RESUMO
CONTEXT: Adefovir dipivoxil is a nucleotide analog that has demonstrated effective antiretroviral activity against human immunodeficiency virus (HIV) with once-daily administration. OBJECTIVE: To determine if adefovir confers antiretroviral or immunologic benefit when added to stable antiretroviral therapy. DESIGN: Multicenter, 24-week, randomized, double-blind, placebo-controlled study. Enrollment was conducted from June 3, 1996, through May 6, 1997. SETTING: Thirty-three US HIV treatment centers. PARTICIPANTS: Of 1171 patients screened, 442 patients infected with HIV receiving stable antiretroviral therapy for at least 8 weeks with plasma HIV RNA greater than 2500 copies/mL and CD4+ cell count above 0.20 x 10(9)/L were randomized. INTERVENTION: Patients were randomized to receive either a single 120-mg/d dose of adefovir dipivoxil (n = 219) or an indistinguishable placebo (n = 223). All patients received L-carnitine, 500 mg/d. Open-label adefovir was offered after 24 weeks and was continued until the end of the study. MAIN OUTCOME MEASURES: Changes in HIV RNA from baseline, based on area under the curve and CD4+ cell levels, adverse events, and effect of baseline genotypic resistance on response to adefovir. RESULTS: Patients assigned to adefovir demonstrated a 0.4-log10 decline from baseline in HIV RNA compared with no change in the placebo group (P<.001), which continued through 48 weeks. CD4+ cell counts did not change. During the initial 24 weeks, elevated hepatic enzyme levels (P<.001), gastrointestinal tract complaints (P<.001), and weight loss (P<.001) were associated with use of adefovir. Between 24 weeks and 48 weeks elevations in serum creatinine occurred in 60% of patients, usually returning to baseline after discontinuation of adefovir. Patients with lamivudine or lamivudine and zidovudine resistance mutations demonstrated anti-HIV effects with adefovir (P< or =.01 vs placebo group). CONCLUSIONS: This study suggests that once-daily adefovir therapy reduces HIV RNA and is active against isolates resistant to lamivudine or lamivudine and zidovudine. Nephrotoxicity occurred when treatment extended beyond 24 weeks but was reversible.
Assuntos
Adenina/análogos & derivados , Fármacos Anti-HIV/uso terapêutico , Infecções por HIV/tratamento farmacológico , Organofosfonatos , Inibidores da Transcriptase Reversa/uso terapêutico , Adenina/uso terapêutico , Adulto , Fármacos Anti-HIV/farmacologia , Contagem de Linfócito CD4 , Método Duplo-Cego , Resistência Microbiana a Medicamentos , Quimioterapia Combinada , Feminino , Genótipo , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , HIV-1/genética , Humanos , Lamivudina/farmacologia , Masculino , RNA Viral , Carga Viral , Zidovudina/farmacologiaRESUMO
9-(2-Phosphonylmethoxyethyl)-N6-cyclopropyl-2,6-diaminopurine (cpr-PMEDAP) is an acyclic nucleotide analog of the [9-(2-phosphonylmethoxyethyl)-] (PME) series containing a cyclopropyl substituent on the N6 position of the 2,6-diaminopurine (DAP) base. Growth inhibition assays in a broad range of tumor cell lines demonstrated that this analog had potent antiproliferative activity with IC50 values similar to those of the structurally related guanine analog 9-(2-phosphonylmethoxyethyl)guanine (PMEG). A substantially lower growth inhibitory effect was observed for the 2,6-diaminopurine analog, PMEDAP. To dissect the basis for these varying potencies, the metabolism of the three analogs was examined in a human pancreatic carcinoma cell line, BxPC-3. HPLC analysis of the intracellular metabolites demonstrated that the cpr-PMEDAP was deaminated to PMEG and subsequently phosphorylated to PMEG mono- and diphosphates (PMEGp and PMEGpp). The level of PMEGpp generated from cpr-PMEDAP-treated cells was 50% greater than the level generated from cells incubated with PMEG. The presence of PMEG in the DNA of cells incubated with cpr-PMEDAP confirmed that the cpr-PMEDAP was converted to PMEG. In contrast, PMEDAP was not deaminated to PMEG, but directly phosphorylated to PMEDAPp and PMEDAPpp. The adenylate deaminase inhibitor 2'-deoxycoformycin (dCF) inhibited the conversion of cpr-PMEDAP in a rat liver cytosolic extract and increased the IC50 value for growth inhibition by 40-fold. The antiproliferative activities of PMEG and PMEDAP were unaffected by dCF. Thus, it appears that cpr-PMEDAP, but not PMEDAP, is converted by an adenylate deaminase-like enzyme and functions as a prodrug of PMEG.
Assuntos
Adenina/análogos & derivados , Antineoplásicos/farmacologia , Guanina/análogos & derivados , Compostos Organofosforados/farmacologia , Pró-Fármacos/farmacologia , Adenina/metabolismo , Adenina/farmacologia , Animais , Antivirais/metabolismo , Antivirais/farmacologia , Divisão Celular/efeitos dos fármacos , Desaminação , Didesoxinucleosídeos/metabolismo , Guanina/farmacologia , Humanos , Pró-Fármacos/metabolismo , Ratos , Células Tumorais CultivadasRESUMO
In a recent phase II clinical study, 13 chronic hepatitis B-infected patients treated daily with 30 mg adefovir dipivoxil for 12 weeks displayed a median 4.1-log10 decrease in plasma hepatitis B virus (HBV)-DNA levels. The decline of viral load during therapy displayed a biphasic kinetic profile that was modeled to determine the efficacy of inhibition of viral production, as well as kinetic constants for the clearance of free virus and the loss of infected cells. Viral production was suppressed with an efficacy of 0.993 +/- 0.008, indicating that only 0.7% of viral production persisted during therapy. The initial, faster phase of viral load decline reflects the clearance of HBV particles from plasma with a half-life of 1.1 +/- 0.3 days, translating to a 48% daily turnover of the free virus. The second, slower phase of viral load decline closely mirrors the rate-limiting process of infected cell loss, with a half-life of 18 +/- 7 days. The duration of therapy required to completely eliminate the virus from plasma or suppress it to levels sufficient to induce seroconversion is a function of the half-life of the free virus, the half-life of infected cells, and the efficacy of inhibition of virus production from infected cells. These quantitative analyses provide a more detailed picture of the dynamics of HBV infection and therapy, and can be used to compare the efficacy of various doses and inhibitors of HBV replication for the treatment of HBV infections.
Assuntos
Adenina/análogos & derivados , Antivirais/uso terapêutico , DNA Viral/sangue , Vírus da Hepatite B/isolamento & purificação , Hepatite B Crônica/tratamento farmacológico , Organofosfonatos , Carga Viral , Adenina/uso terapêutico , Meia-Vida , Antígenos de Superfície da Hepatite B/sangue , Vírus da Hepatite B/fisiologia , Hepatite B Crônica/sangue , Humanos , Cinética , Modelos Biológicos , Fatores de TempoRESUMO
The use of regimens that use nucleoside analogues for the treatment of chronic hepatitis B virus infection is often limited because of their high relapse rates. This is thought to be due to the persistence of virus in nonhepatocyte reservoirs and/or the viral covalently closed circular (CCC) DNA species in the nucleus of infected hepatocytes. We have evaluated the novel nucleoside analogue 9-(2-phosphonylmethoxyethyl)adenine (PMEA) in the duck model of hepatitis B. Eight Pekin-Aylesbury ducks congenitally infected with the duck hepatitis B virus (DHBV) were treated with PMEA at a dosage of 15 mg/kg of body weight/day via the intraperitoneal route for 4 weeks. At the end of the treatment period, four animals were killed and the remainder were monitored for a further 4-week drug-free period before analysis. The results were compared with those for eight age-matched, untreated controls. The levels of viremia, the total intrahepatic DHBV load, and CCC DNA, viral RNA, and protein levels were measured by Southern hybridization, Northern hybridization, and immunoblotting of the appropriate specimen, respectively. Viral proteins and DNA were also measured by immunohistochemistry (IHC) and in situ hybridization (ISH) of sections of liver and pancreatic tissue. PMEA treatment reduced the viremia to undetectable levels, while the total viral DNA load in the liver was reduced by 95% compared to the control level. Viral RNA and protein levels decreased by approximately 30%. ISH and IHC confirmed the PMEA-related intrahepatic changes and established that the amount of virus in bile duct epithelial cells (BDEC) was reduced by 70% during therapy. During the follow-up period all parameters of active virological replication returned to those for the age-matched controls. PMEA had no significant effect upon the number of virus-infected islet or acinar cells in the pancreas. PMEA at a dosage of 15 mg/kg/day has potent activity against DHBV found within hepatocytes and BDEC and inhibits DHBV replication in BDEC.
Assuntos
Adenina/análogos & derivados , Antivirais/farmacologia , Vírus da Hepatite B do Pato/efeitos dos fármacos , Organofosfonatos , Replicação Viral/efeitos dos fármacos , Adenina/farmacologia , Animais , Biomarcadores , Sondas de DNA , DNA Viral/biossíntese , DNA Viral/sangue , Depressão Química , Patos , Immunoblotting , Imuno-Histoquímica , Hibridização In Situ , Fígado/patologia , Fígado/virologia , Pâncreas/patologia , Pâncreas/virologia , RNA Viral/biossíntese , RNA Viral/sangue , Proteínas Virais/biossíntese , Proteínas Virais/genéticaRESUMO
To determine whether adefovir is active against lamivudine-resistant hepatitis B virus (HBV), the inhibition constants of adefovir diphosphate and lamivudine triphosphate for wild-type and mutant human HBV DNA polymerases, which contain amino acid substitutions associated with lamivudine resistance, were compared. Recombinant wild-type and mutant human HBV DNA polymerases were expressed and substantially purified using a baculovirus expression system and immunoaffinity chromatography. HBV DNA polymerase mutants M552I, M552V, and L528M/M552V showed resistance to lamivudine triphosphate with inhibition constants (Ki) increased by 8.0-fold, 19.6-fold, and 25.2-fold compared with that of wild-type HBV DNA polymerase. However, these mutants remained sensitive to adefovir diphosphate with the inhibition constants increasing by 1.3-fold and 2.2-fold or decreasing by 0.79-fold. The L528M single mutation, identified in patients with increasing HBV DNA levels during therapy with famciclovir, also remained sensitive to adefovir diphosphate with the inhibition constant increased by only 2.3-fold.
Assuntos
Adenina/análogos & derivados , Fármacos Anti-HIV/farmacologia , DNA Polimerase Dirigida por DNA/genética , Vírus da Hepatite B/genética , Lamivudina/farmacologia , Mutação/fisiologia , Organofosfonatos , Adenina/farmacologia , Sequência de Aminoácidos , Baculoviridae , Resistência a Medicamentos/fisiologia , Humanos , Dados de Sequência Molecular , Proteínas RecombinantesRESUMO
Agrin is a basal lamina molecule that directs key events in postsynaptic differentiation, most notably the aggregation of acetylcholine receptors (AChRs) on the muscle cell surface. Agrin's AChR clustering activity is regulated by alternative mRNA splicing. Agrin splice forms having inserts at two sites (y and z) in the C-terminal region are highly active, but isoforms lacking these inserts are weakly active. The biochemical consequences of this alternative splicing are unknown. Here, the binding of four recombinant agrin isoforms to heparin, to alpha-dystroglycan (a component of an agrin receptor), and to myoblasts was tested. The presence of a four-amino acid insert at the y site is necessary and sufficient to confer heparin binding ability to agrin. Moreover, the binding of agrin to alpha-dystroglycan is inhibited by heparin when this insert is present. Agrin binding to the cell surface showed analogous properties: heparin inhibits the binding of only those agrin isoforms containing this four-amino acid insert. The results show that alternative splicing of agrin regulates its binding to heparin and suggest that agrin's interaction with alpha-dystroglycan may be modulated by cell surface glycosaminoglycans in an isoform-dependent manner.
Assuntos
Agrina/metabolismo , Processamento Alternativo , Membrana Celular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Heparina/metabolismo , Glicoproteínas de Membrana/metabolismo , Agrina/biossíntese , Agrina/isolamento & purificação , Animais , Anticorpos Monoclonais , Sítios de Ligação , Linhagem Celular , Chlorocebus aethiops , Cromatografia de Afinidade , Distroglicanas , Distrofina/metabolismo , Homeostase , Músculos , Ligação Proteica , Codorniz , RNA Mensageiro/biossíntese , Radioimunoensaio , Ratos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Torpedo , TransfecçãoRESUMO
Combinatorial libraries of nucleic acids are developing into novel sources for lead compounds in drug development. In order to diversify the pool of ss DNA sequences, we have used a modified nucleotide, 5-(1-pentynyl)-2'-deoxyuridine, in place of thymidine in a random nucleic acid library and screened this library against human thrombin. Previously, we described this screening method to identify a novel structural inhibitor (an aptamer) of the coagulation protease thrombin (Bock, L. et. al. (1992) Nature 355 564-566). Using the modified nucleic acid library, we have now isolated a second pool of thrombin inhibitors with strikingly different sequence composition compared to the selection using natural bases. This second class of aptamers is dependent on the presence of the modified nucleotide for protein binding and clotting inhibition. Our method represents a potential strategy to enhance the diversity of libraries for in vitro selection, and thereby increasing the utility of this technique in the identification of molecules with novel biochemical properties.
Assuntos
Desoxiuridina/análogos & derivados , Trombina/genética , Sequência de Bases , Primers do DNA , DNA de Cadeia Simples/química , Biblioteca Gênica , Humanos , Cinética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Oligodesoxirribonucleotídeos/síntese química , RNA/química , Trombina/metabolismoRESUMO
A novel thrombin inhibitor based on single-stranded (ss) deoxynucleotides with the sequence GGTTGGTGTGGTTGG (thrombin aptamer) has been recently discovered. In this study, we tested its efficacy in inhibiting clot-bound thrombin activity and platelet thrombus formation in an ex vivo whole artery angioplasty model. The thrombin aptamer showed a specific dose-dependent inhibition of thrombin-induced platelet aggregation (0.5 U/mL) in human platelet-rich plasma, with an IC50 of approximately 70 to 80 nmol/L. In an in vitro clot-bound thrombin assay system, heparin, used at clinically relevant concentrations of 0.2 U/mL and 0.4 U/mL, was ineffective in inhibiting clot-bound thrombin (6.5% and 34.9% inhibition at 0.2 U/mL and 0.4 U/mL, respectively). In contrast, the thrombin aptamer at an equivalent anticoagulant concentration inhibited clot-bound thrombin (79.7% inhibition). In an ex vivo whole artery angioplasty model, the thrombin aptamer markedly suppressed the generation of fibrinopeptide A (FPA), whereas heparin at 2 U/mL was ineffective. Compared with a scrambled ssDNA control, the thrombin aptamer reduced platelet deposition by 34.5% +/- 5% (mean +/- SEM, n = 4, P = .09) at low shear rates (approximately 200 s-1) and 61.3% +/- 11% (mean +/- SEM, n = 4, P = .05) at high shear rates (approximately 850 s-1). Thrombin aptamers based on ssDNA molecules represent a new class of thrombin inhibitors with potent anticoagulant and antithrombotic properties.
Assuntos
Anticoagulantes/farmacologia , Oligonucleotídeos , Polinucleotídeos/farmacologia , Trombina/antagonistas & inibidores , Trombose/prevenção & controle , Angioplastia , Aptâmeros de Nucleotídeos , Sequência de Bases , Relação Dose-Resposta a Droga , Fibrinopeptídeo A/biossíntese , Humanos , Dados de Sequência Molecular , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacologiaRESUMO
Thrombin is a serine protease that plays a pivotal role in thrombosis and hemostasis, and is a major target for anticoagulation and cardiovascular disease therapy. Using a novel in vitro selection/amplification technique, we have identified a new class of thrombin inhibitors based on single-stranded DNA (ssDNA) oligodeoxyribonucleotides (oligos). These thrombin inhibitors are the first example of the use of this technique to obtain ssDNA oligos that bind a target protein that does not interact physiologically with nucleic acid. Here, we review how iterative selection and amplification were used to identify short ssDNA sequences that bind and inhibit thrombin (Bock et al., Nature 355 (1992) 564-566), and the tertiary structure of one aptamer sequence (Wang et al., Biochemistry 32 (1993) 1899-1904). Results from in vitro and in vivo studies are also summarized (Griffin et al., Blood 81 (1993) 3271-3276). The discovery of a new class of thrombin inhibitors using this technology demonstrates the power of this new approach for rapid drug discovery and development.
Assuntos
DNA de Cadeia Simples , Trombina/antagonistas & inibidores , Anticoagulantes/farmacologia , Sequência de Bases , Fibrina , Fibrinogênio , Meia-Vida , Humanos , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Estrutura Molecular , OligorribonucleotídeosRESUMO
A new class of thrombin inhibitors based on sequence-specific single-stranded DNA oligonucleotides (thrombin aptamer) has recently been identified. The aptamer-binding site on thrombin was examined by a solid-phase plate binding assay and by chemical modification. Binding assay results demonstrated that the thrombin aptamer bound specifically to alpha-thrombin but not to gamma-thrombin and that hirudin competed with aptamer binding, suggesting that thrombin's anion-binding exosite was important for aptamer-thrombin interactions. To identify lysine residues of thrombin that participated in the binding of the thrombin aptamer, thrombin was modified with fluorescein 5'-isothiocyanate in the presence or absence of the thrombin aptamer, reduced, carboxymethylated, and digested with endoproteinase Arg-C. The digestion products were analyzed by reversed-phase high performance liquid chromatography and the peptide maps compared. Four peptides with significantly decreased modification in the presence of the aptamer were identified and subjected to N-terminal sequence analysis. Results indicated that B chain Lys-21 and Lys-65, both located within the anion-binding exosite, are situated within or in close proximity to the aptamer-binding site of human alpha-thrombin. The thrombin aptamer binds to the anion-binding exosite and inhibits thrombin's function by competing with exosite binding substrates fibrinogen and the platelet thrombin receptor.
Assuntos
DNA de Cadeia Simples/metabolismo , Trombina/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , DNA de Cadeia Simples/química , Fluoresceína-5-Isotiocianato , Hirudinas/metabolismo , Humanos , Lisina/análise , Dados de Sequência MolecularRESUMO
Using a novel in vitro selection/amplification technique, we have recently identified a new class of thrombin inhibitors based on single-stranded DNA oligonucleotides. One oligonucleotide, GGTTGGTGTGGTTGG (thrombin, aptamer), showed potent anticoagulant activity in vitro. We have initiated pharmacologic studies in cynomolgus monkeys to study the thrombin aptamer's in vivo anticoagulant properties. Upon infusion of the thrombin aptamer, anticoagulation was rapidly achieved, with a plateau reached within 10 minutes. There was a linear dose-response relationship between thrombin aptamer infusion rate and prolongation of plasma prothrombin time. Ten minutes after the infusion was stopped, no prolongation of prothrombin time was observed, indicating that the thrombin aptamer has an extremely short in vivo half-life, estimated to be 108 +/- 14 seconds. In addition, inhibition of thrombin-induced platelet aggregation in platelet-rich plasma was observed ex vivo without an effect on collagen-induced aggregation, indicating that the inhibition was specific for thrombin and not due to a nonspecific inhibitory effect on platelets. To exploit the short in vivo half-life of the thrombin aptamer, its ability to achieve regional anticoagulation in an extracorporeal hemofiltration circuit in sheep was tested. Doubling of the prothrombin time in the circuit was observed, whereas the systemic prothrombin time was minimally prolonged. We conclude that the thrombin aptamer is a potent anticoagulant in vivo, and specifically inhibits thrombin-induced platelet aggregation ex vivo. The rapid onset of action and short half-life in vivo suggest that the thrombin aptamer may be useful in anticoagulation with extracorporeal circuits and may have distinct advantages in certain acute clinical settings.
Assuntos
Anticoagulantes/farmacologia , Oligonucleotídeos , Polinucleotídeos/farmacologia , Trombina/antagonistas & inibidores , Animais , Aptâmeros de Nucleotídeos , Sequência de Bases , DNA de Cadeia Simples/química , Meia-Vida , Hemofiltração , Cinética , Macaca fascicularis , Masculino , Dados de Sequência Molecular , Agregação Plaquetária/efeitos dos fármacos , Polinucleotídeos/genética , Polinucleotídeos/farmacocinética , Tempo de Protrombina , Ovinos , Trombina/genética , Trombina/farmacologiaRESUMO
Aptamers are double-stranded DNA or single-stranded RNA molecules that bind specific molecular targets. Large randomly generated populations can be enriched in aptamers by in vitro selection and polymerase chain reaction. But so far single-stranded DNA has not been investigated for aptamer properties, nor has a target protein been considered that does not interact physiologically with nucleic acid. Here we describe the isolation of single-stranded DNA aptamers to the protease thrombin of the blood coagulation cascade and report binding affinities in the range 25-200 nM. Sequence data from 32 thrombin aptamers, selected from a pool of DNA containing 60 nucleotides of random sequence, displayed a highly conserved 14-17-base region. Several of these aptamers at nanomolar concentrations inhibited thrombin-catalysed fibrin-clot formation in vitro using either purified fibrinogen or human plasma.
Assuntos
DNA de Cadeia Simples/isolamento & purificação , Trombina/antagonistas & inibidores , Sequência de Bases , Coagulação Sanguínea/efeitos dos fármacos , Clonagem Molecular , Relação Dose-Resposta a Droga , Humanos , Técnicas In Vitro , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Homologia de Sequência do Ácido Nucleico , Tempo de TrombinaAssuntos
Clonagem Molecular/métodos , Fator VII/genética , Sequência de Aminoácidos , Sequência de Bases , Fator VII/biossíntese , Fator VII/história , História do Século XX , Humanos , Dados de Sequência Molecular , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/históriaRESUMO
We have identified a 15-nucleotide site within a G-free transcription cassette that forms triple helix with sequence-specific oligodeoxyribonucleotides. When oligodeoxynucleotides were added to template DNA prior to in vitro transcription, a significant fraction of transcripts were truncated at a site corresponding to the region of triple helix formation. Kinetic analysis of the transcription products demonstrated that these truncated transcripts could be elongated to full length upon prolonged incubation. When an alkylating base was incorporated into the oligodeoxynucleotide to form covalent triple helix, most of the transcripts remained truncated. We conclude that triple helix formation can stall or, in the case of covalent crosslinking, can block RNA polymerase II and thus may provide a method for the specific inhibition of gene expression.
Assuntos
DNA/genética , Mutagênese Insercional , Transcrição Gênica , Sequência de Bases , Ligação de Hidrogênio , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Sondas de OligonucleotídeosRESUMO
A new cell line, SUP-HD1, was established from the pleural effusion of a patient with nodular sclerosing Hodgkin's disease (NSHD). The SUP-HD1 cells had the characteristic morphology of Reed-Sternberg cells and contained acid phosphatase and nonspecific esterase. The cells lacked the Epstein-Barr virus (EBV) genome and reacted with monoclonal antibodies (MoAbs) against CD15 (Leu-M1), CD25 (Tac), CD71 (OKT9), Ki67, and HLA-Dr. However, the SUP-HD1 cells were nonreactive with MoAbs that specifically identify T lymphocytes, B lymphocytes, and macrophage/myeloid cells. Karyotype analysis of the cell line showed clonal abnormalities involving 1p13, 7p15, 8q22, and 11q23, chromosomal locations, at which breakpoints have been reported in HD. Southern blot analysis demonstrated rearrangement of the immunoglobulin heavy chain and kappa light chain genes as well as the gene for the beta chain of the T-cell receptor (TCR). Transcriptional analysis showed expression of RNAs for kappa light chain, interferon-gamma (IFN-gamma), and interleukin-2 receptor (IL-2R) but not IL-2. The SUP-HD1 cells lacked cytoplasmic and surface immunoglobulin heavy chain, but a small amount of cytoplasmic kappa light chain was detected. The presence of nuclear factor kappa B (NF kappa B), a B-lymphocyte-associated transcription factor, was demonstrated in stimulated and unstimulated cells. In addition, the SUP-HD1 cell line, produced IFN-gamma, a T-lymphocyte-associated lymphokine. Based on these data, the SUP-HD1 cells appear to be aberrant lymphocytes with characteristics of both activated B and T lymphocytes. Elaboration of lymphokines such as IFN-gamma by the malignant cells may represent one explanation for the unique clinical and pathologic features of HD.
Assuntos
Doença de Hodgkin/patologia , Interferon gama/biossíntese , Células Tumorais Cultivadas , Adulto , Animais , Antígenos CD/análise , Antígenos de Neoplasias/análise , Aberrações Cromossômicas/patologia , Transtornos Cromossômicos , DNA de Neoplasias/genética , Rearranjo Gênico do Linfócito B , Rearranjo Gênico da Cadeia beta dos Receptores de Antígenos dos Linfócitos T , Doença de Hodgkin/metabolismo , Humanos , Interferon gama/genética , Cariotipagem , Masculino , Camundongos , Camundongos Nus , Microscopia Eletrônica , Transplante de Neoplasias , Derrame Pleural/patologia , RNA Mensageiro/genética , Fatores de Transcrição/análise , Células Tumorais Cultivadas/metabolismoRESUMO
Molecules involved in the antigen receptor-dependent regulation of early T cell activation genes were investigated with the use of functional sequences of the T cell activation-specific enhancer of interleukin-2 (IL-2). One of these sequences forms a protein complex, NFAT-1, specifically with nuclear extracts of activated T cells. This complex appeared 10 to 25 minutes before the activation of the IL-2 gene. Studies with inhibitors of protein synthesis indicated that the time of synthesis of the activator of the IL-2 gene in Jurkat T cells corresponds to the time of appearance of NFAT-1. NFAT-1, or a very similar protein, bound functional sequences of the long terminal repeat (LTR) of the human immunodeficiency virus type 1; the LTR of this virus is known to be stimulated during early T cell activation. The binding site for this complex activated a linked promoter after transfection into antigen receptor-activated T cells but not other cell types. These characteristics suggest that NFAT-1 transmits signals initiated at the T cell antigen receptor.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Elementos Facilitadores Genéticos , Ativação Linfocitária , Proteínas Nucleares/fisiologia , Receptores de Antígenos de Linfócitos T/fisiologia , Sequências Reguladoras de Ácido Nucleico , Linfócitos T/fisiologia , Fatores de Transcrição/fisiologia , Sítios de Ligação , HIV/genética , Humanos , Técnicas In Vitro , Interleucina-2/genéticaRESUMO
cDNA clones encoding human factor V have been isolated from an oligo(dT)-primed human fetal liver cDNA library prepared with vector Charon 21A. The cDNA sequence of factor V from three overlapping clones includes a 6672-base-pair (bp) coding region, a 90-bp 5' untranslated region, and a 163-bp 3' untranslated region within which is a poly(A) tail. The deduced amino acid sequence consists of 2224 amino acids inclusive of a 28-amino acid leader peptide. Direct comparison with human factor VIII reveals considerable homology between proteins in amino acid sequence and domain structure: a triplicated A domain and duplicated C domain show approximately equal to 40% identity with the corresponding domains in factor VIII. As in factor VIII, the A domains of factor V share approximately 40% amino acid-sequence homology with the three highly conserved domains in ceruloplasmin. The B domain of factor V contains 35 tandem and approximately 9 additional semiconserved repeats of nine amino acids of the form Asp-Leu-Ser-Gln-Thr-Thr/Asn-Leu-Ser-Pro and 2 additional semiconserved repeats of 17 amino acids. Factor V contains 37 potential N-linked glycosylation sites, 25 of which are in the B domain, and a total of 19 cysteine residues.
Assuntos
Fator V/genética , Sequência de Aminoácidos , Sequência de Bases , Ceruloplasmina/genética , DNA/genética , Fator VIII/genética , Vetores Genéticos , Humanos , Fígado/análise , Homologia de Sequência do Ácido NucleicoRESUMO
Factor VIII (antihemophilic factor) is a high molecular weight plasma glycoprotein that participates in the blood clotting cascade. The recent cloning and sequence analysis of the cDNA encoding human factor VIII revealed an obvious domain structure for the protein, which can be represented as A1-A2-B-A3-C1-C2. We now report the DNA sequence analysis of porcine exons encoding the entire B domain and part of the A2 and A3 domains. We found an unusually high degree of porcine-human amino acid sequence divergence in the B region compared with the limited sequence available for other regions of the porcine factor VIII molecule. In addition to sequence divergence, there are numerous gaps in the porcine B domain totalling over 200 amino acids. Recombinant DNA techniques were used to effect the removal of large segments of DNA encoding the B domain from the full-length human factor VIII cDNA. These constructs directed the synthesis of biologically active factor VIII when introduced into mammalian cells despite the deletion of up to 38% of the factor VIII molecule.