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1.
Osteoarthritis Cartilage ; 24(10): 1786-1794, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27266646

RESUMO

OBJECTIVE: The anterior cruciate ligament transection (ACLT) rabbit osteoarthritis (OA) model confers permanent knee instability and induces joint degeneration. The degeneration process is complex, but includes chondrocyte apoptosis and OA-like loss of cartilage integrity. Previously, we reported that activation of a volume-sensitive Cl(-) current (ICl,vol) can mediate cell shrinkage and apoptosis in rabbit articular chondrocytes. Our objective was therefore to investigate whether ICl,vol was activated in the early stages of the rabbit ACLT OA model. DESIGN: Adult Rabbits underwent unilateral ACLT and contralateral arthrotomy (sham) surgery. Rabbits were euthanized at 2 or 4 weeks. Samples were analyzed histologically and with assays of cell volume, apoptosis and electrophysiological characterization of ICl,vol. RESULTS: At 2 and 4 weeks post ACLT cartilage appeared histologically normal, nevertheless cell swelling and caspase 3/7 activity were both significantly increased compared to sham controls. In cell-volume experiments, exposure of chondrocytes to hypotonic solution led to a greater increase in cell size in ACLT compared to controls. Caspase-3/7 activity, an indicator of apoptosis, was elevated in both ACLT 2wk and 4wk. Whole-cell currents were recorded with patch clamp of chondrocytes in iso-osmotic and hypo-osmotic external solutions under conditions where Na(+), K(+) and Ca(2+) currents were minimized. ACLT treatment resulted in a large increase in hypotonic-activated chloride conductance. CONCLUSION: Changes in chondrocyte ion channels take place prior to the onset of apparent cartilage loss in the ACLT rabbit model of OA. Further studies are needed to investigate if pharmacological inhibition of ICl,vol decreases progression of OA in animal models.


Assuntos
Condrócitos , Animais , Ligamento Cruzado Anterior , Lesões do Ligamento Cruzado Anterior , Cartilagem Articular , Modelos Animais de Doenças , Osteoartrite , Osteoartrite do Joelho , Coelhos
2.
Int J Immunogenet ; 42(3): 204-7, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25809846

RESUMO

The two-nucleotide deletion recently detected in the mannose-binding lectin 2 gene in purebred and crossbred domestic pigs was not found among 68 wild boars representing 4 populations from Europe and Asia. This suggests that the deletion is a result of breeding and/or genetic drift/bottle necks.


Assuntos
Lectina de Ligação a Manose/genética , Sus scrofa/genética , Animais , Áustria , República Tcheca , Frequência do Gene , Haplótipos , Mutação INDEL , Japão , Dados de Sequência Molecular , Polimorfismo de Nucleotídeo Único , Deleção de Sequência , Suécia
3.
Anim Genet ; 46(5): 571-5, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26202474

RESUMO

Because of increasing litter size in Western pig breeds, additional teats are desirable to increase the capacity for nursing offspring. We applied genome-wide SNP markers to detect QTL regions that affect teat number in a Duroc population. We phenotyped 1024 animals for total teat number. A total of 36 588 SNPs on autosomes were used in the analysis. The estimated heritability for teat number was 0.34 ± 0.05 on the basis of a genomic relationship matrix constructed from all SNP markers. Using a BayesC method, we identified a total of 18 QTL regions that affected teat number in Duroc pigs; 9 of the 18 regions were newly detected.


Assuntos
Estudo de Associação Genômica Ampla , Glândulas Mamárias Animais , Locos de Características Quantitativas , Sus scrofa/genética , Animais , Teorema de Bayes , Cruzamento , Mapeamento Cromossômico , Feminino , Marcadores Genéticos , Tamanho da Ninhada de Vivíparos , Fenótipo , Polimorfismo de Nucleotídeo Único , Sus scrofa/classificação
4.
Int J Immunogenet ; 40(2): 131-9, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22672630

RESUMO

The single nucleotide polymorphism (SNP) G949T in the mannose-binding lectin ( MBL ) 1 gene has been associated with low MBL-A concentration in serum and detected at different frequencies in various European pig populations. However, the origin of this SNP is not known. Part of the MBL1 gene was sequenced in 12 wild boar/Large White crossbred pigs from the second backcross (BC 2 ) generation in a family material originating from two wild boar x Large White intercrosses. Also, MBL-A serum concentration was measured in the entire BC 2 generation (n = 45). Furthermore, the genotypes of 68 wild boars from Sweden, Austria, the Czech Republic, and Japan were determined in regard to five previously described SNPs in MBL1 . The T allele of G949T was present among the BC 2 animals. MBL-A serum concentration in the BC 2 animals showed a bimodal distribution, with one-third of the animals at levels between 0.7 and 1.6 µg mL(-1) and the remaining pigs at levels around 13 µg mL(-1) . There was a co-variation between the presence of the T allele and low MBL-A concentration in serum. The genotyping of the wild boars revealed differences between populations. The T allele of G949T was not detected in the Austrian and Japanese samples and is thus unlikely to be an original feature of wild boars. In contrast, it was present at high frequency (0.35) among the Swedish wild boars, probably representing a founder effect. Five MBL1 haplotypes were resolved. Only two of these were present among the Japanese wild boars compared to four in each of the European populations. This difference may reflect differences in selection pressure and population history.


Assuntos
Lectina de Ligação a Manose/sangue , Lectina de Ligação a Manose/genética , Sus scrofa/genética , Animais , Áustria , Sequência de Bases , República Tcheca , Frequência do Gene , Genótipo , Haplótipos , Japão , Polimorfismo de Nucleotídeo Único , Receptores de Reconhecimento de Padrão/genética , Análise de Sequência de DNA/veterinária , Suécia
5.
Anim Genet ; 44(2): 130-8, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22590984

RESUMO

The phylogeography of the porcine X chromosome has not been studied despite the unique characteristics of this chromosome. Here, we genotyped 59 single nucleotide polymorphisms (SNPs) in 312 pigs from around the world, representing 39 domestic breeds and wild boars in 30 countries. Overall, widespread commercial breeds showed the highest heterozygosity values, followed by African and American populations. Structuring, as inferred from FST and analysis of molecular variance, was consistently larger in the non-pseudoautosomal (NPAR) than in the pseudoautosomal regions (PAR). Our results show that genetic relationships between populations can vary widely between the NPAR and the PAR, underscoring the fact that their genetic trajectories can be quite different. NPAR showed an increased commercial-like genetic component relative to the PAR, probably because human selection processes to obtain individuals with high productive parameters were mediated by introgressing boars rather than sows.


Assuntos
Filogenia , Sus scrofa/genética , Cromossomo X/genética , Análise de Variância , Animais , Teorema de Bayes , Simulação por Computador , Análise Discriminante , Feminino , Frequência do Gene , Genética Populacional , Masculino , Filogeografia , Polimorfismo de Nucleotídeo Único/genética , Análise de Componente Principal , Fatores Sexuais , Especificidade da Espécie , Sus scrofa/classificação
6.
Anim Genet ; 41(2): 113-21, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19793267

RESUMO

Herein, we report the variability among 57 porcine homologs of murine coat colour-related genes. We identified single nucleotide polymorphisms (SNPs) and insertions/deletions (InDels) within 44 expressed gene sequences by aligning eight pig complementary DNA (cDNA) samples. The sequence alignment revealed a total of 485 SNPs and 15 InDels. The polymorphisms were then validated by performing matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) with reference DNA samples obtained from 384 porcine individuals. Of the 384 individuals, three parents of the experimental F(2) family were included to detect polymorphisms between them for linkage mapping. We also genotyped previously reported polymorphisms of 12 genes, and one SNP each in three genes that were detected by performing a BLAST search of the Trace database. A total of 211 SNPs and three InDels were successfully genotyped from our porcine DNA panel. We detected SNPs in 33 of the 44 genes among the parents of an experimental F(2) family and then constructed a linkage map of the 33 genes for this family. The linkage assignment of each gene to the porcine chromosomes was consistent with the location of the BAC clone in the porcine genome and the corresponding gene sequence. We confirmed complete substitutions of EDNRB and MLPH in the Jinhua and Clawn miniature breeds, respectively. Furthermore, we identified polymorphic alleles exclusive to each pig group: 13 for Jinhua, two for Duroc, three for Meishan, four for the Japanese wild boar, one for the Clawn miniature pig and four for the Potbelly pig.


Assuntos
Cor de Cabelo/genética , Polimorfismo Genético , Suínos/genética , Animais , Mapeamento Cromossômico , Mutação INDEL , Camundongos , Polimorfismo de Nucleotídeo Único , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
7.
Science ; 272(5264): 1020-3, 1996 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-8638125

RESUMO

The adenomatous polyposis coli gene (APC) is mutated in familial adenomatous polyposis and in sporadic colorectal tumors, and its product binds to the adherens junction protein beta-catenin. Overexpression of APC blocks cell cycle progression. The APC-beta-catenin complex was shown to bind to DLG, the human homolog of the Drosophila discs large tumor suppressor protein. This interaction required the carboxyl-terminal region of APC and the DLG homology repeat region of DLG. APC colocalized with DLG at the lateral cytoplasm in rat colon epithelial cells and at the synapse in cultured hippocampal neurons. These results suggest that the APC-DLG complex may participate in regulation of both cell cycle progression and neuronal function.


Assuntos
Proteínas do Citoesqueleto/metabolismo , Proteínas de Drosophila , Hormônios de Inseto/metabolismo , Transativadores , Proteínas Supressoras de Tumor , Proteína da Polipose Adenomatosa do Colo , Sequência de Aminoácidos , Animais , Ciclo Celular , Células Cultivadas , Colo/química , Colo/citologia , Proteínas do Citoesqueleto/análise , Proteínas do Citoesqueleto/química , Drosophila , Células Epiteliais , Epitélio/química , Imunofluorescência , Hipocampo/química , Hipocampo/citologia , Humanos , Hormônios de Inseto/análise , Hormônios de Inseto/química , Camundongos , Dados de Sequência Molecular , Neurônios/química , Neurônios/citologia , Ligação Proteica , Proteínas Recombinantes de Fusão/metabolismo , Sinapses/química , beta Catenina
8.
Osteoarthritis Cartilage ; 16(9): 1083-91, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18378169

RESUMO

OBJECTIVES: A collagen scaffold has been long used in order to enhance the regeneration of articular cartilage. In the present study, we investigate the effectiveness of a concentration-gradient (CG) collagen that is designed to recruit efficiently the mesenchymal stem cells (MSCs) to the central region of the full-thickness cartilage defects via haptotaxis. METHODS: The present study used Cellmatrix (0.3% type I collagen; Nitta gelatin, Osaka, Japan) as the collagen material. We prepared 33%CG collagen gel and 50%CG collagen gel. No gradient collagen gel served as negative control. Full-thickness cartilage defects were created at the patella groove of the rabbit knee, to which the three different collagen gels were transplanted. Bromodeoxyuridine (BrdU) positive, proliferating cells were enumerated and localized, whereas the histological grading score for cartilage regeneration was counted. The expression of type I and type II collagens was evaluated by immunohistochemistry. We also confirmed that the MSCs migrate toward the collagen substrate of higher concentration in a stringently in vitro haptotactic manner. RESULTS: Enumeration of the BrdU-positive cells demonstrated that 33%CG collagen gel recruited a significantly larger number of proliferating cells to the central region of the cartilage defect. The histological grading score for the regenerated cartilage treated with 33%CG collagen gel was superior to the other groups. CONCLUSIONS: CG collagen scaffold recruits effectively the MSCs to the center of full-thickness cartilage defect and enhances regeneration of the full-thickness cartilage defect.


Assuntos
Cartilagem Articular/patologia , Condrócitos/metabolismo , Condrogênese/fisiologia , Colágeno/metabolismo , Matriz Extracelular/metabolismo , Cicatrização/fisiologia , Animais , Cartilagem Articular/lesões , Cartilagem Articular/transplante , Matriz Extracelular/patologia , Imuno-Histoquímica , Transplante de Células-Tronco Mesenquimais/métodos , Coelhos , Engenharia Tecidual/métodos , Alicerces Teciduais/estatística & dados numéricos
9.
J Thromb Haemost ; 5(12): 2352-9, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17922804

RESUMO

BACKGROUND: Fibrin polymerization is mediated by interactions between knobs 'A' and 'B' exposed by thrombin cleavage, and holes 'a' and 'b' always present in fibrinogen. The role of A:a interactions is well established, but the roles of knob:hole interactions A:b, B:b or B:a remain ambiguous. OBJECTIVES: To determine whether A:b or B:b interactions have a role in thrombin-catalyzed polymerization, we examined a series of fibrinogen variants with substitutions altering holes 'a': gamma364Ala, gamma364His or gamma364Val. METHODS: We examined thrombin- and reptilase-catalyzed fibrinopeptide release by high-performance liquid chromatography, fibrin clot formation by turbidity, fibrin clot structure by scanning electron microscopy (SEM) and factor (F) XIIIa-catalyzed crosslinking by sodium dodecylsulfate polyacrylamide gel electrophoresis. RESULTS: Thrombin-catalyzed fibrinopeptide A release was normal, but fibrinopeptide B release was delayed for all variants. The variant fibrinogens all showed markedly impaired thrombin-catalyzed polymerization; polymerization of gamma364Val and gamma364His were more delayed than gamma364Ala. There was absolutely no polymerization of any variant with reptilase, which exposed only knobs 'A'. SEM showed that the variant clots formed after 24 h had uniform, ordered fibers that were thicker than normal. Polymerization of the variant fibrinogens was inhibited dose-dependently by the addition of either Gly-Pro-Arg-Pro (GPRP) or Gly-His-Arg-Pro (GHRP), peptides that specifically block holes 'a' and 'b', respectively. FXIIIa-catalyzed crosslinking between gamma-chains was markedly delayed for all the variants. CONCLUSION: These results demonstrate that B:b interactions are critical for polymerization of variant fibrinogens with impaired holes 'a'. Based on these data, we propose a model wherein B:b interactions participate in protofibril formation.


Assuntos
Batroxobina/metabolismo , Fibrinogênio/metabolismo , Fibrinopeptídeo A/metabolismo , Fibrinopeptídeo B/metabolismo , Trombina/metabolismo , Sítios de Ligação , Ligação Competitiva , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Fator XIIIa/metabolismo , Fibrinogênio/química , Fibrinogênio/genética , Fibrinopeptídeo A/química , Fibrinopeptídeo B/química , Cinética , Microscopia Eletrônica de Varredura , Modelos Biológicos , Mutação , Nefelometria e Turbidimetria , Oligopeptídeos/metabolismo , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína
10.
Nucleic Acids Res ; 29(18): 3835-40, 2001 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-11557815

RESUMO

DNA helicase B is a major DNA helicase in mouse FM3A cells. A temperature-sensitive mutant defective in DNA replication, tsFT848, isolated from FM3A cells, has a heat-labile DNA helicase B. In this study, we purified DNA helicase B from mouse FM3A cells and determined partial amino acid sequences of the purified protein. By using a DNA probe synthesized according to one of the partial amino acid sequences, a cDNA was isolated, which encoded a 121.5 kDa protein containing seven conserved motifs for DNA/RNA helicase superfamily members. A database search revealed similarity between DNA helicase B and the alpha subunit of exodeoxyribonuclease V of a number of prokaryotes including Escherichia coli RecD protein, but no homologous protein was found in yeast. The cDNA encoding DNA helicase B from tsFT848 was sequenced and a mutation was found between DNA/RNA helicase motifs IV and V.


Assuntos
Adenosina Trifosfatases/genética , DNA Helicases/genética , Replicação do DNA/genética , DNA Complementar/genética , Proteínas de Escherichia coli , Adenosina Trifosfatases/isolamento & purificação , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Animais , Clonagem Molecular , DNA Helicases/isolamento & purificação , DNA Helicases/metabolismo , DNA Complementar/química , Escherichia coli/enzimologia , Exodesoxirribonuclease V , Exodesoxirribonucleases/genética , Camundongos , Dados de Sequência Molecular , Mutação , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Temperatura , Células Tumorais Cultivadas
11.
Biochim Biophys Acta ; 1398(3): 377-81, 1998 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-9655940

RESUMO

We cloned a cDNA encoding the mouse homologue to human Bloom's syndrome gene (BLM). The deduced amino acid sequence of mouse Blm showed 76% identity to the human sequence with very high homology in seven consecutive domains characteristic of DNA and RNA helicases. The expression of mBLM mRNA was examined in various tissues. Extremely high expression was observed in the testis as compared with other tissues. The mBLM mRNA level in the testis began to increase 12-14 days after birth, corresponding to the appearance of cells in the pachytene phase.


Assuntos
Adenosina Trifosfatases/genética , Síndrome de Bloom/genética , DNA Helicases/genética , Testículo/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar , Expressão Gênica , Humanos , Masculino , Camundongos , Dados de Sequência Molecular , RNA Mensageiro , RecQ Helicases , Homologia de Sequência de Aminoácidos
12.
J Thromb Haemost ; 3(5): 983-90, 2005 May.
Artigo em Inglês | MEDLINE | ID: mdl-15869595

RESUMO

BACKGROUND AND OBJECTIVES: Analysis of dysfibrinogens has improved our understanding of molecular defects and their effects on the function of intact fibrinogen. To eliminate the influence of plasma heterozygous molecules, we synthesized and analyzed recombinant-variant fibrinogens. METHODS: We synthesized two recombinant-variant fibrinogens with a single amino acid substitution at the 15Gly residue in the Bbeta-chain: namely, Bbeta15Cys and Bbeta15Ala. RESULTS: Western blotting analysis of purified fibrinogen revealed the existence of a small amount of a dimeric form only for Bbeta15Cys fibrinogen. For Bbeta15Cys fibrinogen, functional analysis indicated (a) no thrombin-catalyzed fibrinopeptide B (FPB) release and (b) markedly impaired lateral aggregation in thrombin- and reptilase-catalyzed fibrin polymerizations. For Bbeta15Ala fibrinogen, such analysis indicated slight impairments of both thrombin-catalyzed FPB release and lateral aggregation in thrombin-catalyzed fibrin polymerization, but nearly normal lateral aggregation in reptilase-catalyzed fibrin polymerization. These impaired lateral aggregations were accompanied by thinner fibrin fiber diameters (determined by scanning electron microscopy of the corresponding fibrin clots). CONCLUSION: We conclude that a region adjacent to Bbeta15Gly plays important roles in lateral aggregation not only in desA fibrin polymerization, but also in desAB fibrin polymerization, and we speculate that the marked functional differences between Bbeta15A and Bbeta15C fibrinogens in FPB release and fibrin polymerization might not only be due to the presence of a substituted cysteine residue in Bbeta15C fibrinogen, but also to the existence of disulfide-bonded forms. Finally, our data indicate that the Bbeta15Gly residue plays important roles in FPB release and lateral aggregation of protofibrils.


Assuntos
Fibrinogênio/química , Fibrinopeptídeo B/química , Proteínas Recombinantes/química , Alanina/química , Animais , Batroxobina/química , Western Blotting , Catálise , Cromatografia Líquida de Alta Pressão , Cisteína/química , Dimerização , Dissulfetos/química , Eletroforese em Gel de Poliacrilamida , Fibrina/química , Fibrina/ultraestrutura , Glicina/química , Heterozigoto , Humanos , Immunoblotting , Cinética , Microscopia Eletrônica de Varredura , Mutagênese , Ligação Proteica , Venenos de Serpentes , Trombina/química , Fatores de Tempo
13.
Exp Hematol ; 24(2): 116-22, 1996 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-8641332

RESUMO

We investigated the properties of granulocyte-macrophage (GM) progenitors obtained from patients with juvenile chronic myelogenous leukemia (JCML). CD34+ bone marrow cells from a patient with JCML, unlike normal bone marrow cells, generated a large number of cells in serum-containing liquid culture without additional hematopoietic factors. In serum-deprived culture, only granulocyte colony-stimulating factor (G-CSF) had a modest stimulatory effect on GM colony growth in normal controls. In contrast, stem cell factor (SCF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin-3 (IL-3), as well as G-CSF, when tested individually, generated significant numbers of GM colonies in some JCML patients. All two-factor combinations generated significantly more GM colonies in JCML compared with normal controls. In particular, GM-CSF plus SCF exerted an interaction equivalent to the all-factor combination in most patients. Significant differences in the size and constituent cells of GM colonies stimulated by GM-CSF plus SCF were also observed. These results suggest that one possible mechanism for the excessive cell production in JCML is the strong proliferation of GM progenitors induced by hematopoietic factors, especially SCF. According to immunofluorescent analysis, however, it is unlikely that this multiplication is due to an increase in the cell surface expression of c-kit receptors on JCML progenitors.


Assuntos
Fatores de Crescimento de Células Hematopoéticas/farmacologia , Células-Tronco Hematopoéticas/patologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Antígenos CD34 , Medula Óssea/patologia , Divisão Celular/efeitos dos fármacos , Pré-Escolar , Meios de Cultura Livres de Soro/farmacologia , Sinergismo Farmacológico , Feminino , Sangue Fetal/fisiologia , Fator Estimulador de Colônias de Granulócitos/farmacologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Lactente , Interleucina-3/farmacologia , Masculino , Proteínas de Neoplasias/biossíntese , Proteínas Proto-Oncogênicas c-kit/biossíntese , Proteínas Recombinantes/farmacologia , Fator de Células-Tronco/farmacologia , Células Tumorais Cultivadas , Ensaio Tumoral de Célula-Tronco
14.
FEBS Lett ; 362(2): 201-4, 1995 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-7536689

RESUMO

Nerve growth factor (NGF) induces tyrosine phosphorylation of various cellular proteins to activate multiple signal transduction pathways. We show that one of these proteins is paxillin, a cytoskeletal component associated with adhesion plaques. Phospho-amino acid analysis showed that NGF stimulated phosphorylation of its serine in addition to tyrosine residues. Tyrosine phosphorylation of paxillin by NGF was blocked by the pretreatment of the cells with cytochalasin D, an inhibitor of actin polymerization. These results suggest that phosphorylation of paxillin is involved in the signaling pathway of NGF in PC12 cells.


Assuntos
Moléculas de Adesão Celular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Fatores de Crescimento Neural/farmacologia , Células PC12/metabolismo , Fosfoproteínas/metabolismo , Tirosina/análogos & derivados , Animais , Western Blotting , Citocalasina D/farmacologia , Técnicas de Imunoadsorção , Cinética , Paxilina , Fosforilação , Fosfosserina/metabolismo , Fosfotirosina , Ratos , Transdução de Sinais , Tirosina/metabolismo
15.
J Thromb Haemost ; 2(3): 468-75, 2004 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15009465

RESUMO

BACKGROUND AND OBJECTIVES: Analysis of dysfibrinogens has provided useful information aiding our understanding of molecular defects in fibrin polymerization. We have already reported impaired fibrin polymerization in a variant fibrinogen (gammaArg275Cys), the Cys being located in the D:D interface. Since this substitution occurred in a heterozygous individual, interpretation of the functional analysis was complicated. We tried to resolve this complication by synthesizing a recombinant variant fibrinogen. METHODS: A variant gamma-chain expression plasmid was transfected into Chinese hamster ovary cells expressing normal human fibrinogen Aalpha- and Bbeta-chains. The recombinant variant fibrinogen (gamma275C) was purified using an immunoaffinity column, and we compared its structure and functions with those of normal recombinant fibrinogen (gamma275R) and plasma variant fibrinogen. RESULTS: Mass analyses showed the existence of disulfide-linked Cys in both patient and recombinant variant fibrinogens. Functional analyses indicated that both fibrin polymerization and gamma-gamma dimer formation were markedly impaired in the variant fibrinogen. The impairments were much more pronounced in gamma275C than in plasma variant fibrinogen. In addition, scanning electron microscopic observation of fibrin clots made from gamma275C revealed less dense fibrin fiber bundles and larger fiber diameter than in those made from gamma275R, and also the existence of many aberrant fibrin fibers with tapered ends. CONCLUSIONS: These results indicate that gammaArg275 has an important residue affecting the structure and function of the gamma-chain C-terminal domain. However, the variant D:D interface can interact with that of the normal fibrinogen existing in a heterozygous patient with dysfibrinogenemia.


Assuntos
Dissulfetos/metabolismo , Fibrinogênio/química , Fibrinogênio/metabolismo , Substituição de Aminoácidos , Arginina , Sequência de Bases , Sítios de Ligação , Catálise , Cisteína , Primers do DNA , Fator XIIIa/metabolismo , Fibrina/química , Fibrina/metabolismo , Fibrina/ultraestrutura , Fibrinogênio/ultraestrutura , Humanos , Cinética , Microscopia Eletrônica de Varredura , Peso Molecular , Mutagênese Sítio-Dirigida , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Trombina/metabolismo
16.
J Thromb Haemost ; 2(8): 1359-67, 2004 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15304042

RESUMO

BACKGROUND AND OBJECTIVES: We have previously reported that recombinant gamma 275Cys fibrinogen exhibits a marked impairment of functions as well as aberrant fibrin clot and bundle structures, as compared with wild-type, gamma 275Arg, and plasma fibrinogen from a heterozygous proband. Since gamma Arg275His mutations have also been reported in 10 families, we synthesized recombinant gamma 275His fibrinogen and gamma 275Ala fibrinogen (as a control) and analyzed and compared them with gamma 275Cys and gamma 275Arg. METHODS: A variant gamma-chain expression plasmid was transfected into Chinese hamster ovary cells expressing normal human fibrinogen A alpha- and B beta-chains. After purification of the recombinant variant fibrinogens, we performed functional analyzes for thrombin-catalyzed fibrin polymerization and factor XIIIa (FXIIIa)-catalyzed gamma-gamma dimer formation from fibrin or fibrinogen and also ultrastructural analysis of fibrin clots and bundles. RESULTS: By comparison with both gamma 275His and gamma 275Ala fibrinogens, recombinant gamma 275Cys fibrinogen exhibited a more impaired gamma-gamma dimer formation from fibrin or fibrinogen, a more aberrant fibrin clot structure, and thicker fibers in fibrin bundles. In 1 : 1 mixtures of gamma 275Arg and gamma 275Cys fibrinogens or gamma 275Arg and gamma 275His fibrinogens, thrombin-catalyzed fibrin polymerization and both fibrin clot and fiber structures showed some compensation (as compared with gamma 275Cys or gamma 275His alone). CONCLUSION: These results strongly suggest that an amino acid substitution of gamma 275Arg alone disrupts D:D interactions in thrombin-catalyzed fibrin polymerization and the formation of fibrin bundles and fibrin clots. Moreover, the existence of a subsequent disulfide-linked Cys in gamma 275C fibrinogen augments the impairment caused by a His or Ala substitution.


Assuntos
Fator XIIIa/química , Fibrina/química , Fibrinogênio/genética , Trombina/metabolismo , Alanina/química , Animais , Células CHO , Catálise , Cricetinae , Reagentes de Ligações Cruzadas/farmacologia , Eletroforese em Gel de Poliacrilamida , Fibrina/ultraestrutura , Fibrinogênio/química , Histidina/química , Humanos , Microscopia Eletrônica de Varredura , Plasmídeos/metabolismo , Polímeros/química , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacologia , Espectrofotometria , Trombina/química , Fatores de Tempo , Raios Ultravioleta
17.
J Thromb Haemost ; 1(2): 275-83, 2003 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-12871501

RESUMO

We found two heterozygous dysfibrinogenemias, designated fibrinogen Kosai and fibrinogen Ogasa. Kosai was associated with arteriosclerosis obliterans but Ogasa showed no bleeding or thrombotic tendencies. The plasma fibrinogen concentrations from the two propositi (Ogasa and Kosai) were much lower when determined by the thrombin-time method (0.94 and 1.06 g L(-1), respectively) than when determined by the immunological method (2.87 and 2.72 g L(-1), respectively). We performed DNA sequencing and functional analyses to clarify the relationship between the structural and functional abnormalities. Genetic analysis of PCR-amplified DNA from the propositi identified the heterozygous substitution Bbeta15Gly-->Cys (GGT-->TGT). Western blotting analysis of purified fibrinogen revealed the existence of albumin-fibrinogen complexes. Functional analyses indicated that compared with the normal control, the propositi's fibrinogen released only half the normal amount of fibrinopeptide B and showed markedly impaired polymerization. In addition, the observation of thinner fibers in fibrin clots (by scanning electron microscopy) indicated markedly defective lateral aggregation in the variant fibrinogens. The impaired functions may be due to the substitution of Cys for Bbetao15Gly plus the existence of some additional disulfide-bonded forms.


Assuntos
Afibrinogenemia/sangue , Afibrinogenemia/genética , Fibrinogênios Anormais/genética , Fibrinopeptídeo B/metabolismo , Adulto , Substituição de Aminoácidos , Batroxobina/farmacologia , Feminino , Fibrinogênios Anormais/química , Fibrinogênios Anormais/fisiologia , Humanos , Técnicas In Vitro , Microscopia Eletrônica de Varredura , Pessoa de Meia-Idade , Mutação Puntual , Trombina/farmacologia
18.
Transplantation ; 60(10): 1109-12, 1995 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-7482717

RESUMO

Hepatic artery thrombosis after orthotopic liver transplantation is a serious complication, especially in children. We report our experience with intensive anticoagulant therapy during and after living-related liver transplantation in pediatric recipients. Twenty-four patients between 5 months and 15 years of age were studied. The mean diameter of the anastomosed hepatic arteries was 2.7 mm. The anticoagulant therapy consisted of low-molecular-weight heparin, antithrombin III concentrates, prostaglandin E1, fresh frozen plasma, and a protease inhibitor. The profiles of the coagulation and fibrinolytic systems were monitored by measuring several parameters, including plasma levels of thrombin-antithrombin III complex, antithrombin III, plasmin-alpha 2 plasmin inhibitor complex, fibrin degradation product D-dimer, tissue type-plasminogen activator, and plasminogen activator inhibitor-1. Acceleration of the coagulation system and delayed recovery of the fibrinolytic system were observed during the early postoperative days. The plasma level of antithrombin III activity was maintained within the normal range by the administration of antithrombin III concentrates. None of the recipients developed hepatic artery thrombosis. Children have been reported to be at a greater risk of developing hepatic artery thrombosis than adults due to the small diameters of their hepatic arteries and the postoperative hypercoagulable state. We believe that the intensive anticoagulation therapy described in this study, the main concept of which is the early correction of imbalance between the coagulant and anticoagulant systems, could become a model for the prevention of hepatic artery thrombosis in pediatric liver transplantation patients.


Assuntos
Artéria Hepática , Transplante de Fígado/efeitos adversos , Trombose/prevenção & controle , Adolescente , Anticoagulantes/uso terapêutico , Antitrombina III/análise , Criança , Pré-Escolar , Humanos , Lactente
19.
Neuroscience ; 50(1): 99-106, 1992 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-1407563

RESUMO

An antiserum against basic fibroblast growth factor was characterized by immunoblot experiments and used to investigate immunohistochemically the projection fields and fine structures of basic fibroblast growth factor-containing cerebellar Purkinje cells. The antiserum demonstrated clearly purified basic fibroblast growth factor and basic fibroblast growth factor-like molecules of the same molecular weight in homogenates of the adult rat cerebellum. Light and electron microscopic immunohistochemistry revealed that a large number of Purkinje cells, if not all, send immunoreactive dendrites to the molecular layer and basic fibroblast growth factor-containing axons to the deep cerebellar and lateral vestibular nuclei, where basic fibroblast growth factor nerve terminals form synapses with the soma and dendrites of neurons labeled weakly with basic fibroblast growth factor. Nerve cells with basic fibroblast growth factor had immunoreaction deposits mainly in free ribosomes, those attached to the endoplasmic reticulum and in the nuclear euchromatin. These findings suggest that basic fibroblast growth factor is present in cerebellar Purkinje cells and undergoes two modes of transport, one to axon terminals and the other to nuclear euchromatin, known as the RNA transcription zone.


Assuntos
Cerebelo/citologia , Fator 2 de Crescimento de Fibroblastos/análise , Células de Purkinje/citologia , Animais , Axônios/ultraestrutura , Cerebelo/ultraestrutura , Immunoblotting , Imuno-Histoquímica , Masculino , Microscopia Imunoeletrônica , Terminações Nervosas/ultraestrutura , Células de Purkinje/ultraestrutura , Ratos , Ratos Wistar
20.
Neuroscience ; 59(3): 651-62, 1994 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-7516507

RESUMO

The development of basic fibroblast growth factor-like immunoreactivity was investigated in the nuclei, cell bodies and processes of Purkinje cells with attention to basic fibroblast growth factor-containing neuronal input to the deep cerebellar nuclei. Immunoblot analysis with the use of the antisera against basic fibroblast growth factor revealed that crude homogenate of the developing rat cerebellum exhibits a main band with the same molecular weight (18,000 mol. wt) as basic fibroblast growth factor in all the postnasal stages examined. Cerebellar cells were not labeled with the antisera during embryonic life. Under light microscopy, basic fibroblast growth factor-like immunoreactivity was detected initially in cortical cells located close to deep cerebellar fissures of the newborn rat but not in superficial cortical regions. It was difficult to determine whether or not they are Purkinje cells at the fusiform stage. On postnatal day 7, immunoreactive Purkinje cells were identified throughout the cerebellar cortex, and they expressed basic fibroblast growth factor-like immunoreactivity mainly in the apical cytoplasm and proximal dendrites. From postnatal day 14 to postnatal day 28, basic fibroblast growth factor-like immunoreactivity was noted not only throughout the cytoplasm of Purkinje cells but also in the nuclei of the immunopositive cells. Our statistical analysis showed that Purkinje cells with nuclear immunoreaction peaked on postnatal day 21. At these stages, nerve fibers immunoreactive for basic fibroblast growth factor were numerous in the cerebellar medulla and deep cerebellar nuclei. After postnatal day 42, Purkinje cells with intense immunoreactivity in the nuclei showed a marked decrease in number, and immunoreactive structures were distributed in the cerebellum in a fashion similar to that in adult rats. Electron microscopy demonstrated that immunoreactivity was located mainly in the apical cytoplasm of Purkinje cells on postnatal day 7 and throughout the cytoplasm and in the nuclear euchromatin from postnatal day 14 to postnatal day 28, as was expected from light-microscopic observations. Immunoreactivity, even though distributed diffusely in the cytoplasm, was absent from the lumen of endoplasmic reticulum and mitochondria. A small population of Purkinje cell axon terminals forming synapses with the soma and dendrites of deep cerebellar nucleus neurons began to express basic fibroblast growth factor on postnatal day 21. This is much later than the starting age for synaptogenesis between Purkinje cells and deep cerebellar nucleus neurons. The age-dependent changes in the localization of basic fibroblast growth factor within Purkinje cell nucleus, soma and processes suggest a complex transport system of this factor within Purkinje cells during postnatal development.


Assuntos
Envelhecimento/fisiologia , Cerebelo/crescimento & desenvolvimento , Fator 2 de Crescimento de Fibroblastos/metabolismo , Células de Purkinje/fisiologia , Análise de Variância , Animais , Animais Recém-Nascidos , Axônios/fisiologia , Axônios/ultraestrutura , Eletroforese em Gel de Poliacrilamida , Fator 1 de Crescimento de Fibroblastos/análise , Fator 2 de Crescimento de Fibroblastos/análise , Immunoblotting , Imuno-Histoquímica , Masculino , Microscopia Imunoeletrônica , Células de Purkinje/citologia , Células de Purkinje/ultraestrutura , Ratos , Ratos Wistar
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