RESUMO
Progressive supranuclear palsy - Richardson syndrome (PSP-RS) was first described in 1964 by Steele et al. Tau pathology has not been reported in the hypoglossal nuclei of PSP-RS patients, whereas Steele et al. described gliosis with no remarkable neuronal losses in the hypoglossal nucleus. This study aimed to investigate the distribution and degree of tau pathology-associated neurodegeneration, with an emphasis on the hypoglossal nucleus, in patients with PSP-RS. Six clinicopathologically proven PSP-RS cases were included in this study. All patients were clinicopathologically and immunohistochemically re-evaluated. This study confirmed the following neuropathological characteristics of PSP-RS: (1) neurodegeneration usually affects the striatonigral system and cerebellar dentate nucleus; (2) the cerebellar afferent system in PSP-RS is affected by absent-to-mild neurodegeneration; and (3) the extent of tau distribution throughout the central nervous system is greater than the extent of neurodegeneration. Furthermore, we found that subthalamic neurodegeneration was more prominent in the ventromedial region than in the dorsolateral region. Nevertheless, the tau pathology showed no remarkable differences between these two sites. Interestingly, the tau pathology was frequently observed in the hypoglossal nuclei of PSP-RS patients. Gradient neurodegeneration of the subthalamus and tau pathology in the hypoglossal nucleus could be regarded as essential pathological features of PSP-RS.
Assuntos
Biomarcadores , Degeneração Neural/patologia , Subtálamo/patologia , Paralisia Supranuclear Progressiva/diagnóstico , Tauopatias/patologia , Idoso , Idoso de 80 Anos ou mais , Autopsia , Biomarcadores/análise , Biomarcadores/metabolismo , Cerebelo/patologia , Progressão da Doença , Feminino , Humanos , Nervo Hipoglosso/patologia , Masculino , Pessoa de Meia-Idade , Degeneração Neural/diagnóstico , Neurônios/metabolismo , Neurônios/patologia , Paralisia Supranuclear Progressiva/patologia , Tauopatias/diagnóstico , Proteínas tau/análise , Proteínas tau/metabolismoRESUMO
Intravenous immunoglobulin (IVIg) preparations are reportedly effective in inhibiting the relapse of multiple sclerosis (MS), but few reports have investigated the effect of IVIg on dendritic cells (DCs), which are thought to be involved in such relapses. In the system that uses monokines to differentiate DCs from peripheral blood monocytes (Mo-DCs), we investigated the effect of immunoglobulin G (IgG) on these antigen-presenting cells. Using monocytes derived from healthy volunteers, IgG partially inhibited the expression of CD1a, a marker of immature DCs (imDCs), and CD40 and CD80, which are markers associated with T cell activation. In contrast, IgG enhanced the expression of CD83, a marker of mature DCs (mDCs). Furthermore, IgG markedly inhibited the expression of CD49d [very late activation antigen (VLA)-4 alpha4-integrin], the adhesion molecule required for mDCs to cross the blood-brain barrier. We obtained similar results on all the aforementioned cell surface molecules investigated in both healthy controls and MS patients. In addition, IgG treatment of cells from both healthy controls and MS patients inhibited the production of interleukin (IL)-12, a cytokine associated with mDC differentiation, but did not inhibit the production of IL-10. These results suggested the possibility that IgG treatment, apart from its known ability to regulate inflammation, may help to prevent relapses of MS by controlling DC maturation, consequently inhibiting invasion of immune cells into the central nervous system and affecting the cytokine profile.
Assuntos
Células Dendríticas/imunologia , Imunoglobulina G/imunologia , Esclerose Múltipla Recidivante-Remitente/imunologia , Adulto , Antígenos CD/metabolismo , Diferenciação Celular/imunologia , Células Cultivadas , Células Dendríticas/citologia , Feminino , Humanos , Imunoglobulinas Intravenosas/imunologia , Integrina alfa4/metabolismo , Interleucina-10/biossíntese , Interleucina-12/biossíntese , Masculino , Pessoa de Meia-Idade , Monócitos/citologia , Monócitos/imunologiaRESUMO
Human type-2 tissue factor pathway inhibitor (TFPI-2), also known as placental protein 5, is a 32 kDa serine proteinase inhibitor consisting of three tandemly arranged Kunitz-type inhibitor domains homologous to tissue factor pathway inhibitor. TFPI-2 strongly inhibits a wide variety of serine proteinases including trypsin, chymotrypsin, plasmin, kallikrein and blood coagulation factor XIa. In this study, we have isolated and characterized a genomic clone from an artificial chromosome genomic library that encodes the entire human TFPI-2 gene. The human TFPI-2 gene spans approximately 7 kb and consists of five exons and four introns. Each Kunitz-type domain is encoded by a single exon, similar to that observed for murine TFPI-2 and other Kunitz-type proteinase inhibitors. A total of 535 bp of the 3'-flanking region contain two probable polyadenylation sites (AATAAA) at +4297 and +4314. A single transcription initiation site was identified by oligo-capping and reverse transcription-PCR analysis. Transient transfection of reporter plasmids containing segments of the 5'-flanking region into human transformed bone marrow endothelial cells and glioblastoma cells identified an 85 bp region (-224 to -139) sufficient for transcription of the human TFPI-2 gene.
Assuntos
Glicoproteínas/genética , Proteínas da Gravidez/genética , Regiões Promotoras Genéticas , Inibidores de Serina Proteinase/genética , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação/genética , Linhagem Celular , Primers do DNA/genética , DNA Complementar/genética , DNA Complementar/metabolismo , Éxons , Expressão Gênica , Genoma Humano , Humanos , Íntrons , Dados de Sequência Molecular , Mapeamento por Restrição , Deleção de Sequência , Fatores de Transcrição/metabolismo , TransfecçãoRESUMO
BACKGROUND: We have recently found that a hormonally active form of vitamin D, 1alpha,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], exerts anticoagulant effects by upregulating the expression of an anticoagulant glycoprotein, thrombomodulin (TM), and downregulating the expression of a critical coagulation factor, tissue factor (TF), in monocytic cells including human peripheral monocytes. In this study, we investigated the counteracting effects of 1,25(OH)(2)D(3) and its potent analogs on TF induction and TM downregulation by tumor necrosis factor and oxidized LDL in monocytic cells and the modulatory effects of potent analogs on TF and TM expression. METHODS AND RESULTS: Effects of 1,25(OH)(2)D(3) and its potent synthetic analogs (22R)-22-methyl-20-epi-1,25(OH)(2)D(3) (KY3) and 22-oxacalcitriol on TF and TM antigen levels, cell surface activities, and mRNA levels in monocytic cells were examined. 1, 25(OH)(2)D(3) and its potent analogs showed anticoagulant effects in monocytic cells by downregulating TF and upregulating TM expression, counteracting the effects of tumor necrosis factor and oxidized LDL. KY3 was most potent in its regulatory effect on TF and TM expression. CONCLUSIONS: Because KY3 has the highest affinity for vitamin D receptor, our findings suggest that TF and TM regulation by 1, 25(OH)(2)D(3) analogs is also mediated by vitamin D receptor. The 1, 25(OH)(2)D(3) analogs KY3 and 22-oxacalcitriol may have the potential to serve as an agent for preventing and treating atherosclerotic and other cytokine-mediated thrombotic diseases and as a tool for studying the molecular mechanisms of TF and TM regulation.
Assuntos
Calcitriol/análogos & derivados , Calcitriol/farmacologia , Lipoproteínas LDL/farmacologia , Trombomodulina/efeitos dos fármacos , Tromboplastina/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Linhagem Celular , Células Cultivadas , Regulação para Baixo/efeitos dos fármacos , Humanos , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Trombomodulina/metabolismo , Tromboplastina/metabolismo , Células U937/efeitos dos fármacos , Células U937/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
Using the epidermis-specific cytokeratin 14 promoter to deliver HGF exclusively from epidermal keratinocytes, we have examined the potential of hepatocyte growth factor (HGF) secreted from the normal environment to control morphogenesis. The transgenic mice displayed a significant increase of the number of melanocytes and their precursors in embryos starting not later than 16.5 dpc, and then after birth an explosive increase of dermal melanocytes started within 1 week, and these melanocytes were maintained throughout the entire life of the mice. Thus, HGF acts as a paracrine agent to promote survival, proliferation and differentiation of melanocyte precursors in vivo, and eventually causes melanocytosis. Loss of E-cadherin expression in dermal melanocyte precursors suggests that HGF caused dermal localization of melanocytes and their precursors by down-regulation of E-cadherin molecules.
Assuntos
Fator de Crescimento de Hepatócito/genética , Queratinócitos/fisiologia , Melanócitos/fisiologia , Dermatopatias/genética , Animais , Animais Recém-Nascidos , Caderinas/genética , Caderinas/metabolismo , Orelha Externa , Regulação da Expressão Gênica no Desenvolvimento , Fator de Crescimento de Hepatócito/metabolismo , Oxirredutases Intramoleculares/metabolismo , Queratinas/genética , Melanócitos/patologia , Camundongos , Camundongos Transgênicos , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-kit/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Pele/embriologia , Pele/crescimento & desenvolvimento , Pele/patologia , Dermatopatias/patologia , Pigmentação da Pele/genética , Fator de Células-Tronco/metabolismoRESUMO
A photoactive reaction center complex was isolated from a thermophilic green sulfur bacterium, Chlorobium tepidum under anaerobic conditions. The electron transfer occurred from heme c to the photo-oxidized reaction center chlorophyll, P840+, with a half time (t1/2) of 110 or 340 microseconds at 24 or 12 degrees C, respectively. Optical measurements under multiflash excitations indicated that two hemes function as the immediate electron donors to P840+. SDS-PAGE analysis of the RC complex in combination with the N-terminal amino acid sequence analyses revealed five subunit bands; a core protein (65 kDa), the light harvesting bacteriochlorophyll alpha protein (41 kDa), a protein with 2[4Fe-4S] clusters (31 kDa), monoheme cytochrome c (22 kDa), and a 18-kDa protein whose function is unknown. The reaction center complex, thus, contains two molecules of cytochrome c per P840.
Assuntos
Bactérias/metabolismo , Grupo dos Citocromos c/metabolismo , Transporte de Elétrons , Fotossíntese/fisiologia , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Sequência de Aminoácidos , Temperatura Baixa , Meia-Vida , Luz , Complexos de Proteínas Captadores de Luz , Modelos Biológicos , Dados de Sequência Molecular , Complexo de Proteínas do Centro de Reação Fotossintética/química , Complexo de Proteínas do Centro de Reação Fotossintética/efeitos da radiação , Análise de Sequência , EspectrofotometriaRESUMO
Tissue factor pathway inhibitor-2 (TFPI-2), also known as placental protein 5, is a 32 kDa extracellular matrix-associated serine proteinase inhibitor consisting of three tandemly-arranged Kunitz-type domains. Two overlapping genomic clones containing sequences encoding murine TFPI-2 were isolated from a lambda FIXII 129 SVJ mouse genomic library, and the complete nucleotide sequence of the gene was determined. The murine TFPI-2 gene spans approximately 9.3 kilobases and consists of five exons and four introns. The nucleotide sequences surrounding all the exon-intron boundaries are highly conserved and obey the GT-AG rule. Each Kunitz-type domain is encoded by a single exon, similar to that observed for other Kunitz-type proteinase inhibitors. A total of 1,577 bp of the 3'-flanking region contains a probable polyadenylation site (ATTAAA) at +5,759 and an apparent cleavage or termination site (CATTG) at +6,170. The 5'-flanking region of the murine TFPI-2 gene contains a prototypical TATA box, a GC box and two CAAT boxes. In addition, several candidate transcription factor binding sites responsible for placenta-, endothelial cell-, and smooth muscle cell-specific expression of the TFPI-2 gene were also identified.
Assuntos
Glicoproteínas/genética , Proteínas da Gravidez/genética , Análise de Sequência de DNA , Sequência de Aminoácidos , Animais , Sequência de Bases , Genoma , Camundongos , Dados de Sequência Molecular , Fatores de Transcrição/genéticaRESUMO
A patient with clinical features consistent with pulmonary embolism showed no improvement despite therapy with tissue-plasminogen activator and full-dose heparin. Transvenous catheter suction biopsy was successful in establishing an antemortem histologic diagnosis of primary pulmonary artery leiomyosarcoma. Urgent surgical intervention was performed.
Assuntos
Biópsia/métodos , Leiomiossarcoma/patologia , Artéria Pulmonar/patologia , Neoplasias Vasculares/patologia , Idoso , Anticoagulantes/uso terapêutico , Cateterismo , Diagnóstico Diferencial , Feminino , Heparina/uso terapêutico , Humanos , Leiomiossarcoma/cirurgia , Ativadores de Plasminogênio/uso terapêutico , Pneumonectomia , Artéria Pulmonar/cirurgia , Embolia Pulmonar/diagnóstico , Embolia Pulmonar/tratamento farmacológico , Sucção , Ativador de Plasminogênio Tecidual/uso terapêutico , Neoplasias Vasculares/cirurgiaRESUMO
A new method was developed for analysis of biodegradable polyesters, which involves alkali hydrolysis of polyesters to the corresponding hydroxyacids and determination of the hydroxyacids by high-performance liquid chromatography. It can be used to monitor weight change in polyesters and change in molecular ratio of the hydroxyacid constituents of polyesters during in vitro and in vivo degradation.
Assuntos
Poliésteres/análise , Animais , Biodegradação Ambiental , Cromatografia Líquida de Alta Pressão/métodos , Hidrólise , Microesferas , Ratos , Ratos Sprague-DawleyRESUMO
We present a female patient who received an allogeneic bone marrow transplantation for primary refractory Philadelphia-positive acute biphenotypic leukemia. Since leukemic blasts were persistently present in peripheral blood and bone marrow, in spite of the evidence for engraftment of male donor hematopoiesis, we performed donor leukocyte transfusions and discontinued immunosuppression. An initial complete remission was obtained 15 weeks after allogeneic bone marrow transplantation, and lasted for 24 weeks. We concluded that the prominent mechanism for the eradication of the refractory leukemic clone in the patient was the graft-versus-leukemia effect.
Assuntos
Transplante de Medula Óssea , Imunossupressores/efeitos adversos , Transfusão de Leucócitos , Metilprednisolona/efeitos adversos , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Prednisolona/efeitos adversos , Adulto , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Medula Óssea/patologia , Transplante de Medula Óssea/imunologia , Células Clonais/imunologia , Células Clonais/patologia , Terapia Combinada , Resistencia a Medicamentos Antineoplásicos , Evolução Fatal , Feminino , Doença Enxerto-Hospedeiro/tratamento farmacológico , Doença Enxerto-Hospedeiro/etiologia , Reação Enxerto-Hospedeiro , Humanos , Imunossupressores/administração & dosagem , Masculino , Metilprednisolona/administração & dosagem , Células Neoplásicas Circulantes , Células-Tronco Neoplásicas/imunologia , Células-Tronco Neoplásicas/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/sangue , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Prednisolona/administração & dosagem , Recidiva , Indução de Remissão , Doadores de Tecidos , Transplante HomólogoRESUMO
Recently, we found N1,N8-diacetylspermidine (Ac2Spd) and N1,N12-diacetylspermine (Ac2Spm) in human urine, and noted that their amount increased significantly in patients with urogenital malignancies. Previous findings that simultaneous reference to these diacetylpolyamines is useful in distinguishing cancer patients from healthy persons were confirmed by more recent analytical data on urine samples from several cancer patients. Further examination revealed that urinary Ac2Spm and Ac2Spd tended to decrease when cancer patients were treated and entered partial remission. In cases where the Ac2Spm and Ac2Spd levels were normal or near-normal after treatments, the prognosis of the patients was generally good. In contrast, when their level remained far above the normal limits after apparently effective treatment, the prognosis of the patients was poor. When a patient is in remission for more than 3 years, urinary levels of both Ac2Spm and Ac2Spd are stabilized and stay below the normal limits, with rare exceptions. The recurrence of a cancer as well as the complication of a second one during the period of follow-up examination was accompanied by elevation of urinary diacetylpolyamines. These observations indicate that urinary Ac2Spm and Ac2Spd are useful as prognostic indicators after treatment and during follow-up examination of cancer patients.
Assuntos
Biomarcadores Tumorais/uso terapêutico , Neoplasias/urina , Espermidina/análogos & derivados , Espermina/análogos & derivados , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias/diagnóstico , Neoplasias/terapia , Prognóstico , Espermidina/urina , Espermina/urina , Fatores de TempoRESUMO
N1,N8-Diacetylspermidine (Ac2Spd) and N1,N12 diacetylspermine (Ac2Spm), the occurrence of which in healthy human urine was demonstrated recently, increased much more frequently and markedly than total polyamines, acetylputrescine, N1-acetylspermidine and N8-acetylspermidine in patients with urogenital malignancies. Ac2Spd was hardly elevated in cases of benign disease, while Ac2Spm only infrequently stayed within normal limits in patients with malignant disorders. Urine samples from more than 90% of healthy persons, but fewer than 10% of patients with malignancies, gave values within normal limits for both Ac2Spd and Ac2Spm. Simultaneous reference to these diacetylpolyamines is therefore useful in distinguishing patients with malignancies from healthy persons.
Assuntos
Neoplasias/urina , Espermidina/urina , Espermina/análogos & derivados , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Espermina/urinaRESUMO
N1,N12-Diacetylspermine (DiAcSpm)-specific antibodies were raised in rabbits, using N-acetylspermine coupled to mercaptosuccinylated BSA via N-(4-maleimidobutyryloxy)-succinimide as an antigen. Highly DiAcSpm-specific antibodies were enriched from crude sera through a series of affinity-based fractionations. A competitive ELISA system, intended for measuring DiAcSpm in solution, was constructed using this antibody preparation, with N-acetylspermine coupled to a synthetic peptide via N-(8-maleimidocapryloxy)-succinimide as a solid phase antigen. The Ki value for DiAcSpm with this competitive ELISA system was 33 nM, and the cross-reactivity with DiAcSpm, AcSpm, DiAcSpd, N1-AcSpd, and N8-AcSpd was 100, 0.29, 0.20, 0.033, and 0.055%, respectively. This procedure can be applied to the determination of DiAcSpm in human urine samples, giving highly reproducible results. The coefficients of variation obtained were 6.7 and 4.2% for within-run and between-run precision, respectively. The correlation coefficient between DiAcSpm concentrations in urine estimated by ELISA and those by HPLC analysis was calculated to be 0. 99, and the regression equation was expressed as y = 1.04x + 0.026 microM.
Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Espermina/análogos & derivados , Sequência de Aminoácidos , Cromatografia de Afinidade , Cromatografia Líquida de Alta Pressão , Reações Cruzadas , Ensaio de Imunoadsorção Enzimática/normas , Humanos , Soros Imunes , Dados de Sequência Molecular , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Espermina/imunologia , Espermina/urinaRESUMO
We developed an improved system for the simultaneous measurement of free and acetylated polyamines, which comprised a HPLC pump, a separation column, an enzyme reactor, and an electrochemical detector, connected in series. Polyamines were separated with an isocratic elution system, and the separated polyamines were introduced into the enzyme reactor, in which they were deacetylated and oxidized to generate hydrogen peroxide. The amount of hydrogen peroxide generated was then determined with the electrochemical detector. Analysis of a mixture of nine standard polyamines including both free forms and acetylated derivatives with this method revealed that the analytical variables were satisfactory. For the analysis of polyamines in urine, pretreatment of samples with a weakly acidic ion exchange resin was necessary to reduce the interfering substances present in the urine. On successive determinations of polyamines in a urine sample, the coefficients of variation obtained were below 5.4%, except that for spermine (27.6%), and the analytical recovery rates were above 90%, except that for acetylputrescine (78.5%). The correlation coefficient between the total polyamine content in urine estimated by our method and that obtained by means of a commercially available enzymatic assay system was calculated to be 0.98, and the regression equation was expressed as y = 0.81 x + 0.89.
Assuntos
Poliaminas/urina , Putrescina/análogos & derivados , Espermina/urina , Acetilação , Cromatografia Líquida de Alta Pressão , Eletroquímica , Enzimas Imobilizadas , Humanos , Poliaminas/metabolismo , Putrescina/urina , Análise de RegressãoRESUMO
N8-Acetylspermidine was coupled to mercaptosuccinylated BSA using a bifunctional cross-linker, N-(4-maleimidobutyryloxy)succinimide, and the resulting conjugate was used to raise N1,N8-diacetylspermidine (DiAcSpd)-specific antibodies in rabbits. DiAcSpd-specific antibodies were enriched from crude sera through a series of affinity-based fractionations using ligands with structures mimicking those of DiAcSpd and monoacetylspermidines. With the N8-acetylspermidine-BSA conjugate as a solid phase antigen in a competitive ELISA system, the selectivity for DiAcSpd over other polyamine species was high, but competition by DiAcSpd added to the fluid phase was too weak for the system to be applicable to measurement of the concentration of DiAcSpd in human urine. In contrast, with the N1-acetylspermidine-BSA conjugate adsorbed on the ELISA plate, DiAcSpd efficiently competed for the same antibody, thus yielding a sensitive competitive ELISA system for measuring DiAcSpd. The Ki value for DiAcSpd with the latter competitive ELISA system was 54 nM, and the cross-reactivity with DiAcSpd, N1,N12-diacetylspermine, N8-acetylspermidine, N1-acetylspermidine, and acetylputrescine was 100, 1.2, 0.74, 0.12, and 0.08%, respectively. The DiAcSpd-specific antibodies and the competitive ELISA system developed in this study will prove to be useful for analyzing the urinary level of DiAcSpd, that was recently shown to be a promising diagnostic and prognostic indicator of malignant disorders.
Assuntos
Ensaio de Imunoadsorção Enzimática , Espermidina/análogos & derivados , Especificidade de Anticorpos , Reações Antígeno-Anticorpo , Ligação Competitiva , Cromatografia de Afinidade , Humanos , Sensibilidade e Especificidade , Espermidina/imunologia , Espermidina/urinaRESUMO
An analytical system developed for fractionating free and monoacetylated polyamines [Hiramatsu, K. et al. (1994) J. Biochem. 115, 584-589] was proved useful also in detecting diacetylpolyamines, namely N1,N8-diacetylspermidine (diAcSpd) and N1,N12-diacetylspermine (diAcSpm). Detection limits were 0.9 and 0.6 pmol (S/N = 5) for diAcSpd and diAcSpm, respectively. Analytical recovery and within-run variation were also satisfactory. Human urine samples were found to contain diAcSpd and diAcSpm. These polyamines were identified on the basis of the following observations: (i) their retention times were coincident with those of authentic samples; (ii) they were deacetylated to N8-acetylspermidine and monoacetyl- and free spermine, respectively, by acetylpolyamine amidohydrolase; and (iii) they were practically inert to direct oxidation by bacterial polyamine oxidase as were authentic samples. The amounts of eleven polyamine species including diAcSpd and diAcSpm in urine samples from 52 healthy persons were determined. Mean values for the major polyamine components were consistent with those reported by others. Although the amounts of diAcSpd and diAcSpm were very small, comprising only 1.4 and 0.46% of total polyamines, respectively, these two compounds were found to be always present in healthy human urine as regular constituents. Moreover, variation in their content among individuals was small, suggesting that excretion of these components in urine is strictly regulated.
Assuntos
Poliaminas Biogênicas/urina , Espermidina/análogos & derivados , Espermina/análogos & derivados , Biomarcadores/urina , Fracionamento Químico , Humanos , Valores de Referência , Espermidina/urina , Espermina/urinaRESUMO
Rhesus monkey cDNA for tissue factor pathway inhibitor (TFPI) was cloned by means of the reverse transcriptase-polymerase chain reaction, using liver mRNA, and its nucleotide sequence was determined by sequencing five independent clones. Monkey TFPI was found to have a signal peptide of 28 amino acid residues and to be a mature protein of 276 amino acid residues, in which three and seventeen amino acid residue substitutions compared to human TFPI were found, respectively. All the cysteine residues, three putative carbohydrate-linked asparagine residues, and the P1 amino acid residues of each of the three Kunitz inhibitor domains were conserved in the two species. Recombinant monkey TFPI (rTFPI) was isolated from the culture medium of transformed Chinese hamster ovary cells. Amino acid sequence analysis and immunoblotting analysis, using polyclonal and monoclonal antibodies, showed that the carboxyl-terminal basic part of Rhesus monkey rTFPI had been truncated. The inhibitory activity of monkey rTFPI was compared with that of human rTFPI without the carboxyl-terminal basic part. The prothrombin time of human plasma was slightly more prolonged by the addition of monkey rTFPI than by that of human rTFPI. However, no significant differences were found between the potencies of human and monkey rTFPI as to the inhibition of factor Xa and tissue factor-factor VIIa complex.
Assuntos
DNA Complementar/genética , Lipoproteínas/química , Macaca mulatta , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Humanos , Lipoproteínas/genética , Dados de Sequência Molecular , Proteínas Recombinantes/química , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
Dystrophin mRNA expressed in peripheral lymphocytes of individuals with X-linked Duchenne muscular dystrophy (DMD) has been used as a source material for mutation analysis. Here we present the first report of failure of isolation of nonsense dystrophin mRNA in lymphocytes but success in skeletal muscle in a female carrier of DMD. The mutation responsible for dystrophin-negative muscle fibers of the carrier was analysed by direct sequencing of the reverse transcription PCR product of dystrophin mRNA. In her peripheral lymphocytes, no nucleotide change was detected in the 14 kb long mRNA. Remarkably, a novel nucleotide change of C1682T in exon 12, changing glutamine codon to stop codon (Q492X) was found to be present in her skeletal muscle. This change was heterozygous. Analysis of her genomic DNA disclosed heterozygous C and T nucleotides at nt 1682, confirming the genomic origin of the nonsense mutation. Although dystrophin cDNA prepared from lymphocytes was sequenced again after subcloning, mutation-retaining clone could not be isolated. This lymphocyte-specific disappearance of nonsense mRNA strongly suggested tissue-specific skewing of X-inactivation. However, both paternal and maternal dystrophin alleles were shown to be equally expressed in lymphocytes as well as in muscle, indicating no skewing of X-inactivation in lymphocytes. We concluded that the dystrophin mRNA of the DMD carrier was destabilized in lymphocytes. Our results indicated that analysis of mRNA in lymphocytes is not enough for exact carrier diagnosis of Duchenne muscular dystrophy.
Assuntos
Códon sem Sentido/genética , Distrofina/genética , Linfócitos/química , Músculo Esquelético/química , Distrofia Muscular de Duchenne/genética , Adulto , Distrofina/análise , Feminino , Triagem de Portadores Genéticos , Humanos , RNA Mensageiro/análise , RNA Mensageiro/genéticaRESUMO
Human type-2 tissue factor pathway inhibitor (TFPI-2), also known as placental protein 5, is a 32-kDa serine proteinase inhibitor consisting of three tandemly arranged Kunitz-type domains homologous to tissue factor pathway inhibitor. TFPI-2 inhibits a variety of serine proteinases involved in coagulation and fibrinolysis through an arginine residue (R24) in its first Kunitz-type domain, which constitutes a putative P1 residue for the substrate recognition sites of these proteinases. As recent studies have shown that this P1 residue to be a glutamine in murine TFPI-2, we constructed, expressed, and purified a human TFPI-2 mutant with glutamine substituted for arginine at position 24 (R24Q TFPI-2). R24Q TFPI-2 lost approximately 90% of its inhibitory activity towards bovine trypsin and virtually all inhibitory activity towards human plasmin and the factor VIIa-tissue factor complex, emphasizing the importance of the P1 Arg24 residue in the inhibition of these serine proteinases. However, whereas wild-type TFPI-2 is a relatively weak inhibitor of human factor Xa amidolytic activity (IC50 approximately 1 microM), R24Q TFPI-2 exhibited enhanced inhibitory activity towards the amidolytic and coagulant activities of this proteinase with a Ki of 18 nM. While the molecular basis for the enhanced inhibition of human factor Xa by R24Q TFPI-2 is unknown, these data provide suggestive evidence that murine TFPI-2 may function as a serine proteinase inhibitor in spite of the absence of a P1 Arg or Lys residue.
Assuntos
Glicoproteínas/antagonistas & inibidores , Proteínas da Gravidez/antagonistas & inibidores , Inibidores de Serina Proteinase/farmacologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Eletroforese em Gel de Poliacrilamida , Glicoproteínas/genética , Glicoproteínas/isolamento & purificação , Humanos , Lactente , Dados de Sequência Molecular , Mutação Puntual , Proteínas da Gravidez/genética , Proteínas da Gravidez/isolamento & purificaçãoRESUMO
The very rapid clearance of human recombinant tissue factor pathway inhibitor (h-rTFPI) may result from its binding to vascular proteogly can and LDL receptor-related protein (LRP). To investigate the effect of factor Xa on the clearance of h-rTFPI, we developed a specific ELISA for h-rTFPI/factor Xa complex, and compared the pharmacokinetic parameters of h-rTFPI/factor Xa complex and the clearance rate of the cellular proteogly can-associated h-rTFPI/factor Xa complex with those of h-rTFPI by itself in rabbits. We found that the h-rTFPI/factor Xa complex disappeared from circulation at a rapid rate of clearance, having pharmacokinetic parameters similar to those of non-complexed h-rTFPI. After the rapid disappearance of the h-rTFPI complex from plasma, an intravenous injection of heparin resulted in a release of h-rTFPI/factor Xa complex into plasma. However, the recovery of heparin-releasable h-rTFPI/factor Xa decreased significantly in a time-dependent manner. Therefore, we examined the half-life of proteogly can-associated h-rTFPI/factor Xa and determined it to be 51 min, which was significantly shorter than that of h-rTFPI by itself (107 min). These results suggest that a complex formation of h-rTFPI with factor Xa promotes a clearance of proteogly can-associated h-rTFPI existing in the liver and kidney.