest; checkpoint kinases; RNA stability

Interstitial cells of Cajal (ICC) are responsible for the spontaneous and omnipresent electrical activity in the stomach. A quantitative description of the intracellular processes whose coordinated activity is believed to generate electrical slow waves has been developed and is presented here. In line with recent experimental evidence, the model describes how the interplay between the mitochondria and the endoplasmic reticulum in cycling intracellular [Ca.sup.2+] provides the primary regulatory signal for the initiation of the slow wave. The major ion channels that have been identified as influencing slow wave activity have been modeled according to data obtained from isolated ICC. The model has been validated by comparing the simulated profile of the slow waves with experimental recordings and shows good correspondence in terms of frequency, amplitude, and shape in both control and pharmacologically altered conditions. interstitial cells of Cajal; mathematical model; gastric electrophysiology

[alpha] Thalassemia/mental retardation X linked (ATRX) is switch/sucrose nonfermenting-type ATPase localized at pericentromeric heterochromatin in mouse and human cells. Human ATRX mutations give rise to mental retardation syndromes characterized by developmental delay, facial dysmorphisms, cognitive deficits, and microcephaly and the loss of ATRX in the mouse brain leads to reduced cortical size. We find that ATRX is required for normal mitotic progression in human cultured cells and in neuroprogenitors. Using live cell imaging, we show that the transition from prometaphase to metaphase is prolonged in ATRX-depleted cells and is accompanied by defective sister chromatid cohesion and congression at the metaphase plate. We also demonstrate that loss of ATRX in the embryonic mouse brain induces mitotic defects in neuroprogenitors in vivo with evidence of abnormal chromosome congression and segregation. These findings reveal that ATRX contributes to chromosome dynamics during mitosis and provide a possible cellular explanation for reduced cortical size and abnormal brain development associated with ATRX deficiency.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2006.08.035 Byline: Jane C. Quinn (a), Michael Molinek (a), Ben S. Martynoga (a), Paulette A. Zaki (a), Andrea Faedo (b), Alessandro Bulfone (b), Robert F. Hevner (c), John D. West (d), David J. Price (a) Keywords: Chimera; Pax6; Proliferation; Telencephalon; Mouse; Tbr2; Mash1; Nkx2.1; Gsh2; Dlx2; Apical progenitor cell; Basal progenitor cell Abstract: Many cerebral cortical neurons and glia are produced by apical progenitors dividing at the ventricular surface of the embryonic dorsal telencephalon. Other neurons are produced by basal progenitor cells, which are derived from apical progenitors, dividing away from the ventricular surface. The transcription factor Pax6 is expressed in apical progenitors and is downregulated in basal progenitors, which upregulate the transcription factor Tbr2. Here we show that Pax6.sup.-/- cells are under-represented in the cortex of Pax6.sup.+/+ aPax6.sup.-/- chimeras early in corticogenesis, indicating that Pax6 is required for the production of normal numbers of cortical cells. We provide evidence that this underproduction is attributable to an early depletion of the progenitor pool caused by greater than normal proportions of newly divided cells exiting the cell cycle. We show that most progenitor cells dividing away from the ventricular surface in Pax6.sup.-/- embryos fail to express the transcription factor Tbr2 and that Pax6 is required cell autonomously for Tbr2 expression in the developing cortex of Pax6.sup.+/+ aPax6.sup.-/- chimeras. Transcription factors normally expressed ventrally in the telencephalic ganglionic eminences (Mash1, Dlx2 and Gsh2) are upregulated cell autonomously in mutant cells in the developing cortex of Pax6.sup.+/+ aPax6.sup.-/- chimeras; Nkx2.1, which is expressed only in the medial ganglionic eminence, is not. These data indicate that early functions of Pax6 in developing cortical cells are to repress expression of transcription factors normally found in the lateral ganglionic eminence, to prevent precocious differentiation and depletion of the progenitor pool, and to induce normal development of cortical basal progenitor cells. Author Affiliation: (a) Genes and Development Group, Department of Biomedical Sciences, Centres for Integrative Physiology and Neuroscience Research, University of Edinburgh, Hugh Robson Building, George Square, Edinburgh, EH8 9XD, UK (b) Stem Cell Research Institute, Dibit, H. S. Raffaele, Via Olgettina 58, 20132 Milan, Italy (c) Department of Pathology, University of Washington, Seattle, Washington, WA 98104, USA (d) Division of Reproductive and Developmental Sciences, Genes and Development Group, University of Edinburgh, Hugh Robson Building, George Square, Edinburgh, EH8 9XD, UK Article History: Received 7 June 2006; Revised 11 August 2006; Accepted 15 August 2006

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2006.09.012 Byline: Antonio Fernandez-Guerra (a), Antoine Aze (a), Julia Morales (b), Odile Mulner-Lorillon (b), Bertrand Cosson (b), Patrick Cormier (b), Cynthia Bradham (c), Nikki Adams (d), Anthony J. Robertson (e), William F. Marzluff (f), James A. Coffman (e), Anne-Marie Geneviere (a) Keywords: Sea urchin; Cell cycle; Replication; Mitosis; Checkpoint; Kinases Abstract: A search of the Strongylocentrotus purpuratus genome for genes associated with cell cycle control and DNA metabolism shows that the known repertoire of these genes is conserved in the sea urchin, although with fewer family members represented than in vertebrates, and with some cases of echinoderm-specific gene diversifications. For example, while homologues of the known cyclins are mostly encoded by single genes in S. purpuratus (unlike vertebrates, which have multiple isoforms), there are additional genes encoding novel cyclins of the B and K/L types. Almost all known cyclin-dependent kinases (CDKs) or CDK-like proteins have an orthologue in S. purpuratus; CDK3 is one exception, whereas CDK4 and 6 are represented by a single homologue, referred to as CDK4. While the complexity of the two families of mitotic kinases, Polo and Aurora, is close to that found in the nematode, the diversity of the NIMA-related kinases (NEK proteins) approaches that of vertebrates. Among the nine NEK proteins found in S. purpuratus, eight could be assigned orthologues in vertebrates, whereas the ninth is unique to sea urchins. Most known DNA replication, DNA repair and mitotic checkpoint genes are also present, as are homologues of the pRB (two) and p53 (one) tumor suppressors. Interestingly, the p21/p27 family of CDK inhibitors is represented by one homologue, whereas the INK4 and ARF families of tumor suppressors appear to be absent, suggesting that these evolved only in vertebrates. Our results suggest that, while the cell cycle control mechanisms known from other animals are generally conserved in sea urchin, parts of the machinery have diversified within the echinoderm lineage. The set of genes uncovered in this analysis of the S. purpuratus genome should enhance future research on cell cycle control and developmental regulation in this model. Author Affiliation: (a) Observatoire Oceanologique de Banyuls-Laboratoire Arago, CNRS-UMR7628/UPMC, 66650 Banyuls-sur-Mer, France (b) Station Biologique de Roscoff, CNRS-UMR7150/UPMC, 29680 Roscoff, France (c) Developmental, Cellular, and Molecular Biology Group, Duke University, Durham, NC, USA (d) Department of Biological Sciences, California Polytechnic State University, San Luis Obispo, CA, USA (e) Mount Desert Island Biological Laboratory, Salisbury Cove, ME, USA (f) Program in Molecular Biology, University of North Carolina, Chapel Hill, NC, USA Article History: Received 30 May 2006; Revised 6 September 2006; Accepted 7 September 2006

Adult skeletal muscle is able to repeatedly regenerate because of the presence of satellite cells, a population of stem cells resident beneath the basal lamina that surrounds each myofiber. Little is known, however, of the signaling pathways involved in the activation of satellite cells from quiescence to proliferation, a crucial step in muscle regeneration. We show that sphingosine-1-phosphate induces satellite cells to enter the cell cycle. Indeed, inhibiting the sphingolipid-signaling cascade that generates sphingosine-1-phosphate significantly reduces the number of satellite cells able to proliferate in response to mitogen stimulation in vitro and perturbs muscle regeneration in vivo. In addition, metabolism of sphingomyelin located in the inner leaflet of the plasma membrane is probably the main source of sphingosine-1-phosphate used to mediate the mitogenic signal. Together, our observations show that sphingolipid signaling is involved in the induction of proliferation in an adult stem cell and a key component of muscle regeneration.