RESUMO
BACKGROUND: Capnoperitoneum (CP) compromises hemodynamic function during laparoscopy. Three therapeutic concepts were evaluated with an aim to minimize the hemodynamic reaction to CP: First, a controlled increase of intrathoracic blood volume (ITBV) by intravenous fluids; second, partially reduced sympathetic activity by the beta1-blocker esmolol; and third, a decrease in mean arterial pressure (MAP) by the vasodilator sodium nitroprusside. METHODS: For this study, 43 pigs were assigned to treatment with fluid and sodium nitroprusside (group A) or with esmolol (group B). In both groups, the pigs were assigned to head-up, head-down, or supine position, resulting in three different subgroups. Invasive hemodynamic monitoring was established including left heart catheter and cardiac oxygen lung water determination (COLD) measurements. Measurements were documented before CP with the animals in supine position, after induction of a 14-mmHg CP with the animals in each body position, after a 10% reduction in MAP by vasodilation, and after an increase in ITBV of about 30% by infusion of 6% hydroxyethylstarch solution. RESULTS: Increasing ITBV improved hemodynamic function in all body positions during CP. Esmolol reduced cardiac output and myocardial contractility. Sodium nitroprusside did not improve hemodynamic function in any body position. CONCLUSIONS: Optimizing volume load is effective for minimizing hemodynamic changes during CP in the head-up and in head-down positions. In general, beta(1)-blockers cannot be recommended because they might additionally compromise myocardial contractility and suppress compensatory reaction of the sympathetic nerve system. Vasodilation has not improved hemodynamic parameters during CP.
Assuntos
Hemodinâmica , Pneumoperitônio Artificial/efeitos adversos , Medicina Preventiva/métodos , Antagonistas Adrenérgicos beta/uso terapêutico , Animais , Pressão Sanguínea/efeitos dos fármacos , Volume Sanguíneo , Dióxido de Carbono , Hidratação/métodos , Injeções Intravenosas , Nitroprussiato/uso terapêutico , Propanolaminas/uso terapêutico , Suínos , Sistema Nervoso Simpático/efeitos dos fármacos , Sistema Nervoso Simpático/fisiopatologia , Vasodilatadores/uso terapêuticoRESUMO
Functional deficiency or absence of the human von Willebrand factor (VWF)-cleaving protease (VWF-cp), recently termed ADAMTS13, has been shown to cause acquired and congenital thrombotic thrombocytopenic purpura (TTP), respectively. As a first step towards developing a small animal model of TTP, we have cloned the complete (non-truncated) murine Adamts13 gene from BALB/c mice liver poly A+ mRNA. Murine ADAMTS13 is a 1426-amino-acid protein with a high homology and similar structural organization to the human ortholog. Transient expression of the murine Adamts13 cDNA in HEK 293 cells yielded a protein with a molecular weight of approximately 180 kDa which degraded recombinant murine VWF (rVWF) in a dose-dependent manner. The cleavage products of murine rVWF had the expected size of 140 and 170 kDa. Murine ADAMTS13 was inhibited by EDTA and the plasma from a TTP patient.
Assuntos
Metaloendopeptidases/genética , Metaloendopeptidases/metabolismo , Proteínas ADAM , Proteína ADAMTS13 , Sequência de Aminoácidos , Aminoácidos/química , Animais , Northern Blotting , Western Blotting , Linhagem Celular , Clonagem Molecular , Primers do DNA/química , DNA Complementar/metabolismo , Bases de Dados Genéticas , Relação Dose-Resposta a Droga , Ácido Edético/farmacologia , Humanos , Metaloendopeptidases/química , Camundongos , Camundongos Endogâmicos BALB C , Modelos Genéticos , Dados de Sequência Molecular , Filogenia , Poli A/metabolismo , Reação em Cadeia da Polimerase , Ligação Proteica , Estrutura Terciária de Proteína , Púrpura Trombocitopênica Trombótica/sangue , RNA/metabolismo , RNA Mensageiro/metabolismo , Proteínas Recombinantes/química , Homologia de Sequência de Aminoácidos , Distribuição Tecidual , Transfecção , Fator de von Willebrand/metabolismoRESUMO
We have investigated the requirements necessary for high-level production of the hepatitis B virus (HBV strain ayw) large surface glycoprotein preS1 with vaccinia virus (VV) recombinants. In earlier studies, only nanogram amounts of preS1 could be obtained from cells infected with an appropriate recombinant VV carrying the preS1 gene under the transcriptional control of a conventional VV promoter (p7.5). Here, we report that the use of an improved promoter system, i.e., the bacteriophage T7 polymerase/VV hybrid expression system (T7/EMC system) in combination with a G-C conversion at position 5 of the preS1 open reading frame, deleting the myristylation motif of the polypeptide, results in an at least 12-fold increase in preS1 expression compared to the wild-type preS1 expressed with the strongest homologous VV promoter system known so far. Although the T7/EMC promoter system was most effective, improved expression of the modified preS1 (preS1dMyr) is independent from the promoter system used, from the insertion locus of the modified preS1 within the VV genome and also from the cell line used for expression studies.
Assuntos
Regulação Viral da Expressão Gênica , Vetores Genéticos , Antígenos de Superfície da Hepatite B/genética , Vírus da Hepatite B/genética , Ácidos Mirísticos/metabolismo , Precursores de Proteínas/genética , Vaccinia virus/genética , Adaptação Fisiológica , Animais , Linhagem Celular , Chlorocebus aethiops , Conversão Gênica , Humanos , Ácido Mirístico , Fases de Leitura Aberta , Proteínas Recombinantes de Fusão/genética , Células VeroRESUMO
The 'Modified Vaccinia Ankara' (MVA) strain is a potential live vaccine vector. The use of the hemagglutinin (ha) gene of the MVA strain as an insertion site for foreign genes was evaluated. To identify the molecular basis of the hemagglutinin-negative (HA-) phenotype of MVA, the ha gene and the region around this gene were sequenced. Amino acid (aa) sequence comparisons with functional hemagglutinins of other vaccinia strains predicted a functional polypeptide. The late part of the promoter region of the ha gene, however, was deleted, causing the apparent loss of the ha gene function. Nevertheless, insertion of foreign DNA into the ha gene allowed generation of functional recombinant viruses, indicating that the ha-gene region is a suitable insertion site.
Assuntos
Vetores Genéticos , Hemaglutininas Virais/genética , Vaccinia virus/genética , Sequência de Bases , DNA Viral , Estudos de Avaliação como Assunto , Genes Virais , Dados de Sequência Molecular , Deleção de SequênciaRESUMO
Wild-type full-length cDNA of von Willebrand factor (vWF) was expressed in CHO cells. Recombinant vWF (rvWF) was obtained and its molecular composition investigated by two-dimensional electrophoresis. Results of first dimension electrophoresis under non-reducing conditions showed that rvWF-dimer represents a mixture of three different species. Second dimension electrophoresis under reducing conditions revealed, that these protein species represent (i) homo-dimers of either two unprocessed or two fully processed, mature vWF polypeptides, and (ii) the hetero-dimer of unprocessed and mature rvWF monomers. Compared with vWF-dimers from human plasma, which contained predominantly proteolytically degraded polypeptides, recombinant vWF-dimers were shown to consist of non-proteolyzed subunits only.
Assuntos
Fator de von Willebrand/química , Animais , Células CHO , Cricetinae , Eletroforese em Gel de Ágar , Eletroforese em Gel de Poliacrilamida , Humanos , Conformação Proteica , Proteínas Recombinantes/químicaRESUMO
Thorough analysis of multimer composition and molecular structure of recombinant von Willebrand factor (r-vWF) produced by recombinant CHO cells demonstrated r-vWF to be more intact and less proteolytically degraded than plasma-derived vWF (pd-vWF) [B. Fischer et al. (1994) FEBS Lett. 351, 345-348]. In contrast to pd-vWF, r-vWF preparations consisted of pro-vWF (vWF containing covalently attached propeptide) as well as mature vWF subunits forming homo- and hetero-multimers. In order to ensure complete propeptide processing, a r-vWF-producing CHO cell clone was transfected with the cDNA of the human propeptide processing enzyme Furin. A r-vWF/r-Furin co-expressing cell clone was cultivated at industrial scale in high cell density perfusion fermenters. r-vWF obtained from these cells was fully processed. Analysis of r-vWF by multimer analysis revealed a multimer pattern equal in number of high molecular weight multimer to pd-vWF, but absence of satellite bands. Two-dimensional electrophoretic analysis of both the primary dimer and the complete multimer pattern of r-vWF showed that the recombinant coagulation factor was composed exclusively of intact and mature subunits. Since the triplet structure typical to pd-vWF is known to reflect proteolytic degradation, r-vWF thus exhibits an integrity far superior compared to pd-vWF.
Assuntos
Proteínas Recombinantes/biossíntese , Subtilisinas/biossíntese , Fator de von Willebrand/biossíntese , Fator de von Willebrand/química , Animais , Células CHO , Células Clonais , Cricetinae , DNA Complementar , Eletroforese em Gel de Ágar , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Fermentação , Furina , Expressão Gênica , Humanos , Immunoblotting , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Subtilisinas/metabolismo , Transfecção/métodos , Fator de von Willebrand/isolamento & purificaçãoRESUMO
Von Willebrand factor (vWF) is synthesized in endothelial cells as pre-provWF and processed intracellularly to propeptide (vWFpp) and mature vWF. Building on previous studies indicating that recombinant provWF when infused into animals can also be processed extracellularly in vivo, we investigated the processing of provWF in vitro. Incubation of a recombinant provWF (rpvWF) preparation with canine and human vWF-deficient plasma induced a time-dependent decrease in provWF antigen and an increase in vWFpp antigen without changing total vWF antigen or collagen-binding activity. Multimer analysis showed the gradual transformation of the provWF multimers to mature vWF multimers and cleaved vWFpp was visualized on autoradiograms of SDS-polyacrylamide electrophoresis gels using 125-labeled provWF. Processing was facilitated by CaCl2 but prevented by a thrombin inhibitor and did not occur in prothrombin-depleted plasma. When recombinant provWF was incubated with increasing amounts of purified thrombin, the extent of provWF processing was dose-dependent. The specific cleavage of vWFpp was confirmed by immunoblots using an anti-vWFpp antibody and by amino terminal amino-acid analysis. Binding of provWF to collagen decreased the thrombin concentration necessary for propeptide removal to a concentration in the range of that found during blood clotting. Meizothrombin, an intermediate of prothrombin activation, was also able to induce dose-dependent removal of the propeptide from rpvWF. Hirudin preconditioning of vWF-deficient mice attenuated processing of infused rpvWF suggesting that thrombin plays a part in the processing events in vivo.
Assuntos
Precursores de Proteínas/metabolismo , Trombina/farmacologia , Fator de von Willebrand/biossíntese , Fator de von Willebrand/metabolismo , Animais , Biopolímeros , Eletroforese das Proteínas Sanguíneas , Cloreto de Cálcio/farmacologia , Colágeno/metabolismo , Doenças do Cão/sangue , Doenças do Cão/genética , Cães , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Precursores Enzimáticos/farmacologia , Hirudinas/farmacologia , Humanos , Camundongos , Camundongos Knockout , Proteínas Recombinantes de Fusão/metabolismo , Especificidade da Espécie , Trombina/antagonistas & inibidores , Doenças de von Willebrand/sangue , Doenças de von Willebrand/genética , Doenças de von Willebrand/veterinária , Fator de von Willebrand/química , Fator de von Willebrand/genéticaRESUMO
In humans, type III von Willebrand disease is caused by deletions or nonsense mutations. In dogs, the underlying genetic defects have not been determined yet. We searched for the genetic defect in four related type III deficient Dutch Kooiker dogs obtained from one breeder. Mutation analysis was performed with total RNA isolated from platelets or whole blood. The complete coding region of the vWf gene was amplified by RT-PCR and sequenced by the cycle sequencing technique. Two homozygous mutations were found, a G-->A transition at the first position of the donor splice site sequence of intron 16 (TGgtaagt-->TGataagt) and a missense mutation at nt 208 (G-->A) (1). The splice site defect resulted in the generation of a transcript containing 46bp of intron sequence and a stop codon at amino acid position 729 in the propeptide region of the vWf protein. This mutation seems to be causative for the type III phenotype. The effect of the missense mutation in exon 3 which causes a change of Val to Ile on the vWD phenotype is unclear. Probably, this transition represents a polymorphism occurring in Dutch Kooiker dogs. Both mutations were not present in 5 healthy mongrel dogs.
Assuntos
Doenças do Cão/genética , Doenças de von Willebrand/veterinária , Fator de von Willebrand/genética , Animais , Sequência de Bases , Doenças do Cão/sangue , Cães , Feminino , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Mutação Puntual , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Doenças de von Willebrand/sangue , Doenças de von Willebrand/genéticaRESUMO
Serum from rodents and felines has been found very effective in complement-dependent lysis of HIV-1, even in nonimmunized animals, but the effector molecules in animal serum and target structures on HIV-1 envelope gp120/160 responsible for complement activation were not determined. We have found that the natural anti-carbohydrate-specific IgM antibodies present in baby rabbit serum were able to lyse effectively the CD4+ T cells coated with the whole virus or with a recombinant gp120/160, irrespectively of the virus strain or glycoprotein expression system. When the high mannose-type glycans on gp160 were enzymatically removed by endoglycosidase F or blocked with the specific lectins, the complement activation and subsequent cell lysis were abolished. IgM-depleted baby rabbit serum was not able to lyse the gp120/160- and/or whole virus-coated target cells. These results suggest that the target structures for complement-activating and naturally occurring IgM antibodies in baby rabbit serum are high-mannose residues on HIV-1 envelope glycoprotein.
Assuntos
Ativação do Complemento/imunologia , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/imunologia , Proteína gp160 do Envelope de HIV/imunologia , Imunoglobulina M/imunologia , Manose/imunologia , Polissacarídeos/imunologia , Animais , Animais Recém-Nascidos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Células CHO , Linhagem Celular , Cricetinae , Humanos , Coelhos , OvinosRESUMO
The effect of patient preimmunization virus sequences on CTL responses during gp160 immunization were studied. Ten HLA-A2+, HIV+ asymptomatic patients with CD4+ T cells >500/mm3 were given two courses of HIV-1 MN rgp160 vaccine over a 2-year period. Envelope epitope-specific CTL responses, using PBMCs, were measured against peptide-coated autologous B lymphoblastoid cell lines. Optimum CTL epitopes were determined by HLA-A2-binding affinity of 9- to 10-mer peptides containing the HLA-A2.1-binding motif. Ten of the high- or intermediate-binding peptides were conserved among >50% of reported clade B HIV strains. These peptide-specific CTL activities and the patient virus sequences in peptide-coding regions were monitored. Six patients showed envelope peptide-specific CTL responses, which correlated with the presence of whole envelope antigen-specific CTL responses. Five of these patients, who showed responses to epitopes in the gp41 region (aa 814-824), had preimmunization virus similar to the vaccine sequence in this region. Three patients who did not show these epitope-specific responses had initially different sequences in the HIV gene encoding that region. The epitope-specific CTL responses appear to reflect recall responses, as only patients infected with virus containing the vaccine sequence developed them and they could be recalled with a second set of vaccine injections. This appears to be reminiscent of the concept of T cell "original antigenic sin." This vaccine was also immunogenic as measured by gp160-specific lymphocyte-proliferative responses. However, increased immune responses did not impact the HIV load or CTL epitope sequences during therapy.
Assuntos
Vacinas contra a AIDS/imunologia , Infecções por HIV/imunologia , HIV-1/genética , Linfócitos T Citotóxicos/imunologia , Vacinas Sintéticas/imunologia , Sequência de Aminoácidos , Ciclo Celular , Epitopos/química , Epitopos/imunologia , HIV-1/imunologia , Humanos , RNA Viral/genética , Linfócitos T Citotóxicos/citologia , Carga ViralRESUMO
The goal of the present study was to investigate the antigen-specific T-cell response to the recombinant HIV envelope glycoprotein (gp160) and to test the effect of various adjuvant formulations on the efficiency of T-cell priming as well as on magnitude and longevity of the gp160-specific T-cell response. Our studies revealed that, in combination with an appropriate adjuvant (lipid-based adjuvant or mineral carrier complex), immunization with recombinant gp160 led to the appearance of gp160-primed T cells. The T-cell response obtained was substantial (proliferative response of greater than 100,000 delta dpm after one primary and two booster immunizations), gp160-specific (proliferation only in response to gp160, no proliferation after addition of a mock gp160 preparation), and long-lasting (T cell responses of greater than 50,000 delta dpm were observed more than one year after the last booster). The results presented here differ from those of previous studies in that they show the presence of substantial and long-lasting T-cell memory toward the immunogen gp160. Therefore further investigations on the use of these preparations as HIV candidate vaccines appear to be justified.
Assuntos
Produtos do Gene env/imunologia , HIV-1/imunologia , Imunização , Memória Imunológica , Precursores de Proteínas/imunologia , Linfócitos T/imunologia , Adjuvantes Imunológicos/farmacologia , Animais , Epitopos/imunologia , Feminino , Anticorpos Anti-HIV/biossíntese , Proteína gp160 do Envelope de HIV , Masculino , Pan troglodytes , Proteínas Recombinantes/imunologia , Linfócitos T/microbiologia , Células Vero , Vacinas ViraisRESUMO
The human immunodeficiency virus (HIV-1) envelope gene was expressed in large-scale microcarrier cultures of Vero cells using a system involving coinfection with two recombinant vaccinia viruses. One recombinant contained the bacteriophage T7 RNA polymerase gene under control of a vaccinia virus promoter. The second contained the HIV-1 gp160 gene flanked by T7 promoter and termination sequences. The protein was expressed on the surface of infected cells, and it was shown to have a molecular weight of 160 kD and to react with gp41 and gp120 specific monoclonal antibodies. After purification by successive affinity and ion-exchange chromatography, the protein was demonstrated to be present in a particulate form with a diameter in the range of 15-30 nm. When injected into goats a high-titer gp160 specific antibody response was elicited and group-specific neutralizing activity could be demonstrated in vitro. The immunogenicity of the protein was also studied in conjunction with a number of adjuvant formulations, and the highest potency in mice was obtained using a preparation with 0.2% Al(OH)3 and 0.25% deoxycholate.
Assuntos
HIV-1/imunologia , Proteínas dos Retroviridae/imunologia , Vacinas Sintéticas/imunologia , Vacinas/imunologia , Proteínas do Envelope Viral/imunologia , Adjuvantes Imunológicos , Animais , Cabras , Proteína gp160 do Envelope de HIV , Camundongos , Testes de Neutralização , Proteínas dos Retroviridae/biossíntese , Proteínas dos Retroviridae/isolamento & purificação , Vacinas Sintéticas/isolamento & purificação , Vaccinia virus/genética , Células Vero , Proteínas do Envelope Viral/biossíntese , Proteínas do Envelope Viral/isolamento & purificaçãoRESUMO
Human immunodeficiency virus type 1 (HIV-1) may enter the blood stream as free virus or via infected lymphocytes, which poses problems for vaccine development. The classical vaccine designs, attenuated, inactivated, or subunit vaccine will be discussed with regard to HIV. Development of a recombinant subunit vaccine appears to be the most promising approach. Forthcoming results from experiments involving inoculation of chimpanzees should allow evaluation of the feasibility of using a subunit vaccine based on the env-glycoprotein. Also the use of live recombinant vaccinia merits further investigation.
Assuntos
HIV/imunologia , Vacinas Virais , Animais , Humanos , Vacinas Atenuadas , Vacinas SintéticasRESUMO
Recombinant human coagulation factors II and IX and recombinant human protein S were expressed by the Vaccinia virus expression system in Vero cells and isolated from the serum-free media. All three recombinant proteins were purified by the same universal strategy. After concentration by anion exchange chromatography, recombinant coagulation factors were purified by anion exchange filtration in the presence of Ca2+. The addition of Ca2+ to the vitamin K-dependent coagulation factors modified their physico-chemical behavior during anion exchange chromatography and resulted in the separation of the recombinant coagulation proteins from contaminating proteins.
Assuntos
Fator IX/isolamento & purificação , Proteína S/isolamento & purificação , Protrombina/isolamento & purificação , Proteínas Recombinantes/isolamento & purificação , Animais , Coagulação Sanguínea , Cálcio , Chlorocebus aethiops , Cromatografia em Gel/métodos , Cromatografia por Troca Iônica/métodos , Meios de Cultura Livres de Soro , Eletroforese em Gel de Poliacrilamida/métodos , Fator IX/análise , Fator IX/biossíntese , Vetores Genéticos , Humanos , Proteína S/análise , Proteína S/biossíntese , Protrombina/análise , Protrombina/biossíntese , Proteínas Recombinantes/análise , Proteínas Recombinantes/biossíntese , Transfecção/métodos , Vaccinia virus , Células VeroRESUMO
The expression of a modified human coagulation factor VIII cDNA in a human liver-derived cell line is described. A B-domain deleted FVIII (rVIIIdB928) cDNA controlled by a strong viral promoter/enhancer was linked to a dominant selection-/amplification marker and transfected into the human hepatic cell line SK-HEP-1. By means of this system, up to 3.5 U rFVIIIdB928/10(6) cells x 24 h could be detected immediately after selection without gene amplification. This level is orders of magnitude higher than that obtained in Chinese hamster ovary (CHO) f1p4s under the same conditions. Efficient expression of rFVIIIdB928 in SK-HEP-1 cells was temperature dependent, a 4-fold higher level of activity was achieved in culture supernatants at decreased incubation temperatures of 28 degrees C. This system allows the production of high amounts of recombinant rFVIIIdB928 without time and labour consuming gene amplification procedures.
Assuntos
Fator VIII/isolamento & purificação , Fígado/metabolismo , Linhagem Celular , Fator VIII/genética , Amplificação de Genes/genética , Hemofilia A/genética , Humanos , Proteínas Recombinantes/isolamento & purificação , Deleção de Sequência/genética , Temperatura , Trombina/metabolismo , Transfecção/genéticaRESUMO
von Willebrand factor (vWF) from normal human plasma was purified and separated into three fractions containing high, medium, and low molecular weight vWF multimers. vWF fractions were tested for (1) vWF-antigen (vWF:Ag); (2) vWF-ristocetin cofactor activity (vWF:RiCof); (3) vWF-collagen binding activity (vWF:CBA); and (4) a monoclonal antibody-binding ELISA (mAB-binding ELISA), based on the vWF binding to immobilized monoclonal antibody directed to the glycoprotein Ib-binding region within the A1 domain of vWF. The three different fractions of vWF showed a correlation between multimer size and vWF:RiCof/vWG:Ag and vWF:CBA/vWF:Ag, respectively. In contrast, results obtained with the mAB-binding ELISA showed identical levels of mAB-binding/vWF:Ag, without regard for the multimer size present in the tested fraction. Our results therefore suggest that in the case of structurally normal vWF the mAB-binding ELISA reflects the concentration of vWF:Ag rather than vWF function. It is feasible that while the mAB-binding ELISA may show reduced levels for abnormal vWF protein, structurally altered within the A1 domain of vWF as in some patients with vWD type 2, this assay does not appear to be suitable for functional analysis of structurally intact vWF.
Assuntos
Fator de von Willebrand/análise , Anticorpos Monoclonais , Eletroforese em Gel de Ágar , Ensaio de Imunoadsorção Enzimática , Humanos , Peso Molecular , Conformação Proteica , Relação Estrutura-Atividade , Fator de von Willebrand/fisiologiaRESUMO
The smallest circulating von Willebrand factor (vWF) molecule is a dimer composed of two identical subunits containing binding sites for heparin, collagen, platelet glycoproteins and coagulation factor VIII (FVIII). Interdimeric disulfide linking leads to multimers composed of up to 40 dimers. vWF serves as a carrier of FVIII and is required for normal interactions of platelets with the subendothelium of the injured vessel wall. Von Willebrand factor was purified from human plasma cryoprecipitate and fermentation supernatant of recombinant CHO cells by anion exchange chromatography. Heparin affinity chromatography was used to isolate vWF polymers of different degree of multimerization. Analysis of collagen binding and platelet aggregation revealed that these activities increase with increasing degree of multimerization of vWF. Binding of FVIII to vWF was studied by real-time biospecific interaction analysis and surface plasmon technology. The binding data showed that the binding of FVIII is independent of vWF multimerization. Using recombinant FVIII and recombinant vWF, real-time biospecific interaction analysis resulted in a potential stoichiometry of 2 to 2.5 vWF-subunits per bound FVIII molecule. The kinetic analysis of the vWF-FVIII interaction resulted in a binding rate constant of about 3 x 10(6) M-1 s-1 and an equilibrium dissociation constant of about 0.4 x 10(-9) M.
Assuntos
Colágeno/metabolismo , Fator VIII/metabolismo , Agregação Plaquetária/efeitos dos fármacos , Proteínas Recombinantes de Fusão/farmacologia , Fator de von Willebrand/farmacologia , Animais , Anticorpos Monoclonais/imunologia , Biopolímeros , Técnicas Biossensoriais , Células CHO , Cromatografia de Afinidade , Cricetinae , Cricetulus , Meios de Cultura Livres de Soro , Dimerização , Fermentação , Heparina/metabolismo , Humanos , Cinética , Substâncias Macromoleculares , Coelhos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Fator de von Willebrand/química , Fator de von Willebrand/imunologia , Fator de von Willebrand/isolamento & purificação , Fator de von Willebrand/metabolismoRESUMO
Factor X/Xa plays a pivotal role in the coagulation cascade and exhibits a therapeutic potential for the treatment of factor X-deficient as well as FVIII and FIX inhibitor patients. This report describes the establishment of Chinese hamster ovary cell clones expressing recombinant human factor X up to 120 microg/mL x day and 78 microg/10(6) cells x day, that is to 100-fold higher levels than reported previously. Although propeptide removal and single chain precursor to light and heavy chain processing as well as vitamin K-dependent gamma-carboxylation became impaired at these expression levels, up to 25% of the recombinant human factor X produced was active. This represents the highest functional activity ever reported for a vitamin K-dependent protein at such an expression level. Expression of recombinant human factor X in Chinese hamster ovary cells lacking the endoprotease Furin revealed that propeptide removal still occurred, whereas single chain precursor to light/heavy chain processing was abolished. This suggests that a protease different from Furin mediates propeptide removal, a unique finding compared with the other vitamin K-dependent coagulation factors. In contrast, exposure of incompletely processed rFX molecules to soluble recombinant Furin in vitro mediated both of these cleavage reactions despite the absence of a typical argP4-xP3-lys/argP2-argP1 Furin cleavage site in the propeptide, indicating relaxed specificity in vitro. Concomitantly with the degree of processing, the functional activity of recombinant human factor X increased. Interestingly, Furin was shown to even perform correct N-terminal proteolytic trimming of FX molecules truncated amino-terminal to the P3 residue in vitro. Depending on the absence or presence of warfarin in the culture media, as well as on the processing state, four distinct recombinant human factor X light chain isoforms were observed and their structure characterized. One of these light chain forms correlated with the functional activity. Finally, the distribution of the individual light chain isoforms suggests that gamma-carboxylation may be a prerequisite for propeptide removal.
Assuntos
Fator X/biossíntese , Fator X/efeitos dos fármacos , Subtilisinas/farmacologia , Animais , Células CHO/enzimologia , Células CHO/metabolismo , Clonagem Molecular , Cricetinae , Eletroforese em Gel de Poliacrilamida , Furina , Humanos , Peso Molecular , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Estrutura Quaternária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Análise de Sequência de Proteína , Relação Estrutura-Atividade , Subtilisinas/metabolismoRESUMO
The uneven geographic distribution of physicians has been identified as a significant problem for the delivery of health care services. The present study examined one of the factors that contribute to the distribution of physicians; how far they move from their residency sites to establish their first practices. In 1989, the authors selected a random sample of 701 U.S. residency programs in the ten specialties with the most practitioners, and measured the distance each of these physicians moved to his or her first practice location. Of the 701 programs, 58.5% provided usable information about 2,612 physicians. Of these physicians, over 40% had moved less than 10 miles from their residencies, and over 50% had moved less than 75 miles. Comparisons among the physicians from the various specialties showed that the primary care physicians moved significantly shorter distances than did those from the other specialties. In the last two decades, many efforts have been made to increase the geographic distribution of physicians. The evidence from this study suggests that so far as the distances that physicians move from their practice sites are concerned, little has changed. Recent graduates of residency programs show no more tendency to move far from their residency sites than did their counterparts 30 years ago, as reported in the literature.
Assuntos
Internato e Residência/normas , Área de Atuação Profissional/estatística & dados numéricos , Médicos/provisão & distribuição , Especialização/estatística & dados numéricos , Estados UnidosRESUMO
The effects of the infusion of a human recombinant von Willebrand factor (vWF) preparation in pigs homozygous for von Willebrand disease (vWD) were evaluated on serial measurements of von Willebrand factor antigen and activity, FVIII activity, vWF multimer analysis, in-vivo bleeding time and platelet adhesion and thrombus formation on collagen at high shear rates in an ex-vivo model of experimental thrombosis. Plasma-derived human and porcine vWF were used for comparison. Before infusion, the pigs were characterized by undetectable plasma vWF levels, a low level of FVIII, prolonged bleeding time, severely impaired platelet adhesion and thrombus formation. After infusion of the human recombinant vWF, in-vivo recovery of vWF activity ranged from 58% to 82%, depending on the dose infused, and its half-life was longer than for the plasma-derived concentrates. The highest-molecular-weight forms of human recombinant vWF were removed from the circulation gradually. Infusion of the three vWF concentrates produced inconsistent effects on bleeding time and moderate improvement of platelet adhesion and thrombus formation. After infusion, a prolonged increase of FVIII (> 48 h) was observed, suggesting that human recombinant vWF is able to bind and to stabilize porcine factor VIII and that porcine vWD is a good model for studying such interactions.