RESUMO
Hydration status is critical for erythrocyte survival and is mainly determined by intracellular cation content. Active pumps, passive transporters, and ion channels are the key components of volume homeostasis, whereas water passively fits ionic movements. Whenever cation content increases, erythrocyte swells, whereas it shrinks when cation content decreases. Thus, inappropriate cation leak causes erythrocyte hydration disorders, hemolytic anemia, and characteristic red cell shape abnormalities named stomatocytosis. All types of stomatocytosis either overhydrated or dehydrated are linked to inherited or de novo mutations in genes encoding ion transporters or channels. Although intracellular ion content can be assessed by experimental methods, laboratory diagnosis is guided by a combination of red blood cell parameters and deformability measurement when possible, and confirmed by sequencing of the putative genes. A better knowledge of the mechanisms underlying erythrocyte hydration imbalance will further lead to therapeutic improvements.
Assuntos
Volume de Eritrócitos , Desequilíbrio Hidroeletrolítico/diagnóstico , Anemia Hemolítica/diagnóstico , Humanos , Transporte de ÍonsRESUMO
INTRODUCTION: Hereditary spherocytosis (HS), hereditary elliptocytosis (HE), and hereditary stomatocytosis (HSt) are inherited red cell disorders caused by defects in various membrane proteins. The heterogeneous clinical presentation, biochemical and genetic abnormalities in HS and HE have been well documented. The need to raise the awareness of HSt, albeit its much lower prevalence than HS, is due to the undesirable outcome of splenectomy in these patients. METHODS: The scope of this guideline is to identify the characteristic clinical features, the red cell parameters (including red cell morphology) for these red cell disorders associated, respectively, with defective cytoskeleton (HS and HE) and abnormal cation permeability in the lipid bilayer (HSt) of the red cell. The current screening tests for HS are described, and their limitations are highlighted. RESULTS: An appropriate diagnosis can often be made when the screening test result(s) is reviewed together with the patient's clinical/family history, blood count results, reticulocyte count, red cell morphology, and chemistry results. SDS-polyacrylamide gel electrophoresis of erythrocyte membrane proteins, monovalent cation flux measurement, and molecular analysis of membrane protein genes are specialist tests for further investigation. CONCLUSION: Specialist tests provide additional evidence in supporting the diagnosis and that will facilitate the management of the patient. In the case of a patient's clinical phenotype being more severe than the affected members within the immediate family, molecular testing of all family members is useful for confirming the diagnosis and allows an insight into the molecular basis of the abnormality such as a recessive mode of inheritance or a de novo mutation.
Assuntos
Anemia Hemolítica Congênita/diagnóstico , Anemia Hemolítica Congênita/etiologia , Membrana Eritrocítica/metabolismo , Anemia Hemolítica Congênita/complicações , Eliptocitose Hereditária/diagnóstico , Membrana Eritrocítica/química , Humanos , Esferocitose Hereditária/diagnósticoRESUMO
Venous thromboembolism [TE] is a multifactorial disease and antithrombin deficiency [ATD] constitutes a major risk factor. In the present study the prevalence of ATD and the clinical presentation at TE onset in a cohort of paediatric index cases are reported. In 319 unselected paediatric patients (0.1-18 years) from 313 families, recruited between July 1996 and December 2013, a comprehensive thrombophilia screening was performed along with recording of anamnestic data. 21 of 319 paediatric patients (6.6%), corresponding to 16 of 313 families (5.1%), were AT-deficient with confirmed underlying AT gene mutations. Mean age at first TE onset was 14 years (range 0.1 to 17). Thrombotic locations were renal veins (n=2), cerebral veins (n=5), deep veins (DVT) of the leg (n=9), DVT & pulmonary embolism (n=4) and pelvic veins (n=1). ATD co-occurred with the factor-V-Leiden mutation in one and the prothrombin G20210A mutation in two children. In 57.2% of patients a concomitant risk factor for TE was identified, whereas 42.8% of patients developed TE spontaneously. A second TE event within primarily healthy siblings occurred in three of 313 families and a third event among siblings was observed in one family. In an unselected cohort of paediatric patients with symptomatic TE, the prevalence of ATD adjusted for family status was 5.1%. Given its clinical implication for patients and family members, thrombophilia testing should be performed and the benefit of medical or educational interventions should be evaluated in this high risk population.
Assuntos
Proteínas Antitrombina/genética , Trombofilia/epidemiologia , Tromboembolia Venosa/epidemiologia , Adolescente , Criança , Pré-Escolar , Estudos de Coortes , Fator V/genética , Testes Genéticos , Humanos , Lactente , Educação de Pacientes como Assunto , Prevalência , Protrombina/genética , Risco , Trombofilia/genética , Tromboembolia Venosa/genéticaAssuntos
Anemia Hemolítica Congênita/complicações , Anemia Hemolítica Congênita/genética , Hidropisia Fetal/etiologia , Canais Iônicos/genética , Mutação , Anemia Hemolítica Congênita/diagnóstico , Segregação de Cromossomos , Heterozigoto , Humanos , Hidropisia Fetal/diagnóstico , Hidropisia Fetal/genética , Linhagem , FenótipoRESUMO
BACKGROUND: Heparin and its analogs, mediating their anticoagulant activity through antithrombin (AT) activation, remain largely used for the preventive and curative treatment of thrombosis. The major adverse reaction of these drugs is the bleeding risk associated with overdose. Unfractionnated heparin (UFH) can be efficiently and rapidly neutralized by protamine sulfate, but this reversal partially neutralizes low-molecular-weight heparin (LMWH) and is inefficient in reversing fondaparinux. To secure administration of AT-mediated anticoagulants and counteract bleeding disorders, we previously designed a recombinant inactive AT as an antidote to heparin derivatives. OBJECTIVES: To get around the limited production level of recombinant AT, we propose in this study an alternative strategy to produce a chemically modified inactive AT, exhibiting increased heparin affinity, as an antagonist of heparin analogs. METHODS: Plasma-derived AT was chemically modified with 2,3 butanedione, a diketone known to specifically react with the arginine side chain. The chemical reaction was conducted in the presence of heparin to preserve basic residues within the heparin binding site from modifications. RESULTS: AT treated by butanedione and selected for its high heparin affinity (AT-BD) was indeed modified on reactive Arg393 and thus exhibited decreased anticoagulant activity and increased heparin affinity. AT-BD was able to neutralize anticoagulant activity of heparin derivatives in vitro and in vivo and was devoid of intrinsic anticoagulant activity, as assessed by activated partial thromboplastin time assay. CONCLUSIONS: AT-BD appears to be as efficient as protamine to neutralize UFH in vivo but could be more largely used because it also reverses fondaparinux and LMWH.
Assuntos
Anticoagulantes/química , Antitrombinas/uso terapêutico , Antagonistas de Heparina/química , Polissacarídeos/antagonistas & inibidores , Animais , Antitrombinas/química , Arginina/química , Diacetil/química , Desenho de Fármacos , Feminino , Fondaparinux , Hemorragia , Heparina/química , Humanos , Espectrometria de Massas , Camundongos , Tempo de Tromboplastina Parcial , Polissacarídeos/química , Proteínas Recombinantes/química , RiscoAssuntos
Deficiência de Antitrombina III/genética , Antitrombina III/genética , Coagulação Sanguínea/genética , Mutação , Trombina/metabolismo , Trombose/genética , Adulto , Antitrombina III/metabolismo , Deficiência de Antitrombina III/sangue , Testes de Coagulação Sanguínea , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Fenótipo , Trombose/sangue , Adulto JovemRESUMO
The photofragmentation of protonated tryptophan has been investigated in a unique experimental setup, in which ion and neutral issued from the photofragmentation are detected in coincidence, in time and in position. From these data are extracted the kinetic energy, the number of neutral fragments associated with an ion, their masses, and the order of the fragmentation steps. Moreover, the fragmentation time scale ranging from tens of nanoseconds to milliseconds is obtained. From all these data, a comprehensive fragmentation mechanism is proposed.
Assuntos
Modelos Químicos , Modelos Moleculares , Fotoquímica/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Triptofano/química , Triptofano/efeitos da radiação , Simulação por Computador , Luz , Conformação Molecular/efeitos da radiação , PrótonsRESUMO
PURPOSE: To evaluate the potential of cCancer-t/Testis antigens (CTAs) as targets for immunotherapy of bladder cancer, we evaluated the expression of 9 CTA genes or families of genes in normal urothelia, bladder tumours and bladder cancer human bladder tissuescell lines. As expression of most CTAs is controlled by epigenetic mechanisms, we also evaluated the effect of the DNA methylase inhibitor 5-aza-2'-deoxycytidine (5-AZA-DC), and/or theand histone deacetylase inhibitors Trichostatin A (TSA) on their expression in bladder cancer cell lines. MATERIAL AND METHODS: Expression of NY-ESO-1/LAGE-1, MAGE-A, MAGE-C1, BAGE, HOM-TES-85, SCP-1, SSX-1, SSX-2 and SSX-4 was analyzed by semi-quantitative RT-PCR and Western blotting on 10 normal urothelia, 23 24 superficial and 223 invasive tumours and on 10 cell lines treated with 5-aza-2'-deoxycytidine (5-AZA-DC) and/or Trichostatin A (TSA). RESULTS: Expression of all CTA genes could be observed in at least 1 tumour except for HOM-TES-85 for which mRNA was never detected. MAGE-A, BAGE and NY-ESO-1/LAGE-1 mRNAs were the most frequently detected, respectively in 5677%, 212% and 89% of superficial and in 6461%, 4139% and 276% of invasive tumours. With the exception of MAGE-A, CTA transcripts were rarely detected in the cell lines. However, expression of all CTA genes, except SCP-1, could be induced at various levels by the drugs and 5-AZA-DC was a much more potent inducer than TSA. CONCLUSION: These data suggest that immunotherapy of bladder cancer could target CTAs, especially those expressed at higher frequency such as MAGE-A, BAGE and NY-ESO-1/LAGE-1. Moreover, their induction by chemotherapeutic agents such as 5-AZA-DC, provides a potential pretreatment aimed at inducing the immunogenicity of the tumours.
Assuntos
Antígenos de Neoplasias/biossíntese , Neoplasias Testiculares/imunologia , Neoplasias da Bexiga Urinária/metabolismo , Linhagem Celular Tumoral , Humanos , MasculinoAssuntos
Antitrombinas/genética , Antitrombinas/metabolismo , Variação Genética , Heparina/metabolismo , Adulto , Substituição de Aminoácidos , Antitrombinas/química , Antitrombinas/deficiência , Sítios de Ligação/genética , Feminino , Heterozigoto , Humanos , Técnicas In Vitro , Fatores de Risco , Trombose/sangue , Trombose/genéticaRESUMO
Fatal cerebral hemorrhage involving the left thalamus in a neonate was attributed to deep cerebral vein thrombosis. Although antithrombin levels were at the lower end of the normal range, family and genetic studies showed constitutional type I antithrombin deficiency related to a novel missense mutation in the antithrombin gene.
Assuntos
Antitrombinas/deficiência , Hemorragia Cerebral/genética , Mutação Puntual , Antitrombinas/genética , Evolução Fatal , Humanos , Recém-Nascido , Masculino , Trombose Venosa/complicaçõesRESUMO
Sequencing of reverse-transcriptase genes and recombinant virus assays were performed on paired isolates from antiretroviral drug-naive patients randomized to stavudine and didanosine (group 1; n = 21) or zidovudine and lamivudine (group 2; n = 21) at baseline and after > or = 12 months of follow-up. The T215Y mutation emerged in 13 (61.9%) and 2 (9.5%) isolates in groups 1 and 2, respectively (P < .0001). Furthermore, in group 1, mutations associated with multidideoxynucleoside resistance were selected in 3 isolates. In group 2, all isolates carried the M184V mutation. The median fold changes in susceptibilities to zidovudine, stavudine, and lamivudine were 16.4 and 1, 2.2 and 0.6, and 4.5 and > 38 in groups 1 and 2, respectively (P < .0001, all comparisons). These results suggest that the combination of stavudine and didanosine is associated more frequently with the emergence of zidovudine resistance and a decrease in susceptibility to stavudine than the combination of zidovudine and lamivudine.
Assuntos
Didanosina/uso terapêutico , Resistência Microbiana a Medicamentos/genética , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , HIV-1/genética , Lamivudina/uso terapêutico , Inibidores da Transcriptase Reversa/uso terapêutico , Estavudina/uso terapêutico , Zidovudina/uso terapêutico , Adulto , Didanosina/farmacologia , Didesoxinucleosídeos/farmacologia , Quimioterapia Combinada , Feminino , Seguimentos , Genótipo , Infecções por HIV/virologia , HIV-1/isolamento & purificação , Humanos , Lamivudina/farmacologia , Masculino , Testes de Sensibilidade Microbiana , Fenótipo , Mutação Puntual , Inibidores da Transcriptase Reversa/farmacologia , Estavudina/farmacologia , Zidovudina/farmacologiaRESUMO
Microcytic anemia (mk) mice and Belgrade (b) rats are severely iron deficient because of impaired intestinal iron absorption and defective iron metabolism in peripheral tissues. Both animals carry a glycine to arginine substitution at position 185 in the iron transporter known as Nramp2/DMT1 (divalent metal transporter 1). DMT1 messenger RNA (mRNA) and protein expression has been examined in the gastrointestinal tract of mk mice. Northern blot analysis indicates that, by comparison to mk/+ heterozygotes, mk/mk homozygotes show a dramatic increase in the level of DMT1 mRNA in the duodenum. This increase in RNA expression is paralleled by a concomitant increase of the 100-kd DMT1 isoform I protein expression in the duodenum. Immunohistochemical analyses show that, as for normal mice on a low-iron diet, DMT1 expression in enterocytes of mk/mk mice is restricted to the duodenum. However, and in contrast to normal enterocytes, little if any expression of DMT1 is seen at the apical membrane in mk/mk mice. These results suggest that the G185R mutation, which was shown to impair the transport properties of DMT1, also affects the membrane targeting of the protein in mk/mk enterocytes. This loss of function of DMT1 is paralleled by a dramatic increase in expression of the defective protein in mk/mk mice. This is consistent with a feedback regulation of DMT1 expression by iron stores. (Blood. 2000;96:3964-3970)
Assuntos
Anemia/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Transporte de Cátions , Duodeno/química , Proteínas de Ligação ao Ferro , Proteínas de Membrana/genética , Alelos , Anemia/etiologia , Animais , Transporte Biológico , Proteínas de Transporte/imunologia , Duodeno/citologia , Duodeno/ultraestrutura , Immunoblotting , Imuno-Histoquímica , Ferro da Dieta/farmacologia , Camundongos , Camundongos Mutantes , Microvilosidades , RNA Mensageiro/metabolismo , Distribuição TecidualRESUMO
Mutations at the natural resistance-associated macrophage protein 1 (Nramp1) locus cause susceptibility to infection with antigenically unrelated intracellular pathogens. Nramp1 codes for an integral membrane protein expressed in the lysosomal compartment of macrophages, and is recruited to the membrane of phagosomes soon after the completion of phagocytosis. To define whether Nramp1 functions as a transporter at the phagosomal membrane, a divalent cation-sensitive fluorescent probe was designed and covalently attached to a porous particle. The resulting conjugate, zymosan-FF6, was ingested by macrophages and its fluorescence emission was recorded in situ after phagocytosis, using digital imaging. Quenching of the probe by Mn(2+) was used to monitor the flux of divalent cations across the phagosomal membrane in peritoneal macrophages obtained from Nramp1-expressing (+/+) and Nramp1-deficient (-/-) macrophages. Phagosomes from Nramp1(+/+) mice extrude Mn(2+) faster than their Nramp(-/-) counterparts. The difference in the rate of transport is eliminated when acidification of the phagosomal lumen is dissipated, suggesting that divalent metal transport through Nramp1 is H(+) dependent. These studies suggest that Nramp1 contributes to defense against infection by extrusion of divalent cations from the phagosomal space. Such cations are likely essential for microbial function and their removal from the phagosomal microenvironment impairs pathogenesis, resulting in enhanced bacteriostasis or bactericidal activity.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Transporte de Cátions , Membranas Intracelulares/metabolismo , Proteínas de Ligação ao Ferro , Macrófagos Peritoneais/metabolismo , Manganês/metabolismo , Proteínas de Membrana/metabolismo , Fagossomos/imunologia , Fagossomos/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Cátions Bivalentes/metabolismo , Etilenodiaminas/farmacologia , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/metabolismo , Fura-2/metabolismo , Concentração de Íons de Hidrogênio , Membranas Intracelulares/efeitos dos fármacos , Macrófagos Peritoneais/citologia , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/imunologia , Camundongos , Camundongos Knockout , Microscopia de Fluorescência , Mutação , Espectrometria de Fluorescência , Tapsigargina/farmacologia , Zimosan/análogos & derivados , Zimosan/síntese química , Zimosan/metabolismoRESUMO
We have investigated the molecular bases of familial antithrombin deficiency in eight French families. Eight mutations in the antithrombin coding exons were identified, seven of which were novel mutations. In all cases, individuals were heterozygous for the mutation. We found two small frameshift deletions in exon 3a, leading to type I deficiency. Five missense mutations in exons 3b or 5 also caused type I deficiency and their potential consequences on the antithrombin three-dimensional structure were analysed. The last mutation in exon 4 was associated with a type II 'reactive site' deficiency: a dysfunctional antithrombin that is affected in its interaction with thrombin was present in circulation.
Assuntos
Deficiência de Antitrombina III/genética , Fibrina/deficiência , Mutação de Sentido Incorreto , Trombose/genética , Sítios de Ligação/genética , Éxons , Fibrina/genética , Deleção de Genes , Heterozigoto , Humanos , Reação em Cadeia da PolimeraseRESUMO
Nramp2, also known as DMT1 and DCT1, is a 12-transmembrane (TM) domain protein responsible for dietary iron uptake in the duodenum and iron acquisition from transferrin in peripheral tissues. Nramp2/DMT1 produces by alternative splicing two isoforms differing at their C terminus (isoforms I and II). The subcellular localization, mechanism of action, and destination of divalent cations transported by the two Nramp2 isoforms are not completely understood. Stable CHO transfectants expressing Nramp2 isoform II modified by addition of a hemaglutinin epitope in the loop defined by the TM7-TM8 interval were generated. Immunofluorescence with permeabilized and intact cells established that Nramp2 isoform II is expressed at the plasma membrane and demonstrated the predicted extracytoplasmic location of the TM7-TM8 loop. Using the fluorescent, metal-sensitive dye calcein, and a combination of membrane-permeant and -impermeant iron chelators, Nramp2 transport was measured and quantitated with respect to kinetic parameters and at steady state. Iron transport at the plasma membrane was time- and pH-dependent, saturable, and proportional to the amount of Nramp2 expression. Iron uptake by Nramp2 at the plasma membrane was into the nonferritin-bound, calcein-accessible so-called "labile iron pool." Ion selectivity experiments show that Nramp2 isoform II can also transport Co(2+) and Cd(2+) but not Mg(2+) into the calcein-accessible pool. Parallel experiments with transfectants expressing the lysosomal Nramp1 homolog do not show any divalent cation transport activity, establishing major functional differences between Nramp1 and Nramp2. Monitoring the effect of Nramp2 on the calcein-sensisitve labile iron pool allows a simple, rapid, and nonisotopic approach to the functional study of this protein.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Transporte de Cátions , Cátions Bivalentes/metabolismo , Membrana Celular/metabolismo , Citoplasma/metabolismo , Fluoresceínas/metabolismo , Proteínas de Ligação ao Ferro , Ferro/metabolismo , Proteínas de Membrana/metabolismo , Animais , Ligação Competitiva , Células CHO , Cádmio/metabolismo , Proteínas de Transporte/genética , Cobalto/metabolismo , Cricetinae , Transporte de Íons , Radioisótopos de Ferro , Proteínas de Membrana/genética , Camundongos , Isoformas de Proteínas , Proteínas Recombinantes de Fusão/metabolismo , TransfecçãoRESUMO
BACKGROUND: Sevoflurane, with its low pungency and low blood and tissue solubility, is an attractive anaesthetic in paediatric outpatient surgery. Propofol-anaesthesia is recognised for its rapid and clear-headed emergence. This study was designed to compare emergence and recovery characteristics of sevoflurane and propofol anaesthesia for tonsillectomy in children. METHODS: Children aged 3-10 years, undergoing elective tonsillectomy, were randomly assigned to receive propofol (n=25, induction with 3 mg x kg(-1), maintenance with 100-250 microg x kg(-1) min(-1)) or sevoflurane anaesthesia (n=25, induction 7 vol.%, maintenance 2-3 vol.%). Tracheal intubation was performed with alfentanil 20 microg x kg(-1) and atracurium 0.5 mg x kg(-1). Ventilation was controlled to maintain normocapnia and all patients received N2O/O2 (60:40 vol.%) for induction and maintenance of anaesthesia. At the end of surgery infiltration of the operative sites with bupivacaine 2 mg x kg(-1) was provided for postoperative analgesia. Emergence, recovery, discharge times, and incidence of side effects were compared between the two groups. RESULTS: Time to extubation (14 vs 15 min), time to response to simple verbal command (21 vs 21 min) and time to discharge from the recovery room (45 vs 50 min) were similar in the sevoflurane and propofol groups, respectively. There was a significantly greater incidence of postoperative agitation in the sevoflurane group (46%) compared with the propofol group (9%) (P=0.008). This did not, however, delay discharge from the recovery room. The incidence of nausea and vomiting was not significantly different (8% vs 0%; P=0.49). CONCLUSION: In children, recovery from anaesthesia with sevoflurane results in a higher incidence of agitation compared with propofol.
Assuntos
Anestésicos/farmacologia , Éteres Metílicos/farmacologia , Propofol/farmacologia , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Éteres Metílicos/efeitos adversos , Propofol/efeitos adversos , Agitação Psicomotora/etiologia , Sevoflurano , Fatores de Tempo , TonsilectomiaRESUMO
The labile iron pool (LIP) of animal cells has been implicated in cell iron regulation and as a key component of the oxidative-stress response. A major mechanism commonly implied in the downregulation of LIP has been the induced expression of ferritin (FT), particularly the heavy subunits (H-FT) that display ferroxidase activity. The effects of H-FT on LIP and other physiological parameters were studied in murine erythroleukemia (MEL) cells stably transfected with H-FT subunits. Clones expressing different levels of H-FT displayed similar concentrations of total cell iron (0.3 +/- 0.1 mmol/L) and of reduced/total glutathione. However, with increasing H-FT levels the cells expressed lower levels of LIP and reactive oxygen species (ROS) and ensuing cell death after iron loads and oxidative challenges. These results provide direct experimental support for the alleged roles of H-FT as a regulator of labile cell iron and as a possible attenuator of the oxidative cell response. H-FT overexpression was of no apparent consequence to the cellular proliferative capacity. However, concomitant with the acquisition of iron and redox regulatory capacities, the H-FT-transfectant cells commensurately acquired multidrug resistance (MDR) properties. These properties were identified as increased expression of MDR1 mRNA (by reverse transcription polymerase chain reaction [RT-PCR]), P-glycoprotein (Western immunoblotting), drug transport activity (verapamil-sensitive drug efflux), and drug cytotoxicity associated with increased MDR1 or PgP. Although enhanced MDR expression per se evoked no significant changes in either LIP levels or ROS production, it might be essential for the survival of H-FT transfectants, possibly by expediting the export of cell-generated metabolites.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Resistência a Múltiplos Medicamentos/fisiologia , Eritrócitos/metabolismo , Ferritinas/biossíntese , Estresse Oxidativo/fisiologia , Animais , Eritrócitos/fisiologia , Ferro/metabolismo , Camundongos , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transfecção , Células Tumorais CultivadasRESUMO
The present study was designed to analyze the thrombomodulin proximal promoter region spanning nucleotides -293 to -12 to search for polymorphisms that could modify thrombomodulin gene expression in patients with venous thromboembolic disease. The study population comprised 205 patients and 394 healthy subjects of similar age and sex distribution. No polymorphisms and only 1 point mutation (G-33A) were found. The G-33A mutation was present at the heterozygous state in 2 patients and in 1 control. Being more frequent in the patients (0.97%) than in the controls (0.25%), the G-33A mutation might be a risk factor for venous thrombosis. To investigate the effect of this mutation on the thrombomodulin promoter activity, the proximal promoter region of the gene (bearing or not bearing the G-33A mutation) was inserted into a promotorless expression vector, upstream of the firefly luciferase gene, and transiently transfected into EA.hy926 endothelial cells. Under the conditions of the assay, the G-33A mutation mildly decreased the promoter activity. This study confirms that abnormalities of the thrombomodulin proximal promoter are not frequent in patients with venous thromboembolism.
Assuntos
Mutação Puntual , Regiões Promotoras Genéticas/genética , Tromboembolia/genética , Trombomodulina/genética , Trombose Venosa/genética , Adenina , Adulto , Estudos de Casos e Controles , Feminino , França , Testes Genéticos , Guanina , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Polimorfismo Genético , Trombomodulina/biossínteseRESUMO
Solving the structure of the stable complex between a serine protease inhibitor (serpin) and its target has been a long standing goal. We describe herein the characterization of a monoclonal antibody that selectively recognizes antithrombin in complex with either thrombin, factor Xa, or a synthetic peptide corresponding to residues P14 to P9 of the serpin's reactive center loop (RCL, ultimately cleaved between the P1 and P'1 residues). Accordingly, this antibody reacts with none of the monomeric conformers of antithrombin (native, latent, and RCL-cleaved) and does not recognize heparin-activated antithrombin or antithrombin bound to a non-catalytic mutant of thrombin (S195A, in which the serine of the charge stabilizing system has been swapped for alanine). The neoepitope encompasses the motif DAFHK, located in native antithrombin on strand 4 of beta-sheet A, which becomes strand 5 of beta-sheet A in the RCL-cleaved and latent conformers. The inferences on the structure of the antithrombin-protease stable complex are that either a major remodeling of antithrombin accompanies the final elaboration of the complex or that, within the complex, at the most residues P14 to P6 of the RCL are inserted into beta-sheet A. These conclusions limit drastically the possible locations of the defeated protease within the complex.
Assuntos
Antitrombinas/metabolismo , Autoanticorpos/imunologia , Serina Endopeptidases/metabolismo , Sequência de Aminoácidos , Animais , Antitrombinas/química , Antitrombinas/imunologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Conformação Molecular , Dados de Sequência Molecular , Serina Endopeptidases/químicaRESUMO
We describe a 73-yr-old woman anaesthetized for a laminectomy. She suffered from hepatic failure with mild encephalopathy complicated by several exacerbations associated with sedative and opioid therapy. The challenge for anaesthesia management was to provide adequate analgesia and avoid causing hepatic encephalopathy during and after the surgery. We used remifentanil to provide intraoperative and postoperative analgesia, because it has a short duration of action and does not require hepatic metabolism. We closely monitored the respiratory and the neurological status throughout the administration and conclude that remifentanil can provide perioperative analgesia in patients at risk of developing hepatic encephalopathy.