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1.
Eur J Nutr ; 59(8): 3581-3590, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32078064

RESUMO

PURPOSE: Endothelial dysfunction and inflammation are conditions which fuel atherosclerosis and ischaemic heart disease. We have previously reported reduced cardiovascular (CV) mortality following supplementation with selenium and coenzyme Q10 to 443 elderly individuals with low selenium status (mean 67 µg/L) for 4 years. Here, we wanted to evaluate a possible association between the supplementation and the plasma concentrations of the von Willebrand factor (vWf), and the plasminogen activator inhibitor-1 (PAI-1), as they, besides other functions, are also strongly associated with endothelial function. METHODS: In this sub-study, 308 individuals (active substance: 157, placebo: 151) were included. Blood samples were drawn after 6 and 36 months and vWf and PAI-1 were determined in plasma by ELISA. Changes in concentrations of the biomarkers were evaluated by the use of T tests, repeated measures of variance, and ANCOVA analyses. RESULTS: The active treatment group presented a lower level of vWf after 36 months compared with the placebo group (1.08 U/mL vs. 5.10 U/mL; p = 0.0007). The results were validated through the repeated measures of variance evaluation. The PAI-1 levels showed an equally significant decrease in the active group (26.2 ng/mL vs. 49.2 ng/mL; p = 0.0002) and were also validated through repeated measures of variance evaluation. CONCLUSION: In this sub-study on elderly receiving selenium and coenzyme Q10, or placebo we found significantly lower levels of vWf and PAI-1 in the active treatment group as compared to the placebo group. We interpret this as a better endothelial function because of the intervention, which accords with a previous finding of reduced CV mortality.


Assuntos
Doenças Cardiovasculares , Selênio , Idoso , Suplementos Nutricionais , Humanos , Inibidor 1 de Ativador de Plasminogênio , Estudos Prospectivos , Ativador de Plasminogênio Tecidual , Ubiquinona/análogos & derivados , Fator de von Willebrand
2.
Acta Anaesthesiol Scand ; 59(10): 1303-10, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26079310

RESUMO

BACKGROUND: During fluid infusion therapy, plasma proteins are diluted and leak from the intravascular space, which alters the colloid osmotic pressure (COP) and potentially affects coagulation. We hypothesised that acetated Ringer's and starch solution, alone or in combination, influence these mechanisms differently. MATERIALS AND METHODS: On different occasions, 10 male volunteers were infused with 20 ml/kg acetated Ringer's and 10 ml/kg 6% hyroxyethyl starch 130/0.4 (Voluven(®) ) alone or in combination (first with starch solution followed by Ringer's solution). Blood samples were collected every 30-min for measurements of COP, blood haemoglobin, platelets, and plasma concentrations of albumin, immunoglobulins (IgG and IgM), coagulation factor VII (FVII), fibrinogen, cystatin C, activated partial thromboplastin time (APTT) and prothrombin international normalised ratio (PT-INR). Changes were compared with the haemoglobin-derived plasma dilution. RESULTS: The COP increased by 8.4% (SD 3) with starch and decreased by 26.2% (7.9) with Ringer's. These infusions diluted the plasma by 23.4% (5.3) and 18.7% (4.9) respectively. The COP changes in the combined experiment followed the same pattern as the individual infusions. Albumin and IgG changes in excess of the plasma dilution were very subtle. The intravascular contents of the IgM and platelets decreased, whereas FVII, fibrinogen and cystatin C increased. PT-INR increased by 1/3 of the plasma dilution, whereas changes in APTT did not correlate with the plasma dilution. CONCLUSIONS: The starch increased COP and only minor capillary leak occurred in healthy volunteers. The fluid-induced plasma dilution correlated with mild impairment of the extrinsic coagulation pathway but not of the intrinsic pathway.


Assuntos
Proteínas Sanguíneas/metabolismo , Permeabilidade Capilar , Hidratação , Derivados de Hidroxietil Amido/farmacologia , Soluções Isotônicas/farmacologia , Adolescente , Adulto , Coloides , Humanos , Masculino , Pressão Osmótica , Tempo de Tromboplastina Parcial , Adulto Jovem
3.
Hum Reprod ; 28(7): 1846-52, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23592222

RESUMO

STUDY QUESTION: Do thrombin generation and haemostatic parameters differ during the two phases of the menstrual cycle? SUMMARY ANSWER: Total thrombin concentration is higher during the luteal phase compared with the follicular phase of the menstrual cycle. WHAT IS KNOWN ALREADY: The coagulation cascade is affected by many variables, such as fluctuations in the levels of sex hormones. The studies on the variations in haemostatic parameters during the menstrual cycle have produced diverse results. STUDY DESIGN, SIZE, DURATION: Thrombin generation and selected haemostatic parameters (fibrinogen, factor II, factor VII, factor VIII, factor X, von Willebrand factor, antithrombin and D-dimer) were measured during the two phases of a normal menstrual cycle in 102 healthy women not taking any form of hormone medication. PARTICIPANTS/MATERIALS, SETTING, METHODS: The study cohort consisted of 102 healthy women with regular menstrual cycles. Thrombin generation was measured by the calibrated automated thrombogram method. Progesterone and sex hormone-binding globulin were measured by chemiluminescence enzyme immunoassays. Estradiol was measured by a sensitive radioimmunoassay. Fibrinogen was measured by a clotting method, antithrombin was measured by a chromogenic method and factor II, factor VII, factor VIII, factor X, von Willebrand factor and D-dimer were measured by photometric methods. MAIN RESULTS AND THE ROLE OF CHANCE: It was shown that the total amount of generated thrombin (Endogenous Thrombin Potential) was significantly higher during the luteal compared with the follicular phase (P = 0.027). Factor X was significantly higher during the follicular phase (P = 0.028). Progesterone exhibited significant associations (measured by the least squares regression analysis) with fibrinogen and factor X during the follicular phase (P = 0.043 and P = 0.033, respectively) and with factors II and VII during the luteal phase (P = 0.034 and P = 0.024, respectively). The validity of the results from the regression analysis was further confirmed by performing correlation analyses (Pearson correlation matrix) for haemostatic markers for the luteal and follicular phases (accepted correlation level >0.8). LIMITATIONS, REASONS FOR CAUTION: The wide confidence interval for the differences in endogenous thrombin potential during the two phases could imply that the size of the cohort may not be sufficient to fully evaluate the biological variations. Additionally, the haemostatic markers were not shown to have significant associations with thrombin generation, suggesting that the increased thrombin concentration during the luteal phase would be mediated by another mechanism, as yet unidentified. WIDER IMPLICATIONS OF THE FINDINGS: The associations between progesterone and the haemostatic markers, as shown for both phases of the menstrual cycle, suggest a previously unknown or undefined yet potentially significant role for progesterone in the coagulation system. However, it has been shown that the use of progestogen-only preparations does not affect the coagulation system, which is partly the reason why they are considered safe for women with thrombophilia or previous thrombotic event. Further studies are required in order to demonstrate whether our results can be extrapolated for synthetic progestins, which might have significant implication on the indications for their use.


Assuntos
Fase Folicular/metabolismo , Fase Luteal/metabolismo , Trombina/metabolismo , Adulto , Coagulação Sanguínea/fisiologia , Feminino , Fibrinogênio/metabolismo , Humanos , Progesterona/metabolismo , Análise de Regressão , Globulina de Ligação a Hormônio Sexual/metabolismo
4.
Platelets ; 24(5): 407-11, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-22891819

RESUMO

Stroke is worldwide a leading cause of death and disability. Its etiology is regarded as heterogeneous. Platelets are implicated in its pathophysiology, but our understanding of their specific role is incomplete. Only sparse and conflicting information exists about platelet reactivity and activity in acute stroke. Some scientists take the view that platelets activate in conjunction with acute cerebral infarctions. Others put forward evidence corroborating the contrary notion. Increased soluble P-selectin as a sign of platelet and/or endothelial activity seems to be a feature of the disease. The latter point of view is opposed by other researchers. Due to these conflicting opinions, this study is devoted to platelet characteristics in acute cerebral infarctions. We studied subjects (n = 72; age 74 ± 10(SD) years; 31 females) having acute stroke. As controls served atrial fibrillation (AF) patients (n = 58; age 69 ± 7(SD) years; 12 females) subject to electrical cardioversion, a flow cytometer was put to use for measuring platelet reactivity and activity. After agonist provocation, both platelet bound P-selectin and fibrinogen were employed as estimates of platelet reactivity. Dilutions of a thrombin-receptor-activating peptide (TRAP-6) (74 and 57 µmol/l) (P-selectin and fibrinogen) and ADP (8.5 and 1.7 µmol/l) (fibrinogen only) were put to use as platelet agonists. Membrane-bound P-selectin without agonist stimulation served as a measure of in vivo platelet activation. Soluble P-selectin, as determined from a commercial ELISA, was used to assess platelet and/or endothelial activity. In acute stroke neither platelet-bound P-selectin nor fibrinogen after stimulation, i.e. reactivity, differed from AF controls. In contrast, lower platelet activity as judged from surface attached and circulating P-selectin without agonist stimulation proved to be a feature of cerebral infarctions. The p-values were p < 0.001 and p < 0.01, respectively. It is concluded that acute stroke is not associated with platelet reactivity platelets circulate less activated during the disease. It is evident that the mechanisms reflecting platelet reactivity and activity being investigated in this study play minor roles in stroke pathophysiology. New powerful platelet inhibitory drugs are currently introduced. To avoid major bleeding studies on platelet, behavior in acute stroke are necessary before including these medications in stroke treatment protocols.


Assuntos
Plaquetas/metabolismo , Infarto Cerebral/etiologia , Idoso , Idoso de 80 Anos ou mais , Fibrilação Atrial/complicações , Fibrilação Atrial/metabolismo , Feminino , Fibrinogênio/metabolismo , Humanos , Inflamação/metabolismo , Contagem de Leucócitos , Masculino , Pessoa de Meia-Idade , Neutrófilos/citologia , Neutrófilos/metabolismo , Selectina-P/metabolismo , Ativação Plaquetária/fisiologia , Contagem de Plaquetas , Estudos Prospectivos , Ligação Proteica , Acidente Vascular Cerebral/metabolismo
5.
Curr Opin Cell Biol ; 5(3): 424-33, 1993 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-8352959

RESUMO

Multiple DNA repair processes are required to maintain the integrity of the cellular genome. Recent advances, including elucidation of three-dimensional structures of DNA repair enzymes, and the cloning and characterization of DNA repair genes implicated in human inherited disease, have given new insights into the surprising complexity of cellular responses to DNA damage.


Assuntos
Reparo do DNA , Animais , Síndrome de Cockayne/genética , DNA/metabolismo , Guanina/análogos & derivados , Guanina/toxicidade , Humanos , Metilação , Xeroderma Pigmentoso/genética
6.
Acta Anaesthesiol Scand ; 55(6): 677-85, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21574963

RESUMO

BACKGROUND: Colloid fluids influence the coagulation system by diluting the plasma and, potentially, by exerting other effects that are unique for each fluid product. We hypothesised that changes in the coagulation measured at the end of surgery would be mainly governed by differences in half-life between the colloid fluids. METHODS: Eighty-four patients were randomised to receive one of four colloids: HES 130/0.42/6:1 (Venofundin(®)), 130/0.4/9:1 (Voluven(®)), 200/0.5/5:1 (Haes-steril(®)) and 6% dextran 70 (Macrodex(®)). Blood samples were taken just before and after a preoperative 500 ml bolus, and also after subsequent elective hip replacement surgery. Volume expansion was estimated from the blood dilution and coagulation assessed by ROTEM, activated partial thromboplastin time, prothrombin international normalised ratio (PT-INR), D-dimer and thrombin-antithrombin complex (TAT). RESULTS: The blood volume expansion amounted to approximately 600 ml for all four colloids directly after infusion. Voluven(®) and Haes-steril(®) prolonged the aPT time and Venofundin(®) increased TAT. Although all colloids increased PT-INR and D-dimer, the ROTEM analyses showed that they consistently shortened the clotting time and weakened the clot strength. These effects were mainly unchanged after surgery, during which the haemorrhage averaged 500-600 ml. Macrodex(®) produced a stronger volume support at the end of the surgery (91% of infused volume; P<0.001) than the three starch solutions (42-60%). CONCLUSIONS: All tested colloid fluids induced a mild hypercoagulable state with faster clotting, but with weaker clot strength. The additive influence of surgery was relatively small, and postoperative changes in coagulation were mainly due to differences in the half-life of each colloid.


Assuntos
Artroplastia de Quadril , Coagulação Sanguínea/efeitos dos fármacos , Volume Sanguíneo/efeitos dos fármacos , Dextranos/farmacologia , Derivados de Hidroxietil Amido/farmacologia , Substitutos do Plasma/farmacologia , Idoso , Coloides , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
7.
Science ; 286(5446): 1897-905, 1999 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-10583946

RESUMO

Faithful maintenance of the genome is crucial to the individual and to species. DNA damage arises from both endogenous sources such as water and oxygen and exogenous sources such as sunlight and tobacco smoke. In human cells, base alterations are generally removed by excision repair pathways that counteract the mutagenic effects of DNA lesions. This serves to maintain the integrity of the genetic information, although not all of the pathways are absolutely error-free. In some cases, DNA damage is not repaired but is instead bypassed by specialized DNA polymerases.


Assuntos
Dano ao DNA , Reparo do DNA , DNA/metabolismo , Animais , DNA/efeitos da radiação , Adutos de DNA/metabolismo , DNA Glicosilases , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Humanos , Mutagênicos , N-Glicosil Hidrolases/química , N-Glicosil Hidrolases/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Raios Ultravioleta
8.
Science ; 152(3721): 512-4, 1966 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-5910188

RESUMO

Transfer ribonucleic acid, isolated under conditions in which the original macromolecular structure is never denatured, is indistinguishable from transfer ribonucleic acid prepared by conventional methods involving denaturing steps. This finding is consistent with the absence of direct genetic control of the formation of macromolecular structure of transfer ribonucleic acid.


Assuntos
RNA de Transferência , Fenômenos Químicos , Química , Cromatografia , Diálise , Técnicas In Vitro , Saccharomyces
9.
Science ; 291(5507): 1284-9, 2001 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-11181991

RESUMO

Cellular DNA is subjected to continual attack, both by reactive species inside cells and by environmental agents. Toxic and mutagenic consequences are minimized by distinct pathways of repair, and 130 known human DNA repair genes are described here. Notable features presently include four enzymes that can remove uracil from DNA, seven recombination genes related to RAD51, and many recently discovered DNA polymerases that bypass damage, but only one system to remove the main DNA lesions induced by ultraviolet light. More human DNA repair genes will be found by comparison with model organisms and as common folds in three-dimensional protein structures are determined. Modulation of DNA repair should lead to clinical applications including improvement of radiotherapy and treatment with anticancer drugs and an advanced understanding of the cellular aging process.


Assuntos
Reparo do DNA/genética , Genes , Genoma Humano , DNA/metabolismo , Dano ao DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Bases de Dados Factuais , Desoxirribonucleases/genética , Desoxirribonucleases/metabolismo , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , N-Glicosil Hidrolases/genética , N-Glicosil Hidrolases/metabolismo , Polimorfismo Genético , Rad51 Recombinase , Recombinação Genética , Transdução de Sinais
10.
Trends Biochem Sci ; 20(10): 405-11, 1995 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-8533153

RESUMO

There are one million molecules of poly(ADP-ribose) polymerase (PARP) in mammalian cell nuclei and the enzyme is found in most eukaryotes, with the notable exception of yeasts. In response to DNA damage caused by ionizing radiation or alkylating agents, PARP binds to strand interruptions in DNA and undergoes rapid automodification with synthesis of long branched polymers of highly negatively charged poly(ADP-ribose). DNA repair occurs after dissociation of modified PARP from DNA strand breaks. Biochemical data with enzyme-depleted extracts and studies of enzyme-deficient mice show that PARP does not participate directly in DNA repair. Possible roles for poly(ADP-ribose) synthesis are discussed.


Assuntos
Dano ao DNA , Poli(ADP-Ribose) Polimerases/genética , Biossíntese de Proteínas , Animais , Apoptose , Glicosídeo Hidrolases/genética , Histonas , Modelos Genéticos , NAD/genética , NAD/metabolismo , Filogenia , Poli(ADP-Ribose) Polimerases/biossíntese
12.
Curr Opin Genet Dev ; 7(2): 158-69, 1997 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-9115419

RESUMO

The major DNA excision repair pathways of base excision repair for endogenous DNA lesions and nucleotide excision repair for DNA damage inflicted by ultraviolet light have been reconstructed with purified mammalian proteins and details of these repair mechanisms are emerging. Similar data are becoming available with regard to mismatch repair for correction of replication errors. Deletion of individual DNA repair proteins in knockout mice provides information on the roles of such factors in vivo and recent three-dimensional structures of several repair enzymes explain their detailed modes of action.


Assuntos
Reparo do DNA , Animais , Síndrome de Cockayne/genética , DNA Helicases/metabolismo , Enzimas Reparadoras do DNA , Proteínas de Ligação a DNA , Humanos , Camundongos , Ácidos Nucleicos Heteroduplexes , Proteínas de Ligação a Poli-ADP-Ribose , Proteínas/metabolismo , Fatores de Transcrição , Transcrição Gênica
13.
Platelets ; 19(5): 352-8, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18791941

RESUMO

Recent studies indicate connections between periodontitis and atherothrombosis, and the periodontal pathogen Porphyromonas gingivalis has been found within atherosclerotic lesions. P. gingivalis-derived proteases, designated gingipains activate human platelets, probably through a "thrombin-like" activity on protease-activated receptors (PARs). However, the potential interplay between P. gingivalis and other physiological platelet activators has not been investigated. The aim of this study was to elucidate consequences and mechanisms in the interaction between P. gingivalis and the stress hormone epinephrine. By measuring changes in light transmission through platelet suspensions, we found that P. gingivalis provoked aggregation, whereas epinephrine alone never had any effect. Intriguingly, pre-treatment of platelets with a low, sub-threshold number of P. gingivalis (i.e. a density that did not directly provoke platelet aggregation) resulted in a marked aggregation response when epinephrine was added. This synergistic action was not inhibited by the cyclooxygenas inhibitor aspirin. Furthermore, fura-2-measurements revealed that epinephrine caused an intracellular Ca(2+) mobilization in P. gingivalis pre-treated platelets, whereas epinephrine alone had no effect. Inhibition of the arg-specific gingipains, but not the lys-specific gingipains, abolished the aggregation and the Ca(2+) response provoked by epinephrine. Similar results were achieved by separate blockage of platelet alpha(2)-adrenergic receptors and PARs. In conclusion, the present study shows that a sub-threshold number of P. gingivalis sensitizes platelets to epinephrine. We suggest that P. gingivalis-derived arg-specific gingipains activates a small number of PARs on the surface of the platelets. This leads to an unexpected Ca(2+) mobilization and a marked aggregation response when epinephrine subsequently binds to the alpha(2)-adrenergic receptor. The present results are consistent with a direct connection between periodontitis and stress, and describe a novel mechanism that may contribute to pathological platelet activation.


Assuntos
Adesinas Bacterianas/fisiologia , Plaquetas/microbiologia , Cisteína Endopeptidases/fisiologia , Epinefrina/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Porphyromonas gingivalis/fisiologia , Apirase/farmacologia , Aspirina/farmacologia , Plaquetas/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Inibidores de Ciclo-Oxigenase/farmacologia , Cisteína Endopeptidases Gingipaínas , Humanos , Técnicas In Vitro , Leupeptinas/farmacologia , Nefelometria e Turbidimetria , Oligopeptídeos/farmacologia , Fragmentos de Peptídeos/farmacologia , Inibidores da Agregação Plaquetária/farmacologia , Porphyromonas gingivalis/enzimologia , Porphyromonas gingivalis/patogenicidade , Inibidores de Proteases/farmacologia , Pirróis/farmacologia , Quinazolinas/farmacologia , Receptores Ativados por Proteinase/efeitos dos fármacos , Receptores Ativados por Proteinase/fisiologia , Virulência , Ioimbina/farmacologia
14.
J Bone Joint Surg Br ; 90(3): 388-92, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18310767

RESUMO

Thrombin has many biological properties similar to those of growth factors. In a previous study, we showed that thrombin improves healing of the rat tendo Achillis. Low molecular weight heparin (LMWH) inhibits the activity and the generation of thrombin. We therefore considered that LMWH at a thromboprophylactic dose might inhibit tendon repair. Transection of the tendo Achillis was carried out in 86 rats and the healing tested mechanically. Low molecular weight heparin (dalateparin) was either injected a few minutes before the operation and then given continuously with an osmotic mini pump for seven days, or given as one injection before the operation. In another experiment ,we gave LMWH or a placebo by injection twice daily. The anti-factor Xa activity was analysed. Continuous treatment with LMWH impaired tendon healing. After seven days, this treatment caused a 33% reduction in force at failure, a 20% reduction in stiffness and a 67% reduction in energy uptake. However, if injected twice daily, LMWH had no effect on tendon healing. Anti-factor Xa activity was increased by LMWH treatment, but was normal between intermittent injections. Low molecular weight heparin delays tendon repair if given continuously, but not if injected intermittently, probably because the anti-factor Xa activity between injections returns to normal, allowing sufficient thrombin stimulation for repair. These findings indicate the need for caution in the assessment of long-acting thrombin and factor Xa inhibitors.


Assuntos
Tendão do Calcâneo/lesões , Tendão do Calcâneo/cirurgia , Anticoagulantes/efeitos adversos , Heparina de Baixo Peso Molecular/efeitos adversos , Animais , Autoanticorpos/sangue , Esquema de Medicação , Fator Xa/análise , Injeções , Modelos Animais , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Fatores de Tempo , Cicatrização
15.
Colloids Surf B Biointerfaces ; 62(2): 214-9, 2008 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-18006286

RESUMO

Surface mediated immune complement activation can be detected by a variety of antibody utilizing methods such as ELISA, fluorescence- or radiolabelling techniques, QCM, and ellipsometry. In the present work we investigated how the common anticoagulants heparin, dalteparin, fondaparinux and sodium citrate affected the binding of anti-complement factor 3c (anti-C3c) on a model complement activator surface, immobilised IgG, after incubation in human blood serum. The results show, as expected, that different anticoagulants affect the antibody binding differently. Increasing amounts of heparin, dalteparin and sodium citrate in normal serum resulted in a decreasing anti-C3c binding. The antibody deposition was not sensitive for the fondaparinux concentration. Surprisingly high concentrations of anti-coagulantia were needed to completely eradicate the antibody binding. Experiments in EGTA-serum showed that anticoagulants interfered directly with both the classical and alternative pathways. Control C3a-des arg ELISA measurements show that the lowered antibody surface binding was not a result of complement depletion in serum. Kallikrein generation by hydrophilic glass surfaces was not affected by high anticoagulant concentrations.


Assuntos
Anticoagulantes/sangue , Anticoagulantes/farmacologia , Ativação do Complemento/efeitos dos fármacos , Proteínas do Sistema Complemento/análise , Proteínas Sanguíneas/metabolismo , Colorimetria , Complemento C3/metabolismo , Complemento C3a/metabolismo , Dalteparina/farmacologia , Ácido Egtázico , Ensaio de Imunoadsorção Enzimática , Fondaparinux , Heparina/farmacologia , Humanos , Imunoensaio , Imunoglobulina G/química , Técnicas In Vitro , Indicadores e Reagentes , Calicreínas , Peso Molecular , Polissacarídeos/farmacologia , Ressonância de Plasmônio de Superfície , Propriedades de Superfície
16.
J Thromb Haemost ; 16(1): 142-149, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29065247

RESUMO

Essentials Two candidate International Standards for thromboplastin (coded RBT/16 and rTF/16) are proposed. International Sensitivity Index (ISI) of proposed standards was assessed in a 20-centre study. The mean ISI for RBT/16 was 1.21 with a between-centre coefficient of variation of 4.6%. The mean ISI for rTF/16 was 1.11 with a between-centre coefficient of variation of 5.7%. SUMMARY: Background The availability of International Standards for thromboplastin is essential for the calibration of routine reagents and hence the calculation of the International Normalized Ratio (INR). Stocks of the current Fourth International Standards are running low. Candidate replacement materials have been prepared. This article describes the calibration of the proposed Fifth International Standards for thromboplastin, rabbit, plain (coded RBT/16) and for thromboplastin, recombinant, human, plain (coded rTF/16). Methods An international collaborative study was carried out for the assignment of International Sensitivity Indexes (ISIs) to the candidate materials, according to the World Health Organization (WHO) guidelines for thromboplastins and plasma used to control oral anticoagulant therapy with vitamin K antagonists. Results Results were obtained from 20 laboratories. In several cases, deviations from the ISI calibration model were observed, but the average INR deviation attributabled to the model was not greater than 10%. Only valid ISI assessments were used to calculate the mean ISI for each candidate. The mean ISI for RBT/16 was 1.21 (between-laboratory coefficient of variation [CV]: 4.6%), and the mean ISI for rTF/16 was 1.11 (between-laboratory CV: 5.7%). Conclusions The between-laboratory variation of the ISI for candidate material RBT/16 was similar to that of the Fourth International Standard (RBT/05), and the between-laboratory variation of the ISI for candidate material rTF/16 was slightly higher than that of the Fourth International Standard (rTF/09). The candidate materials have been accepted by WHO as the Fifth International Standards for thromboplastin, rabbit plain, and thromboplastin, recombinant, human, plain.


Assuntos
Anticoagulantes/uso terapêutico , Coagulação Sanguínea/efeitos dos fármacos , Monitoramento de Medicamentos/normas , Coeficiente Internacional Normatizado/normas , Tempo de Protrombina/normas , Tromboplastina/normas , Animais , Calibragem , Humanos , Ensaio de Proficiência Laboratorial , Variações Dependentes do Observador , Valor Preditivo dos Testes , Coelhos , Proteínas Recombinantes/normas , Padrões de Referência , Reprodutibilidade dos Testes
18.
Curr Biol ; 4(12): 1069-76, 1994 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-7535646

RESUMO

BACKGROUND: The base excision-repair pathway is the major cellular defence mechanism against spontaneous DNA damage. The enzymes involved have been highly conserved during evolution. Base excision-repair has been reproduced previously with crude cell-free extracts of bacterial or human origin. To further our understanding of base excision-repair, we have attempted to reconstitute the pathway in vitro using purified enzymes. RESULTS: We report here the successful reconstitution of the base excision-repair pathway with five purified enzymes from Escherichia coli: uracil-DNA glycosylase, a representative of the DNA glycosylases that remove various lesions from DNA; the AP endonuclease IV that specifically cleaves at abasic sites; RecJ protein which excises a 5' terminal deoxyribose-phosphate residue; DNA polymerase I; and DNA ligase. The reaction proceeds with high efficiency in the absence of additional factors in the reconstituted system. Four of the enzymes are absolutely required for completion of the repair reaction. An unusual feature we have discovered is that the pathway branches after enzymatic incision at an abasic DNA site. RecJ protein is required for the major reaction, which involves replacement of only a single nucleotide at the damaged site; in its absence, an alternative pathway is observed, with generation of longer repair patches by the 5' nuclease function of DNA polymerase I. CONCLUSIONS: Repair of uracil in DNA is achieved by a very short-patch excision-repair process involving five different enzymes. No additional protein factors seem to be required. There is a minor, back-up pathway that uses replication factors to generate longer repair patches.


Assuntos
DNA Glicosilases , Reparo do DNA/fisiologia , Proteínas de Escherichia coli , Escherichia coli/enzimologia , Exodesoxirribonucleases , Proteínas de Bactérias/metabolismo , Sequência de Bases , DNA Ligases/metabolismo , DNA Polimerase I/metabolismo , DNA Liase (Sítios Apurínicos ou Apirimidínicos) , Desoxirribonuclease IV (Fago T4-Induzido) , Escherichia coli/genética , Liases/metabolismo , Dados de Sequência Molecular , N-Glicosil Hidrolases/metabolismo , Oligodesoxirribonucleotídeos/metabolismo , Uracila-DNA Glicosidase
19.
Curr Biol ; 5(5): 496-9, 1995 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-7583098

RESUMO

The repair of double-stranded breaks in DNA and the recombination of antibody gene V(D)J segments share a common pathway involving the Ku protein, which binds DNA ends, and its associated protein kinase.


Assuntos
Anticorpos/genética , Antígenos Nucleares , DNA Helicases , Reparo do DNA/fisiologia , Proteínas de Ligação a DNA/fisiologia , Proteínas Nucleares/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Animais , Proteína Quinase Ativada por DNA , Humanos , Autoantígeno Ku , Modelos Químicos , Recombinação Genética
20.
Curr Biol ; 8(25): 1395-8, 1998.
Artigo em Inglês | MEDLINE | ID: mdl-9889105

RESUMO

DNA ligase IV is the most recently identified member of a family of enzymes joining DNA strand breaks in mammalian cell nuclei [1] [2]. The enzyme occurs in a complex with the XRCC4 gene product [3], an interaction mediated via its unique carboxyl terminus [4] [5]. Cells lacking XRCC4 are hypersensitive to ionising radiation and defective in V(D)J recombination [3] [6], implicating DNA ligase IV in the pathway of nonhomologous end-joining (NHEJ) of DNA double-strand breaks mediated by XRCC4, the Ku70/80 heterodimer and the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) in mammalian cells (reviewed in [7]). The phenotype of a null mutant of the Saccharomyces cerevisiae DNA ligase IV homologue indicates that the enzyme is non-essential and functions in yeast NHEJ [8] [9] [10]. Unlike other mammalian DNA ligases for which cDNAs have been characterised, DNA ligase IV is encoded by an intronless gene (LIG4). Here, we show that targeted disruption of LIG4 in the mouse leads to lethality associated with extensive apoptotic cell death in the embryonic central nervous system. Thus, unlike Ku70/80 and DNA-PKcs [11] [12] [13] [14], DNA ligase IV has an essential function in early mammalian development.


Assuntos
Sistema Nervoso Central/enzimologia , DNA Ligases/fisiologia , Embrião de Mamíferos/enzimologia , Animais , Apoptose , Sistema Nervoso Central/embriologia , Sistema Nervoso Central/patologia , DNA Ligase Dependente de ATP , DNA Ligases/genética , Embrião de Mamíferos/patologia , Marcação de Genes , Genes Letais , Genótipo , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
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