RESUMO
Hemophilia B, or factor IX deficiency, is an X-linked recessive disorder occurring in about 1 in 25,000 males. Affected individuals are at risk for spontaneous bleeding into many organs; treatment mainly consists of the transfusion of clotting factor concentrates prepared from human blood or recombinant sources after bleeding has started. Small- and large-animal models have been developed and/or characterized that closely mimic the human disease state. As a preclinical model for gene therapy, recombinant adeno-associated viral vectors containing the human or canine factor IX cDNAs were infused into the livers of murine and canine models of hemophilia B, respectively. There was no associated toxicity with infusion in either animal model. Constitutive expression of factor IX was observed, which resulted in the correction of the bleeding disorder over a period of over 17 months in mice. Mice with a steady-state concentration of 25% of the normal human level of factor IX had normal coagulation. In hemophilic dogs, a dose of rAAV that was approximately 1/10 per body weight that given to mice resulted in 1% of normal canine factor IX levels, the absence of inhibitors, and a sustained partial correction of the coagulation defect for at least 8 months.
Assuntos
Dependovirus , Fator IX/genética , Terapia Genética , Vetores Genéticos , Hemofilia B/terapia , Animais , Anticorpos/sangue , Tempo de Sangramento , Transformação Celular Viral , Modelos Animais de Doenças , Cães , Humanos , Fígado , Camundongos , Camundongos Endogâmicos C57BL , Recombinação GenéticaRESUMO
The cDNA for human gamma-glutamyl carboxylase, which accomplishes the post-translational modification required for the activity of all of the vitamin K-dependent proteins, was cloned. The enzyme is a 758-residue integral membrane protein and appears to have three transmembrane domains near its amino terminus. The hydrophilic COOH-terminal half of the carboxylase has 19.3 percent identity with soybean seed lipoxygenase. Expression of the cloned cDNA resulted in an increase in carboxylase activity in microsomes of transfected cells compared to mock-transfected cells.
Assuntos
Carbono-Carbono Ligases , Ligases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Bovinos , Clonagem Molecular , DNA/genética , Humanos , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/química , Reação em Cadeia da Polimerase , Proteínas Recombinantes , Alinhamento de SequênciaRESUMO
The chromatic dimensions of human color vision have a neural basis in the retina. Ganglion cells, the output neurons of the retina, exhibit spectral opponency; they are excited by some wavelengths and inhibited by others. The hypothesis that the opponent circuitry emerges from selective connections between horizontal cell interneurons and cone photoreceptors sensitive to long, middle, and short wavelengths (L-, M-, and S-cones) was tested by physiologically and anatomically characterizing cone connections of horizontal cell mosaics in macaque monkeys. H1 horizontal cells received input only from L- and M-cones, whereas H2 horizontal cells received a strong input from S-cones and a weaker input from L- and M-cones. All cone inputs were the same sign, and both horizontal cell types lacked opponency. Despite cone type selectivity, the horizontal cell cannot be the locus of an opponent transformation in primates, including humans.
Assuntos
Percepção de Cores/fisiologia , Interneurônios/fisiologia , Células Fotorreceptoras Retinianas Cones/fisiologia , Células Ganglionares da Retina/fisiologia , Animais , Dendritos/ultraestrutura , Humanos , Interneurônios/citologia , Macaca fascicularis , Macaca mulatta , Macaca nemestrina , Estimulação Luminosa , Transdução de Sinais , Vias VisuaisRESUMO
We report the clinical course of 2 patients with central diabetes insipidus and evolving to panyhypopituitarism which prompted the diagnosis of an isolated pituitary stalk thickening (PST). In both patients, all etiological investigations were normal and the first biopsy revealed an isolated lymphocytic infiltrate with no sign of malignancy. Close clinical follow-up accompanied by serial brain MRIs was proposed to determine a precise diagnosis and for early detection and treatment of neoplastic disease. In our first case, the diagnosis of germinoma was made 9 months after the PST diagnosis owing to tumor progression. In the second case, the time course was even longer with the diagnosis of germinoma 6 years following initial presentation. In these cases, it is speculated that the lymphocytic infiltrates represent the first sign of a host reaction to an occult germinoma. To our knowledge, this is the third case reported of lymphocytic infiltrates preceding a germinoma in a prepubertal girl, and the only case reported in a prepubertal boy. These cases underline the difficulties in establishing the diagnosis of germinoma in a patient with isolated PST.
Assuntos
Hormônio do Crescimento Humano/uso terapêutico , Hipopituitarismo/etiologia , Linfócitos/patologia , Hipófise/patologia , Criança , Pré-Escolar , Desamino Arginina Vasopressina/uso terapêutico , Diabetes Insípido Neurogênico/complicações , Diabetes Insípido Neurogênico/tratamento farmacológico , Diagnóstico Diferencial , Feminino , Germinoma/diagnóstico , Hormônio do Crescimento Humano/deficiência , Humanos , Hipopituitarismo/diagnóstico , Imageamento por Ressonância Magnética , Masculino , Adeno-Hipófise/patologia , Tiroxina/uso terapêuticoRESUMO
The propeptide sequences of the vitamin K-dependent clotting factors serve as a recognition site for the enzyme gamma-glutamylcarboxylase, which catalyzes the carboxylation of glutamic acid residues at the NH2 terminus of the mature protein. We describe a mutation in the propeptide of Factor IX that results in warfarin sensitivity because of reduced affinity of the carboxylase for the Factor IX precursor. The proband has a Factor IX activity level of > 100% off warfarin and < 1% on warfarin, at a point where other vitamin K-dependent factors were at 30-40% activity levels. Direct sequence analysis of amplified genomic DNA from all eight exons and exon-intron junctions showed a single guanosine-->adenosine transition at nucleotide 6346 resulting in an alanine to threonine change at residue -10 in the propeptide. To define the mechanism by which the mutation resulted in warfarin sensitivity, we analyzed wild-type and mutant recombinant peptides in an in vitro carboxylation reaction. The peptides that were analyzed included the wild-type sequence, the Ala-10-->Thr sequence, and Ala-10-->Gly, a substitution based on the sequence in bone gamma-carboxyglutamic acid protein. Measurement of C02 incorporation at a range of peptide concentrations yielded a Vmax of 343 cpm/min/reaction for the wild-type peptide, and Vmax values of 638 and 726 for A-10T and A-10G respectively, a difference of only twofold. The Km values, on the other hand, showed a 33-fold difference between wild-type and the variants, with a value of 0.29 microM for wild-type, and 10.9 and 9.50 microM, respectively, for A-10T and A-10G. Similar kinetic experiments showed no substantial differences between wild-type and mutant peptides in kinetic parameters of the carboxylase-peptide complexes for reduced vitamin K. We conclude that the major defect resulting from the Factor IX Ala-l0-->Thr mutation is a reduction in affinity of the carboxylase for the mutant propeptide. These studies delineate a novel mechanism for warfarin sensitivity. In addition, the data may also explain the observation that bone Gla protein is more sensitive to warfarin than the coagulation proteins.
Assuntos
Anticoagulantes/farmacologia , Carbono-Carbono Ligases , Fator IX/genética , Mutação , Precursores de Proteínas/genética , Varfarina/farmacologia , Sequência de Aminoácidos , Anticoagulantes/uso terapêutico , Éxons/genética , Fator IX/metabolismo , Humanos , Cinética , Ligases/metabolismo , Masculino , Microssomos/metabolismo , Pessoa de Meia-Idade , Dados de Sequência Molecular , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Alinhamento de Sequência , Vitamina K/metabolismo , Varfarina/uso terapêutico , População BrancaRESUMO
Factor X (FX) is a vitamin K-dependent plasma protein required for the intrinsic and extrinsic pathways of blood coagulation. FXSanto Domingo is a hereditary FX deficiency which is characterized clinically by a severe bleeding diathesis. The proposita has a FX activity of less than 1% and a FX antigen of less than 5%. We have determined the molecular basis of the defect in the FXSanto Domingo gene by amplification of all eight exons with polymerase chain reaction and subsequent sequence analysis. The patient is homozygous for a G----A transition in exon I at codon -20 (numbering the alanine at the NH2 terminus of the mature protein as +1), resulting in the substitution of arginine for glycine in the carboxy-terminal part of the signal peptide. This amino acid change occurs near the presumed cleavage site of the signal peptidase. We hypothesized that the mutation might prevent cleavage by the signal peptidase which in turn would impair proper secretion of the FX protein. To test this hypothesis, we compared the expression of wild type and mutant FX cDNA in a human kidney cell line. Wild type and mutant constructs in the expression vector pCMV4 were introduced into the human embryonic kidney cell line 293 by calcium phosphate transfection. FX antigen levels in the supernatant of the cells harboring the wild type construct were 2.4 micrograms/10(7) cells per 24 h, whereas antigen levels in media from cells containing the FXSanto Domingo construct were undetectable. No FX antigen was detected in the cell lysates of cells transfected with the mutant construct. To insure that the difference in protein levels was not due to a difference in steady state levels of mRNA, Northern analysis was performed on RNA from the cell lysates of both constructs. The results showed a transcript of the same size, present in roughly equal amounts, in both cases. Thus, the defect in the signal sequence of FXSanto Domingo exerts its effect posttranscriptionally. FXSanto Domingo is the first described example of a bleeding diathesis due to a mutation in the signal sequence.
Assuntos
Deficiência do Fator X/genética , Mutação , Adolescente , Sequência de Aminoácidos , Sequência de Bases , Fator X/análise , Fator X/genética , Feminino , Humanos , Dados de Sequência Molecular , FenótipoRESUMO
Using the techniques of molecular biology, we made a chimeric Factor IX by replacing the first epidermal growth factor-like domain with that of Factor VII. The resulting recombinant chimeric molecule, Factor IXVIIEGF1, had at least a twofold increase in functional activity in the one-stage clotting assay when compared to recombinant wild-type Factor IX. The increased activity was not due to contamination with activated Factor IX, nor was it due to an increased rate of activation by Factor VIIa-tissue factor or by Factor XIa. Rather, the increased activity was due to a higher affinity of Factor IXVIIEGF1 for Factor VIIIa with a Kd for Factor VIIIa about one order of magnitude lower than that of recombinant wild-type Factor IXa. In addition, results from animal studies show that this chimeric Factor IX, when infused into a dog with hemophilia B, exhibits a greater than threefold increase in clotting activity, and has a biological half-life equivalent to recombinant wild-type Factor IX.
Assuntos
Fator de Crescimento Epidérmico/metabolismo , Fator IX/metabolismo , Hemofilia B/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Clorometilcetonas de Aminoácidos/farmacologia , Animais , DNA Recombinante/metabolismo , Compostos de Dansil/farmacologia , Cães , Fator de Crescimento Epidérmico/genética , Fator IX/genética , Fator IX/imunologia , Fator IX/isolamento & purificação , Fator IXa/metabolismo , Fator VIIIa/metabolismo , Fator VIIa/farmacologia , Fator XIa/farmacologia , Fator Xa/metabolismo , Inibidores do Fator Xa , Humanos , Tempo de Tromboplastina Parcial , Proteínas Recombinantes de Fusão/isolamento & purificaçãoRESUMO
BACKGROUND: Warfarin is a mainstay of therapy for conditions associated with an increased risk of thromboembolic events. However, the use of this common agent is fraught with complications and little is known regarding inter-individual variation in warfarin response. OBJECTIVE: We tested for association between single nucleotide polymorphisms (SNPs) in VKORC1 and CYP2C9 and average weekly warfarin dose required to maintain patients at their desired anticoagulation target. METHODS: The sample consisted of 93 European-American patients from anticoagulation clinics at the University of North Carolina at Chapel Hill. Data on mean weekly warfarin dose were collected over a mean treatment period of 20.6 months. ANCOVA models were used and haplotype analysis was performed. RESULTS: Three of six VKORC1 SNPs were found to be very strongly associated with the average warfarin dose required to achieve the target international normalised ratio (INR; p<0.0001). The mean weekly dose by genotype ranged from approximately 27 to 47 mg. There was no evidence for an association between either of the two CYP2C9 polymorphisms studied, CYP2C9*2 and CYP2C9*3. CYP2C9*3 was significantly (p = 0.05) associated with average warfarin dosage after adjustment for VKORC1*1173. CONCLUSIONS: These results are of considerable clinical interest and confirm recently published results regarding the role of these two genes in modifying warfarin metabolism and maintenance dosage. The consistent findings regarding the role of VKORC1 and CYP2C9 in warfarin metabolism and maintenance dosage represent a clinically useful proof of principal for the use of pharmacogenomic information in medicine and may lead to improved understanding of warfarin's actions.
Assuntos
Anticoagulantes/administração & dosagem , Predisposição Genética para Doença , Oxigenases de Função Mista/genética , Polimorfismo de Nucleotídeo Único/genética , Varfarina/administração & dosagem , Adulto , Idoso , Idoso de 80 Anos ou mais , Hidrocarboneto de Aril Hidroxilases/genética , Citocromo P-450 CYP2C9 , Feminino , Frequência do Gene/genética , Haplótipos , Humanos , Coeficiente Internacional Normatizado , Masculino , Pessoa de Meia-Idade , Vitamina K Epóxido Redutases , População BrancaRESUMO
Vitamin K-dependent carboxylation, an essential posttranslational modification catalyzed by gamma-glutamyl carboxylase, is required for the biological functions of proteins that control blood coagulation, vascular calcification, bone metabolism, and other important physiological processes. Concomitant with carboxylation, reduced vitamin K (KH2) is oxidized to vitamin K epoxide (KO). KO must be recycled back to KH2 by the enzymes vitamin K epoxide reductase and vitamin K reductase in a pathway known as the vitamin K cycle. Our current knowledge about the enzymes of the vitamin K cycle is mainly based on in vitro studies of each individual enzymes under artificial conditions, which are of limited usefulness in understanding how the complex carboxylation process is carried out in the physiological environment. In this chapter, we review the current in vitro activity assays for vitamin K cycle enzymes. We describe the rationale, establishment, and application of cell-based assays for the functional study of these enzymes in the native cellular milieu. In these cell-based assays, different vitamin K-dependent proteins were designed and stably expressed in mammalian cells as reporter proteins to accommodate the readily used enzyme-linked immunosorbent assay for carboxylation efficiency evaluation. Additionally, recently emerged genome-editing techniques TALENs and CRISPR-Cas9 were used to knock out the endogenous enzymes in the reporter cell lines to eliminate the background. These cell-based assays are easy to scale up for high-throughput screening of inhibitors of vitamin K cycle enzymes and have been successfully used to clarify the genotypes and their clinical phenotypes of enzymes of the vitamin K cycle.
Assuntos
Ensaios Enzimáticos/métodos , NAD(P)H Desidrogenase (Quinona)/química , Vitamina K Epóxido Redutases/química , Vitamina K/química , Animais , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Processamento de Proteína Pós-Traducional/genética , Vitamina K/antagonistas & inibidores , Vitamina K/metabolismo , Vitamina K 1/análogos & derivados , Vitamina K 1/químicaRESUMO
Integration of the analytical framework and experimental tools of metabolic engineering with emerging technologies such as DNA microarrays and directed evolution stands to dramatically improve the approaches by which strain improvement and biocatalyst design are pursued in the future. Progress in genomics and applied molecular biology, together with increasing emphasis on renewable resource utilization for chemical production, has advanced metabolic engineering to the forefront of biotechnological interest.
Assuntos
Microbiologia Industrial , Biologia MolecularRESUMO
Vitamin K-dependent proteins require carboxylation of certain glutamates for their biological functions. The enzymes involved in the vitamin K-dependent carboxylation include: gamma-glutamyl carboxylase (GGCX), vitamin K epoxide reductase (VKOR) and an as-yet-unidentified vitamin K reductase (VKR). Due to the hydrophobicity of vitamin K, these enzymes are likely to be integral membrane proteins that reside in the endoplasmic reticulum. Therefore, structure-function studies on these enzymes have been challenging, and some of the results are notably controversial. Patients with naturally occurring mutations in these enzymes, who mainly exhibit bleeding disorders or are resistant to oral anticoagulant treatment, provide valuable information for the functional study of the vitamin K cycle enzymes. In this review, we discuss: (i) the discovery of the enzymatic activities and gene identifications of the vitamin K cycle enzymes; (ii) the identification of their functionally important regions and their active site residues; (iii) the membrane topology studies of GGCX and VKOR; and (iv) the controversial issues regarding the structure and function studies of these enzymes, particularly, the membrane topology, the role of the conserved cysteines and the mechanism of active site regeneration of VKOR. We also discuss the possibility that a paralogous protein of VKOR, VKOR-like 1 (VKORL1), is involved in the vitamin K cycle, and the importance of and possible approaches for identifying the unknown VKR. Overall, we describe the accomplishments and the remaining questions in regard to the structure and function studies of the enzymes in the vitamin K cycle.
Assuntos
Coagulação Sanguínea , Carbono-Carbono Ligases/metabolismo , NAD(P)H Desidrogenase (Quinona)/metabolismo , Vitamina K Epóxido Redutases/metabolismo , Vitamina K/metabolismo , Sequência de Aminoácidos , Animais , Carbono-Carbono Ligases/química , Carbono-Carbono Ligases/genética , Regulação Enzimológica da Expressão Gênica , Genótipo , Humanos , Modelos Moleculares , Dados de Sequência Molecular , NAD(P)H Desidrogenase (Quinona)/genética , Fenótipo , Conformação Proteica , Relação Estrutura-Atividade , Vitamina K Epóxido Redutases/química , Vitamina K Epóxido Redutases/genéticaRESUMO
The 3'-terminus of 26 S rRNA from the sea urchin Lytechinus variegatus has been determined by oligonucleotide fingerprinting, S1 nuclease mapping and terminal nucleotide analysis. There are two species of 26 S rRNA of approximately equal abundance, one 19 nucleotides longer than than other.
Assuntos
RNA Ribossômico/genética , Ouriços-do-Mar/genética , Animais , Sequência de Bases , Endonucleases , Endonucleases Específicas para DNA e RNA de Cadeia SimplesRESUMO
Ribosomal RNA genes (rDNA) from Lytechinus variegatus were isolated by selective heat denaturation of main band DNA followed by single-strand specific nuclease (S1) treatment to remove the single-stranded DNA. After S1 nuclease treatment the partially purified fraction contained 10% rDNA, representing a 50-fold purification. Preparative CsCl centrifugation of this fraction resulted in highly purified rDNA with an average molecular weight of 1.9 - 10(7) and no single-strand breaks. High molecular weight sea urchin DNA was refractory to selective heat denaturation. DNA with an average molecular weight of greater than or equal to 2.9 - 10(7) was only 60-80% denatured after heating 13 degrees C above the Tm, whereas, DNA with an average molecular weight of less than or equal to 1.9 - 10(7) was 98% denatured. This phenomenon appears not to be due to time, buffer, or pH, but is dependent on size.
Assuntos
DNA de Cadeia Simples/isolamento & purificação , DNA/isolamento & purificação , RNA Ribossômico/biossíntese , Ouriços-do-Mar/metabolismo , Transcrição Gênica , Animais , Genes , Peso Molecular , Desnaturação de Ácido Nucleico , Ribonucleases , Ouriços-do-Mar/análise , TemperaturaRESUMO
A sea urchin ribosomal DNA 1.9 kilobase BamHI fragment adjacent to the 5' end of the 18 S gene has been mapped with the restriction enzymes, XhoI, EcoRi, SmaI and HinfI. A 270 basepair fragment which most likely contains the 5' end of the presumed primary transcript of rRNA was identified by hybridization of [32P]DNA fragments to total nuclear RNA separated on methylmercury hydroxide gels and bound to diazobenzyloxymethyl paper. Under these denaturing conditions the size of Lytechinus variegatus precursor rRNA was determined to be 7.2 kilobases (33 S).
Assuntos
Clonagem Molecular , Genes , RNA Ribossômico/genética , Animais , Enzimas de Restrição do DNA , DNA Recombinante/metabolismo , Escherichia coli/genética , Peso Molecular , Plasmídeos , Capuzes de RNA/genética , RNA de Transferência/genética , Ouriços-do-MarRESUMO
The binding of Ca2+ induces a conformational change in factor IX which can be monitored with conformation specific antibodies. Anti-FIX:Mg(II) antibodies recognize a conformational epitope (FIX') that can be induced by several metal ions such as Ca2+, Mg2+, Mn2+ and Ba2+, while anti-FIX:Ca(II) antibodies recognize a conformational epitope (FIX*) that can be only induced by Ca2+ and Sr2+ ions (Liebman et al., J. Biol. Chem., vol. 262 (1987) pp. 7605-7612). The latter conformation is essential for the function of factor IX. In this study we tried to identify residues in the Gla-domain of factor IX which are involved in binding to anti-factor IX:Mg(II) and anti-factor IX:Ca(II) antibodies. For this we substituted residues in recombinant human factor IX for those of factor X or factor VII. The substitution of residues 1-40 of factor IX by those of factor VII eliminated binding to both types of antibodies. Re-introduction of factor IX specific residues increased the binding to conformation specific anti-factor IX antibodies, but reduced the binding to conformation specific anti-factor VII antibodies, indicating that the structural integrity of the Gla-domain was not seriously affected by the mutations. We provide evidence that residues 33, 39 and 40 of human factor IX are important for binding to anti-factor IX:Mg(II) antibodies, while residues 1-11 are important for binding to anti-factor IX:Ca(II) antibodies.
Assuntos
Anticorpos/química , Epitopos/química , Fator IX/química , Fator IX/imunologia , Conformação Proteica , Sequência de Aminoácidos , Sítios de Ligação de Anticorpos , Linhagem Celular , Fator IX/metabolismo , Humanos , Rim , Cinética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , TransfecçãoRESUMO
Mutations in the high-affinity sulfonylurea receptor (SUR)-1 cause one of the severe recessively inherited diffuse forms of congenital hyperinsulinism or, when associated with loss of heterozygosity, focal adenomatosis. We hypothesized that SUR1 mutations would render the beta-cell insensitive to sulfonylureas and to glucose. Stimulated insulin responses were compared among eight patients with diffuse hyperinsulinism (two mutations), six carrier parents, and ten normal adults. In the patients with diffuse hyperinsulinism, the acute insulin response to intravenous tolbutamide was absent and did not overlap with the responses seen in either adult group. There was positive, albeit significantly blunted, acute insulin response to intravenous dextrose in the patients with diffuse hyperinsulinism. Graded infusions of glucose, to raise and then lower plasma glucose concentrations over 4 h, caused similar rises in blood glucose but lower peak insulin levels in the hyperinsulinemic patients. Loss of acute insulin response to tolbutamide can identify children with diffuse SUR1 defects. The greater response to glucose than to tolbutamide indicates that ATP-sensitive potassium (KATP) channel-independent pathways are involved in glucose-mediated insulin release in patients with diffuse SUR1 defects. The diminished glucose responsiveness suggests that SUR1 mutations and lack of KATP channel activity may contribute to the late development of diabetes in patients with hyperinsulinism independently of subtotal pancreatectomy.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Hiperinsulinismo/congênito , Hiperinsulinismo/genética , Insulina/metabolismo , Mutação/fisiologia , Canais de Potássio Corretores do Fluxo de Internalização , Canais de Potássio/genética , Receptores de Droga/genética , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Glucose/farmacologia , Heterozigoto , Humanos , Hipoglicemiantes/farmacologia , Injeções Intravenosas , Secreção de Insulina , Masculino , Valores de Referência , Receptores de Sulfonilureias , Tolbutamida/farmacologiaRESUMO
Post-translational modification of glutamate to gamma carboxyl glutamate is required for the activity of vitamin K-dependent proteins. Carboxylation is accomplished by the enzyme gamma glutamyl carboxylase (GGCX) which requires the propeptide-containing substrate and three co-substrates: reduced vitamin K, CO2, and O2. Most propeptides bind tightly to GGCX and all of the Glu residues that will be modified are modified during one binding event. Complete carboxylation is thus dependent upon the rate of carboxylation and the dissociation rate constant of the substrate from the GGCX enzyme. If the propeptide is released before carboxylation is complete, partially carboxylated vitamin K-dependent proteins are produced. The rate of carboxylation is mainly controlled by the level of reduced vitamin K available for the reactions while the dissociation rate constant is dependent upon both the propeptide and the Gla domain of the substrate. In addition, there are allosteric effects that increase the rate of dissociation of the fully carboxylated substrates. Carboxylation requires the abstraction of a proton from the 4-carbon of glutamate by reduced vitamin K and results in the conversion of vitamin K to vitamin K epoxide. The vitamin K epoxide must be recycled to vitamin K before it can be reused, a reaction catalyzed by the enzyme vitamin K epoxide reductase (VKOR). The gene for VKOR has recently been identified but the enzyme itself has not been purified to homogeneity. It appears, however, that most of the variability observed in patients response to warfarin may be attributed to variability in the VKOR gene.
Assuntos
Vitamina K/fisiologia , Sequência de Aminoácidos , Animais , Sítios de Ligação , Carbono/química , Citoplasma/metabolismo , Variação Genética , Ácido Glutâmico/química , Humanos , Cinética , Oxigenases de Função Mista/química , Modelos Biológicos , Modelos Químicos , Dados de Sequência Molecular , Oxigênio/metabolismo , Processamento de Proteína Pós-Traducional , Prótons , Fatores de Tempo , Vitamina K/metabolismo , Vitamina K Epóxido Redutases , Varfarina/farmacologiaRESUMO
The presence of tissue factor is essential for factor VIIa (FVIIa) to reach its full catalytic potential. The previous work in this laboratory demonstrated that substitution of the EGF1 domain of factor VIIa with that of factor IX (FVII((IXegf1))a) results in a substantial decrease in TF-binding affinity and catalytic activity. Supporting simulations of the solution structures of Ca(2+)-bound factor VIIa and FVII((IXegf1))a with tissue factor are provided. Mutants are generated, based on the simulation model, to study the effect of EGF1 substitution on catalytic activity. The simulations show larger Gla-EGF1 and EGF1-EGF2 inter-domain motions for FVII((IXegf1))a than for factor VIIa. The catalytic domain of the chimeric factor VIIa has been disturbed and several surface loops in the catalytic domain of FVII((IXegf1))a (Loop 170s (170-182), Loop 1 (185-188) and Loop 2 (221A-225)) manifest larger position fluctuations than wild-type. The position of Loop 140s (142-152) of FVII((IXegf1))a, near the N terminus insertion site of the catalytic domain, shifts relative to factor VIIa, resulting in a slight alteration of the active site. The results suggest that these four loops mediate the effect of the EGF1 domain substitution on the S1 site and catalytic residues. To test the model, we prepared mutations of these surface loops, including four FVII mutants, D186A, K188A, L144A and R147A, a FVII mutant with multiple mutations (MM3: L144A+R147A+D186A) and a FVII mutant with Loop 170s partially deleted, Loop 170s(del). The catalytic activities towards a small peptidyl substrate decreased 2.4, 4.5 and 9-fold for Loop 170s(del)a (a, activated), L144Aa and D186Aa, respectively, while MM3a lost almost all catalytic activity. The combined results of the simulations and mutants provide insight into the mechanism by which tissue factor enhances factor VIIa catalytic activity.
Assuntos
Domínio Catalítico , Fator de Crescimento Epidérmico/química , Fator VIIa/química , Fator VIIa/metabolismo , Tromboplastina/metabolismo , Substituição de Aminoácidos/genética , Apoproteínas/química , Apoproteínas/genética , Apoproteínas/metabolismo , Sítios de Ligação , Catálise , Quimotripsina/química , Simulação por Computador , Fator VII/química , Fator VII/genética , Fator VII/metabolismo , Fator VIIa/genética , Humanos , Ligação de Hidrogênio , Cinética , Movimento (Física) , Mutação/genética , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Software , Soluções , Relação Estrutura-Atividade , Tromboplastina/químicaRESUMO
It has been asserted that any comprehensive understanding of cocaine abuse and its treatment will require attention to both behavioral and pharmacological variables. Although the preclinical literature evaluating the effects of pharmacological variables on cocaine self-administration has been extensively reviewed, no comprehensive review of the effects of environmental variables on cocaine self-administration has been published. The present review summarizes and critiques the preclinical findings on environmental determinants of cocaine self-administration. The influence of environmental variables on the effects of pharmacological interventions on cocaine self-administration are also described. Several environmental variables have been shown to affect cocaine self-administration, including unit dose, schedule of cocaine delivery, schedules of nondrug stimuli, behavioral history, conditioned stimuli, food deprivation, exposure to stress, and rearing environment. Among these variables, unit dose, schedule of cocaine delivery, availability of alternative nondrug reinforcers, food deprivation, and rearing environment have also been shown to alter pharmacological treatment effects on cocaine self-administration. Thus, drug effects on cocaine self-administration are malleable and dependent upon the environmental context within which they occur. Suggestions for future research on the effects of these and other environmental variables on cocaine self-administration and its pharmacological treatment are presented.
Assuntos
Transtornos Relacionados ao Uso de Cocaína/tratamento farmacológico , Transtornos Relacionados ao Uso de Cocaína/psicologia , Cocaína/farmacologia , Inibidores da Captação de Dopamina/farmacologia , Animais , Cocaína/administração & dosagem , Inibidores da Captação de Dopamina/administração & dosagem , Humanos , AutoadministraçãoRESUMO
The 1191-bp sequence which includes the 5' end of 18S X rDNA and its adjacent spacer have been determined for a cloned fragment of sea urchin rDNA. The 5' end of 33S precursor rRNA (pre-rRNA) has been located by S1 nuclease mapping and primer extension. Pre-rRNA appears to be initiated at an A, 658 bp upstream from the 5' terminus of 18S rRNA. The first nucleotide of 18S rRNA was also analyzed by S1 nuclease mapping and found to correspond to T in the nontranscribed DNA strand. Comparisons of the transcription initiation region in rDNA and the 5' end of 18S X rDNA with the corresponding regions in other eukaryotes reveal no significant nucleotide sequence homology in the precursor portion while the 5' end of 18S X rDNA is highly conserved.