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Three alternative strategies for the calculation of the IR intensity of crystalline systems, as determined by Born charges, have been implemented in the Crystalcode, using a Gaussian type basis set. One uses the Berry phase (BP) algorithm to compute the dipole moment; another does so, instead, through well localized crystalline orbitals (Wannier functions, WF); and the third is based on a coupled perturbed Hartree–Fock or Kohn–Sham procedure (CP). In WF and BP, the derivative of the dipole moment with respect to the atomic coordinates is evaluated numerically, whereas in CP it is analytical. In the three cases, very different numerical schemes are utilized, so that the equivalence of the obtained IR intensities is not ensured a priori but instead is the result of the high numerical accuracy of the many computational steps involved. The main aspects of the three schemes are briefly recalled, and the dependence of the results on the computational parameters (number of kpoints in reciprocal space, tolerances for the truncation of the Coulomb and exchange series, and so on) is documented. It is shown that in standard computational conditions the three schemes produce IR intensities that differ by less than 1%; this difference can be reduced by an order of magnitude by acting on the parameters that control the accuracy of the calculation. A large unit cell system (80 atoms per cell) is used to document the relative cost of the three schemes. Within the current implementation the BP strategy, despite its seminumerical nature, is the most efficient choice. That is because it is the oldest implementation, and it is based on the simplest of the three algorithms. Thus, parallelism and other schemes for improving efficiency have, so far, been implemented to a lesser degree in the other two cases.

We have investigated the host/guest molecular interactions and the H-bonding clustering in the H2O/HKUST-1 system. In-situFourier transform infrared spectroscopy was the experimental technique used to gather information at the molecular level. Measurements collected under sorption equilibrium conditions and in a dynamic regime were interpreted with the aid of a detailed QMvibrational analysis of the host/guest adduct and of a series of H2O clusters. The local arrangement of the adduct formed by H2O and the coordinatively unsaturated sites was characterized in terms of structure and interaction energy, and signatures for this adduct were predicted by theory in the ν(OH) range and experimentally confirmed. Within the metal organic framework nanocavities, six-membered water clusters having specific conformations were found to be the prevailing species. Cyclic trimers were also identified, their stabilization being ascribed to nanoconfining effects. The analysis of the δ(HOH) range elucidated the nature of the complex band shape and confirmed the results obtained in the ν(OH) interval. Low-temperature measurements allowed us to exclude the crystallization of the water clusters in the nanocavities.

This work is a synthesis of research into seismic risk perception in the Pollino area (southern Italy). Over the last 3 years, there has been an ongoing earthquake swarm affecting this area that straddles the border between the regions of Calabria and Basilicata. The perception of seismic risk is an important element in environmental planning. If land is considered in terms of reciprocal interaction between humans and their physical space, Geoethics can find a synthesis between humanistic and scientific knowledge with regard to the theme of disasters. Geoethics can especially help in terms of educating the population of an area about integrated risk management. It is believed that improved communications, awareness of risk complexity and levels of preparation would increase a community's resilience and allow for more effective planning. With this premise, a questionnaire was given to students in primary and secondary education, and to a sample of adults in some of the villages affected by the Pollino earthquake swarm. A comparison with people's mental representation of risk regarding great earthquakes of the past, such as those of Calabria in 1783, helps us to clarify the relationship between an extreme event and a disaster.

BACKGROUND: The rabbit Vx2 liver tumor is a fast-growing carcinoma model commonly used to study tumor behaviours under cancer treatments. The reduction of tumor viability and the degree of induced necrosis are the common criteria to evaluate the efficacy of cancer treatments. Currently, it is not easy to perform a rapid and reproducible tissue quantification by histopathological analysis.OBJECTIVE: Our objective was to use infrared-imaging combined with linear-discriminant-analysis model (LDA) to automatically quantify the necrotic effect induced by doxorubicin-eluting-implants (DEI) on a Vx2 liver tumors model.METHODS: Three rabbits were subjected to DEI treatment and compared to a control group (CTRL) of nine rabbits. Tumor bearing livers were resected, fixed in formalin and embedded in paraffin. On two consecutive sections, we performed hematoxylin–eosin–saffron (HES) staining and infrared-imaging. Infrared-images were then subjected to the LDA-model analysis.RESULTS: The LDA and HES images strongly correlated for viable and necrotic tumor tissues. For the DEI group, the model determined that the surface of viable tumor represented 0.24±5% of the tumor (CTRL: 55.71±17%, P<0.0001) and the necrotic tissue 66.46±20% of the tumor (CTRL: 18.45±12%, P<0.0001).CONCLUSIONS: Our results showed that infrared-imaging coupled with LDA-model analysis could be a helpful to easily and objectively assess the treatment efficacy.

Metabolic syndrome is a risk factor for nephrolithiasis. This study was performed to evaluate the clinical and biochemical profile of calcium-oxalate nephrolithiasis in stone formers with metabolic syndrome.A total of 526 recurrent stone formers, 184 of them with metabolic syndrome, and 214 controls were examined. Stone formers with metabolic syndrome showed higher sodium excretion [mean (95% confidence interval), 196 (176–218) vs 160 (150–168) mmol/24h; p<0.01] and lower citrate excretion [2.23 (1.99–2.58) vs 2.84 (2.51–3.17) mmol/24 h; p<0.01] compared to controls, whereas stone formers without metabolic syndrome showed higher calcium and oxalate excretion [5.43 (5.01–5.82) vs 3.58 (2.84–4.19) and 0.34 (0.32–0.36) vs 0.26 (0.20–0.31) mmol/24h for calcium and oxalate, respectively; p<0.01] and lower citrate excretion [2.18 (1.98–2.38) vs 2.84 (2.51–3.17) mmol/24 h; p<0.01] compared to controls.The biochemical profiles was significantly different between stone formers with metabolic syndrome and those without. Dietary habits play a central role in the pathogenesis of nephrolithiasis in stone formers with metabolic syndrome.

Befitting oxygen's key role in life's processes, hypoxia engages multiple signaling systems that evoke pervasive adaptations. Using surrogate genetics in a powerful biological model, we dissect a poorly understood hypoxia-sensing and signal transduction system. Hypoxia triggers NO-dependent accumulation of cyclic GMP and translocation of cytoplasmic GFP-Relish (an NFκB/Rel transcription factor) to the nucleus in DrosophilaS2 cells. An enzyme capable of eliminating NO interrupted signaling specifically when it was targeted to the mitochondria, arguing for a mitochondrial NO signal. Long pretreatment with an inhibitor of nitric oxide synthase (NOS), L-NAME, blocked signaling. However, addition shortly before hypoxia was without effect, suggesting that signaling is supported by the prior action of NOS and is independent of NOS action during hypoxia. We implicated the glutathione adduct, GSNO, as a signaling mediator by showing that overexpression of the cytoplasmic enzyme catalyzing its destruction, GSNOR, blocks signaling, whereas knockdown of this activity caused reporter translocation in the absence of hypoxia. In downstream steps, cGMP accumulated, and calcium-dependent signaling was subsequently activated via cGMP-dependent channels. These findings reveal the use of unconventional steps in an NO pathway involved in sensing hypoxia and initiating signaling.

Befitting oxygen's key role in life's processes, hypoxia engages multiple signaling systems that evoke pervasive adaptations. Using surrogate genetics in a powerful biological model, we dissect a poorly understood hypoxia-sensing and signal transduction system. Hypoxia triggers NO-dependent accumulation of cyclic GMP and translocation of cytoplasmic GFP-Relish (an NFκB/Rel transcription factor) to the nucleus in Drosophila S2 cells. An enzyme capable of eliminating NO interrupted signaling specifically when it was targeted to the mitochondria, arguing for a mitochondrial NO signal. Long pretreatment with an inhibitor of nitric oxide synthase (NOS), L-NAME, blocked signaling. However, addition shortly before hypoxia was without effect, suggesting that signaling is supported by the prior action of NOS and is independent of NOS action during hypoxia. We implicated the glutathione adduct, GSNO, as a signaling mediator by showing that overexpression of the cytoplasmic enzyme catalyzing its destruction, GSNOR, blocks signaling, whereas knockdown of this activity caused reporter translocation in the absence of hypoxia. In downstream steps, cGMP accumulated, and calcium-dependent signaling was subsequently activated via cGMP-dependent channels. These findings reveal the use of unconventional steps in an NO pathway involved in sensing hypoxia and initiating signaling.

This article examines the themes of choice and agency in the context of motherhood as it appears in the Pāli Buddhist texts. I focus on two characters, Māya, the Buddha’s birth mother and Visākhā, one of his best known female lay disciples. Both of these women are known primarily because of their connection to the Buddha, and their spiritual path is neglected, or even disdained, because they are considered as secondary and auxiliary characters whose only purpose is to support the men they surround (Gross, 1995). In this article, I propose a “matricentric” feminist approach (O’Reilly, 2014) to challenge this attitude, and the patriarchal hierarchy that underlies it. Based on this radical interpretation of Buddhist principles, I show how the Buddhist path appears, in the Pāli texts, as a “mothering path” that a number of women, like Māya and Visākhā, choose and undertake as a spiritual path, thereby recognizing women’s agency as mothers and the validity and value of mothering in a soteriological context.

Interleukin (IL)‐5 is a hematopoietic cytokine able to regulate differentiation, survival, and effector functions of eosinophils. It binds specifically to its receptor, which is composed of a cytokine‐specific α‐chain and a β‐chain shared with the receptors for IL‐3 and the granulocyte macrophage‐colony stimulating factor. The molecular mechanisms by which IL‐5 modulates eosinophil survival remain unclear. In this study, we demonstrate that IL‐5 withdrawal induces eosinophil apoptosis through a mitochondria‐dependent pathway, independently of Fas receptor activation. The lipid kinase phosphatidylinositol‐3 kinase plays a crucial role in the maintenance of eosinophil survival, as inhibition of its activity results in apoptosis. IL‐5 induces phosphorylation and thus, inhibition of the Forkhead transcription factor FOXO3a and glycogen synthase kinase 3 (GSK‐3). We analyzed expression of FOXO3a‐dependent transcriptional targets: Fas ligand or Bim (a proapoptotic Bcl‐2 family member), but neither was detected in apoptotic eosinophils. We further show that GSK‐3 is activated after IL‐5 withdrawal, and inhibition of its activity rescues eosinophils from apoptosis. β‐catenin, a direct GSK‐3 substrate, is present in the nucleus of IL‐5‐stimulated eosinophils, but it is translocated to the plasma membrane in the absence of cytokine in a GSK‐3‐dependent manner. This is the first report describing a potential role for GSK‐3 and β‐catenin in regulating eosinophil survival and suggests a novel mechanism by which IL‐5 inhibits the constitutive apoptotic program in these cells.

The central‐zone vibrational spectrum of α‐quartz (SiO2) is calculated by building the Hessian matrix numerically from the analytical gradients of the energy with respect to the atomic coordinates. The nonanalytical part is obtained with a finite field supercell approach for the high‐frequency dielectric constant and a Wannier function scheme for the evaluation of Born charges. The results obtained with four different Hamiltonians, namely Hartree–Fock, DFT in its local (LDA) and nonlocal gradient corrected (PBE) approximation, and hybrid B3LYP, are discussed, showing that B3LYP performs far better than LDA and PBE, which in turn provide better results than HF, as the mean absolute difference from experimental frequencies is 6, 18, 21, and 44 cm−1, respectively, when a split valence basis set containing two sets of polarization functions is used. For the LDA results, comparison is possible with previous calculations based on the Density Functional Perturbation Theory and usage of a plane‐wave basis set. The effects associated with the use of basis sets of increasing size are also investigated. It turns out that a split valence plus a single set of dpolarization functions provides frequencies that differ from the ones obtained with a double set of dfunctions and a set of ffunctions on all atoms by on average less than 5 cm−1. © 2004 Wiley Periodicals, Inc. J Comput Chem 25: 1873–1881, 2004

The vibrational spectrum of calcite (CaCO 3) is evaluated at an ab initio periodic quantum-mechanical level by using the CRYSTAL package. A localized basis set of Gaussian-type functions and the B3LYP hybrid Hamiltonian are adopted. The dynamical matrix is obtained by differentiating numerically the analytical first derivatives of the energy. The accuracy with respect to all computational parameters is documented. The calculated frequencies are compared with available IR and RAMAN data (16 and 5 peaks, respectively), the mean absolute error being less than 12 cm −1 (frequencies range from 100 to 1600 cm −1). Overall, the agreement with experiment is very satisfactory, and shows that simulation can produce at a relatively low cost the full spectra of crystalline compounds of mineralogical interest.

Chemotherapy resistance remains a major obstacle to successful treatment and better outcome in cancer patients. The advent of whole genome experimental strategies, such as DNA microarrays, has transformed the way researchers approach cancer research. There is considerable hope that microarray technology will lead to the identification of new targets for therapeutic intervention, a better understanding of the disease process, and, ultimately, to higher survival rates and more personalized medicine. The question at hand is what is the best approach to apply these new technologies to the study of anticancer drug resistance, and how can the results obtained in the laboratory be quickly moved to a clinical setting? This review offers an overview of the microarray technology, including its recently associated strategies, such as array comparative genomic hybridization and promoter arrays. It also highlights some recent examples of microarray studies, which represent a first step toward a better understanding of drug resistance in cancer and, ultimately, personalized medicine.

The problem of numerical accuracy in the calculation of vibrational frequencies of crystalline compounds from the hessian matrix is discussed with reference to α‐quartz (SiO2) as a case study and to the specific implementation in the CRYSTAL code. The Hessian matrix is obtained by numerical differentiation of the analytical gradient of the energy with respect to the atomic positions. The process of calculating vibrational frequencies involves two steps: the determination of the equilibrium geometry, and the calculation of the frequencies themselves. The parameters controlling the truncation of the Coulomb and exchange series in Hartree–Fock, the quality of the grid used for the numerical integration of the Exchange‐correlation potential in Density Functional Theory, the SCF convergence criteria, the parameters controlling the convergence of the optimization process as well as those controlling the accuracy of the numerical calculation of the Hessian matrix can influence the obtained vibrational frequencies to some extent. The effect of all these parameters is discussed and documented. It is concluded that with relatively economical computational conditions the uncertainty related to these parameters is smaller than 2–4 cm−1. In the case of the Local Density Approximation scheme, comparison is possible with recent calculations performed with a Density Functional Perturbation Theory method and a plane‐wave basis set. © 2004 Wiley Periodicals, Inc. J Comput Chem 25: 888–897, 2004

Genome-wide monitoring of gene expression using DNA microarrays represents one of the latest breakthroughs in experimental molecular biology and provides unprecedented opportunity to explore the biological processes underlying human diseases by providing a comprehensive survey of a cell’s transcriptional landscape. In the cancer field, this revolutionary technology allows the simultaneous assessment of the transcription of tens of thousands of genes, and of their relative expression between normal cells and malignant cells. As microarray analysis emerges from its infancy, there is widespread hope that microarrays will significantly impact on our ability to explore the genetic changes associated with cancer etiology and development, and ultimately lead to the discovery of new biomarkers for disease diagnosis and prognosis prediction, and of new therapeutic tools. This review provides an overview of microarray technology, specifically in the context of cancer research and describes some of its recent applications to the study of cancer. In addition, the challenges of translating microarray findings into molecular cancer diagnosis and prognosis tools, with the potential of altering clinical practice through individualized cancer care and ultimately of contributing to the battle against cancer, are discussed.

The serine/threonine kinase protein kinase B (PKB/c-Akt) acts downstream of the lipid kinase phosphoinositide 3-kinase (PI3K) and functions as an essential mediator in many growth-factor-induced cellular responses such as cell cycle regulation, cell survival and transcriptional regulation. PI3K activation generates 3′-phosphorylated phosphatidylinositol lipids(PtdIns3P) and PKB activation requires PtdIns3P-dependent membrane translocation and phosphorylation by upstream kinases. However PKB activation and function is also regulated by interaction with other proteins. Here we show binding of PKB to periplakin, a member of the plakin family of cytolinker proteins. Interaction between PKB and periplakin was mapped to part of the pleckstrin homology (PH) domain of PKB, which is probably not involved in lipid binding, and indeed binding to periplakin did not affect PKB activation. We therefore investigated the possibility that periplakin may act as a scaffold or localization signal for PKB. In cells endogenous periplakin localizes to different cellular compartments, including plasma membrane,intermediate filament structures, the nucleus and mitochondria. Overexpression of the C-terminal part of periplakin, encompassing the PKB binding region,results in predominant intermediate filament localization and little nuclear staining. This also resulted in inhibition of nuclear PKB signalling as indicated by inhibition of PKB-dependent Forkhead transcription factor regulation. These results suggest a possible role for periplakin as a localization signal in PKB-mediated signalling.

A feeding trial was conducted to evaluate the effect of aflatoxin (AF)-contaminated diets on growth and hematological and immunological parameters. Low doses of aflatoxins (140 and 280 ppb) were included in a corn-soybean diet provided for ad libitum consumption to 36 weanling piglets for a period of 4 wk. A "dose-related" decrease in weight gain was observed in treated animals. This effect was significant (P < 0.05) in the 280 ppb-treated group compared to the control group. Ingestion of AF-contaminated feed at either level had no effect on total red blood cell numbers or on their relative number of lymphocytes, monocytes, neutrophils, basophils, and eosinophils in blood. Likewise, AF did not alter globulin, albumins, or total protein concentrations in serum, nor did AF alter the expression of regulatory cytokines produced by either Th1 (IL-2) or Th2 (IL-4) lymphocyte subsets in phytohemagglutinin-stimulated blood samples. By contrast, AF had a biphasic effect on total white blood cell number; the low dose of AF (140 ppb) decreased the total number of white blood cells, whereas the high dose (280 ppb) had the opposite effect. Consumption of AF also increased the concentration of gamma-globulin in the serum. A reduced immune response induced by Mycoplasma agalactiae in the 280-ppb-treated group was also observed. Cytokine mRNA expression in phytohemagglutinin-stimulated blood cells indicated that AF decreased proinflammatory (IL-1beta, TNF-alpha) and increased anti-inflammatory (IL-10) cytokine mRNA expression. These results demonstrate that low doses of AF depress growth and alter many aspects of humoral and cellular immunity in pigs.

AFX-like Forkhead transcription factors, which are controlled by phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB) signaling, are involved in regulating cell cycle progression and cell death. Both cell cycle arrest and induction of apoptosis are mediated in part by transcriptional regulation of p27kip1. Here we show that the Forkheads AFX (FOXO4) and FKHR-L1 (FOXO3a) also directly control transcription of the retinoblastoma-like p130 protein and cause upregulation of p130 protein expression. Detailed analysis of p130 regulation demonstrates that following Forkhead-induced cell cycle arrest, cells enter G0and become quiescent. This is shown by a change in phosphorylation of p130 to G0-specific forms and increased p130/E2F-4 complex formation. Most importantly, long-term Forkhead activation causes a sustained but reversible inhibition of proliferation without a marked increase in apoptosis. As for the activity of the Forkheads, we also show that protein levels of p130 are controlled by endogenous PI3K/PKB signaling upon cell cycle reentry. Surprisingly, not only nontransformed cells, but also cancer cells such as human colon carcinoma cells, are forced into quiescence by Forkhead activation. We therefore propose that Forkhead inactivation by PKB signaling in quiescent cells is a crucial step in cell cycle reentry and contributes to the processes of transformation and regeneration.

ABSTRACTAFX-like Forkhead transcription factors, which are controlled by phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB) signaling, are involved in regulating cell cycle progression and cell death. Both cell cycle arrest and induction of apoptosis are mediated in part by transcriptional regulation of p27kip1. Here we show that the Forkheads AFX (FOXO4) and FKHR-L1 (FOXO3a) also directly control transcription of the retinoblastoma-like p130 protein and cause upregulation of p130 protein expression. Detailed analysis of p130 regulation demonstrates that following Forkhead-induced cell cycle arrest, cells enter G0and become quiescent. This is shown by a change in phosphorylation of p130 to G0-specific forms and increased p130/E2F-4 complex formation. Most importantly, long-term Forkhead activation causes a sustained but reversible inhibition of proliferation without a marked increase in apoptosis. As for the activity of the Forkheads, we also show that protein levels of p130 are controlled by endogenous PI3K/PKB signaling upon cell cycle reentry. Surprisingly, not only nontransformed cells, but also cancer cells such as human colon carcinoma cells, are forced into quiescence by Forkhead activation. We therefore propose that Forkhead inactivation by PKB signaling in quiescent cells is a crucial step in cell cycle reentry and contributes to the processes of transformation and regeneration.

The use of DNA microarrays for the analysis of complex biological samples is becoming a mainstream part of biomedical research. One of the most commonly used methods compares the relative abundance of mRNA in two different samples by probing a single DNA microarray simultaneously. The simplicity of this concept sometimes masks the complexity of capturing and processing microarray data. On the basis of the analysis of many of our microarray experiments, we identified the major causes of distortion of the microarray data and the sources of noise. In this study, we provide a systematic statistical approach for extraction of true expression ratios from raw microarray data, which we describe as an unfolding process. The results of this analysis are presented in the form of a model describing the relationship between the measured fluorescent intensities and the concentrations of mRNA transcripts. We developed and tested several algorithms for inference of the model parameters for the microarray data. Special emphasis is given to the statistical robustness of these algorithms, in particular resistance to outliers. We also provide methods for measurement of noise and reproducibility of the microarray experiments.

ABSTRACTVirus infection induces an antiviral response that is predominantly associated with the synthesis and secretion of soluble interferon. Here, we report that herpes simplex virus type 1 virions induce an interferon-independent antiviral state in human embryonic lung cells that prevents plaquing of a variety of viruses. Microarray analysis of 19,000 human expressed sequence tags revealed induction of a limited set of host genes, the majority of which are also induced by interferon. Genes implicated in controlling the intracellular spread of virus and eliminating virally infected cells were among those induced. Induction of the cellular response occurred in the absence of de novo cellular protein synthesis and required viral penetration. In addition, this response was only seen when viral gene expression was inhibited, suggesting that a newly synthesized viral protein(s) may function as an inhibitor of this response.

ABSTRACTInterleukin-3 (IL-3), IL-5, and granulocyte-macrophage colony-stimulating factor regulate the survival, proliferation, and differentiation of hematopoietic lineages. Phosphatidylinositol 3-kinase (PI3K) has been implicated in the regulation of these processes. Here we investigate the molecular mechanism by which PI3K regulates cytokine-mediated proliferation and survival in the murine pre-B-cell line Ba/F3. IL-3 was found to repress the expression of the cyclin-dependent kinase inhibitor p27KIP1through activation of PI3K, and this occurs at the level of transcription. This transcriptional regulation occurs through modulation of the forkhead transcription factor FKHR-L1, and IL-3 inhibited FKHR-L1 activity in a PI3K-dependent manner. We have generated Ba/F3 cell lines expressing a tamoxifen-inducible active FKHR-L1 mutant [FKHR-L1(A3):ER*]. Tamoxifen-mediated activation of FKHR-L1(A3):ER* resulted in a striking increase in p27KIP1promoter activity and mRNA and protein levels as well as induction of the apoptotic program. The level of p27KIP1appears to be critical in the regulation of cell survival since mere ectopic expression of p27KIP1was sufficient to induce Ba/F3 apoptosis. Moreover, cell survival was increased in cytokine-starved bone marrow-derived stem cells from p27KIP1null-mutant mice compared to that in cells from wild-type mice. Taken together, these observations indicate that inhibition of p27KIP1transcription through PI3K-induced FKHR-L1 phosphorylation provides a novel mechanism of regulating cytokine-mediated survival and proliferation.

Interleukin-3 (IL-3), IL-5, and granulocyte-macrophage colony-stimulating factor regulate the survival, proliferation, and differentiation of hematopoietic lineages. Phosphatidylinositol 3-kinase (PI3K) has been implicated in the regulation of these processes. Here we investigate the molecular mechanism by which PI3K regulates cytokine-mediated proliferation and survival in the murine pre-B-cell line Ba/F3. IL-3 was found to repress the expression of the cyclin-dependent kinase inhibitor p27KIP1through activation of PI3K, and this occurs at the level of transcription. This transcriptional regulation occurs through modulation of the forkhead transcription factor FKHR-L1, and IL-3 inhibited FKHR-L1 activity in a PI3K-dependent manner. We have generated Ba/F3 cell lines expressing a tamoxifen-inducible active FKHR-L1 mutant [FKHR-L1(A3):ER*]. Tamoxifen-mediated activation of FKHR-L1(A3):ER* resulted in a striking increase in p27KIP1promoter activity and mRNA and protein levels as well as induction of the apoptotic program. The level of p27KIP1appears to be critical in the regulation of cell survival since mere ectopic expression of p27KIP1was sufficient to induce Ba/F3 apoptosis. Moreover, cell survival was increased in cytokine-starved bone marrow-derived stem cells from p27KIP1null-mutant mice compared to that in cells from wild-type mice. Taken together, these observations indicate that inhibition of p27KIP1transcription through PI3K-induced FKHR-L1 phosphorylation provides a novel mechanism of regulating cytokine-mediated survival and proliferation.

RNA polymerase II lacking the Rpb9 subunit uses alternate transcription initiation sites in vitroandin vivoand is unable to respond to the transcription elongation factor TFIIS in vitro. Here, we show thatRPB9has a synthetic phenotype with the TFIIS gene. Disruption of RPB9in yeast also resulted in sensitivity to 6-azauracil, which is a phenotype linked to defects in transcription elongation. Expression of the TFIIS gene on a high-copy plasmid partially suppressed the 6-azauracil sensitivity of Δrpb9cells. We set out to determine the relevant cellular role of yeast Rpb9 by assessing the ability of 20 different site-directed and deletion mutants of RPB9to complement the initiation and elongation defects of Δrpb9cells in vivo. Rpb9 is composed of two zinc ribbons. The N-terminal zinc ribbon restored the wild-type pattern of initiation start sites, but was unable to complement the growth defects associated with defects in elongation. Most of the site-directed mutants complemented the elongation-specific growth phenotypes and reconstituted the normal pattern of transcription initiation sites. The anti-correlation between the growth defects of cells disrupted for RPB9and the selection of transcription start sites suggests that this is not the primary cellular role for Rpb9. Genome-wide transcription profiling of Δrpb9cells revealed only a few changes, predominantly in genes related to metabolism.

Cytosolic phospholipase A2(cPLA2) plays a critical role in various neutrophil functions including the generation of leukotrienes and platelet‐activating factor release. Enzyme activity is regulated both by translocation to the membrane in a Ca2+‐dependent manner and serine phosphorylation by members of the mitogen‐activated protein kinase (MAPK) family. In this report, we have investigated the role of granulocyte/macrophage colony‐stimulating factor (GM‐CSF)‐mediated signalling pathways in the regulation of cPLA2. GM‐CSF‐induced cPLA2phosphorylation was not affected by pharmacological inhibition of p38 MAPK, phosphatidylinositol 3‐kinase or Src. However, inhibition of extracellular signal‐regulated kinase (ERK) MAPK activation resulted in a partial inhibition of cPLA2phosphorylation, revealed in a slower onset of phosphorylation. A cell line stably transfected with the GM‐CSF receptor was used to further analyze GM‐CSF‐mediated cPLA2phosphorylation. Mutation of tyrosine residues 577 and 612 resulted in a delayed cPLA2phosphorylation similar to the pharmacological ERK inhibition. Furthermore, inhibition of p38 MAPK in cells bearing the double mutant βc577/612 completely abrogated GM‐CSF‐induced cPLA2phosphorylation. We conclude that GM‐CSF can mediate cPLA2phosphorylation through the redundant activation of both p38 and ERK MAP kinases.

Cytosolic phospholipase A2(cPLA2) plays a critical role in various neutrophil functions including the generation of leukotrienes and platelet-activating factor release. Enzyme activity is regulated both by translocation to the membrane in a Ca2+-dependent manner and serine phosphorylation by members of the mitogen-activated protein kinase (MAPK) family. In this report, we have investigated the role of granulocyte/macrophage colony-stimulating factor (GM-CSF)-mediated signalling pathways in the regulation of cPLA2. GM-CSF-induced cPLA2phosphorylation was not affected by pharmacological inhibition of p38 MAPK, phosphatidylinositol 3-kinase or Src. However, inhibition of extracellular signal-regulated kinase (ERK) MAPK activation resulted in a partial inhibition of cPLA2phosphorylation, revealed in a slower onset of phosphorylation. A cell line stably transfected with the GM-CSF receptor was used to further analyze GM-CSF-mediated cPLA2phosphorylation. Mutation of tyrosine residues 577 and 612 resulted in a delayed cPLA2phosphorylation similar to the pharmacological ERK inhibition. Furthermore, inhibition of p38 MAPK in cells bearing the double mutant βc577/612 completely abrogated GM-CSF-induced cPLA2phosphorylation. We conclude that GM-CSF can mediate cPLA2phosphorylation through the redundant activation of both p38 and ERK MAP kinases.