PCAF regulates the stability of the transcriptional regulator and cyclin-dependent kinase inhibitor p27Kip1

P27(Kip1) (p27) is a member of the Cip/Kip family of cyclin-dependent kinase inhibitors. Recently, a new function of p27 as transcriptional regulator has been reported. It has been shown that p27 regulates the expression of target genes mostly involved in splicing, cell cycle, respiration and translation. We report here that p27 directly binds to the transcriptional coactivator PCAF by a region including amino acids 91-120. PCAF associates with p27 through its catalytic domain and acetylates p27 at lysine 100. Our data showed that overexpression of PCAF induces the degradation of p27 whereas in contrast, the knockdown of PCAF stabilizes the protein. A p27 mutant in which K100 was substituted by arginine (p27-K100R) cannot be acetylated by PCAF and has a half-life much higher than that of p27WT. Moreover, p27-K100R remains stable along cell-cycle progression. Ubiquitylation assays and the use of proteasome inhibitors indicate that PCAF induces p27 degradation via proteasome. We also observed that knockdown of skp2 did not affect the PCAF induced degradation of p27. In conclusion, our data suggest that the p27 acetylation by PCAF regulates its stability.

The transcriptional co-activator PCAF regulates cdk2 activity.

Cyclin dependent kinases (cdks) regulate cell cycle progression and transcription. We report here that the transcriptional co-activator PCAF directly interacts with cdk2. This interaction is mainly produced during S and G2/M phases of the cell cycle. As a consequence of this association, PCAF inhibits the activity of cyclin/cdk2 complexes. This effect is specific for cdk2 because PCAF does not inhibit either cyclin D3/cdk6 or cyclin B/cdk1 activities. The inhibition is neither competitive with ATP, nor with the substrate histone H1 suggesting that somehow PCAF disturbs cyclin/cdk2 complexes. We also demonstrate that overexpression of PCAF in the cells inhibits cdk2 activity and arrests cell cycle progression at S and G2/M. This blockade is dependent on cdk2 because it is rescued by the simultaneous overexpression of this kinase. Moreover, we also observed that PCAF acetylates cdk2 at lysine 33. As this lysine is essential for the interaction with ATP, acetylation of this residue inhibits cdk2 activity. Thus, we report here that PCAF inhibits cyclin/cdk2 activity by two different mechanisms: (i) by somehow affecting cyclin/cdk2 interaction and (ii) by acetylating K33 at the catalytic pocket of cdk2. These findings identify a previously unknown mechanism that regulates cdk2 activity.

Muscle protein waste in tumor-bearing rats is effectively antagonized by a beta 2-adrenergic agonist (clenbuterol). Role of the ATP-ubiquitin-dependent proteolytic pathway.

Tissue protein hypercatabolism (TPH) is a most important feature in cancer cachexia, particularly with regard to the skeletal muscle. The rat ascites hepatoma Yoshida AH-130 is a very suitable model system for studying the mechanisms involved in the processes that lead to tissue depletion, since it induces in the host a rapid and progressive muscle waste mainly due to TPH (Tessitore, L., G. Bonelli, and F. M. Baccino. 1987. Biochem. J. 241:153-159). Detectable plasma levels of tumor necrosis factor-alpha associated with marked perturbations in the hormonal homeostasis have been shown to concur in forcing metabolism into a catabolic setting (Tessitore, L., P. Costelli, and F. M. Baccino. 1993. Br. J. Cancer. 67:15-23). The present study was directed to investigate if beta 2-adrenergic agonists, which are known to favor skeletal muscle hypertrophy, could effectively antagonize the enhanced muscle protein breakdown in this cancer cachexia model. One such agent, i.e., clenbuterol, indeed largely prevented skeletal muscle waste in AH-130-bearing rats by restoring protein degradative rates close to control values. This normalization of protein breakdown rates was achieved through a decrease of the hyperactivation of the ATP-ubiquitin-dependent proteolytic pathway, as previously demonstrated in our laboratory (Llovera, M., C. García-Martínez, N. Agell, M. Marzábal, F. J. López-Soriano, and J. M. Argilés. 1994. FEBS (Fed. Eur. Biochem. Soc.) Lett. 338:311-318). By contrast, the drug did not exert any measurable effect on various parenchymal organs, nor did it modify the plasma level of corticosterone and insulin, which were increased and decreased, respectively, in the tumor hosts. The present data give new insights into the mechanisms by which clenbuterol exerts its preventive effect on muscle protein waste and seem to warrant the implementation of experimental protocols involving the use of clenbuterol or alike drugs in the treatment of pathological states involving TPH, particularly in skeletal muscle and heart, such as in the present model of cancer cachexia.

A T42A Ran Mutation: Differential Interactions with Effectors and Regulators, and Defect in Nuclear Protein Import

Ran, the small, predominantly nuclear GTPase, has been implicated in the regulation of a variety of cellular processes including cell cycle progression, nuclear-cytoplasmic trafficking of RNA and protein, nuclear structure, and DNA synthesis. It is not known whether Ran functions directly in each process or whether many of its roles may be secondary to a direct role in only one, for example, nuclear protein import. To identify biochemical links between Ran and its functional target(s), we have generated and examined the properties of a putative Ran effector mutation, T42A-Ran. T42A-Ran binds guanine nucleotides as well as wild-type Ran and responds as well as wild-type Ran to GTP or GDP exchange stimulated by the Ran-specific guanine nucleotide exchange factor, RCC1. T42A-Ran·GDP also retains the ability to bind p10/NTF2, a component of the nuclear import pathway. In contrast to wild-type Ran, T42A-Ran·GTP binds very weakly or not detectably to three proposed Ran effectors, Ran-binding protein 1 (RanBP1), Ran-binding protein 2 (RanBP2, a nucleoporin), and karyopherin ß (a component of the nuclear protein import pathway), and is not stimulated to hydrolyze bound GTP by Ran GTPase-activating protein, RanGAP1. Also in contrast to wild-type Ran, T42A-Ran does not stimulate nuclear protein import in a digitonin permeabilized cell assay and also inhibits wild-type Ran function in this system. However, the T42A mutation does not block the docking of karyophilic substrates at the nuclear pore. These properties of T42A-Ran are consistent with its classification as an effector mutant and define the exposed region of Ran containing the mutation as a probable effector loop.

Filamin depletion blocks endoplasmic spreading and destabilizes force-bearing adhesions

Cell motility is an essential process that depends on a coherent, cross-linked actin cytoskeleton that physically coordinates the actions of numerous structural and signaling molecules. The actin cross-linking protein, filamin (Fln), has been implicated in the support of three-dimensional cortical actin networks capable of both maintaining cellular integrity and withstanding large forces. Although numerous studies have examined cells lacking one of the multiple Fln isoforms, compensatory mechanisms can mask novel phenotypes only observable by further Fln depletion. Indeed, shRNA-mediated knockdown of FlnA in FlnB¿/¿ mouse embryonic fibroblasts (MEFs) causes a novel endoplasmic spreading deficiency as detected by endoplasmic reticulum markers. Microtubule (MT) extension rates are also decreased but not by peripheral actin flow, because this is also decreased in the Fln-depleted system. Additionally, Fln-depleted MEFs exhibit decreased adhesion stability that appears in increased ruffling of the cell edge, reduced adhesion size, transient traction forces, and decreased stress fibers. FlnA¿/¿ MEFs, but not FlnB¿/¿ MEFs, also show a moderate defect in endoplasm spreading, characterized by initial extension followed by abrupt retractions and stress fiber fracture. FlnA localizes to actin linkages surrounding the endoplasm, adhesions, and stress fibers. Thus we suggest that Flns have a major role in the maintenance of actin-based mechanical linkages that enable endoplasmic spreading and MT extension as well as sustained traction forces and mature focal adhesions.

Errors congènits del metabolisme (ECM).

La terminologia i el concepte d'Error Congènit del Metabolisme (ECM), van ser establerts per A. Garrod a principis de segle. Avui día sabem que estan causats per errors o mutacions en els gens. Degut a la naturalesa del nostre codi genètic, segons el qual les instruccions del DNA són traduïdes a un producte gènic, les proteïnes, que seran les encarregades d'executar-lo; les mutacions del DNA es tradueixen en proteïnes anòmales amb la corresponent...

Influência da qualidade da água de irrigação na dispersão da argila de Latossolos

O fenômeno de dispersão da argila do solo pode ser provocado pela qualidade da água de irrigação. Objetivou-se estudar o efeito da irrigação com água com diferentes condutividades elétricas (CE), combinadas com diferentes valores de Relação de Adsorção de Sódio (RAS), na dispersão da argila de solos, de diferentes mineralogias, do Estado de Minas Gerais. As amostras foram coletadas no horizonte B de solos das cidades de Viçosa, Belo Horizonte e Barroso, classificados como Latossolo Vermelho-Amarelo distrófico típico (LVA), Latossolo Vermelho perférrico típico (LV) e Latossolo Vermelho distroférrico típico (LVd) destacados, respectivamente, pela presença de argilominerais caulinita, hematita e gibbsita. Os tratamentos corresponderam à percolação, nos três solos, de soluções de NaCl e CaCl2 preparadas de forma a se obter seis diferentes valores de CE (20; 50; 100; 200; 400 e 800 mS m-1) e cinco de RAS (0, 5, 10, 20 e 40 mmol c L-1), em três repetições, dispostos em um delineamento em blocos casualizados. A aplicação das soluções foi feita em permeâmetros de coluna vertical e carga constante. A solução foi aplicada até o momento em que a CE do efluente se aproximou daquela da solução aplicada (C/C0 = 1,0). Para avaliar o efeito das soluções aplicadas na estrutura do solo, foram quantificados os teores de argila dispersa em água (ADA). Os valores de ADA nas amostras de solo submetido à percolação das diferentes soluções salino-sódicas (ADA-S) foram relacionados com a CE e a RAS da solução percolada, ajustando-se superfícies de resposta com este fim. O comportamento dos solos foi diferenciado, no que se refere à dispersividade da argila, o que estava associado à mineralogia, mas, também, ao grau de intemperismo do solo. O LVA apresentou comportamento não dispersivo em todos os tratamentos e, no geral, todas as soluções de percolação, notadamente as de maiores CE, independentemente dos valores de RAS, proporcionaram diminuição no teor de argila dispersa (ADA-S) nos solos LV e LVd.

Expression of the NH(2)-terminal fragment of RasGAP in pancreatic beta-cells increases their resistance to stresses and protects mice from diabetes.

OBJECTIVE: Our laboratory has previously established in vitro that a caspase-generated RasGAP NH(2)-terminal moiety, called fragment N, potently protects cells, including insulinomas, from apoptotic stress. We aimed to determine whether fragment N can increase the resistance of pancreatic beta-cells in a physiological setting. RESEARCH DESIGN AND METHODS: A mouse line, called rat insulin promoter (RIP)-N, was generated that bears a transgene containing the rat insulin promoter followed by the cDNA-encoding fragment N. The histology, functionality, and resistance to stress of RIP-N islets were then assessed. RESULTS: Pancreatic beta-cells of RIP-N mice express fragment N, activate Akt, and block nuclear factor kappaB activity without affecting islet cell proliferation or the morphology and cellular composition of islets. Intraperitoneal glucose tolerance tests revealed that RIP-N mice control their glycemia similarly as wild-type mice throughout their lifespan. Moreover, islets isolated from RIP-N mice showed normal glucose-induced insulin secretory capacities. They, however, displayed increased resistance to apoptosis induced by a series of stresses including inflammatory cytokines, fatty acids, and hyperglycemia. RIP-N mice were also protected from multiple low-dose streptozotocin-induced diabetes, and this was associated with reduced in vivo beta-cell apoptosis. CONCLUSIONS: Fragment N efficiently increases the overall resistance of beta-cells to noxious stimuli without interfering with the physiological functions of the cells. Fragment N and the pathway it regulates represent, therefore, a potential target for the development of antidiabetes tools.

Relações de aporte de sedimento e implicação de sua utilização no pagamento por serviço ambiental em bacias hidrográfias

Em razão de sua simplicidade, a relação de aporte de sedimento (RAS), dada pela razão entre a produção de sedimento no exutório e a erosão total na bacia, é comumente usada no estudo e na determinação da produção de sedimentos em bacias hidrográficas. Como há uma série de complexidades ligando a produção de sedimentos (Y) e a erosão a montante (E) na bacia, foram desenvolvidas distintas equações de RAS. Programas de pagamento por serviço ambiental relativos ao abatimento de sedimentação, como o Produtor de Água, frequentemente requerem a estimativa da redução da produção de sedimentos nas bacias hidrográficas, em função de implantação do controle de erosão a montante. Os objetivos do presente trabalho foram calcular a RAS da bacia do ribeirão Pipiripau (DF), usando oito equações existentes na literatura, e estimar a provável redução em Y, em função de uma redução de 50 % da erosão na bacia, bem como avaliar as eventuais dificuldades em sua aplicação. Os valores de RAS obtidos variaram entre 0,12 e 0,52, com média de 0,24 e desvio-padrão de 0,14. Duas das equações apresentaram alta sensibilidade às variáveis, indicando que haveria significativo potencial de propagação de erro se elas fossem usadas. Partindo-se de uma redução de erosão de 50 % na bacia (117.500 t ano-1), a redução no aporte de sedimento usando as RAS das oito equações variou de 14 a 61 mil t ano-1. Essa significativa variação em Y indica considerável incerteza na estimativa de serviços ambientais relativos à redução de aporte de sedimento, em programas de controle de erosão.

Opposing roles for calcineurin and ATF3 in squamous skin cancer.

Calcineurin inhibitors such as cyclosporin A (CsA) are the mainstay of immunosuppressive treatment for organ transplant recipients. Squamous cell carcinoma (SCC) of the skin is a major complication of treatment with these drugs, with a 65 to 100-fold higher risk than in the normal population. By contrast, the incidence of basal cell carcinoma (BCC), the other major keratinocyte-derived tumour of the skin, of melanoma and of internal malignancies increases to a significantly lesser extent. Here we report that genetic and pharmacological suppression of calcineurin/nuclear factor of activated T cells (NFAT) function promotes tumour formation in mouse skin and in xenografts, in immune compromised mice, of H-ras(V12) (also known as Hras1)-expressing primary human keratinocytes or keratinocyte-derived SCC cells. Calcineurin/NFAT inhibition counteracts p53 (also known as TRP53)-dependent cancer cell senescence, thereby increasing tumorigenic potential. ATF3, a member of the 'enlarged' AP-1 family, is selectively induced by calcineurin/NFAT inhibition, both under experimental conditions and in clinically occurring tumours, and increased ATF3 expression accounts for suppression of p53-dependent senescence and enhanced tumorigenic potential. Thus, intact calcineurin/NFAT signalling is critically required for p53 and senescence-associated mechanisms that protect against skin squamous cancer development.

Evolving team compositions by agent swapping

Optimizing collective behavior in multiagent systems requires algorithms to find not only appropriate individual behaviors but also a suitable composition of agents within a team. Over the last two decades, evolutionary methods have emerged as a promising approach for the design of agents and their compositions into teams. The choice of a crossover operator that facilitates the evolution of optimal team composition is recognized to be crucial, but so far, it has never been thoroughly quantified. Here, we highlight the limitations of two different crossover operators that exchange entire agents between teams: restricted agent swapping (RAS) that exchanges only corresponding agents between teams and free agent swapping (FAS) that allows an arbitrary exchange of agents. Our results show that RAS suffers from premature convergence, whereas FAS entails insufficient convergence. Consequently, in both cases, the exploration and exploitation aspects of the evolutionary algorithm are not well balanced resulting in the evolution of suboptimal team compositions. To overcome this problem, we propose combining the two methods. Our approach first applies FAS to explore the search space and then RAS to exploit it. This mixed approach is a much more efficient strategy for the evolution of team compositions compared to either strategy on its own. Our results suggest that such a mixed agent-swapping algorithm should always be preferred whenever the optimal composition of individuals in a multiagent system is unknown.

Sorafenib fourth-line treatment in imatinib-, sunitinib-, and nilotinib-resistant metastatic GIST : a retrospective analysis : 10564

Background: Gastrointestinal stromal tumors (GISTs) are rare mesenchymal tumors usually caused by mutations in the KIT or PDGFRA gene. Advanced disease generally cannot be cured by surgery nor by tyrosine kinase inhibitors (TKI), but TKIs have considerably improved outcome for patients (pts) with advanced GIST. Patients failing TKI treatment with imatinib (IM), sunitinib (SU) or nilotinib (NI) have a poor prognosis. Sorafenib is a multi kinase inhibitor that blocks not only receptor tyrosine kinases such as KIT, VEGFR and PDGFR but also serine/threonine kinases along the RAS/RAF/MEK/ERK pathway. Recently, clinical activity of sorafenib in third-line treatment in patients with GIST after IM and SU failure has been shown (Wiebe et al. ASCO 2008, #10502). Methods: We report herein preliminary data of 32 pts treated with sorafenib in nine European centers. Centers were selected based on their previous and known experience in GIST and reported all pts treated. Pts received sorafenib after failure of IM, SU and NI in fourth-line treatment. Baseline characteristics and treatment details have been retrieved via questionary. Results: Median age at sorafenib treatment start was 62 years (range 33-81 y), and the majority of pts were male (63 %). Primary tumor site was gastric or small intestine in 25% and 41% of pts, respectively. All pts had failed IM, SU, NI. 19 % of pts achieved partial remission and 44% disease stabilization. Approximately half of the pts had an improvement of symptoms and/or performance. Half of the pts were on treatment longer than 4 months (actuarial data) and 41% of pts continue to receive sorafenib. Median progression-free survival is 20 weeks and median overall survival 42 weeks (Kaplan-Meier), at a median follow-up of 22 weeks (range 3-54). Conclusions: This is the largest series assessing efficacy of sorafenib fourth-line treatment for IM, SU and NI refractory GIST reported yet. Sorafenib displays significant clinical activity in this heavily pretreated group of patients.

Putting Public Service Motivation into Context. A Balance Between Universalism and Particularism

Research on Public Service Motivation (PSM) has increased enormously in the last 20 years. Besides the analysis of the antecedents of PSM and its impact on organizations and individuals, many open questions about the nature of PSM itself still remain. This article argues that the theoretical construct of PSM should be contextualized by integrating the political and administrative contexts of public servants when investigating their specific attitudes towards working in a public environment. It also challenges the efficacy of the classic four-dimensional structure of PSM when it is applied to a specific context. The findings of a confirmatory factor analysis from a dataset of 3754 employees of 279 Swiss municipalities support the appropriateness of contextualizing parts of the PSM construct. They also support the addition of an extra dimension called, according to previous research, Swiss democratic governance. With regard to our results, there is a need for further PSM research to set a definite measure of PSM, particularly in regard to the international diffusion of empirical research on PSM.Points for practitionersThis study shows that public service motivation is a relevant construct for practitioners and may be used to better assess whether public agents are motivated by values or not. Nevertheless, it stresses also that the measurement of PSM must be adapted to the institutional context as well. Public managers interested in understanding better the degree to which their employees are motivated by public values must be aware that the measurement of this PSM construct has to be contextualized. In other words, PSM is also a function of the institutional environment in which organizations operate.

A Cell Permeable Rasgap-derived Peptide Sensitizes Cancer Cells To Radiotherapy

Purpose/Objective(s): Radiotherapy is an effective treatment modality against cancer. Despite recent technical progresses in radiation delivery precision, toxicity to healthy tissues remains the main limiting factor. RasGAP is a regulator of the Ras and Rho pathway; it has either a pro- or anti-apoptotic activity depending on the level of caspase expressed in the cell. The RasGAP derived peptide: TAT-RasGAP317 - 326 is the minimal sequence known to sensitize cancer cells, but not healthy cells, to genotoxin-induced apoptosis. In this study the TAT-RasGAP317 - 326 radio-sensitizing effect was tested in vitro and in vivo.Materials/Methods: Two weeks clonogenic forming assays with 5 human cancer cells (PANC-1, HCT116, U87, U251 and HeLa) and a non tumorigenic cell line (HaCaT) were performed. Cells were exposed to 0, 1, 2 and 4 Gy with or without 20 mMTAT-RasGAP317 - 326. Twenty mMTAT peptide was also used as control. TAT-RasGAP317 - 326 effect was also tested in tumor xenograft mouse models. Mice bearing HCT116 tumors (WT or p53 mutant) received 1.65 mg/kg TAT-RasGAP317 - 326 i.p. injected and were locally irradiated for 10 days with 3 Gy. Tumor volume was then followed during a minimum of 20 days. Control mice were treated with a single modality, either with TAT-RasGAP317 - 326 or with radiotherapy.Results: At all the tested radiation doses TAT-RasGAP317 - 326 showed a significant supra additive radio-sensitizing effect on all the tested tumor cell lines. Furthermore, it showed no sensitizing effect on the non tumorigenic cell line. In vivo, TAT-RasGAP317 - 326 also showed a significantly radio-sensitizing effect as shown by a significant higher reduction in tumor volume as much as by a significant tumor growth delay.Conclusions: Taken together our data suggest that TAT-RasGAP317 - 326 has a radio-sensitizing effect on in vivo and in vitro tumors without any effect on healthy tissues. Therefore TAT-RasGAP317 - 326 should be considered as a novel and attractive sensitizer compound allowing an improvement of the therapeutic interval.