RESUMO
OBJECTIVES: The Medical Outcomes Study 36 item Short-Form Health Survey (SF-36) is one of the most commonly used patient reported outcome measure. This study aimed to examine the relationship between SF-36 version 2 (SF-36V2) summary scores and Friedreich ataxia (FRDA) clinical characteristics, and to investigate the responsiveness of the scale, in comparison with the Friedreich Ataxia Rating Scale (FARS), over 1, 2 and 3 years. MATERIALS AND METHODS: Descriptive statistics were used to examine the characteristics of the cohort at baseline and years 1, 2 and 3. Correlations between FRDA clinical characteristics and SF-36V2 summary scores were reported. Responsiveness was measured using paired t tests. RESULTS: We found significant correlations between the physical component summary (PCS) of the SF-36V2 and various FRDA clinical parameters but none for the mental component summary. No significant changes in the SF-36V2 were seen over 1 or 2 years; however, PCS scores at Year 3 were significantly lower than at baseline (-3.3, SD [7.6], P=.01). FARS scores were found to be significantly greater at Years 1, 2 and 3 when compared to baseline. CONCLUSIONS: Our findings suggest that despite physical decline, individuals with FRDA have relatively stable mental well-being. This study demonstrates that the SF-36V2 is unlikely to be a useful tool for identifying clinical change in FRDA therapeutic trials.
Assuntos
Ataxia de Friedreich/patologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Ataxia de Friedreich/epidemiologia , Ataxia de Friedreich/terapia , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Avaliação de Resultados em Cuidados de SaúdeRESUMO
In systemic sclerosis (SSc), mouth and face involvement leads to problems in oral health-related quality of life (OHRQoL). Mouth Handicap in Systemic Sclerosis scale (MHISS) is a 12-item questionnaire specifically quantifying mouth disability in SSc, organized in 3 subscales. Our aim was to validate Italian version of MHISS, by assessing its test-retest reliability and internal and external consistency in Italian SSc patients. Forty SSc patients (7 dSSc, 33 lSSc; age and disease duration: 57.27 ± 11.41, 9.4 ± 4.4 years; 22 with sicca syndrome) were evaluated with MHISS. MHISS was translated following a forward-backward translation procedure, with independent translations and counter-translation. Test-retest reliability was evaluated, comparing the results of two administrations, with intraclass correlation coefficient (ICC). Internal consistency was assessed by Cronbach's α and external consistency by comparison with mouth opening. MHISS has a good test-retest reliability (ICC: 0.93) and internal consistency (Cronbach's α:0.99). A good external consistency was confirmed by correlation with mouth opening (rho: -0,3869, p: 0.0137). Total MHISS score was 17.65 ± 5.20, with scores of subscale 1 (reduced mouth opening) of 6.60 ± 2.85 and scores of subscales 2 (sicca syndrome) and 3 (aesthetic concerns) of 7.82 ± 2.59 and 3.22 ± 1.14. Total and subscale 2 scores are higher in dSSc than in lSSc. This result may be due to the higher presence of sicca syndrome in dSSc than in lSSc (p = 0.0109). Our results support validity and reliability in Italian SSc patients of MHISS, specifically measuring SSc OHRQoL.
Assuntos
Avaliação da Deficiência , Idioma , Saúde Bucal , Qualidade de Vida , Escleroderma Sistêmico/complicações , Inquéritos e Questionários/normas , Traduções , Idoso , Feminino , Humanos , Itália , Masculino , Pessoa de Meia-Idade , Boca/fisiopatologia , Avaliação de Resultados em Cuidados de Saúde , Reprodutibilidade dos Testes , Escleroderma Sistêmico/reabilitaçãoRESUMO
OBJECTIVES: In fibromyalgia syndrome (FMS) defined rehabilitation guidelines are yet to be validated. Our aim is to evaluate the efficacy of the Rességuier method (RM) in FMS. METHODS: Forty-one patients were randomly assigned to Interventional (22 pts) and Observational (19 pts) Group (IG and OG). The study lasted 8 months. Patients were assessed at baseline (T0) after a 2-month rehabilitation (T1) and at a 6-month follow-up (T2) (only IG) with SF-36 Physical (PSI) and Mental Synthetic Index (MSI), Regional Pain Scale (RPS), Fibromyalgia Impact Questionnaire (FIQ), Number Rating Scales 0-10 to measure pain, movement quality, sleep, relax ability, analgesics number/per week. OG patients maintained their lifestyle for the duration of the study. RM aims to obtain patient awareness and control of bodily perceptions, thus reaching a modulation of responses to pain. Therapist controls patient attention and perception by verbal and manual contacts and leads them to perform bodily and respiratory active and conscious movements. RESULTS: In IG, at T1 all items were improved: PSI and MSI (p<0.001 and =0.001), FIQ (p<0.0001), RPS (p<0.001), pain (p<0.0001), movement quality (p=0.001), relax ability (p<0.0001), sleep (p<0.001); analgesics number/per week was reduced (p<0.001). All results obtained at T1, except FIQ, were maintained at T2. In OG at T1 versus T0, no difference in any of the assessed parameters was observed. CONCLUSION: In FMS patients, the rehabilitation with RM improves HRQoL, FMS-related disability and perceived pain, thus reducing the assumption of analgesics.
Assuntos
Fibromialgia/reabilitação , Fibromialgia/terapia , Terapias Mente-Corpo/métodos , Adulto , Avaliação da Deficiência , Feminino , Fibromialgia/fisiopatologia , Seguimentos , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Medição da Dor , Qualidade de Vida , Sono , Resultado do TratamentoRESUMO
This study aimed to determine the prevalence of anti-platelet use, and the extent to which contraindications to anti-platelet therapy prevent its use, in 726 diabetic patients attending a private clinic. Among those who reported a history of cardiovascular disease (CVD), 87.1% were on anti-platelet therapy. Of those without prior CVD but with at least one CVD risk factor, 59.8% were not on anti-platelet therapy, but only 7.1% of these had a contraindication to anti-platelet therapy. This study showed that high usage of anti-platelet therapy in diabetic patients with prior CVD is achievable, and that contraindications did not explain low use in those without prior CVD.
Assuntos
Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/tratamento farmacológico , Inibidores da Agregação Plaquetária/uso terapêutico , Adulto , Idoso , Idoso de 80 Anos ou mais , Contraindicações , Diabetes Mellitus Tipo 2/terapia , Gerenciamento Clínico , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Contagem de Plaquetas/métodos , Transfusão de Plaquetas , Adulto JovemRESUMO
Friedreich Ataxia (FRDA) is the commonest inherited ataxia. Clinical trials of pharmaceuticals are increasingly being conducted in this condition. This requires the most accurate outcome measures to enable trials to be conducted with a minimum number of subjects in the shortest time frame and to minimize the risk of false negative results. Upper limb function is a major area of morbidity in FRDA. We therefore have compared the performance of three tests of upper limb function in FRDA: the Nine Hole Peg Test (9HPT), Box and Blocks Test (BBT) and Jebsen Taylor Hand Function Test (JTHFT). This study was undertaken to ascertain the best test for inclusion in a Friedreich Ataxia Functional Composite (FAFC) test for use in clinical studies and therapeutic trials. The three tests were administered to the dominant and non-dominant upper limbs of 38 individuals with genetically proven FRDA on two occasions, 12 months apart. The results of testing were correlated with the following disease parameters; age at disease onset, disease duration and score for the Friedreich Ataxia Rating Scale (FARS). The responsiveness to change of each test was assessed by measuring the effect size and calculations of the number of subjects required for similarly powered therapeutic trials. Results for all tests correlated significantly with disease duration and FARS score. The only test scores that changed significantly over 12 months were those for the non-dominant 9HPT and BBT. Scores for these two tests also had the largest effect sizes and required the fewest subjects for similarly powered therapeutic trials. We conclude, therefore, that the non-dominant 9HPT and BBT are the best tests for inclusion in a FAFC. Since the 9HPT has already been suggested for inclusion in a FAFC, we recommend that this test is used but that it is the non-dominant limb that is tested.
Assuntos
Avaliação da Deficiência , Ataxia de Friedreich/patologia , Exame Neurológico/métodos , Índice de Gravidade de Doença , Extremidade Superior/fisiopatologia , Adolescente , Adulto , Feminino , Ataxia de Friedreich/genética , Ataxia de Friedreich/fisiopatologia , Humanos , Masculino , Glicoproteínas de Membrana/genética , Pessoa de Meia-Idade , Avaliação de Resultados em Cuidados de Saúde , Desempenho Psicomotor , Adulto JovemRESUMO
INTRODUCTION: Rehabilitation may contribute to the management of Systemic Sclerosis (SSc) dealing with disabilities due to skin and joint involvement. AIM: to evaluate the efficacy of a district specific and global rehabilitation program tailored for SSc patients. MATERIALS AND METHODS: 20 SSc patients were enrolled and randomly assigned to 2 groups. Interventional group (10 pts) was treated that included hand and face specific rehabilitation and at least a global rehabilitation technique such as hydrokinesytherapy or land-based program, also comprising respiratory exercises. Hand lymphatic drainage was added when necessary. Observational group (10 patients) was only provided with educational advices and medical information about SSc. Patients were evaluated at baseline (T0) and after the 9 weeks treatment period (T1). Interventional group was also assessed after a 9 weeks follow-up (T2). Patients were evaluated by SF-36, HAQ and a purpose-built-questionnaire for global health condition and with Hamis test, Duruöz scale, range of motion, water volumetric test, mouth opening and a purpose-built-questionnaire for hand and face involvement. RESULTS: At the end of the treatment, patients of interventional group improved in all the parameters evaluated. At follow-up, mouth mobility and functionality such as global health status was partially lost, only hand mobility and functionality parameters were maintained. No changes were observed in controls. CONCLUSION: The association and of district-specific and global rehabilitative techniques conceived and tailored for SSc patients improves disability, HRQoL, hand and face disability and functionality, with its effects partially maintained at the follow-up.
Assuntos
Manipulações Musculoesqueléticas , Escleroderma Sistêmico/reabilitação , Avaliação da Deficiência , Feminino , Articulação da Mão/fisiopatologia , Nível de Saúde , Humanos , Masculino , Massagem , Pessoa de Meia-Idade , Exercícios de Alongamento Muscular , Qualidade de Vida , Escleroderma Sistêmico/fisiopatologia , Resultado do TratamentoRESUMO
Accessions from seven wild Solanum species were evaluated in the field for resistance to the Colorado potato beetle, Leptinotarsa decemlineata (Say). The multivariate insect population density data were analyzed using factor analysis. The factors extracted corresponded to relevant phases of the insect's life cycle and provided information on the mode of resistance (antixenosis and antibiosis) of the plant species. S. berthaultii, S. capsicibaccatum, S. jamesii, S. pinnatisectum, and S. trifidum demonstrated both antixenosis and antibiosis but expressed different levels of resistance. The mode of resistance of S. polyadenium seemed to be antibiosis and that of S. tarijense antixenosis. Genetic variability and heritability of insect resistance traits within accessions was trivial or inconsistent for all Solanum species studied.
Assuntos
Besouros , Variação Genética , Controle Biológico de Vetores , Solanaceae/genética , Animais , Genótipo , Controle Biológico de Vetores/métodosRESUMO
Twelve C-terminal residues of human glutathione S-transferase A1-1 form a helix in the presence of glutathione-conjugate, or substrate alone, and partly cover the active site. According to X-ray structures, the helix is disordered in the absence of glutathione, but it is not known if it is helical and delocalized, or in a random-coil conformation. Mutation to a tyrosine of residue 220 within this helix was previously shown to affect the pK(a) of Tyr-9 at the active site, in the apo form of the enzyme, and it was proposed that an on-face hydrogen bond between Tyr-220 and Tyr-9 provided a means for affecting this pK(a). In the current study, X-ray structures of the W21F and of the C-terminal mutation, W21F/F220Y, with glutathione sulfonate bound, show that the C-terminal helix is disordered (or delocalized) in the W21F crystal but is visible and ordered in a novel location, a crystal packing crevice, in one of three monomers in the W21F/F220Y crystal, and the proposed hydrogen bond is not formed. Fluorescence spectroscopy studies using an engineered F222W mutant show that the C-terminus remains delocalized in the absence of glutathione or when only the glutathione binding site is occupied, but is ordered and localized in the presence of substrate or conjugate, consistent with these and previous crystallographic studies. Proteins 2001;42:192-200.
Assuntos
Glutationa Transferase/química , Animais , Sítios de Ligação , Cristalografia por Raios X , Glutationa Transferase/genética , Isoenzimas , Modelos Moleculares , Mutação , Fragmentos de Peptídeos/química , Conformação Proteica , Ratos , Espectrometria de FluorescênciaRESUMO
BACKGROUND: An outbreak of trichinosis involving a cohort of 33 returning travelers from a resort island in a neighboring country was suspected, beginning with 2 initial cases who were hospitalized with a syndrome of fever, myalgia and eosinophilia. METHOD: At the initial visit, a full history was obtained and a physical examination was performed for each individual. Also, blood was drawn for full blood count, blood film for malaria parasites and Trichinella serology. Extensive epidemiological investigations identified a total of 84 returning travelers from 6 separate groups, of which 58 (69%) were subjected to a detailed interview, including the kinds of meat consumed on the island. RESULTS: Twenty-five of these 33 persons (75.7 %) fulfilled clinical or serological case definition criteria for trichinosis. IgG antibody for Trichinella spiralis was detected in 8 out of 32 persons who had the test done (25%). Symptoms were generally mild, with only one patient (3%) requiring steroids for prolonged myositis. CONCLUSION: We suspect this outbreak to be due to trichinosis although the source could not be identified. Extensive epidemiological investigations identified a total of 84 returning travelers from 6 separate groups, of which 58 (69%) were subjected to a detailed interview, including the kinds of meat consumed on the island, but the source of the outbreak could not be identified.
Assuntos
Surtos de Doenças , Viagem/estatística & dados numéricos , Trichinella spiralis/imunologia , Triquinelose/epidemiologia , Animais , Anticorpos Anti-Helmínticos/sangue , Estudos de Coortes , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imunoglobulina G/sangue , Masculino , Singapura/epidemiologia , Triquinelose/prevenção & controleRESUMO
The yeast Saccharomyces cerevisiae transcription factor Ste12p is responsible for activating genes in response to MAP kinase cascades controlling mating and filamentous growth. Ste12p is negatively regulated by two inhibitor proteins, Dig1p (also called Rst1p) and Dig2p (also called Rst2p). The expression of a C-terminal Ste12p fragment (residues 216 to 688) [Ste12p(216-688)] from a GAL promoter causes FUS1 induction in a strain expressing wild-type STE12, suggesting that this region can cause the activation of endogenous Ste12p. Residues 262 to 594 are sufficient to cause STE12-dependent FUS1 induction when overexpressed, and this region of Ste12p was found to bind Dig1p but not Dig2p in yeast extracts. In contrast, recombinant glutathione S-transferase-Dig2p binds to the Ste12p DNA-binding domain (DBD). Expression of DIG2, but not DIG1, from a GAL promoter inhibits transcriptional activation by an Ste12p DBD-VP16 fusion. Furthermore, disruption of dig1, but not dig2, causes elevated transcriptional activation by a LexA-Ste12p(216-688) fusion. Ste12p has multiple regions within the C terminus (flanking residue 474) that can promote multimerization in vitro, and we demonstrate that these interactions can contribute to the activation of endogenous Ste12p by overproduced C-terminal fragments. These results demonstrate that Dig1p and Dig2p do not function by redundant mechanisms but rather inhibit pheromone-responsive transcription through interactions with separate regions of Ste12p.
Assuntos
Proteínas Fúngicas/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Feromônios/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
Intracellular protein transport between the endoplasmic reticulum (ER) and the Golgi apparatus and within the Golgi apparatus is facilitated by COP (coat protein)-coated vesicles. Their existence in plant cells has not yet been demonstrated, although the GTP-binding proteins required for coat formation have been identified. We have generated antisera against glutathione-S-transferase-fusion proteins prepared with cDNAs encoding the Arabidopsis Sec21p and Sec23p homologs (AtSec21p and AtSec23p, respectively). The former is a constituent of the COPI vesicle coatomer, and the latter is part of the Sec23/24p dimeric complex of the COPII vesicle coat. Cauliflower (Brassica oleracea) inflorescence homogenates were probed with these antibodies and demonstrated the presence of AtSec21p and AtSec23p antigens in both the cytosol and membrane fractions of the cell. The membrane-associated forms of both antigens can be solubilized by treatments typical for extrinsic proteins. The amounts of the cytosolic antigens relative to the membrane-bound forms increase after cold treatment, and the two antigens belong to different protein complexes with molecular sizes comparable to the corresponding nonplant coat proteins. Sucrose-density-gradient centrifugation of microsomal cell membranes from cauliflower suggests that, although AtSec23p seems to be preferentially associated with ER membranes, AtSec21p appears to be bound to both the ER and the Golgi membranes. This could be in agreement with the notion that COPII vesicles are formed at the ER, whereas COPI vesicles can be made by both Golgi and ER membranes. Both AtSec21p and AtSec23p antigens were detected on membranes equilibrating at sucrose densities equivalent to those typical for in vitro-induced COP vesicles from animal and yeast systems. Therefore, a further purification of the putative plant COP vesicles was undertaken.
Assuntos
Arabidopsis/metabolismo , Proteínas de Plantas/metabolismo , Citosol/metabolismo , Substâncias Macromoleculares , Proteínas de Membrana/metabolismo , Proteínas de Plantas/imunologia , Proteínas Recombinantes de Fusão/imunologia , Frações Subcelulares/metabolismoRESUMO
Meiotic mutant (2n) gametes formed by first-division restitution without crossover (FDR-NCO) are expected to be superior to FDR with crossover (FDR-CO) because they transmit to the progeny, without disruption by recombination, almost 100% of the parental genotype. FDR-CO transfers approximately 80% of the parental heterozygosity and a large fraction of the epistatic interactions. Another genetic expectation associated with both FDR gametes is their equivalence for the phenotypic expression of traits controlled by genes residing between centromeres and proximal crossover sites. This set of unique cytogenetic features of FDR mutants was employed here as a tool to infer physical location of quantitative trait loci controlling total tuber yield (TTY) in potato. Two assays were conducted to verify the superiority of FDR-NCO over FDR-CO gametes for TTY by using progenies from 4x-2x factorial crosses. Male clones were 2n-pollen producers by either FDR-CO or FDR-NCO mechanisms. Compared with the 4x parents, TTY of the progenies ranged from 41% to 175% (i.e., high-parent heterosis). However, no significant TTY differences were observed between FDR-CO and FDR-NCO families. In addition, the size of variance components of males was smaller than females and near zero. Our results reinforce the hypothesis that genes controlling yielding ability have a predominant physical location between centromeres and proximal chiasmata. Quantitative trait loci in chromosome regions with reduced levels of recombination may provide a partial explanation for the slow progress in increasing TTY through conventional 4x-4x crosses and for the often high degree of heterosis obtained by introgressing genetic diversity via 4x-2x crosses in potato.
RESUMO
CD8 deficiency is a rare primary immunodeficiency caused by the defect of a tyrosine kinase, ZAP-70, which transduces signals from the T cell receptor. We report here a case of CD8 deficiency, having CD4+ T cells with a unique phenotype. The patient's T cells did not respond to anti-CD3 stimulation in vitro, suggesting that they were naive. However, many CD4+ T cells with activated and memory phenotypes, which expressed CD45RO+, HLA-DR+ and CD25+, were present in the peripheral blood, and these cells accumulated in the perivascular area of his infiltrative erythematous skin lesions. The patient's T cells could be activated by a high concentration of phytohaemagglutinin (PHA), indicating the presence of an alternate signalling pathway which bypasses ZAP-70 and activates CD4+ T cells in vivo. The origin and role of activated CD4+ T cells in the pathogenesis involved in the skin lesions are discussed.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Antígenos CD8/fisiologia , Síndromes de Imunodeficiência/sangue , Pele/imunologia , Pele/patologia , Humanos , Sistema Imunitário/imunologia , Memória Imunológica , Lactente , Ativação Linfocitária , Masculino , Fito-Hemaglutininas/farmacologia , Proteínas Tirosina Quinases/deficiência , Proteínas Tirosina Quinases/imunologia , Receptores de Antígenos de Linfócitos T/deficiência , Proteína-Tirosina Quinase ZAP-70RESUMO
Bovine corneal keratan sulphate has been fragmented using the enzyme endo-beta)-galactosidase and 1H-NMR chemical shift data are reported for five newly isolated tetrasaccharides which derived from the repeat region. They have the structures: GlcNAc(beta1-3)Gal(beta1-4)GlcNAc6S(beta1-3)Gal-ol, GlcNAc6S(beta1-3)Gal(beta1-4)GlcNAc(beta1-3)Gal-ol, GlcNAc(beta1-3)Gal6S(beta1-4)GlcNAc6S(beta1-3)Gal-ol, GlcNAc6S(beta1-3)Gal6S(beta1-4)GlcNAc(beta1-3)Gal-ol, and GlcNAc6S(beta1-3)Gal(beta1-4)GlcNAc6S(beta1-3)Gal-ol. Structures for two trisaccharides formed by a peeling reaction are also given. These are GlcNAc(beta1-3)Gal6S(beta1-4)GlcNAc-ol and GlcNAc6S(beta1-3)Gal(beta1-4)GlcNAc6S-ol where GlcNAc6S-ol represents N-acetylglucosaminitol 6-O-sulphate. Characterisation of such structures and their spectral assignments will be of considerable value for the studies of both selectin ligands and undersulphated keratan sulphates such as those occurring on cell surfaces and in brain tissue.
Assuntos
Acetilglucosamina/química , Córnea/química , Glicosídeo Hidrolases , Sulfato de Queratano/metabolismo , Oligossacarídeos/química , beta-Galactosidase/metabolismo , Acetilglucosamina/metabolismo , Amino Açúcares/química , Animais , Bovinos , Glicosaminoglicanos/química , Sulfato de Queratano/química , Espectroscopia de Ressonância Magnética , Polissacarídeos/químicaRESUMO
We have previously shown that prostaglandin E2 (PGE2) and IL-4 inhibit the priming of IFN-gamma-production during the differentiation of naive CD4+ T cells from human cord blood by different signal-transducing mechanisms. To compare and analyse the molecular mechanisms by which PGE2 and IL-4 inhibit the priming of IFN-gamma production, we investigated the effects of PGE2 and IL-4 on the methylation of the IFN-gamma gene during the in vitro differentiation of naive CD4+ T cells. In human naive CD4+ T cells, which produce primarily IL-2 and a little amount of IFN-gamma, the IFN-gamma gene was methylated. After stimulation via TCR, CD4+ T cells produced IFN-gamma and the CpG dinucleotide contained within the TATA proximal regulatory element of the IFN-gamma gene was partially hypomethylated. Both IL-4 and PGE2 inhibited the hypomethylation of this site and the acquisition of IFN-gamma-producing ability. In contrast to the SnaBI site in the TATA proximal regulatory element, the HpalI site in the first intron of the IFN-gamma gene of the CD4+ T cells from cord blood was completely methylated even after stimulation via TCR. 5-azacytidine restored the IFN-gamma-producing ability of these cells treated with IL-4 and PGE2. These findings suggest that, although the signal transduction that inhibits the priming of IFN-gamma-production is different for each reagent, the protection from hypomethylation of the regulatory region of the IFN-gamma gene is involved in the molecular mechanisms by which these reagents inhibit the priming of IFN-gamma-production during the differentiation of human naive CD4+ T cells.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Metilação de DNA/efeitos dos fármacos , Dinoprostona/farmacologia , Interferon gama/biossíntese , Interferon gama/genética , Interleucina-4/farmacologia , Azacitidina/farmacologia , Linfócitos T CD4-Positivos/citologia , Diferenciação Celular , Sangue Fetal/citologia , Humanos , Íntrons , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Sequências Reguladoras de Ácido NucleicoRESUMO
The keratan sulfate-containing proteoglycans were isolated from fourteen pooled human corneas (thirteen from 61- to 86-year-olds, plus one from a 12-year-old). These proteoglycans were subjected to digestion with the enzyme keratanase II, and the released oligosaccharides, which included nonreducing termini and repeat region oligosaccharides but not linkage regions, were reduced with alkaline borohydride and identified on two separate ion-exchange columns. Both of the latter had been calibrated with samples, most of which had been derived from bovine corneal keratan sulfate (Tai, G.-H., Huckerby, T. N., and Nieduszynski, I. A. (1996) J. Biol. Chem. 271, 23535-23546) and all of which had been fully characterized by NMR spectroscopic analysis. The capping structures identified in human corneal keratan sulfates occurred in the relative proportions: NeuAcalpha(2-6)- >NeuAcalpha(2-3)- >GalNAc(S)beta(1-3)-. The other groups of capping structures which had been identified in bovine corneal keratan sulfate, i.e. NeuGcalpha(2-3)-, NeuGcalpha(2-6)-, GlcNAc(S)beta(1-3)- were absent, although the possibility of the presence of some Galalpha(1-3)- structures could not be excluded. In addition, the human sample showed significantly higher levels of alpha(1-3)-fucosylated repeat region structures than did the bovine sample, and it is not clear whether this reflects a species or age dependence as the bovine corneas were from young animals, whereas the human corneas were predominantly from an older group. The charge densities and keratan sulfate chain sizes of the human and bovine keratan sulfate-containing proteoglycans were seen to be similar.
Assuntos
Córnea/metabolismo , Sulfato de Queratano/metabolismo , Acetilglucosaminidase/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Sequência de Carboidratos , Bovinos , Criança , Cromatografia por Troca Iônica , Humanos , Pessoa de Meia-Idade , Dados de Sequência Molecular , Peso MolecularRESUMO
We investigated the effects of prostaglandin E2 and IL-4 on the acquisition of cytokine-producing ability by naive CD4(+) T cells in human umbilical cord blood. The presence of PGE2 or IL-4 at primary stimulation inhibited the production of IFN-gamma at secondary stimulation, and the combination of these stimuli resulted in cooperative effects. During primary stimulation with anti-CD3, the intracellular cAMP level was elevated in PGE2-treated cells but not in IL-4-treated or control cells. The signal provided by PGE2, but not by IL-4, was inhibited with RpcAMP, indicating that it was mediated by cAMP. After differentiation into Th2-like cells, cAMP levels in PGE2- and IL-4-treated cells were not different. Our results suggest that both PGE2 and IL-4 play important roles with distinct mechanisms in inhibiting the priming of IFN-gamma production of naive CD4(+) T cells.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Dinoprostona/farmacologia , Interferon gama/imunologia , Interleucina-4/farmacologia , Transdução de Sinais/imunologia , Linfócitos T CD4-Positivos/citologia , Diferenciação Celular , Humanos , Interferon gama/biossíntese , Ativação Linfocitária , Transdução de Sinais/efeitos dos fármacos , Células Th2/imunologiaRESUMO
Pim-1 is an oncogene-encoded serine/threonine kinase expressed primarily in cells of the hematopoietic and germ line lineages. Previously identified only in mammals, pim-1 cDNA was cloned and sequenced from the African clawed frog Xenopus laevis. The coding region of Xenopus pim-1 encoded a protein of 324 residues, which exhibited 64% amino acid identity with the full-length human cognate. Xenopus Pim-1 was expressed in bacteria as a glutathione S-transferase (GST) fusion protein and in COS cells. Phosphoamino acid analysis revealed that recombinant Pim-1 autophosphorylated on serine and threonine and to a more limited extent on tyrosine. Electrospray ionization mass spectroscopy was undertaken to locate these phosphorylation sites, and the primary autophosphorylation site of GST-Pim-1 was identified as Ser-190 with Thr-205 and Ser-4 being minor sites. Ser-190, which immediately follows the high conserved Asp-Phe-Gly motif in catalytic subdomain VII, is also featured in more than 20 other protein kinases. To evaluate the importance of the Ser-190 site on the phosphotransferase activity of Pim-1, Ser-190 was mutated to either alanine or glutamic acid, and the constructs were expressed in bacteria as GST fusion proteins and in COS cells. These mutants confirmed that Ser-190 is a major autophosphorylation site of Pim-1 and indicated that phosphorylation of Pim-1 on the Ser-190 residue may serve to activate this kinase.
Assuntos
Fosfotransferases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proto-Oncogenes , Sequência de Aminoácidos , Animais , Sequência de Bases , Células COS , Clonagem Molecular , Primers do DNA , Feminino , Glutationa Transferase , Humanos , Cinética , Mamíferos , Camundongos , Dados de Sequência Molecular , Oócitos/fisiologia , Fosforilação , Reação em Cadeia da Polimerase , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas c-pim-1 , Ratos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Xenopus laevisRESUMO
The 78-kDa protein kinase Mekk1 plays an important role in the stress response pathway that involves the activation of downstream kinases Sek1 and stress-activated protein kinase/c-Jun NH2-terminal kinase. Conserved serine and threonine residues located between the kinase subdomains VII and VIII of many protein kinases are phosphorylated for maximal kinase activation. Two threonine residues within this region in Mekk1 at positions 560 and 572, but not the serine at 557, were shown to be essential for catalytic activity in this study. When these threonine residues were replaced with alanine, there was a significant loss in phosphotransferase activity toward the primary substrate, Sek1, and a large decrease in autophosphorylation activity. Site-directed mutagenesis demonstrated that these threonine residues cannot be replaced with either serine or glutamic acid for preservation of phosphotransferase activity. Further examination of the Mekk1 mutants isolated from 32P-labeled transfected COS cells showed that Thr-560 and Thr-572 were indeed phosphorylated after two-dimensional tryptic-chymotryptic phosphopeptide analysis. Additional determinants in the NH2-terminal domain of Mekk1 also play a role in the regulation of Mekk1 activity. Although Pak3 and PKC can activate Mekk1 in vivo, this interaction is indirect and independent, since there was no direct phosphorylation of Mekk1 by Pak3 or PKC or of Pak3 by PKC, respectively.
Assuntos
MAP Quinase Quinase Quinase 1 , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Treonina/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células COS , Catálise , Ativação Enzimática , Camundongos , Dados de Sequência Molecular , Fosforilação , Proteína Quinase C/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Acetato de Tetradecanoilforbol/farmacologia , Quinases Ativadas por p21RESUMO
A human eosinophilic leukemia cell line, EoL-1, stopped proliferating at the G1 phase, differentiated into eosinophilic granule-containing cells, and died by apoptosis when stimulated with dibutyryl cyclic AMP (dbcAMP). To clarify the effects of dbcAMP, the effects of butyrate and cAMP-increasing reagents, prostaglandin E2 (PGE2) and forskolin, on EoL-1 cellular differentiation and apoptosis were examined and compared. PGE2 and forskolin but not butyrate induced differentiation to eosinophilic granule-containing cells, suggesting that cAMP played a primary role in eosinophilic differentiation of EoL-1 cells. PGE2, forskolin and butyrate, when used alone, did not induce apoptosis of EoL-1 cells significantly at the concentrations used, but sequential stimulation of EoL-1 cells with the cAMP-increasing reagents and butyrate showed that butyrate induced further maturation and apoptosis of cAMP-induced eosinophilic granule-containing cells. These results showed that cAMP and butyrate have different effects on eosinophilic differentiation and apoptosis of EoL-1 cells. The cAMP-increasing reagents and butyrate also showed different effects on expression of members of the bcl-2 family; PGE2 decreased bcl-2 and bax levels, whereas butyrate increased the bcl-2 level. PGE2 or PGE2+butyrate, but not butyrate alone, induced bcl-XS expression. EoL-1 cells constitutively expressed Fas and anti-Fas antibody induced EoL-1 cell death, but the Fas/Fas ligand system was not involved in dbcAMP-induced EoL-1 cell apoptosis. The EoL-1 cell line is thus a useful model in which to examine differentiation and apoptosis of eosinophilic leukemia cells.