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1.
Clin Radiol ; 79(1): 25-32, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-37925365

RESUMO

A high volume of cross-sectional imaging has created a window of opportunity for radiologists to identify renal angiomyolipomas (AMLs). The purpose of this review is to help the reader recognise the spectrum of renal AML appearances using different imaging methods and to gain an understanding of the classic and atypical features for appropriate lesion characterisation. Risk factors for AML growth and rupture will be highlighted. An overview of the imaging features of acute AML rupture will be provided, principally relating to computed tomography (CT) assessment. A series of cases will be presented, including a case of peripartum renal AML rupture during Caesarean section leading to diagnostic dilemma. The indications for intervention and available treatment options will be considered: medical therapy, surgery, and interventional radiology (IR) techniques including their pros and cons. Emergency interventional radiology management with selective transarterial embolisation will be presented and analysed in relation to technique, angiographic appearances (pre and post embolisation) and associated complications.


Assuntos
Angiomiolipoma , Neoplasias Renais , Leucemia Mieloide Aguda , Gravidez , Humanos , Feminino , Neoplasias Renais/diagnóstico por imagem , Neoplasias Renais/terapia , Neoplasias Renais/complicações , Angiomiolipoma/diagnóstico por imagem , Angiomiolipoma/terapia , Cesárea , Tomografia Computadorizada por Raios X , Leucemia Mieloide Aguda/complicações
2.
Hum Reprod Open ; 2023(3): hoad021, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37304815

RESUMO

STUDY QUESTION: Does application of an unbiased method for analysis of magnetic resonance (MR) images reveal any effect on uterine or fibroid volume from treatment of heavy menstrual bleeding (HMB) with three 12-week courses of the selective progesterone receptor modulator ulipristal acetate (SPRM-UPA)? SUMMARY ANSWER: Application of an unbiased method for analysis of MR images showed that treatment of HMB with SPRM-UPA was not associated with a significant reduction in the volume of the uterus or in the volume of uterine fibroids. WHAT IS KNOWN ALREADY: SPRM-UPA shows therapeutic efficacy for treating HMB. However, the mechanism of action (MoA) is not well understood and there have been mixed reports, using potentially biased methodology, regarding whether SPRM-UPA has an effect on the volume of the uterus and fibroids. STUDY DESIGN SIZE DURATION: In a prospective clinical study (with no comparator), 19 women with HMB were treated over a period of 12 months with SPRM-UPA and uterine and fibroid size were assessed with high resolution structural MRI and stereology. PARTICIPANTS/MATERIALS SETTING METHODS: A cohort of 19 women aged 38-52 years (8 with and 11 without fibroids) were treated with three 12-week courses of 5 mg SPRM-UPA given daily, with four weeks off medication in-between treatment courses. Unbiased estimates of the volume of uterus and total volume of fibroids were obtained at baseline, and after 6 and 12 months of treatment, by using the Cavalieri method of modern design-based stereology in combination with magnetic resonance imaging (MRI). MAIN RESULTS AND THE ROLE OF CHANCE: Bland-Altman plots showed good intra-rater repeatability and good inter-rater reproducibility for measurement of the volume of both fibroids and the uterus. For the total patient cohort, two-way ANOVA did not show a significant reduction in the volume of the uterus after two or three treatment courses of SPRM-UPA (P = 0.51), which was also the case when the groups of women with and without fibroids were considered separately (P = 0.63). One-way ANOVA did not show a significant reduction in total fibroid volume in the eight patients with fibroids (P = 0.17). LIMITATIONS REASONS FOR CAUTION: The study has been performed in a relatively small cohort of women and simulations that have subsequently been performed using the acquired data have shown that for three time points and a group size of up to 50, with alpha (Type I Error) and beta (Type II Error) set to 95% significance and 80% power, respectively, at least 35 patients would need to be recruited in order for the null hypothesis (that there is no significant reduction in total fibroid volume) to be potentially rejected. WIDER IMPLICATIONS OF THE FINDINGS: The imaging protocol that we have developed represents a generic paradigm for measuring the volume of the uterus and uterine fibroids that can be readily incorporated in future studies of medical treatments of HMB. In the present study, SPRM-UPA failed to produce a significant reduction in the volume of the uterus or the total volume of fibroids (which were present in approximately half of the patients) after either two or three 12-week courses of treatment. This finding represents a new insight in respect of the management of HMB using treatment strategies that target hormone-dependence. STUDY FUNDING/COMPETING INTERESTS: The UPA Versus Conventional Management of HMB (UCON) trial was funded by the EME Programme (Medical Research Council (MRC) and National Institutes of Health Research (NIHR)) (12/206/52). The views expressed in this publication are those of the authors and not necessarily those of the Medical Research Council, National Institute for Health Research, or Department of Health and Social Care.Medical Research Council (MRC) Centre grants to the Centre for Reproductive Health (CRH) (G1002033 and MR/N022556/1) are also gratefully acknowledged. H.C. has clinical research support for laboratory consumables and staff from Bayer AG and provides consultancy advice (All paid to Institution) for Bayer AG, PregLem SA, Gedeon Richter, Vifor Pharma UK Ltd, AbbVie Inc., and Myovant Sciences GmbH. H.C. has received royalties from UpToDate for an article on abnormal uterine bleeding. L.W. has received grant funding from Roche Diagnostics (Paid to Institution). All other authors have no conflicts to declare. TRIAL REGISTRATION NUMBER: The study reported here is an embedded mechanism of action study (no comparator) within the UCON clinical trial (registration ISRCTN: 20426843).

4.
Nat Commun ; 8(1): 1800, 2017 11 27.
Artigo em Inglês | MEDLINE | ID: mdl-29176716

RESUMO

Selective, robust and cost-effective chemical sensors for detecting small volatile-organic compounds (VOCs) have widespread applications in industry, healthcare and environmental monitoring. Here we design a Pt(II) pincer-type material with selective absorptive and emissive responses to methanol and water. The yellow anhydrous form converts reversibly on a subsecond timescale to a red hydrate in the presence of parts-per-thousand levels of atmospheric water vapour. Exposure to methanol induces a similarly-rapid and reversible colour change to a blue methanol solvate. Stable smart coatings on glass demonstrate robust switching over 104 cycles, and flexible microporous polymer membranes incorporating microcrystals of the complex show identical vapochromic behaviour. The rapid vapochromic response can be rationalised from the crystal structure, and in combination with quantum-chemical modelling, we provide a complete microscopic picture of the switching mechanism. We discuss how this multiscale design approach can be used to obtain new compounds with tailored VOC selectivity and spectral responses.

6.
Nature ; 502(7471): 346-9, 2013 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-24132291

RESUMO

Super-luminous supernovae that radiate more than 10(44) ergs per second at their peak luminosity have recently been discovered in faint galaxies at redshifts of 0.1-4. Some evolve slowly, resembling models of 'pair-instability' supernovae. Such models involve stars with original masses 140-260 times that of the Sun that now have carbon-oxygen cores of 65-130 solar masses. In these stars, the photons that prevent gravitational collapse are converted to electron-positron pairs, causing rapid contraction and thermonuclear explosions. Many solar masses of (56)Ni are synthesized; this isotope decays to (56)Fe via (56)Co, powering bright light curves. Such massive progenitors are expected to have formed from metal-poor gas in the early Universe. Recently, supernova 2007bi in a galaxy at redshift 0.127 (about 12 billion years after the Big Bang) with a metallicity one-third that of the Sun was observed to look like a fading pair-instability supernova. Here we report observations of two slow-to-fade super-luminous supernovae that show relatively fast rise times and blue colours, which are incompatible with pair-instability models. Their late-time light-curve and spectral similarities to supernova 2007bi call the nature of that event into question. Our early spectra closely resemble typical fast-declining super-luminous supernovae, which are not powered by radioactivity. Modelling our observations with 10-16 solar masses of magnetar-energized ejecta demonstrates the possibility of a common explosion mechanism. The lack of unambiguous nearby pair-instability events suggests that their local rate of occurrence is less than 6 × 10(-6) times that of the core-collapse rate.

7.
Nature ; 485(7397): 217-20, 2012 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-22575962

RESUMO

The flare of radiation from the tidal disruption and accretion of a star can be used as a marker for supermassive black holes that otherwise lie dormant and undetected in the centres of distant galaxies. Previous candidate flares have had declining light curves in good agreement with expectations, but with poor constraints on the time of disruption and the type of star disrupted, because the rising emission was not observed. Recently, two 'relativistic' candidate tidal disruption events were discovered, each of whose extreme X-ray luminosity and synchrotron radio emission were interpreted as the onset of emission from a relativistic jet. Here we report a luminous ultraviolet-optical flare from the nuclear region of an inactive galaxy at a redshift of 0.1696. The observed continuum is cooler than expected for a simple accreting debris disk, but the well-sampled rise and decay of the light curve follow the predicted mass accretion rate and can be modelled to determine the time of disruption to an accuracy of two days. The black hole has a mass of about two million solar masses, modulo a factor dependent on the mass and radius of the star disrupted. On the basis of the spectroscopic signature of ionized helium from the unbound debris, we determine that the disrupted star was a helium-rich stellar core.

8.
J Lipid Res ; 47(11): 2525-37, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16914769

RESUMO

Although the immune system, inflammation, and cellular metabolism are linked to diseases associated with dyslipidemias, the mechanism(s) remain unclear. To determine whether there is a mechanistic link between lipid availability and inflammation/immune activation, we evaluated macrophage cell lines incubated under conditions of altered exogenous and endogenous lipid availability. Limiting exogenous lipids results in decreased lysosomal acidity and decreased lysosomal enzymatic activity. Both lysosomal parameters are restored with the addition of oleoyl-CoA, suggesting that fatty acids play a role in the regulation of lysosomal function. Cell surface expression of major histocompatibility complex (MHC)-encoded molecules is also decreased in the absence of exogenous lipids. Additionally, we observe decreased gamma-interferon stimulation of cell surface MHC class II. Using cerulenin to limit the endogenous synthesis of fatty acids results in decreased cell surface expression of MHC class II but does not appear to alter lysosomal acidity, suggesting that lysosomal acidity is dependent on exogenous, but not endogenous, fatty acid availability. Testing these conclusions in an in vivo mouse model, we observed statistically significant, diet-dependent differences in lysosomal acidity and MHC class II cell surface expression. Collectively, these data demonstrate a mechanistic link between lipid availability and early events in the immune response.


Assuntos
Ácidos Graxos/metabolismo , Genes MHC da Classe II , Antígenos de Histocompatibilidade Classe II/metabolismo , Lisossomos/metabolismo , Animais , Membrana Celular/metabolismo , Feminino , Glucosilceramidase/química , Humanos , Sistema Imunitário/metabolismo , Inflamação , Lipídeos/química , Lisossomos/enzimologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL
9.
Aust Dent J ; 47(4): 321-6, 2002 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-12587768

RESUMO

BACKGROUND: This study aimed to estimate the dental treatment needs and oral health status of a sample of older adults in residential aged care facilities in Perth. METHODS: The 348 participants (> or = 65 years) were interviewed and screened in 25 facilities. The screenings were carried out by one examiner using a mirror and a portable light. RESULTS: Over half (52 per cent) of the participants were edentulous and 45 per cent of those edentulous participants for whom a recording was made (n=174) had oral mucosal conditions. The 164 dentate participants had a mean of 12 disease-free standing teeth, a mean decayed, missing or filled teeth (DMFT) of 24.7 (mean DT 0.8, mean FT 5.3) and half of them required the removal of supragingival calculus. Of those with root caries experience (n=127), a mean of 1.3 untreated decayed roots and a mean of 1.9 roots covered in plaque were recorded. The majority of the participants (83 per cent) were pensioners eligible for government subsidized dental care and 47 per cent were reported by the Directors of Nursing to have dementia. CONCLUSIONS: The data collected here demonstrate poor oral health conditions and a substantial treatment need in a neglected population. More people in nursing homes and hostels are keeping their natural teeth compared with a similar population studied 13 years ago.


Assuntos
Assistência Odontológica para Idosos/estatística & dados numéricos , Avaliação Geriátrica/estatística & dados numéricos , Instituição de Longa Permanência para Idosos , Programas de Rastreamento , Avaliação das Necessidades/estatística & dados numéricos , Casas de Saúde , Idoso , Idoso de 80 Anos ou mais , Distribuição de Qui-Quadrado , Índice CPO , Cálculos Dentários/epidemiologia , Cárie Dentária/epidemiologia , Placa Dentária/epidemiologia , Restauração Dentária Permanente/estatística & dados numéricos , Feminino , Acessibilidade aos Serviços de Saúde/estatística & dados numéricos , Humanos , Arcada Edêntula/epidemiologia , Arcada Parcialmente Edêntula/epidemiologia , Masculino , Doenças da Boca/epidemiologia , Índice Periodontal , Cárie Radicular/epidemiologia , Doenças Dentárias/epidemiologia , Austrália Ocidental/epidemiologia
10.
Nature ; 414(6864): 617-9, 2001 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-11740553

RESUMO

The nature of dark matter remains mysterious, with luminous material accounting for at most approximately 25 per cent of the baryons in the Universe. We accordingly undertook a survey looking for the microlensing of stars in the Large Magellanic Cloud (LMC) to determine the fraction of Galactic dark matter contained in massive compact halo objects (MACHOs). The presence of the dark matter would be revealed by gravitational lensing of the light from an LMC star as the foreground dark matter moves across the line of sight. The duration of the lensing event is the key observable parameter, but gives non-unique solutions when attempting to estimate the mass, distance and transverse velocity of the lens. The survey results to date indicate that between 8 and 50 per cent of the baryonic mass of the Galactic halo is in the form of MACHOs (ref. 3), but removing the degeneracy by identifying a lensing object would tighten the constraints on the mass in MACHOs. Here we report a direct image of a microlens, revealing it to be a nearby low-mass star in the disk of the Milky Way. This is consistent with the expected frequency of nearby stars acting as lenses, and demonstrates a direct determination of a lens mass from a microlensing event. Complete solutions such as this for halo microlensing events will probe directly the nature of the MACHOs.

11.
Biochemistry ; 40(34): 10334-41, 2001 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-11513612

RESUMO

The activity of membrane-associated protein kinase C (PKC) is tightly controlled by the physical properties of the membrane lipid bilayer, in particular, curvature stress, which is induced by bilayer-destabilizing lipid components. An important example of this is the weakened lipid headgroup interactions induced by phosphatidylethanolamine (PE) and cholesterol. In this work our previous observation with a mixed isoform PKC showing a biphasic dependence of activity as a function of membrane curvature stress [Slater et al. (1994) J. Biol. Chem. 269, 4866-4871] was here extended to individual isoforms. The Ca(2+)-dependent PKCalpha, PKCbeta, and PKCgamma, along with Ca(2+)-independent PKCdelta, but not PKCepsilon or PKCzeta, displayed a biphasic activity as a function of membrane PE content. The fluorescence anisotropy of N-(5-dimethylaminonaphthalene-1-sulfonyl)dioleoylphosphatidylserine (dansyl-PS), which probes the lipid environment of PKC, also followed a biphasic profile as a function of PE content for full-length PKCalpha, PKCbetaIotaIota, and PKCgamma as did the isolated C1 domain of PKCalpha. In addition, the rotational correlation time of both PKCalpha and PKCdelta C1-domain-associated sapintoxin D, a fluorescent phorbol ester, was also a biphasic function of membrane lipid PE content. These results indicate that the C1 domain acts as a sensor of the bilayer surface properties and that its conformational response to these effects may directly underlie the resultant effects on enzyme activity.


Assuntos
Bicamadas Lipídicas/química , Proteína Quinase C/química , Proteína Quinase C/metabolismo , Animais , Encéfalo/enzimologia , Polarização de Fluorescência , Isoenzimas/química , Isoenzimas/metabolismo , Cinética , Fosfatidilcolinas/química , Fosfatidiletanolaminas/química , Fosfatidilserinas/química , Proteína Quinase C beta , Proteína Quinase C-alfa , Proteína Quinase C-delta , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo
12.
J Econ Entomol ; 94(3): 609-16, 2001 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-11425013

RESUMO

The pollination effectiveness of the commercially reared bumble bee Bombus impatiens Cresson, was compared in field studies to the honey bee, Apis mellifera L., for lowbush blueberry, Vaccinium angustifolium Ait. A preliminary study indicated that B. impatiens had potential as an alternative pollinator. In a 3-yr study, percentage fruit set, percentage harvested berries, berry weight, and seeds per berry were compared in blueberry fields stocked at 7.5 A. mellifera hives per hectare to 5, 7.5, or 10 B. impatiens colonies per hectare. Percentage of harvested berries (yield) was significantly higher in fields stocked with B. impatiens at 10 colonies per hectare. No other parameters measuring pollinator effectiveness were significantly different at 5, 7.5, or 10 colonies per hectare. Flower handling time was significantly faster for B. impatiens and it more frequently collected blueberry pollen. All parameters of pollinator effectiveness were similar for B. impatiens, A. mellifera, and native wild bees in a follow-up study. Overall, B. impatiens was a suitable alternative to A. mellifera.


Assuntos
Abelhas , Comportamento Animal , Pólen , Animais
13.
Biochim Biophys Acta ; 1544(1-2): 207-16, 2001 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-11341930

RESUMO

Protein kinase C (PKC) can be activated by interaction with filamentous actin (F-actin) in the absence of membrane lipids (S.J. Slater, S.K. Milano, B.A. Stagliano, K.J. Gergich, J.P. Curry, F.J. Taddeo and C.D. Stubbs, Biochemistry 39 (2000) 271-280). Here, the effects of ethanol on the F-actin-induced activities of a panel of PKC isoforms consisting of 'conventional' (cPKC) alpha, betaI, gamma, 'novel' (nPKC) delta, epsilon and 'atypical' (aPKC) zeta were investigated using purified PKC and F-actin. Ethanol was found to inhibit the Ca2+- and phorbol ester-dependent activities of cPKCalpha and betaI, and the Ca2+- and phorbol ester-independent activity of cPKCgamma, whereas the activities of nPKCdelta, epsilon and aPKCzeta were unaffected. Although the activities of cPKCalpha and betaI induced by saturating levels of phorbol ester were inhibited by ethanol, the binding of these isozymes to F-actin was unaffected within the same phorbol ester concentration range. Conversely, within submaximal levels of phorbol ester, cPKCalpha and betaI activities were unaffected by ethanol whereas binding to F-actin was inhibited. The potency of the inhibition of F-actin-induced cPKCbetaI activity increased with n-alkanol chain length up to n-hexanol, after which it declined. The results indicate that PKC activities associated with F-actin, and therefore cellular processes involving the actin cytoskeleton, are potential targets for ethanol action. The effects of ethanol on these processes may differ according to the particular regulating PKC isoform, its intracellular localization and the presence of activators and cofactors.


Assuntos
Actinas/metabolismo , Etanol/farmacologia , Proteína Quinase C/metabolismo , Sequência de Aminoácidos , Ativação Enzimática , Ligação Proteica , Acetato de Tetradecanoilforbol/farmacologia
14.
Biochemistry ; 40(20): 6085-92, 2001 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-11352745

RESUMO

Phorbol ester-induced conventional protein kinase C (PKCalpha, -betaIota/IotaIota, and -gamma) isozyme activities are potentiated by 1,2-diacyl-sn-glycerol. This has been attributed to a "cooperative" interaction of the two activators with two discrete sites termed the low- and high-affinity phorbol ester binding sites, respectively [Slater, S. J., Milano, S. K., Stagliano, B. A., Gergich, K. J., Ho, C., Mazurek, A., Taddeo, F. J., Kelly, M. B., Yeager, M. D., and Stubbs, C. D. (1999) Biochemistry 38, 3804-3815]. Here, we report that the 1-O-alkyl ether diglyceride, 1-O-hexadecyl-2-acetyl-sn-glycerol (HAG), like its 1,2-diacyl counterpart, 1-oleoyl-2-acetyl-sn-glycerol (OAG), also potentiated PKCalpha, -betaI/II, and -gamma activities induced by the phorbol ester 4beta-12-O-tetradecanoylphorbol-13-acetate (TPA). Similar to OAG, HAG was found to bind to the low-affinity phorbol ester binding site and to enhance high-affinity phorbol ester binding, and to decrease the level of Ca(2+) required for phorbol ester-induced activity, while being without effect on the Ca(2+) dependence of membrane association. Thus, similar to OAG, HAG may also potentiate phorbol ester-induced activity by interacting with the low-affinity phorbol ester binding site, leading to a reduced level of Ca(2+) required for the activating conformational change. However, HAG was found not to behave like a 1,2-diacyl-sn-glycerol in that alone it did not induce PKC activity, and also in that it enhanced OAG-induced activity. The results reveal HAG to be a member of a new class of "nonactivating" compounds that modulate PKC activity by interacting with the low-affinity phorbol ester binding site.


Assuntos
Diglicerídeos/metabolismo , Éteres de Glicerila/metabolismo , Proteína Quinase C/metabolismo , Acetato de Tetradecanoilforbol/metabolismo , Animais , Sítios de Ligação/efeitos dos fármacos , Cálcio/metabolismo , Cálcio/farmacologia , Diglicerídeos/farmacologia , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Ativação Enzimática/efeitos dos fármacos , Indução Enzimática/efeitos dos fármacos , Éteres de Glicerila/farmacologia , Isoenzimas/metabolismo , Lipossomos/química , Lipossomos/metabolismo , Ésteres de Forbol/metabolismo , Ligação Proteica/efeitos dos fármacos , Proteína Quinase C/biossíntese , Proteína Quinase C beta , Proteína Quinase C-alfa , Proteína Quinase C-delta , Ratos , Acetato de Tetradecanoilforbol/farmacologia
15.
Biochemistry ; 40(14): 4437-45, 2001 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-11284700

RESUMO

Evidence is provided for direct protein-protein interactions between protein kinase C (PKC) alpha, betaI, betaII, gamma, delta, epsilon, and zeta and members of the Rho family of small GTPases. Previous investigations, based on the immunoprecipitation approach, have provided evidence consistent with a direct interaction, but this remained to be proven. In the study presented here, an in vitro assay, consisting only of purified proteins and the requisite PKC activators and cofactors, was used to determine the effects of Rho GTPases on the activities of the different PKC isoforms. It was found that the activity of PKCalpha was potently enhanced by RhoA and Cdc42 and to a lesser extent by Rac1, whereas the effects on the activities of PKCbetaI, -betaII, -gamma, -delta, -epsilon, and -zeta were much reduced. These results indicate a direct interaction between PKCalpha and each of the Rho GTPases. However, the Rho GTPase concentration dependencies for the potentiating effects on PKCalpha activity differed for each Rho GTPase and were in the following order: RhoA > Cdc42 > Rac1. PKCalpha was activated in a phorbol ester- and Ca(2+)-dependent manner. This was reflected by a substantial decrease in the phorbol ester concentration requirements for activity in the presence of Ca(2+), which for each Rho GTPase was induced within a low nanomolar phorbol ester concentration range. The activity of PKCalpha also was found to be dependent on the nature of the GTP- or GDP-bound state of the Rho GTPases, suggesting that the interaction may be regulated by conformational changes in both PKCalpha and Rho GTPases. Such an interaction could result in significant cross-talk between the distinct pathways regulated by these two signaling elements.


Assuntos
Proteína Quinase C/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Cálcio/metabolismo , Cálcio/farmacologia , Cátions Bivalentes/metabolismo , Cátions Bivalentes/farmacologia , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Isoenzimas/classificação , Isoenzimas/metabolismo , Lipídeos de Membrana/metabolismo , Ligação Proteica , Proteína Quinase C/classificação , Proteína Quinase C-alfa , Ratos , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Acetato de Tetradecanoilforbol/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo
16.
Biochemistry ; 39(2): 271-80, 2000 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-10630986

RESUMO

The mechanism of activation of protein kinase C isoforms by filamentous actin (F-actin) was investigated with respect to isozyme specificity and phorbol ester and Ca(2+) dependencies. It was found that the "conventional" (cPKC), alpha, betaI, betaII, and gamma, "novel" (nPKC) delta and epsilon, and "atypical" (aPKC) zeta isoforms were each activated by F-actin with varying potencies. The level of activity along with the affinity for binding to F-actin was further potentiated by the phorbol ester 4beta-12-O-tetradecanoylphorbol 13-acetate (TPA), the potency of which again varied for each isoform. By contrast to the other cPKC isoforms, the level of cPKC-gamma activity was unaffected by TPA, as was also the case for aPKC-zeta. It was found that whereas in the absence of F-actin the soluble form of cPKC-betaI contained two phorbol ester binding sites of low and high affinity, respectively, as previously reported for cPKC-alpha [Slater et al. (1998) J. Biol. Chem. 273, 23160-23168], the F-actin-bound form of the isozyme contained only a single site of relatively low affinity. The level of TPA required to induce cPKC-alpha, -betaI, and -betaII activity and the binding of these isozymes to F-actin was reduced in the presence of Ca(2+). By contrast, the activity of cPKC-gamma was unaffected by Ca(2+), as were the activities of nPKC-delta and -epsilon and aPKC-zeta, as expected. Thus, the interaction with F-actin appears to be a general property of each of the seven PKC isozymes tested. However, isoform specificity may, in part, be directed by differences in the phorbol ester and Ca(2+) dependences, which, with the notable exception of cPKC-gamma, appear to resemble those observed for the activation of each isoform by membrane association. The observation that cPKC isoforms may translocate to F-actin as well as the membrane as a response to an elevation of Ca(2+) levels may allow for the functional coupling of fluctuations of intracellular Ca(2+) levels through cPKC to F-actin cytoskeleton-mediated processes.


Assuntos
Actinas/metabolismo , Proteína Quinase C/metabolismo , Actinas/isolamento & purificação , Animais , Sítios de Ligação , Cálcio/farmacologia , Ativação Enzimática/efeitos dos fármacos , Isoenzimas/metabolismo , Ligação Proteica , Proteína Quinase C/biossíntese , Coelhos , Ratos , Especificidade por Substrato , Acetato de Tetradecanoilforbol/farmacologia
17.
Biochem J ; 344 Pt 2: 451-60, 1999 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-10567228

RESUMO

The fluorescent phorbol ester 12-N-methylanthraniloylphorbol 13-acetate [sapintoxin D (SAPD)] was used as both the activator and the probe for the activating conformational change of the C1 domain of recombinant protein kinase C (PKC)alpha. Fluorescence emission spectra and steady-state anisotropy measurements of SAPD in fully active membrane-associated PKC show that there is a relatively hydrophobic environment and restricted motional freedom characterizing the phorbol-ester-binding site. SAPD also interacts with the membrane lipids so that it was necessary to resort to time-resolved anisotropy measurements to resolve the signals corresponding to PKC-bound SAPD from that associated with buffer and lipid. In the presence of membrane lipids (unilamellar vesicles of phosphatidylcholine and phosphatidylserine, 4:1 molar ratio) and Ca(2+), at a concentration sufficient to activate the enzyme fully, a long correlation time characteristic of highly restricted motion was observed for PKC-associated SAPD. The fraction of SAPD molecules displaying this restricted motion, in comparison with the total SAPD including that in lipids and in buffer, increased with increasing concentrations of Ca(2+) and paralleled the appearance of enzyme activity, whereas the rotational correlation time remained constant. This could be rationalized as an increase in the number of active PKC conformers in the total population of PKC molecules. It therefore seems that there is a distinct conformation of the C1 activator-binding domain associated with the active form of PKC. The addition of SAPD and dioleoyl-sn-glycerol together produced an activity higher than that achievable by either activator alone both at concentrations that alone induced maximal activity for the respective activator; this higher activity was associated with a further restriction in SAPD motion. Increasing the cholesterol concentration, the phosphatidylethanolamine concentration, the sn-2 unsaturation in phosphatidylcholine and the vesicle curvature each also elevated SAPD-induced PKC activity and again increased the PKC-associated SAPD rotational correlation time. In summary, the rotational correlation time of PKC-bound SAPD, extractable from a single time-resolved fluorescence anisotropy measurement, provides a novel probe for the involvement of interactions between the C1 domain and phorbol ester in the modulation of PKC activity.


Assuntos
Ésteres de Forbol/metabolismo , Proteína Quinase C/química , Proteína Quinase C/metabolismo , Sítios de Ligação , Cálcio/farmacologia , Diglicerídeos/metabolismo , Ativação Enzimática , Polarização de Fluorescência , Corantes Fluorescentes/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Movimento (Física) , Estrutura Quaternária de Proteína , Acetato de Tetradecanoilforbol/farmacologia
18.
Alcohol Clin Exp Res ; 23(9): 1552-60, 1999 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-10512323

RESUMO

Protein kinase C (PKC) is involved in the control of many key signaling pathways in cells. Investigations over the past decade have shown that many effects of ethanol on cell function are closely interconnected with PKC. Three distinct areas of investigation have emerged; they are reviewed in this article. In vitro studies show that ethanol and higher alcohols can both inhibit or enhance PKC activity, depending on the experimental conditions. These studies show that alcohols interact directly with PKC, suggesting at least some role of this interaction in intoxication and anesthesia. Most ion channel systems are modulated by ethanol to varying degrees, and inhibition of PKC attenuates this effect; however, the mechanism by which ethanol brings about this effect is not known. Lastly, prolonged or chronic ethanol exposure up-regulates PKC, an effect that has important consequences, for example, in neuronal development; again, the mechanism leading to this process is not understood. The current consensus is that PKC is intimately involved in acute and chronic ethanol action, and the challenge now is to determine the mechanisms involved so that strategies can be developed to control these effects.


Assuntos
Depressores do Sistema Nervoso Central/farmacologia , Etanol/farmacologia , Canais Iônicos/efeitos dos fármacos , Proteína Quinase C/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Animais , Humanos , Canais Iônicos/metabolismo , Proteína Quinase C/metabolismo , Transdução de Sinais/fisiologia
19.
Biochemistry ; 38(12): 3804-15, 1999 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-10090770

RESUMO

Protein kinase Calpha (PKCalpha) has been shown to contain two discrete activator sites with differing binding affinities for phorbol esters and diacylglycerols. The interaction of diacylglycerol with a low-affinity phorbol ester binding site leads to enhanced high-affinity phorbol ester binding and to a potentiated level of activity [Slater, S. J., Ho, C., Kelly, M. B., Larkin, J. D. , Taddeo, F. J., Yeager, M. D., and Stubbs, C. D. (1996) J. Biol. Chem. 271, 4627-4631]. In this study, the mechanism of this enhancement of activity was examined with respect to the Ca2+ dependences of membrane association and accompanying conformational changes that lead to activation. The association of PKCalpha with membranes containing 12-O-tetradecanoylphorbol 13-acetate (TPA) or 1, 2-dioleoylglycerol (DAG), determined from tryptophan to dansyl-PE resonance energy transfer (RET) measurements, was found to occur at relatively low Ca2+ levels (

Assuntos
Diglicerídeos/farmacologia , Isoenzimas/metabolismo , Ésteres de Forbol/farmacologia , Proteína Quinase C/metabolismo , Anisotropia , Cálcio/metabolismo , Relação Dose-Resposta a Droga , Ativação Enzimática , Lipídeos de Membrana/metabolismo , Conformação Proteica , Proteína Quinase C beta , Proteína Quinase C-alfa
20.
J Biol Chem ; 273(36): 23160-8, 1998 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-9722545

RESUMO

The activity of membrane-associated protein kinase C (PKC) has previously been shown to be regulated by two discrete high and low affinity binding regions for diacylglycerols and phorbol esters (Slater, S. J., Ho, C., Kelly, M. B., Larkin, J. D., Taddeo, F. J., Yeager, M. D., and Stubbs, C. D. (1996) J. Biol. Chem. 271, 4627-4631). PKC is also known to interact with both cytoskeletal and nuclear proteins; however, less is known concerning the mode of activation of this non-membrane form of PKC. By using the fluorescent phorbol ester, sapintoxin D (SAPD), PKCalpha, alone, was found to possess both low and high affinity phorbol ester-binding sites, showing that interaction with these sites does not require association with the membrane. Importantly, a fusion protein containing the isolated C1A/C1B (C1) domain of PKCalpha also bound SAPD with low and high affinity, indicating that the sites may be confined to this domain rather than residing elsewhere on the enzyme molecule. Both high and low affinity interactions with native PKCalpha were enhanced by protamine sulfate, which activates the enzyme without requiring Ca2+ or membrane lipids. However, this "non-membrane" PKC activity was inhibited by the phorbol ester 4beta-12-O-tetradecanoylphorbol-13-acetate (TPA) and also by the fluorescent analog, SAPD, opposite to its effect on membrane-associated PKCalpha. Bryostatin-1 and the soluble diacylglycerol, 1-oleoyl-2-acetylglycerol, both potent activators of membrane-associated PKC, also competed for both low and high affinity SAPD binding and inhibited protamine sulfate-induced activity. Furthermore, the inactive phorbol ester analog 4alpha-TPA (4alpha-12-O-tetradecanoylphorbol-13-acetate) also inhibited non-membrane-associated PKC. In keeping with these observations, although TPA could displace high affinity SAPD binding from both forms of the enzyme, 4alpha-TPA was only effective at displacing high affinity SAPD binding from non-membrane-associated PKC. 4alpha-TPA also displaced SAPD from the isolated C1 domain. These results show that although high and low affinity phorbol ester-binding sites are found on non-membrane-associated PKC, the phorbol ester binding properties change significantly upon association with membranes.


Assuntos
Diglicerídeos/farmacologia , Isoenzimas/efeitos dos fármacos , Lactonas/farmacologia , Lipídeos de Membrana/farmacologia , Ésteres de Forbol/farmacologia , Proteína Quinase C/efeitos dos fármacos , Regulação Alostérica , Animais , Briostatinas , Interações Medicamentosas , Indução Enzimática , Corantes Fluorescentes , Isoenzimas/genética , Macrolídeos , Protaminas/farmacologia , Proteína Quinase C/genética , Proteína Quinase C-alfa , Proteína Quinase C-delta , Ratos , Proteínas Recombinantes/efeitos dos fármacos , Solubilidade , Acetato de Tetradecanoilforbol/análogos & derivados , Acetato de Tetradecanoilforbol/farmacologia
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