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1.
Clin Exp Immunol ; 176(1): 112-9, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24028181

RESUMO

The average age of patients receiving renal transplantation is increasing as programmes have been established which support the donation of organs from elderly donors to older recipients. Little is known about the effect of immunosuppressive therapy on the immune system of older patients. In this study, T cell function and the composition of the T cell repertoire were analysed in immunosuppressed renal transplant recipients of different age and cytomegalovirus (CMV) status in comparison to age- and CMV-matched controls. Independent of age and CMV status, the production of interleukin (IL)-2 and interferon (IFN)-γ by T cells was decreased in the patient groups and autologous serum from patients was capable of inhibiting the proliferation of CD3⁺ T cells. CXCR5 expression on T cells was increased in patients versus controls reflecting reduced endogenous IL-2 signalling under immunosuppressive therapy. In CMV-seronegative patients kidney transplantation and immunosuppressive therapy did not induce changes in the CD8⁺ T cell pool, but there was a moderate increase in CD4⁺CD28⁻ effector T cells when compared to age-matched controls. In contrast, latent CMV infection triggered a shift from early to late differentiated CD4⁺ and CD8⁺ T cells in patients and controls. This shift was most pronounced in elderly transplant patients under immunosuppressive therapy. In conclusion, our results demonstrate that immunosuppressive therapy following kidney transplantation is effective in patients older than 65 years. Latent CMV infection, however, accelerates age-related changes in the T cell repertoire in elderly people under immunosuppressive therapy. These patients should therefore be monitored with special care.


Assuntos
Hospedeiro Imunocomprometido/imunologia , Imunossupressores/imunologia , Transplante de Rim/métodos , Linfócitos T/imunologia , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Proliferação de Células/efeitos dos fármacos , Citomegalovirus/imunologia , Infecções por Citomegalovirus/imunologia , Infecções por Citomegalovirus/virologia , Feminino , Citometria de Fluxo , Humanos , Hospedeiro Imunocomprometido/efeitos dos fármacos , Imunossupressores/uso terapêutico , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucina-2/imunologia , Interleucina-2/metabolismo , Masculino , Pessoa de Meia-Idade , Receptores CXCR5/imunologia , Receptores CXCR5/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Fatores de Tempo
2.
Science ; 275(5296): 67-70, 1997 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-8974393

RESUMO

The controversial question of how thiamine diphosphate, the biologically active form of vitamin B1, is activated in different enzymes has been addressed. Activation of the coenzyme was studied by measuring thermodynamics and kinetics of deprotonation at the carbon in the 2-position (C2) of thiamine diphosphate in the enzymes pyruvate decarboxylase and transketolase by use of nuclear magnetic resonance spectroscopy, proton/deuterium exchange, coenzyme analogs, and site-specific mutant enzymes. Interaction of a glutamate with the nitrogen in the 1'-position in the pyrimidine ring activated the 4'-amino group to act as an efficient proton acceptor for the C2 proton. The protein component accelerated the deprotonation of the C2 atom by several orders of magnitude, beyond the rate of the overall enzyme reaction. Therefore, the earlier proposed concerted mechanism or stabilization of a C2 carbanion can be excluded.


Assuntos
Piruvato Descarboxilase/metabolismo , Tiamina Pirofosfato/metabolismo , Transcetolase/metabolismo , Regulação Alostérica , Sítios de Ligação , Catálise , Deutério/metabolismo , Ativação Enzimática , Ácido Glutâmico/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Espectroscopia de Ressonância Magnética , Mutagênese Sítio-Dirigida , Prótons , Piruvato Descarboxilase/química , Piruvatos/metabolismo , Termodinâmica , Tiamina Pirofosfato/química , Transcetolase/química
3.
Biochim Biophys Acta ; 1385(2): 221-8, 1998 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-9655909

RESUMO

Activation of the coenzyme ThDP was studied by measuring the kinetics of deprotonation at the C2 carbon of thiamin diphosphate in the enzymes pyruvate decarboxylase, transketolase, pyruvate dehydrogenase complex, pyruvate oxidase, in site-specific mutant enzymes and in enzyme complexes containing coenzyme analogues by proton/deuterium exchange detected by 1H-NMR spectroscopy. The respective deprotonation rate constant is above the catalytic constant in all enzymes investigated. The fast deprotonation requires the presence of an activator in pyruvate decarboxylase from yeast, showing the allosteric regulation of this enzyme to be accomplished by an increase in the C2-H dissociation rate of the enzyme-bound thiamin diphosphate. The data of the thiamin diphosphate analogues and of the mutant enzymes show the N1' atom and the 4'-NH2 group to be essential for the activation of the coenzyme and a conserved glutamate involved in the proton abstraction mechanism of the enzyme-bound thiamin diphosphate.


Assuntos
Piruvato Descarboxilase/metabolismo , Complexo Piruvato Desidrogenase/metabolismo , Piruvato Oxidase/metabolismo , Tiamina Pirofosfato/metabolismo , Transcetolase/metabolismo , Regulação Alostérica , Escherichia coli/enzimologia , Cinética , Lactobacillus/enzimologia , Ressonância Magnética Nuclear Biomolecular , Leveduras/enzimologia
4.
Orthop Traumatol Surg Res ; 101(8): 947-51, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26589193

RESUMO

INTRODUCTION: Due to poor results and failure encountered in osteosynthesis of peri-articular fracture of the knee, arthroplasty may be suggested to osteopenic elderly subjects. All osteosynthesis techniques entail loss of independence and are associated with elevated mortality. No studies definitively establish better management of such fractures. HYPOTHESIS: Total arthroplasty provides better autonomy after peri-articular fracture of the knee. MATERIAL AND METHOD: Seventy-nine patients aged over 65years were operated on for peri-articular fracture of the knee between April 2008 and March 2013. In 21 cases, treated by a single surgeon, total knee arthroplasty was performed in view of osteopenia or osteoarthritis. Mean age was 79years (range, 68-96years). There were 10 distal femoral and 11 proximal tibial fractures. Mean follow-up was 31months (range, 9-68months). Cases of pathologic fracture, failed osteosynthesis and non-operative management were excluded. All patients showed severe osteopenia on radiology and half already had advanced osteoarthritis. RESULTS: One-year mortality was 14%. At last follow-up, the revision rate was 9.5%. Fifteen patients were followed up. Mean Parker score fell from 7.2 (range, 2-9) preoperatively to 4.6 (range, 0-9) at last follow-up, indicating loss of independence. At follow-up, mean IKS score was 116.6 (range, 0-192) with mean IKS knee score of 78.4 (range, 0-100) and IKS function score of 38.2 (range, 0-100). Mean Oxford score was 36/60 (range, 18-53). Global IKS and IKS function scores were significantly better in case of ASA-2 than ASA-3 (P<0.05). There was no difference between femoral and tibial fractures in terms of IKS or Oxford score or loss of independence. DISCUSSION: Total knee arthroplasty can be considered for peri-articular fracture of the knee in osteopenic geriatric patients. Although surgical revision was less frequent than after osteosynthesis and resumption of weight-bearing was immediate, autonomy was still impaired. Mortality was comparable to other reports. LEVEL OF EVIDENCE: IV, retrospective study.


Assuntos
Artroplastia do Joelho , Fraturas do Fêmur/cirurgia , Vida Independente , Fraturas Intra-Articulares/cirurgia , Articulação do Joelho/cirurgia , Fraturas da Tíbia/cirurgia , Idoso , Idoso de 80 Anos ou mais , Artroplastia do Joelho/métodos , Doenças Ósseas Metabólicas/complicações , Feminino , Fraturas do Fêmur/complicações , Fixação Interna de Fraturas/métodos , Humanos , Articulação do Joelho/fisiopatologia , Masculino , Osteoartrite do Joelho/complicações , Osteoartrite do Joelho/cirurgia , Reoperação , Estudos Retrospectivos , Fraturas da Tíbia/complicações , Suporte de Carga
5.
Protein Sci ; 7(10): 2164-74, 1998 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-9792104

RESUMO

The RNase H domain from HIV-1 (HIV RNase H) encodes an essential retroviral activity. Refolding of the isolated HIV RNase H domain shows a kinetic intermediate detectable by stopped-flow far UV circular dichroism and pulse-labeling H/D exchange. In this intermediate, strands 1, 4, and 5 as well as helices A and D appear to be structured. Compared to its homolog from Escherichia coli, the rate limiting step in refolding of HIV RNase H appears closer to the native state. We have modeled this kinetic intermediate using a C-terminal deletion fragment lacking helix E. Like the kinetic intermediate, this variant folds rapidly and shows a decrease in stability. We propose that inhibition of the docking of helix E to this folding intermediate may present a novel strategy for anti HIV-1 therapy.


Assuntos
HIV-1/enzimologia , Dobramento de Proteína , Ribonuclease H/química , Dicroísmo Circular , Deutério/metabolismo , Desenho de Fármacos , Inibidores Enzimáticos/química , Estabilidade Enzimática/efeitos dos fármacos , Guanidina/farmacologia , Hidrogênio/metabolismo , Cinética , Modelos Moleculares , Mutação/genética , Desnaturação Proteica , Estrutura Secundária de Proteína , Proteínas Virais/química
6.
Protein Sci ; 2(11): 1862-8, 1993 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-8268797

RESUMO

A core-glycosylated form of the dimeric enzyme invertase has been isolated from secretion mutants of Saccharomyces cerevisiae blocked in transport to the Golgi apparatus. This glycosylation variant corresponds to the form that folds and associates during biosynthesis of the protein in vivo. In the present work, its largely homogeneous subunit size and well-defined quaternary structure were utilized to characterize the folding and association pathway of this highly glycosylated protein in comparison with the nonglycosylated cytoplasmic and the high-mannose-glycosylated periplasmic forms of the same enzyme encoded by the suc2 gene. Renaturation of core-glycosylated invertase upon dilution from guanidinium-chloride solutions follows a unibimolecular reaction scheme with consecutive first-order subunit folding and second-order association reactions. The rate constant of the rate-limiting step of subunit folding, as detected by fluorescence increase, is k1 = 1.6 +/- 0.4 x 10(-3) s-1 at 20 degrees C; it is characterized by an activation enthalpy of delta H++ = 65 kJ/mol. The reaction is not catalyzed by peptidyl-prolyl cis-trans isomerase of the cyclophilin type. Reactivation of the enzyme depends on protein concentration and coincides with subunit association, as monitored by size-exclusion high-pressure liquid chromatography. The association rate constant, estimated by numerical simulation of reactivation kinetics, increases from 5 x 10(3) M-1 s-1 to 7 x 10(4) M-1 s-1 between 5 and 30 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)


Assuntos
Glicoproteínas/química , Glicosídeo Hidrolases/química , Dobramento de Proteína , Saccharomyces cerevisiae/enzimologia , Reativadores Enzimáticos , Glicosilação , Temperatura Alta , Cinética , Modelos Químicos , Conformação Proteica , Desnaturação Proteica , Espectrometria de Fluorescência , beta-Frutofuranosidase
7.
Protein Sci ; 1(1): 120-31, 1992 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-1304875

RESUMO

The role of carbohydrate chains for the structure, function, stability, and folding of glycoproteins has been investigated using invertase as a model. The protein is encoded by several different genes, and its carbohydrate moiety is heterogeneous. Both properties complicate physicochemical comparisons. Here we used the temperature-sensitive sec18 secretion mutant of yeast with a single invertase gene (SUC2). This mutant produces the carbohydrate-free internal invertase, the core-glycosylated form, and, at the permissive temperature, the fully glycosylated external enzyme, all with identical protein moieties. The core-glycosylated enzyme resembles the nascent glycoprotein chain that folds in the endoplasmic reticulum. Therefore, it may be considered a model for the in vivo folding of glycoproteins. In addition, because of its uniform glycosylation, it can be used to investigate the state of association of native invertase. Glycosylation is found to stabilize the protein with respect to thermal denaturation and chaotropic solvent components; the stabilizing effect does not differ for the external and the core-glycosylated forms. Unlike the internal enzyme, the glycosylated forms are protected from aggregation. Native internal invertase is a dimer (115 kDa) whereas the core-glycosylated enzyme is a mixture of dimers, tetramers, and octamers. This implies that core-glycosylation is necessary for oligomerization to tetramers and octamers. Dimerization is required and sufficient to generate enzymatic activity; further association does not alter the specific activity of core-glycosylated invertase, suggesting that the active sites of invertase are not affected by the association of the dimeric units. Reconstitution of the glycosylated and nonglycosylated forms of the enzyme after preceding guanidine denaturation depends on protein concentration. The maximum yield (approximately 80%) is obtained at pH 6-8 and protein concentrations < or = 4 micrograms/mL for the nonglycosylated and < or = 40 for the glycosylated forms of the enzyme. The lower stability of the internal enzyme is reflected by a narrower pH range of reactivation and enhanced aggregation. As indicated by the sigmoidal reactivation kinetics at low protein concentration both folding and association are rate-determining.


Assuntos
Glicoproteínas/química , Glicosídeo Hidrolases/química , Saccharomyces cerevisiae/enzimologia , Reativadores Enzimáticos/farmacologia , Estabilidade Enzimática , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Glicosilação , Guanidina , Guanidinas/farmacologia , Temperatura Alta/efeitos adversos , Concentração de Íons de Hidrogênio , Conformação Proteica , Desnaturação Proteica , Dobramento de Proteína , Processamento de Proteína Pós-Traducional , Saccharomyces cerevisiae/genética , Ureia/farmacologia , beta-Frutofuranosidase
8.
FEBS Lett ; 305(3): 203-5, 1992 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-1363729

RESUMO

During refolding and reassociation of chemically denatured non-glycosylated invertase from Saccharomyces cerevisiae, aggregation competes with correct folding, leading to low yields of reactivation (Kern et al. (1992) Protein Sci. 1, 120-131). In the presence of the chaperone GroEL, refolding is completely arrested. This suggests the formation of a stable complex between GroEL and non-native non-glycosylated invertase. Addition of MgATP results in a slow release of active invertase from the chaperone complex. When GroEL/ES and MgATP are present during refolding, the final reactivation yield increases from 14% to 36%. In contrast, refolding of the core-glycosylated and the high-mannose glycosylated forms of invertase is not arrested by GroEL. Only a short lag phase at the beginning of reactivation and a slightly increased reactivation yield (64% to 86% for core-glycosylated and 62% to 76% for external invertase) indicate a weak interaction of the glycosylated forms with the chaperone.


Assuntos
Proteínas de Bactérias/metabolismo , Glicosídeo Hidrolases/metabolismo , Proteínas de Choque Térmico/metabolismo , Chaperonina 60 , Glicosilação , Dobramento de Proteína , beta-Frutofuranosidase
9.
FEBS Lett ; 348(2): 145-8, 1994 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-7913447

RESUMO

The folding of proteins can be assisted by two unrelated groups of helper molecules. Chaperones suppress non-productive side reactions by stoichiometric binding to folding intermediates, and folding enzymes catalyze slow rate-limiting steps of folding. We reinvestigated, whether peptidyl-prolyl-cis/trans-isomerases of the cyclophilin type act simultaneously as chaperones and as folding catalysts in the reactivation of human carbonic anhydrase II, as reported recently [Freskgård, P.-O. et al. (1992) Science 258, 466-468; Rinfret, A. et al. (1994) Biochemistry 33, 1668-1673]. No increase in the yield of native carbonic anhydrase-II could be detected in the presence of three different prolyl isomerases, when reactivation was followed by a sensitive assay for an extended time of 4 h. We conclude that the role of prolyl isomerases in the refolding of carbonic anhydrase can be explained solely by their isomerase activity. There is no need to invoke simultaneous functions as chaperones for these folding catalysts.


Assuntos
Isomerases de Aminoácido/metabolismo , Proteínas de Transporte/metabolismo , Proteínas/metabolismo , Sequência de Aminoácidos , Anidrases Carbônicas/metabolismo , Chaperoninas , Ativação Enzimática , Humanos , Cinética , Dados de Sequência Molecular , Peptidilprolil Isomerase , Dobramento de Proteína
10.
Wien Klin Wochenschr ; 98(14): 457-61, 1986 Jul 18.
Artigo em Alemão | MEDLINE | ID: mdl-3751066

RESUMO

An outbreak of hepatitis A was observed in a shelter for the homeless in Vienna with about 200 inhabitants. Twenty-two cases occurred within a period of 6 months. The outbreak could not be brought under control by measures of general hygiene. However, after the administration of hepatitis A immunoglobulin (120 IU/ml), at a dosage of 0.05 ml/kg body weight, to 102 of the 105 seronegative inhabitants and members of staff, no further clinical cases of hepatitis A were reported from this group. Nevertheless, 8 of these 102 "protected" persons showed signs of subclinical infection at subsequent follow up. Apart from these, 2 further cases of hepatitis A occurred among the non-immunised children at risk, whose parents had refused permission for serological investigation or immunoglobulin administration.


Assuntos
Surtos de Doenças , Hepatite A/transmissão , Pessoas Mal Alojadas , Adolescente , Adulto , Idoso , Áustria , Criança , Pré-Escolar , Surtos de Doenças/prevenção & controle , Desinfecção/métodos , Feminino , Hepatite A/prevenção & controle , Humanos , Imunização Passiva , Lactente , Masculino , Pessoa de Meia-Idade , Habitação Popular
11.
Orthop Traumatol Surg Res ; 99(4): 391-8, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23510631

RESUMO

BACKGROUND: Knee arthrodesis is used to treat patients with failed infected total knee arthroplasty (TKA). Among fixation methods, intramedullary nailing increases the chances of bone union but may carry a risk of infection around the nail. This risk is not well understood, because available case-series studies were not confined to patients with knee infection. HYPOTHESIS: Infection recurrence rates after knee arthrodesis with intramedullary nailing used to treat failed infected TKA are similar to those seen with other fixation methods. METHODS: We retrospectively reviewed 31 cases of knee arthrodesis with fixation by a modular intramedullary nail performed at a subspecialized center treating complex osteoarticular infections (CRIOAC). The antibiotic regimen was determined based on multidisciplinary discussions and microbiological studies of preoperative and intraoperative specimens. Mean follow-up was 50 ± 22 months (range, 28-90 months). Arthrodesis was performed in one stage (n=6) or two stages (n=25). Success was defined as presence, after a postoperative follow-up of at least 24 months, based on the following criteria: normal erythrocyte sedimentation rate and/or C-reactive protein, no wound inflammation or sinus tract, no revision surgery, and no antibiotic treatment. Bone union was not a criterion for a successful arthrodesis procedure. RESULTS: Removal of the fixation material was required in three patients and long-term palliative antibiotic therapy in three patients (fixation material in place with repeated positive specimens) for a total of six failures due to infection (6/31, 19.4%). None of the patients experienced mechanical failure (no breakage of the material and no fixation failure of the nails designed to allow osteointegration). The mean leg length discrepancy was 10 ± 10 mm (range, 5-34 mm) and the mean Oxford score was 41 ± 11 (range, 23-58). The 50-month rate of arthrodesis survival to revision surgery for nail removal was 77.8 ± 4% and the 50-month rate of arthrodesis survival without revision surgery for persistent infection was 74.6 ± 4.2%. DISCUSSION: The infection recurrence rate was higher than with other fixation methods but remained acceptable (19.4%). Use of a modular intramedullary nail prevented major leg-length discrepancies, which are often poorly accepted by the patients, and allowed immediate weight bearing despite the often severe bone loss. LEVEL OF EVIDENCE: Level IV, retrospective cohort study.


Assuntos
Artrite Infecciosa/cirurgia , Artrodese/instrumentação , Artroplastia do Joelho/efeitos adversos , Pinos Ortopédicos , Infecções Relacionadas à Prótese/cirurgia , Infecções Estafilocócicas/cirurgia , Idoso , Idoso de 80 Anos ou mais , Antibacterianos/uso terapêutico , Artrite Infecciosa/tratamento farmacológico , Artrite Infecciosa/microbiologia , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Desenho de Prótese , Infecções Relacionadas à Prótese/tratamento farmacológico , Infecções Relacionadas à Prótese/microbiologia , Reoperação , Estudos Retrospectivos , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/isolamento & purificação , Fatores de Tempo , Resultado do Tratamento
12.
Orthop Traumatol Surg Res ; 99(5): 549-53, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23890966

RESUMO

INTRODUCTION: Dislocation following total hip arthroplasty (THA) may require surgical revision, and is one of the most frequent causes for revision in national registers. The goals of this study were to determine the characteristics of revision THA for dislocation and identify the typical features of hips revised due to dislocation. MATERIALS AND METHODS: A prospective multicenter study (30 centers) was performed in first revision THA performed between January 1, 2010 and December 31, 2011 (multiple revisions were excluded). RESULTS: Two hundred nineteen (10.4%) of all first revisions (2153 cases in 2107 patients) were for dislocation, which was the fifth cause of revision. There were 135 men and 84 women, mean age 65.9 years old (24.3-92.4) at primary THA and 72.9 years old (31.9-98.8) at revision. Revision surgery was performed a mean 7.1 years (± 7.1) after primary THA. The predictive risk factors for dislocation were: a 22.2mm diameter femoral head (risk × 2.4), a posterolateral approach (risk × 1.7), older age (risk × 1.1), an elevated rim liner for primary THA (risk × 6.6). The use of a dual mobility cup did not influence the rate of revision for dislocation (8.8%) compared to the use of a flat rim liner (9.1%). DISCUSSION: The 10.4% rate of revision of THA for dislocation seems markedly lower than the results in the literature both for frequency and ranking. The use of elevated rim or constrained liners designed to decrease the risk of dislocation does not improve results compared to standard liners. LEVEL OF EVIDENCE: Level IV, prospective prognostic study without a control group.


Assuntos
Artroplastia de Quadril/efeitos adversos , Articulação do Quadril , Prótese de Quadril/efeitos adversos , Luxações Articulares/epidemiologia , Luxações Articulares/cirurgia , Adulto , Distribuição por Idade , Idoso , Idoso de 80 Anos ou mais , Artroplastia de Quadril/métodos , Distribuição de Qui-Quadrado , Estudos de Coortes , Feminino , Seguimentos , França , Humanos , Incidência , Luxações Articulares/etiologia , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Estudos Prospectivos , Falha de Prótese , Amplitude de Movimento Articular/fisiologia , Reoperação/estatística & dados numéricos , Medição de Risco , Distribuição por Sexo , Estatísticas não Paramétricas , Resultado do Tratamento
13.
Arztl Jugendkd ; 71(1): 13-20, 1980 Feb.
Artigo em Alemão | MEDLINE | ID: mdl-7395642

RESUMO

PIP: The author examine the relationship between the age at menarche of 384 mothers and their daughters in Gorlitz, the German Democratic Republic (SUMMARY IN ENG)^ieng


Assuntos
Menarca , Adolescente , Adulto , Fatores Etários , Feminino , Humanos , Mães , Estações do Ano , Estatística como Assunto
14.
J Exp Biol ; 205(Pt 18): 2943-54, 2002 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-12177159

RESUMO

Cell suspensions of rainbow trout Oncorhynchus mykiss pseudobranch, prepared by Ca(2+) depletion and mechanical maceration, contained a distinct population of cells that always kept their relatively cuboidal shape and did not round up in suspension or proliferate after adhering to the surface of cell culture dishes. Phasecontrast microscopy revealed an extensive system of basal membrane invaginations, and Na(+)-K(+)-ATPase- and anionexchanger-like immunoreactivity could be localized in cell membranes. The cells were characterized by a high mitochondrial density. Using specific antibodies, V-ATPase subunit B was localized in the plasma membrane. Using a cytosensor microphysiometer, the rate of acid secretion of these cells was measured and compared with the activity of a gill cell preparation. Incubation of pseudobranch cells with bafilomycin A1 (10(-6) mol l(-1)), a specific inhibitor of V-ATPase, reduced the rate of acid secretion by about 10% under control conditions, while no effect of bafilomycin on the rate of acid secretion of gill cells was observed. Application of amiloride (5 x 10(-5) mol l(-1)) reduced the rate of acid secretion in cells of both organs, pseudobranch and gills. Incubation of pseudobranch cells with DIDS (10(-3) mol l(-1)) resulted in a minor increase in the rate of proton secretion, but in cells prepared from the gills of rainbow trout acid secretion was reduced by about 30-40%. It is concluded that pseudobranch cells are equipped with various pathways to secrete protons, and that the anion exchange activity especially of pseudobranch cells appears to be different from that in gills.


Assuntos
Concentração de Íons de Hidrogênio , Macrolídeos , Oncorhynchus mykiss/fisiologia , ATPase Trocadora de Sódio-Potássio/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismo , Animais , Antibacterianos/farmacologia , Membrana Celular/enzimologia , Fenômenos Fisiológicos Celulares , Células/citologia , Células/efeitos dos fármacos , Células Cultivadas , Brânquias/fisiologia , Imuno-Histoquímica , Queratinas/análise , Mitocôndrias/metabolismo , Subunidades Proteicas/análise
15.
J Biol Chem ; 270(2): 740-5, 1995 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-7822304

RESUMO

The kinetics of unfolding and refolding of human carbonic anhydrase II (HCAII) and its catalysis by the peptidyl-prolyl-cis/trans-isomerase cyclophilin were investigated. HCAII contains 15 trans- and 2 cis-prolyl peptide bonds, and, when long-term denatured, virtually all unfolded molecules contain non-native prolyl isomers. In unfolding these molecules (Us) are produced slowly in a biphasic process reflecting the isomerization of several trans-prolines and of one cis-proline. In refolding, the rapid formation of an intermediate of the molten globule type is followed by several slow prolyl isomerizations, which determine the rate of reactivation. By a short 10-s incubation in 5.0 M guanidinium chloride at 2 degrees C, unfolded HCAII species with all prolines still in the native conformation (Uf) could be produced. Surprisingly, only a fraction of Uf refolds rapidly, but the other molecules refold slowly. Evidently, some prolyl peptide bonds isomerize early in refolding, at the stage of the molten globule and as a consequence, molecules with incorrect prolyl isomers are formed in competition with the productive folding of Uf. This fraction of slow-folding molecules is strongly increased when cyclophilin is present, because it accelerates the formation of non-native prolyl isomers as long as the molecules remain in the molten globule state. Later cyclophilin catalyzes the isomerization of these prolyl peptide bonds toward the native state, which are stabilized in their conformation by further folding to the native state. This catalysis is very efficient, because only prolines that are accessible in the molten globule are involved in this sequence of isomerization and reisomerization.


Assuntos
Isomerases de Aminoácido/metabolismo , Anidrases Carbônicas/metabolismo , Proteínas de Transporte/metabolismo , Dobramento de Proteína , Catálise , Ativação Enzimática , Humanos , Cinética , Peptidilprolil Isomerase , Desnaturação Proteica
16.
J Bacteriol ; 170(12): 5830-6, 1988 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-3056918

RESUMO

The bacteriophage T4 dexA gene product is required during infection of Escherichia coli strains carrying a mutation in the optA gene. We purified the DexA protein from cells which overproduced the protein. The protein was assayed for nuclease activity on synthetic di- and oligonucleotide substrates of known sequence and secondary structure. Sequence and structure significantly affected nuclease activity. The properties of the enzyme may explain the requirement for the DexA protein during infection of optA mutant hosts.


Assuntos
Escherichia coli/genética , Exodesoxirribonucleases/metabolismo , Fagos T/genética , Proteínas Virais/genética , Sequência de Bases , Escherichia coli/enzimologia , Exodesoxirribonuclease V , Genes , Genes Virais , Cinética , Dados de Sequência Molecular , Mutação , Sondas de Oligonucleotídeos , Fagos T/enzimologia , Proteínas Virais/isolamento & purificação , Proteínas Virais/metabolismo
17.
J Manipulative Physiol Ther ; 17(9): 596-600, 1994.
Artigo em Inglês | MEDLINE | ID: mdl-7884329

RESUMO

OBJECTIVE: To evaluate chiropractic management of primary nocturnal enuresis in children. DESIGN: A controlled clinical trial for 10 wk preceded by and followed by a 2-wk nontreatment period. SETTING: Chiropractic clinic of the Palmer Institute of Graduate Studies and Research. PARTICIPANTS: Forty-six nocturnal enuretic children (31 treatment and 15 control group), from a group of 57 children initially included in the study, participated in the trial. INTERVENTION: High velocity, short lever adjustments of the spine consistent with the Palmer Package Techniques; or a sham adjustment using an Activator at a nontension setting administered to the examiner's underlying contact point. Two 5th-year chiropractic students under the supervision of two clinic faculty performed the adjustments. MAIN OUTCOME MEASURES: Frequency of wet nights. RESULTS: The post-treatment mean wet night frequency of 7.6 nights/2 wk for the treatment group was significantly less than its baseline mean wet night frequency of 9.1 nights/2 wk (p = 0.05). For the control group, there was practically no change (12.1 to 12.2 nights/2 wk) in the mean wet night frequency from the baseline to the post-treatment. The mean pre- to post-treatment change in the wet night frequency for the treatment group compared with the control group did not reach statistical significance (p = 0.067). Twenty-five percent of the treatment-group children had 50% or more reduction in the wet night frequency from baseline to post-treatment while none among the control group had such reduction. CONCLUSIONS: Results of the present study strongly suggest the effectiveness of chiropractic treatment for primary nocturnal enuresis. A larger study of longer duration with a 6-month follow-up is therefore warranted.


Assuntos
Quiroprática/métodos , Enurese/terapia , Adolescente , Criança , Pré-Escolar , Enurese/psicologia , Feminino , Humanos , Masculino , Estresse Psicológico
18.
Rev Fr Gynecol Obstet ; 81(2): 57-60, 1986 Feb.
Artigo em Francês | MEDLINE | ID: mdl-3008297

RESUMO

The authors offer a diagnostic approach in cases of mammillary discharge and present a study of 38 cases of spontaneous discharge. The diagnosis is based on clinical findings combined with supplementary examinations, beginning with a cytological examination of the discharges, either bilateral or, in most cases, unilateral. The supplementary investigations are standard: mammography, galactography, with more and more frequent recourse to ultrasonography. If warranted on sufficient grounds, biopsy by sectorectomy is carried out to provide a histological diagnosis. The observed results show epithelial vegetation with (38%) and without (37.5%) atypical cells, the histological interpretation of which is: benign papilloma: 50 percent, diffuse papillomatosis: 17 percent, galactophoric carcinoma: 1 case and galactophoric ectasias: 22 percent. This study is compared to the results of Mouriquand et al [10] on induced discharges, where the frequency of papilloma and papillomatosis is slightly inferior by about 5 percent.


Assuntos
Doenças Mamárias/diagnóstico , Exsudatos e Transudatos/análise , Biópsia , Mama/patologia , Doenças Mamárias/metabolismo , Neoplasias da Mama/diagnóstico , Carcinoma Intraductal não Infiltrante/diagnóstico , Exsudatos e Transudatos/citologia , Feminino , Humanos , Mamografia , Papiloma/diagnóstico , Ultrassonografia
19.
J Med Virol ; 19(2): 123-34, 1986 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-3522805

RESUMO

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of respiratory syncytial virus (RSV) antigens in nasopharyngeal secretions (NPS) from children with acute respiratory disease. Antisera against RSV nucleocapsids were used as immunoreagents for this test system. The results obtained by RSV antigen ELISA were compared to those of indirect immunofluorescence (IF) and tissue culture virus isolation (TC). Of the 404 NPS obtained, 278 were tested in parallel by ELISA and IF and 205 by ELISA and TC, and 89 were screened in parallel by all three methods. The sensitivity of ELISA in relation to IF was 86.7%, the specificity 95.7%. Sensitivity and specificity obtained by ELISA were 89.9% and 94.4%, respectively, compared to TC. False-negative results were obtained with all three test systems used.


Assuntos
Antígenos Virais/análise , Nasofaringe/microbiologia , Vírus Sinciciais Respiratórios/análise , Doenças Respiratórias/microbiologia , Adolescente , Criança , Pré-Escolar , Relação Dose-Resposta Imunológica , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Humanos , Lactente , Recém-Nascido , Nasofaringe/imunologia , Vírus Sinciciais Respiratórios/imunologia , Vírus Sinciciais Respiratórios/isolamento & purificação , Doenças Respiratórias/imunologia
20.
Biochemistry ; 34(41): 13594-602, 1995 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-7577948

RESUMO

To investigate the kinetics of the prolyl peptide bond cis/trans isomerization of N-succinyl-Ala-Phe-Pro-Phe-(4)-nitroanilide catalyzed by peptidyl prolyl cis/trans isomerases (PPIases), one-dimensional dynamic 1H NMR spectroscopy was employed. To this end line shape analyses of proton signals were performed at various concentrations of both cytosolic porcine kidney cyclophilin (Cyp18) and peptide substrate. Catalysis of the cis/trans isomerization by Cyp18 is best described by a four-site exchange model, where the four sites represent the cis and trans isomers free in solution and bound to the enzyme. Combination of dynamic NMR spectroscopy with the classical protease-coupled PPIase assay allowed determination of the complete set of the microscopic rate constants describing the four site exchange model. The comparison of the rate constants of cis-->trans isomerization of the peptide free in solution and bound to cyclophilin yields an acceleration factor of 3.5 x 10(5). Dissociation of the Michaelis complexes are of the same order of magnitude as the isomerization rates on the enzyme. Therefore, all microscopic rate constants contribute to the steady state parameters. For the first time, the kcat (620 s-1) and KM (220 microM) value for the trans isomer in addition to the values of the cis isomer (kcat = 680 s-1, KM = 80 microM) could be determined under reversible conditions at pH 6.0 and 10 degrees C. The affinity of Cyp18 for the cis isomer is 4 times higher than for the trans isomer. This results in a shift of the cis/trans equilibrium toward the cis isomer.(ABSTRACT TRUNCATED AT 250 WORDS)


Assuntos
Isomerases de Aminoácido/metabolismo , Proteínas de Transporte/metabolismo , Prolina , Isomerases de Aminoácido/isolamento & purificação , Sequência de Aminoácidos , Animais , Sítios de Ligação , Proteínas de Transporte/isolamento & purificação , Catálise , Chaperoninas/metabolismo , Citosol/enzimologia , Rim/enzimologia , Cinética , Espectroscopia de Ressonância Magnética , Matemática , Modelos Teóricos , Dados de Sequência Molecular , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Peptidilprolil Isomerase , Estereoisomerismo , Especificidade por Substrato , Suínos
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