RESUMO
Pneumatosis Intestinalis (PI) is a rare radiological finding defined as the presence of extra-luminal gas within the intestinal wall. Several anti-tumor drugs can induce a damage of the gastrointestinal walls as an adverse effect, causing loss of mucosal integrity and endoluminal gas diffusion, responsible for PI development. We retrospectively analyzed 8 cases of PI detected through radiological imaging in oncologic patients undergoing various therapeutic regimens: five patients were receiving chemotherapy, two molecular targeted therapy (MTT) and one immunotherapy. Three patients were asymptomatic and pneumatosis was incidentally detected at routinary follow-up CT and then treated conservatively. Five patients presented acute abdomen symptoms and in these cases bowel perforation was the cause of death. Our experience confirms PI and perforation as rare complications of drug toxicity, especially in oncologic patients treated with combinations of different anticancer drugs and documented the second reported case of PI associated with atezolizumab and alectinib single administration.
Assuntos
Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Perfuração Intestinal , Pneumatose Cistoide Intestinal , Humanos , Perfuração Intestinal/induzido quimicamente , Perfuração Intestinal/diagnóstico por imagem , Pneumatose Cistoide Intestinal/induzido quimicamente , Pneumatose Cistoide Intestinal/diagnóstico por imagem , Estudos Retrospectivos , Perfuração EspontâneaRESUMO
BACKGROUND: Neurofibromatosis type 1 is a genetic disorder associated with cognitive deficits, learning disabilities and behavioral problems. These domains appear to have a still controversial debated association with local areas of T2-hyperintensities on MRI images, called unidentified bright objects (UBOs). METHODS: A cohort of 36 children (aged 7-11 years) included consecutively, underwent neuropsychological and behavioral assessment to determine their cognitive and neuropsychological profile, and the frequency of specific learning disabilities. MRI examination was used to determine the impact of UBOs' presence, number, and location on the cognitive, neuropsychological and behavioral profile, and also the presence of optic glioma. RESULTS: The mean full intelligence quotient was 104.6; only one child had mild intellectual disability. Forty one percent of children had a diagnosis of specific learning disabilities and reading was mainly involved. Twenty per cent had attention problems. All children had normal scores in visuo-motor and visuo-perceptual tests. UBOs were present in 94.0% of the MRI examinations. Two children had optic glioma. Children with UBOs in a specific location and children with UBOs elsewhere were statistically compared, no one of the location seemed to have an impact on general cognition measured with full intelligence quotient. The thalamus was associated with problems in calculation and striatum with behavioral problems. An inverse relationship between the number of UBOs and the full intelligence quotient was present, but without a statistical significance. CONCLUSIONS: In this study, the specific location of UBOs did not seem to influence the general cognitive profile and also the relationship between their number and the full intelligence quotient was not significant; these results are still controversial in literature. Finally, the presence of UBOs in the thalamus and striatum may represent a neuroradiological pattern that influences performances in calculation and behavior respectively in children with Neurofibromatosis type 1.
Assuntos
Transtornos Cognitivos/etiologia , Transtornos Cognitivos/patologia , Neurofibromatose 1/patologia , Neurofibromatose 1/psicologia , Adolescente , Criança , Transtornos Cognitivos/epidemiologia , Estudos de Coortes , Feminino , Humanos , Imageamento por Ressonância Magnética , MasculinoRESUMO
Efficient bacterial chromosome segregation typically requires the coordinated action of a three-component machinery, fueled by adenosine triphosphate, called the partition complex. We present a phenomenological model accounting for the dynamic activity of this system that is also relevant for the physics of catalytic particles in active environments. The model is obtained by coupling simple linear reaction-diffusion equations with a proteophoresis, or "volumetric" chemophoresis, force field that arises from protein-protein interactions and provides a physically viable mechanism for complex translocation. This minimal description captures most known experimental observations: dynamic oscillations of complex components, complex separation, and subsequent symmetrical positioning. The predictions of our model are in phenomenological agreement with and provide substantial insight into recent experiments. From a nonlinear physics view point, this system explores the active separation of matter at micrometric scales with a dynamical instability between static positioning and traveling wave regimes triggered by the dynamical spontaneous breaking of rotational symmetry.
Assuntos
Trifosfato de Adenosina/metabolismo , Genoma Bacteriano , Fenômenos Biofísicos , Catálise , Difusão , Modelos Biológicos , Domínios e Motivos de Interação entre ProteínasRESUMO
The aim of this work was to determine the effects of dark and light conditions on the E2, testosterone and thyroid hormones levels and on the gene expression levels (vitellogenin 1, vitellogenin 2, and estradiol receptor one) in European eels (Anguilla anguilla) during ovarian development induced by increasing doses of carp pituitary extracts (CPEs). The subjects were divided into 2 groups: 14-hour light:10-hour dark (Light Group) and 24-hour darkness (Dark Group). All the eels received intramuscular injections with CPE at a dosage of 10 mg/kg body weight (BW) once a week for the first 3 weeks, 20 mg/kg BW fourth-sixth week, 30 mg/kg BW seventh-ninth week, and 40 mg/kg up to the end of the experiment (13th week). Vitellogenin and estradiol receptor expression levels did not show significant differences between the two housing conditions whereas in both groups vitellogenin mRNA increased starting from first CPE injection. Testosterone and 17-beta estradiol plasma levels were significantly greater in the Dark Group compared with the Light Group starting from the ninth and the 13th week, respectively. These results suggest that darkness could be a useful variable for standardizing gonadal maturation in eels kept in captivity.
Assuntos
Enguias/fisiologia , Estrogênios/metabolismo , Fotoperíodo , Testosterona/metabolismo , Extratos de Tecidos/farmacologia , Vitelogeninas/metabolismo , Animais , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Hipófise , Receptores de Estradiol/genética , Receptores de Estradiol/metabolismo , Hormônios Tireóideos/sangue , Hormônios Tireóideos/metabolismo , Extratos de Tecidos/administração & dosagem , Vitelogeninas/genéticaRESUMO
Infantile neuroaxonal dystrophy (INAD) is a rare autosomal recessive neurodegenerative disorder characterized by infantile onset and rapid progression of psychomotor regression and hypotonia evolving into spasticity. The neuroradiologic hallmark of the disease is represented by progressive cerebellar atrophy. Prior to the discovery of mutations in the PLA2G6 gene in family with INAD, the clinical diagnosis of the disease had been confirmed by the presence of spheroid bodies (SB) in a peripheral nerve biopsy. Various studies have found that some patients with mutations lacked SB and some without mutations had SB, indicating incomplete detection using either pathologic or molecular methods (7). This, together with the observation that the spectrum of clinical features associated with mutations in PLA2G6 is broader than previously described, has increased the usefulness of Magnetic Resonance (MR) in INAD diagnosis, particularly in the frequent occurrence of atypical cases, especially in the early stages of the disease. We retrospectively reviewed the MR studies of eight patients in whom clinical and imaging onset met the typical criteria for INAD. Their clinical and MR imaging (MRI) onset and follow-up were evaluated together with the neuroradiological findings reported in the literature in order to identify MRI features useful in differentiating INAD from other diseases with similar clinical onset and to discuss which of them are the most important, thus suggesting INAD diagnosis. Our contribution included the use of Proton Spectroscopy ((1)H-MR), diffusion weighted MR imaging (DWI) and diffusion tensor imaging (DTI) in the follow-up of seven of the eight patients. The literature reviewed included attempts to correlate clinical and MR data with the genotype in the group of patients carrying PLA2G6 mutations. From the limited and inhomogeneous cohort of patients included in our study, a correlation between the MR features, phenotype and genotype was not exhaustive.
RESUMO
Ghrelin, the endogenous ligand of the growth hormone (GH) secretagogue receptor, is considered a pleiotropic regulator involved in a large array of functions, including control of energy balance, regulation of food intake and, more recently, modulation of the reproductive axis. The present study was aimed at determining the changes in plasma concentrations of acyl-ghrelin in pregnant and lactating sows, with special emphasis on the relationship with the levels of GH, leptin, non-esterified fatty acids (NEFA) and insulin-like growth factor (IGF-1). Blood samples were collected via jugular venipuncture from 22 multiparous sow 30, 60 and 90 days after artificial insemination, 7 and 21 days after farrowing and at first oestrus post-weaning. Plasma concentrations of acyl-ghrelin, leptin, GH and IGF-1 were quantified by validated radioimmunoassay; NEFA were determined using a colorimetric procedure. Plasma acyl ghrelin levels were highest at 30 days of pregnancy and decreased thereafter and during lactation. At the beginning of lactation, GH, IGF-1 and NEFA concentrations significantly increased, while a significant reduction occurred in leptin. In conclusion, ghrelin concentrations in sow maternal circulation does not seem to play an important role in maintaining circulating GH levels during lactation; moreover, ghrelin is not associated with leptin, NEFA and IGF-1 levels.
Assuntos
Lactação/metabolismo , Hormônios Peptídicos/sangue , Prenhez/metabolismo , Suínos , Animais , Ácidos Graxos não Esterificados/sangue , Feminino , Grelina , Hormônio do Crescimento/sangue , Fator de Crescimento Insulin-Like I/metabolismo , Lactação/sangue , Leptina/sangue , Gravidez , Prenhez/sangue , Suínos/sangue , Suínos/metabolismo , Suínos/fisiologia , Fatores de Tempo , DesmameRESUMO
Ghrelin is a peripheral circulating hormone, mainly released from the stomach, which can stimulate food intake. We studied fed, fasted and fasted-refed prepuberal gilts in order to outline possible changes in gastric mucosal ghrelin cells and in plasma ghrelin profiles in response to food deprivation. Acyl-ghrelin-immunoreactive cells were numerous in oxyntic glands, less abundant in cardiac glands and least frequent in pyloric glands, with the addition of a minor population of labelled cells in the gastric pit mucosa. When fed and fasted animals were compared (72-h fast versus fed; n = 4 each), no clear-cut differences were revealed in labelled cell numbers, nor in their staining intensity. An RIA for plasma porcine acyl-ghrelin (n-octanoylated at Ser-3), not recognizing des-acyl-ghrelin, was validated. Plasma acyl-ghrelin progressively increased upon fasting (over 6, 12, 24 and 48 h); ghrelin levels significantly (P<0.05) higher than those prefast were reached at 72 h. After refeeding, plasma ghrelin was rapidly restored to basal values by 6 h. In the same animals, plasma insulin was significantly reduced throughout the fasting period (6-72 h), while rapidly increasing after refeeding. Non-esterified fatty acid levels increased during fasting (12-72 h) and rapidly returned to low values after refeeding. In conclusion, the present study demonstrates that starvation and refeeding influence ghrelin plasma level in prepuberal gilts. The absence of detectable changes in ghrelin cells, as seen in immunohistochemistry, could be due to a large intracellular storage of potentially releasable acylghrelin.
Assuntos
Jejum , Mucosa Gástrica/química , Hormônios Peptídicos/metabolismo , Suínos/metabolismo , Animais , Glicemia/análise , Cromatografia Líquida de Alta Pressão , Ingestão de Alimentos , Ácidos Graxos não Esterificados/sangue , Feminino , Grelina , Imuno-Histoquímica/métodos , Insulina/sangue , Hormônios Peptídicos/análise , Hormônios Peptídicos/sangue , Período Pós-Prandial , Radioimunoensaio/métodos , Reprodutibilidade dos Testes , Maturidade Sexual/fisiologiaRESUMO
Reactive oxygen species (ROS) have been found to play important roles in regulating cellular functions. Their action in vivo has been related to specific effects on signal transduction pathways, such as Ras pathway. In order to characterize which elements of Ras pathway are affected by ROS, we have analyzed the action of different oxidizing agents on the ability of GTPase activating protein GAP and nucleotide exchange factor GEF to enhance the intrinsic activities of Ras. The action of these agents on the binding between H-Ras and its effector c-Raf-1 was also investigated. No effects were observed on the intrinsic activities of H-Ras or Ras2p. On the other hand, reversible inhibitions of GEF and GAP actions on Ras were found, whose extent was dependent on the agent used. As tested with the scintillation proximity assay, these agents also inhibited the binding of c-Raf-1 to H-Ras. Our data reveal new potential targets for the action of ROS on Ras pathway and suggest that they can influence the Ras activation state indirectly via regulators and effectors.
Assuntos
Proteínas Ativadoras de GTPase/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Oxidantes/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Proteínas ras/metabolismo , Proteínas Ativadoras de GTPase/efeitos dos fármacos , Fatores de Troca do Nucleotídeo Guanina/antagonistas & inibidores , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Oxirredução , Proteínas Proto-Oncogênicas c-raf/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMO
Activation of the neuronal Ras GDP/GTP exchange factor (GEF) CDC25Mm/GRF1 is known to be associated with phosphorylation of serine/threonine. To increase our knowledge of the mechanism involved, we have analyzed the ability of several serine/threonine kinases to phosphorylate CDC25Mm in vivo and in vitro. We could demonstrate the involvement of cAMP-dependent protein kinase (PKA) in the phosphorylation of CDC25Mm in fibroblasts overexpressing this RasGEF as well as in mouse brain synaptosomal membranes. In vitro, PKA was found to phosphorylate multiple sites on purified CDC25Mm, in contrast to protein kinase C, calmodulin kinase II, and casein kinase II, which were virtually inactive. Eight phosphorylated serines and one threonine were identified by mass spectrometry and Edman degradation. Most of them were clustered around the Ras exchanger motif/PEST motifs situated in the C-terminal moiety (residues 631-978) preceding the catalytic domain. Ser745 and Ser822 were the most heavily phosphorylated residues and the only ones coinciding with PKA consensus sequences. Substitutions S745D and S822D showed that the latter mutation strongly inhibited the exchange activity of CDC25Mm on Ha-Ras. The multiple PKA-dependent phosphorylation sites on CDC25Mm suggest a complex regulatory picture of this RasGEF. The results are discussed in the light of structural and/or functional similarities with other members of this RasGEF family.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , ras-GRF1/metabolismo , Sequência de Bases , Proteínas Quinases Dependentes de AMP Cíclico/química , Primers do DNA , Mutagênese Sítio-Dirigida , Fosforilação , Serina/metabolismoRESUMO
We describe a 11 year-old-boy with Sneddon syndrome, confirmed by skin biopsy, and MR evidence of diffuse cerebral hyperintensity of white matter; he also suffered from pre-perinatal hypoxic-ischemic distress. Arylsulfatase A activity was found reduced because of arylsulfatase A pseudodeficiency. We suggest that the association of pre-perinatal distress, Sneddon syndrome and arylsulfatase A pseudodeficiency is responsible for the diffuse impairment of cerebral white matter, never reported in Sneddon syndrome and similar to described cases of delayed posthypoxic demyelination and arylsulfatase A pseudodeficiency.
Assuntos
Córtex Cerebral/patologia , Leucodistrofia Metacromática/patologia , Fibras Nervosas Mielinizadas/patologia , Síndrome de Sneddon/patologia , Córtex Cerebral/fisiopatologia , Criança , Progressão da Doença , Humanos , Leucodistrofia Metacromática/genética , Imageamento por Ressonância Magnética , Masculino , Linhagem , Pele/patologia , Síndrome de Sneddon/genéticaRESUMO
The properties of variants of elongation factor (EF) Tu mutated at three positions implicated in its GTPase activity are presented. Mutation I60A, which reduces one wing of a "hydrophobic barrier" screening off the nucleophilic water molecule found at the GTP gamma-phosphate, causes a reduction of the intrinsic GTPase activity contrary to prediction and has practically no influence on other properties. Mutation D80N, which in the isolated G-domain of EF-Tu caused a strong stimulation of the intrinsic GTPase, reduces this activity in the intact molecule. However, whereas for wild-type EF-Tu complex formation with aa-tRNA reduces the GTPase, EF-Tu[D80N] shows a strongly increased activity when bound to Phe-tRNA. Moreover, ribosomes or kirromycin can stimulate its GTPase up to the same level as for wild-type. This indicates that a local destabilization of the magnesium binding network does not per se cause an increased GTPase but does affect its tight regulation. Interestingly, mutant D80N sequestrates EF-Ts by formation of a more stable complex. Substitutions T61A and T61N induce low intrinsic GTPase, and the stimulation by ribosome is less for T61A than for T61N but still detectable, while kirromycin stimulates the GTPase of both mutants equally. This provides more evidence that stimulation by kirromycin and ribosomes follows a different mechanism. The functional implications of these mutations are discussed in the context of a transition state mechanism for catalysis. An alternative structural explanation for the strong conservation of Ile-60 is proposed.
Assuntos
Ácido Aspártico/genética , GTP Fosfo-Hidrolases/genética , Isoleucina/genética , Mutagênese Sítio-Dirigida , Fator Tu de Elongação de Peptídeos/genética , Treonina/genética , Sítios de Ligação/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Ésteres , GTP Fosfo-Hidrolases/metabolismo , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Magnésio/química , Magnésio/metabolismo , Modelos Moleculares , Fator Tu de Elongação de Peptídeos/antagonistas & inibidores , Fator Tu de Elongação de Peptídeos/biossíntese , Fator Tu de Elongação de Peptídeos/metabolismo , Peptídeos/síntese química , Piridonas/química , RNA de Transferência de Fenilalanina/metabolismo , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Ribossomos/metabolismoRESUMO
The observation that activated c-Ha-Ras p21 interacts with diverse protein ligands suggests the existence of mechanisms that regulate multiple interactions with Ras. This work studies the influence of the Ras effector c-Raf-1 on the action of guanine nucleotide exchange factors (GEFs) on Ha-Ras in vitro. Purified GEFs (the catalytic domain of yeast Sdc25p and the full-length and catalytic domain of mouse CDC25Mm) and the Ras binding domains (RBDs) of Raf-1 (Raf (1-149) and Raf (51-131)) were used. Our results show that not only the intrinsic GTP/GTP exchange on Ha-Ras but also the GEF-stimulated exchange is inhibited in a concentration-dependent manner by the RBDs of Raf. Conversely, the scintillation proximity assay, which monitors the effect of GEF on the Ras.Raf complex, showed that the binding of Raf and GEF to Ha-Ras.GTP is mutually exclusive. The various GEFs used yielded comparable results. It is noteworthy that under more physiological conditions mimicking the cellular GDP/GTP ratio, Raf enhances the GEF-stimulated GDP/GTP exchange on Ha-Ras, in agreement with the sequestration of Ras.GTP by Raf. Consistent with our results, the GEF-stimulated exchange of Ha-Ras.GTP was also inhibited by another effector of Ras, the RBD (amino acid residues 133-314) of phosphatidylinositol 3-kinase p110alpha. Our data show that Raf-1 and phosphatidylinositol 3-kinase can influence the upstream activation of Ha-Ras. The interference between Ras effectors and GEF could be a regulatory mechanism to promote the activity of Ha-Ras in the cell.
Assuntos
Fosfatidilinositol 3-Quinases/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-raf/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Animais , Ligação Competitiva , Domínio Catalítico , Regulação da Expressão Gênica , Fatores de Troca do Nucleotídeo Guanina , Guanosina Trifosfato/metabolismo , Camundongos , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas c-raf/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Fatores ras de Troca de Nucleotídeo GuaninaRESUMO
During the centuries the pathophysiological concept of illness underwent drastic changes, depending on medical discoveries and religious belief. There is evidences of surgical practice in Mesopotamia, Egypt, and Greece 2000 years B.C., but we have to wait the Roman Empire with Celso, Galeno, and most of all Asclepiade and Temisone to appreciate the endeavor to rationally understand diseases as an alteration among different body constituents and not only as divine will. During the Middle Ages, when medicine was disputed by clergy, Ugo and Teodorico Borgognone perceived by intuition the problem of sepsis in patient recovery. This problem was cleared up six centuries later by Semmelweis with his antisepsis. Discoveries of blood circulation, hemostasis, anesthesia, and antisepsis in the 17th and 19th centuries permitted better disease management and have promoted, since the 1950s, pathophysiology as an autonomous discipline. Now pathophysiological mechanisms are used for comprehension of several diseases, such as gallstone formation, peptic ulcers, cranial trauma, cardiovascular abnormalities, and many others, to suggest to us which are the better surgical or clinical trials to realize, while observing caution about their limits and consequences. By a multidisciplinary approach involving surgeons, physiologists, biologists, and physicians, we are able to facilitate patient recovery and to perform modern, scientific, high-quality disease management.
Assuntos
Cirurgia Geral/história , Fisiologia/história , Procedimentos Cirúrgicos Operatórios , História Antiga , História Medieval , História Moderna 1601- , HumanosRESUMO
BACKGROUND AND OBJECTIVE: Patients with cortical malformations often have intractable seizures and are candidates for epilepsy surgery. Within an unselected series of patients with various forms of cortical malformation, nine patients with multilobar polymicrogyria had electrical status epilepticus during sleep (ESES) accompanied by infrequent focal motor seizures. Eight patients also had intractable atonic drop attack seizures. Because ESES usually is accompanied by a good long-term seizure prognosis, the objective of this study was to examine ESES outcome among patients with a structural lesion that is usually highly epileptogenic and has a low seizure remission trend. METHODS: The nine patients had follow-up periods lasting 4 to 19 years. All underwent brain MRI, serial sleep EEG recordings, and cognitive testing during and after ESES. RESULTS: ESES and drop attack seizures appeared between the ages of 2 and 5 years (mean, 4 years) and ceased between the ages of 5 and 12 years (mean, 8 years). At the last visit patients were 8 to 23 years of age (mean, 14.5 years) and were either seizure free or had very infrequent focal motor seizures during sleep. Three patients were free from antiepileptic drugs. In no patient was definite cognitive deterioration apparent after ESES in comparison with earlier evaluations. CONCLUSIONS: Age-related secondary bilateral synchrony underlying ESES may be facilitated in multilobar polymicrogyria. The good seizure outcome contrasts with that usually found in the presence of cortical malformations. For children with polymicrogyria and drop attack seizures, surgical treatment of the epilepsy should be considered cautiously, and sleep EEG recordings should be performed systematically.
Assuntos
Córtex Cerebral/anormalidades , Eletroencefalografia , Epilepsias Parciais/patologia , Sono/fisiologia , Estado Epiléptico/patologia , Idade de Início , Córtex Cerebral/fisiopatologia , Pré-Escolar , Epilepsias Parciais/fisiopatologia , Feminino , Humanos , Lactente , Imageamento por Ressonância Magnética , Masculino , Estudos Retrospectivos , Estado Epiléptico/fisiopatologia , SíndromeRESUMO
Elongation factor (EF) Tu from Escherichia coli contains three domains, of which domain 1 (N-terminal domain) harbors the site for nucleotide binding and GTP hydrolysis. To analyze the function of domains 2 [middle (M) domain] and 3 [C-terminal (C) domain], EF-Tu(DeltaM) and EF-Tu(DeltaC) were engineered as GST-fused products and purified. Circular dichroism and thermostability showed that both constructs have conserved organized structures. Though inactive in poly(Phe) synthesis the two constructs could bind GDP and GTP with comparable micromolar affinities. Therefore, like the isolated N-terminal domain, they had lost a typical feature of EF-Tu, the >100 times stronger affinity for GDP than for GTP. EF-Tu(DeltaM) and EF-Tu(DeltaC) had an intrinsic GTPase activity comparable to that of wild-type EF-Tu. Ribosomes did not stimulate the GTPase activity of either factor, while kirromycin increased the GTPase activity of both constructs, particularly of EF-Tu(DeltaC), to a level, however, much lower than that of the intact molecule. The interaction with aa-tRNA of both mutants was >90% reduced. As a major result, their GDP-bound form could efficiently respond to EF-Ts. All four EF-Tu-specific antibiotics [kirromycin, pulvomycin, GE2270 A (=MDL 62 879), and enacyloxin IIa] retarded significantly the dissociation of EF-Tu(DeltaC).GTP, showing the same kind of effect as on EF-Tu.GTP, but they were little active on EF-Tu(DeltaM). GTP. Like EF-Tu(DeltaC).GTP, EF-Tu(DeltaM).GTP was, however, able to bind efficiently kirromycin and enacyloxin IIa, as determined via competition with EF-Ts. Together, these results enlight selective functions of domains 2 and 3, particularly toward the interaction with EF-Ts and antibiotics, and emphasize their functional cooperativity for an efficient interaction of EF-Tu with ribosomes and aa-tRNA and for maintaining the differential affinity for GTP and GDP.
Assuntos
Fator Tu de Elongação de Peptídeos/metabolismo , Dicroísmo Circular , Análise Mutacional de DNA , Escherichia coli , Fatores de Elongação Ligados a GTP Fosfo-Hidrolases/metabolismo , Guanosina Difosfato/metabolismo , Temperatura Alta , Ligantes , Modelos Moleculares , Fator Tu de Elongação de Peptídeos/genética , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Peptídeos/metabolismo , Polienos/farmacologia , Desnaturação Proteica , Engenharia de Proteínas , Piridonas/farmacologia , Aminoacil-RNA de Transferência/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Ribossomos/efeitos dos fármacos , Deleção de SequênciaRESUMO
This work studies the coordination of the action of GTPase-activating protein (GAP) and guanine nucleotide exchange factor (GEF) on activated human c-Ha-Ras p21. Purified human p120-GAP was obtained with a new efficient procedure. To distinguish the GTPase-activating effect of p120-GAP from other effects dependent on the interaction with activated Ha-Ras, the nonhydrolyzable GTP analogue guanosine 5'-O-(thiotriphosphate) (GTPgammaS) was used. The results showed that the GTPgammaS/GTPgammaS exchange enhanced by the C-terminal catalytic domain of the yeast GEF Sdc25p (C-Sdc25p) is prevented by p120-GAP. This effect is strictly specific for the activated form of Ha-Ras, the target of GAP; no effect on Ha-Ras.GDP was detectable. The GAP catalytic domain also inhibited C-Sdc25p but to a lower extent. The interfering effect by p120-GAP was also evident in a homologous mammalian system, using full-length mouse RasGEF, its C-terminal half-molecule, or C-terminal catalytic domain. As a consequence of this inhibition, presence of p120-GAP enhanced the regeneration of Ha-Ras.GTPgammaS by GEF at a GDP:GTPgammaS ratio mimicking the in vivo GDP:GTP ratio. Our work describes a novel function of p120-GAP and suggests a mechanism by which GAP protects Ha-Ras.GTP in vivo against unproductive exchanges. This constrain is likely involved in the regulation of the physiological GDP/GTP cycle of Ras and in the action of p120-GAP as downstream effector of Ras. Helix alpha3 is proposed as a Ras element playing a key-role in the interference between GAP and GEF on Ras.
Assuntos
GTP Fosfo-Hidrolases/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Proteínas/metabolismo , Animais , Sítios de Ligação , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Clonagem Molecular , Escherichia coli , Proteínas de Ligação ao GTP/metabolismo , Proteínas Ativadoras de GTPase , Guanosina Difosfato/metabolismo , Humanos , Cinética , Camundongos , Modelos Estruturais , Fosfoproteínas Fosfatases/química , Fosfoproteínas Fosfatases/metabolismo , Conformação Proteica , Proteínas/antagonistas & inibidores , Proteínas/isolamento & purificação , Proteínas Proto-Oncogênicas p21(ras)/química , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Ativadoras de ras GTPase , ras-GRF1RESUMO
This work analyzes the action of enacyloxin Ila, an inhibitor of bacterial protein biosynthesis. Enacyloxin IIa [IC50 on poly(Phe) synthesis approximately 70 nM] is shown to affect the interaction between elongation factor (EF) Tu and GTP or GDP; in particular, the dissociation of EF-Tu-GTP is strongly retarded, causing the Kd of EF- Tu-GTP to decrease from 500 to 0.7 nM. In its presence, the migration velocity of both GTP- and GDP-bound EF-Tu on native PAGE is increased. The stimulation of EF-Tu-GDP dissociation by EF-Ts is inhibited. EF- Tu-GTP can still form a stable complex with aminoacyl-tRNA (aa-tRNA), but it no longer protects aa-tRNA against spontaneous deacylation, showing that the EF-Tu-GTP orientation with respect to the 3' end of aa-tRNA is modified. However, the EF-Tu-dependent binding of aa-tRNA to the ribosomal A-site is impaired only slightly by the antibiotic and the activity of the peptidyl-transferase center, as determined by puromycin reactivity, is not affected. In contrast, the C-terminal incorporation of Phe into poly(Phe)-tRNA bound to the P-site is inhibited, an effect that is observed if Phe-tRNA is bound to the A-site nonenzymatically as well. Thus, enacyloxin IIa can affect both EF-Tu and the ribosomal A-site directly, inducing an anomalous positioning of aa-tRNA, that inhibits the incorporation of the amino acid into the polypeptide chain. Therefore, it is the first antibiotic found to have a dual specificity targeted to EF-Tu and the ribosome.
Assuntos
Proteínas de Bactérias/antagonistas & inibidores , Elongação Traducional da Cadeia Peptídica/efeitos dos fármacos , Fator Tu de Elongação de Peptídeos/antagonistas & inibidores , Inibidores da Síntese de Proteínas/farmacologia , Ribossomos/efeitos dos fármacos , Proteínas de Bactérias/biossíntese , Eletroforese em Gel de Poliacrilamida , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Fatores de Elongação Ligados a GTP Fosfo-Hidrolases/metabolismo , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Cinética , Estrutura Molecular , Polienos/farmacologia , Aminoacil-RNA de Transferência/metabolismoRESUMO
Human and Saccharomyces cerevisiae Ras proteins and their regulators GAP (GTPase activating protein)and GEF (guanine nucleotide exchange factor) display structural similarities and are functionally interchangeable in vivo and in vitro, indicating that the molecular mechanism regulating Ras proteins has been conserved during evolution. As the only exceptions, the two S.cerevisiae GAPs, Ira1p and Ira2p, are strictly specific for yeast Ras proteins and cannot stimulate the GTPase of mammalian Ras. This study searches for the reasons for the different sensitivity to Ira2p of human H-ras p21 and yeast Ras2p. Construction of H-ras/Ras2p chimaeras showed that Gly18 of Ras2p (Ala11 of H-ras p21) is an important determinant for the specificity of Ira2p, revealing for the first time a function for this position. A second even more crucial determinant was found to be the 89-102 region of Ras2p (82-95 of H-ras p21) including the distal part of strand beta4, loop L6 and the proximal part of helix alpha3. It was possible to construct Ras2p's resistant to Ira2p but still sensitive to human p120-GAP and, conversely, a H-ras p21 sensitive to Ira2p. This work helps clarify specific aspects of the conserved molecular mechanism of interaction between Ras proteins and their negative GAP regulators.
Assuntos
Proteínas Fúngicas/farmacologia , Proteínas/farmacologia , Proteínas de Saccharomyces cerevisiae , Proteínas ras/genética , Proteínas ras/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Escherichia coli/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas Ativadoras de GTPase , Humanos , Técnicas In Vitro , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/química , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas Ativadoras de ras GTPase , Proteínas ras/químicaRESUMO
Intramuscolar mixoma is a rare benign neoplasm of mesenchymal origin consisting of stellate shaped cells in an abundant loose, mucopolysaccharide rich matrix. We report two cases of intramuscolar mixoma with review of the literature. Our objectives are to delineate the clinico pathologic characteristic, the nature of constituent cells and the features differing from others benign and malignant neoplasms.
Assuntos
Neoplasias Musculares/patologia , Mixoma/patologia , Músculos do Pescoço , Adulto , Idoso , Diagnóstico Diferencial , Feminino , Neoplasias de Cabeça e Pescoço/diagnóstico , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Neoplasias Musculares/diagnóstico , Neoplasias Musculares/epidemiologia , Mixoma/diagnóstico , Mixoma/epidemiologia , Células-Tronco Neoplásicas/patologia , Coxa da PernaRESUMO
Lipomatous tumor of palatine tonsil are rare. We report a case detected in a 72 year old male with a discussion about differential diagnosis of malignant polipoid lesions of the tonsil and some consideration about the problem of the growth of such neoformation.