Asunto(s)
Creatina/aislamiento & purificación , Hemoperfusión/instrumentación , Indicán/aislamiento & purificación , Membranas Artificiales , Adsorción , Celulosa/análogos & derivados , Celulosa/química , Carbón Orgánico/química , Difusión , Humanos , Polímeros/química , Porosidad , Povidona/química , Sulfonas/químicaRESUMEN
In 2005 the Swedish Association of the Pharmaceutical Industry (LIF) initiated a national environmental classification and information system for pharmaceuticals. This investigation reports the results from a survey, conducted among the persons involved in the start-up process. The aim of this study is to generate knowledge contributing to the clarification of the motivations, expectations, and intentions underlying the development and implementation of the system. The decision to implement a classification and information system for pharmaceuticals was the result of a combination of several driving forces, mainly political pressure and a possibility to increase the industries' goodwill, while at the same time keeping the process under the industries' control. The expected possible effects of the system, other than increased goodwill, are according to this survey assumed to be low. The system offers little guidance for end-users in the substitution of one pharmaceutical for another. One possible reason for this could be that LIF needs to observe the interests of all its members' and should not affect competition. The affiliation of the involved actors correlates to how these actors view and value the system, but this has not hampered the collaborative process to develop and implement it.
Asunto(s)
Industria Farmacéutica , Ambiente , Sistemas de Información/normas , Preparaciones Farmacéuticas/clasificación , Investigación Empírica , Humanos , Intención , Motivación , Gestión de Riesgos , Encuestas y Cuestionarios , SueciaRESUMEN
We have produced by phage-display a disulfide-linked cyclic nonapeptide (inhibitory peptide-01, IP01), CLLRMRSIC, that binds to intracellular adhesion molecule-1 (ICAM-1) and blocks binding to its counter-structure, leukocyte functional antigen-1 (LFA-1). As a first step towards improving its pharmacologic properties, we have performed a structural and functional analysis of this peptide inhibitor to determine the features relevant to ICAM-1 binding. We report here the solution model of our initial product, IP01, as derived from two-dimensional nuclear magnetic resonance (NMR) restraints and molecular modeling. Distance and dihedral angle restraints, generated from nuclear Overhauser effect spectroscopy (NOESY) and one-dimensional-NMR experiments respectively, were used to generate an ensemble of structures using distance geometry and simulated annealing. Molecular dynamic simulations produced three interconverting conformational families consistent with the NMR-derived constraints. We describe these conformations and their mechanism of interconversion. Furthermore, we have measured the IC50 s of a series of inhibitors generated from IP01 through alanine substitution of each residue. These results show that the L2-L3-R4-M5-R6 segment is functionally active, conformationally flexible, and contains a beta-turn involving residues R4-S7, while the C1-C9-I8-S7 segment is less functionally-active but adopts a more defined solution conformation, consistent with a scaffolding function. This model will be useful for designing nonpeptide-based organic inhibitors with improved pharmacologic properties.
Asunto(s)
Proteínas Portadoras/química , Molécula 1 de Adhesión Intercelular/química , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Conformación Molecular , Péptidos Cíclicos/química , Proteínas Portadoras/antagonistas & inhibidores , Antígeno-1 Asociado a Función de Linfocito/química , Péptidos Cíclicos/antagonistas & inhibidores , Conformación ProteicaRESUMEN
CYP2C19 is selective for the 4'-hydroxylation of S-mephenytoin while the highly similar CYP2C9 has little activity toward this substrate. To identify critical amino acids determining the specificity of human CYP2C19 for S-mephenytoin 4'-hydroxylation, we constructed chimeras by replacing portions of CYP2C9 containing various proposed substrate recognition sites (SRSs) with those of CYP2C19 and mutating individual residues by site-directed mutagenesis. Only a chimera containing regions encompassing SRSs 1--4 was active (30% of wild-type CYP2C19), indicating that multiple regions are necessary to confer specificity for S-mephenytoin. Mutagenesis studies identified six residues in three topological components of the proteins required to convert CYP2C9 to an S-mephenytoin 4'-hydroxylase (6% of the activity of wild-type CYP2C19). Of these, only the I99H difference located in SRS 1 between helices B and C reflects a change in a side chain that is predicted to be in the substrate-binding cavity formed above the heme prosthetic group. Two additional substitutions, S220P and P221T residing between helices F and G but not in close proximity to the substrate binding site together with five differences in the N-terminal portion of helix I conferred S-mephenytoin 4'-hydroxylation activity with a K(M) similar to that of CYP2C19 but a 3-fold lower K(cat). Three residues in helix I, S286N, V292A, and F295L, were essential for S-mephenytoin 4'-hydroxylation activity. On the basis of the structure of the closely related enzyme CYP2C5, these residues are unlikely to directly contact the substrate during catalysis but are positioned to influence the packing of substrate binding site residues and likely substrate access channels in the enzyme.
Asunto(s)
Sustitución de Aminoácidos , Hidrocarburo de Aril Hidroxilasas , Sistema Enzimático del Citocromo P-450/metabolismo , Mefenitoína/metabolismo , Oxigenasas de Función Mixta/metabolismo , Esteroide 16-alfa-Hidroxilasa , Esteroide Hidroxilasas/metabolismo , Sustitución de Aminoácidos/genética , Asparagina/genética , Citocromo P-450 CYP2C19 , Citocromo P-450 CYP2C9 , Sistema Enzimático del Citocromo P-450/genética , Histidina/genética , Humanos , Hidroxilación , Isoleucina/genética , Cinética , Oxigenasas de Función Mixta/genética , Mutagénesis Sitio-Dirigida , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Prolina/genética , Proteínas Recombinantes de Fusión/síntesis química , Proteínas Recombinantes de Fusión/metabolismo , Serina/genética , Esteroide Hidroxilasas/genética , Especificidad por Sustrato/genética , Treonina/genéticaRESUMEN
The molecular basis for reversible inhibition of rabbit CYP2B4 and CYP2B5 and rat CYP2B1 by phenylimidazoles was assessed with active-site mutants and new three-dimensional models based on the crystal structure of CYP2C5. 4-Phenylimidazole was 17- to 32-fold more potent toward CYP2B4 and CYP2B1 than CYP2B5. The 3D models, along with site-directed mutagenesis data, revealed the importance of residue 114 for sensitivity to inhibition of all three CYP2B enzymes. Besides Ile 114, Val 367 was also found to be critical for inhibition of CYP2B4 and CYP2B1. The most interesting new insights were obtained from analysis of the CYP2B5 model and the CYP2B5 active-site mutants. Simultaneous substitution of residues 114, 294, 363, and 367 with the corresponding residues of CYP2B4 decreased the IC(50) value for inhibition by 4-phenylimidazole 12-fold. Docking 4-phenylimidazole into the models of CYP2B5 mutants demonstrated that the inhibitor-binding site is strongly influenced by residue-residue interactions, especially between residues 114 and 294. A chlorine substitution at position 4 of the phenyl moiety of 4- and 1-phenylimidazole resulted in IC(50) values 95- and 130-fold lower for CYP2B4 than for CYP2B5, respectively, suggesting that these compounds are selective inhibitors of CYP2B4. Overall, the study revealed that differences in the determinants of inhibition between CYP2B4 and CYP2B5 are caused not only by single residue inhibitor contacts but also by residue-residue interactions. This new generation of CYP2B models may provide valuable information for the design of selective inhibitors of human CYP2B6 and for the development of drugs that avoid drug interactions due to P450 inhibition.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas , Citocromo P-450 CYP2B1/antagonistas & inhibidores , Inhibidores Enzimáticos del Citocromo P-450 , Inhibidores Enzimáticos/farmacología , Imidazoles/farmacología , Esteroide Hidroxilasas/antagonistas & inhibidores , Secuencia de Aminoácidos , Aminoácidos/metabolismo , Sitios de Unión/genética , Citocromo P-450 CYP2B1/genética , Citocromo P-450 CYP2B1/metabolismo , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Familia 2 del Citocromo P450 , Escherichia coli , Modelos Moleculares , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Esteroide Hidroxilasas/genética , Esteroide Hidroxilasas/metabolismo , TransfecciónRESUMEN
In this article, we address the issue of using a continuous speech recognition tool to obtain phonetic or phonological representations of speech. Two experiments were carried out in which the performance of a continuous speech recognizer (CSR) was compared to the performance of expert listeners in a task of judging whether a number of prespecified phones had been realized in an utterance. In the first experiment, nine expert listeners and the CSR carried out exactly the same task: deciding whether a segment was present or not in 467 cases. In the second experiment, we expanded on the first experiment by focusing on two phonological processes: schwa-deletion and schwa-insertion. The results of these experiments show that significant differences in performance were found between the CSR and the listeners, but also between individual listeners. Although some of these differences appeared to be statistically significant, their magnitude is such that they may very well be acceptable depending on what the transcriptions are needed for. In other words, although the CSR is not infallible, it makes it possible to explore large datasets, which might outweigh the errors introduced by the mistakes the CSR makes. For these reasons, we can conclude that the CSR can be used instead of a listener to carry out this type of task: deciding whether a phone is present or not.
Asunto(s)
Procesamiento Automatizado de Datos , Lingüística/métodos , Habla , Bases de Datos Factuales , Humanos , Fonética , Estadísticas no ParamétricasRESUMEN
Reverse transcriptase-polymerase chain reaction was used to amplify a partial cDNA from rabbit lung mRNA that shared 77% protein sequence identity with the mouse pregnane X receptor (PXR). Rapid amplification of cDNA ends from a rabbit kidney lambdaZAP expression library resulted in the isolation of overlapping cDNAs spanning the complete coding sequence. The deduced amino acid sequence of 411 residues exhibited 79% overall amino acid identity with human PXR and 77% identity with mouse PXR. Based on this protein sequence relationship and a similar degree of conservation exhibited by the mouse and human PXR orthologs, the cDNA appears to encode the rabbit PXR ortholog. 5'-rapid amplification of cDNA ends performed on an adaptor-ligated cDNA library from rabbit liver revealed the presence of an alternate mRNA, which differed at the 5'-terminus. RNase protection assays indicated that the alternate mRNA was expressed at >50-fold lower levels in rabbit kidney and liver. Rifampicin treatment of CV-1 cells cotransfected with a rabbit PXR expression plasmid and a luciferase reporter construct containing two copies of the DR3 enhancer from CYP3A23 produced a 6-fold induction of luciferase activity. In contrast, rat PXR was not responsive to this antibiotic under the same conditions. Pregnenolone 16alpha-carbonitrile was an efficacious activator of rat PXR, but failed to significantly activate rabbit PXR at equivalent concentrations. These results indicate that the ligand activation profile of rabbit PXR is distinct from rat PXR and more closely resembles that of human PXR. The rabbit PXR activation profile is consistent with the cytochrome P450 (P450) 3A6 induction profile in rabbits.
Asunto(s)
Antibióticos Antituberculosos/farmacología , Hidrocarburo de Aril Hidroxilasas , Receptores Citoplasmáticos y Nucleares/agonistas , Receptores de Esteroides/agonistas , Rifampin/farmacología , Secuencia de Aminoácidos , Animales , Antiinflamatorios/farmacología , Núcleo Celular/efectos de los fármacos , Sistema Enzimático del Citocromo P-450/biosíntesis , Sistema Enzimático del Citocromo P-450/metabolismo , ADN/biosíntesis , ADN/genética , Dexametasona/farmacología , Inducción Enzimática/efectos de los fármacos , Humanos , Riñón/efectos de los fármacos , Riñón/enzimología , Hígado/efectos de los fármacos , Hígado/enzimología , Ratones , Oxigenasas de Función Mixta/metabolismo , Datos de Secuencia Molecular , Poli A/biosíntesis , Poli A/genética , Receptor X de Pregnano , ARN/biosíntesis , ARN/genética , Conejos , Ratas , Receptores Citoplasmáticos y Nucleares/genética , Receptores de Esteroides/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección/genéticaRESUMEN
Tolbutamide is a sulfonylurea-type oral hypoglycemic agent whose action is terminated by hydroxylation of the tolylsulfonyl methyl moiety catalyzed by cytochrome P-450 (CYP) enzymes of the human CYP2C subfamily. Although most studies have implicated CYP2C9 as the exclusive catalyst of hepatic tolbutamide hydroxylation in humans, there is evidence that other CYP2C enzymes (e.g., CYP2C19) may also participate. To that end, we used an immunochemical approach to assess the role of individual CYP2Cs in microsomal tolbutamide metabolism. Polyclonal antibodies were raised to CYP2C9 purified from human liver, and were then back-adsorbed against recombinant CYP2C19 coupled to a solid-phase support. Western blotting revealed that the absorbed anti-human CYP2C9 preparation reacted with only recombinant CYP2C9 and the corresponding native protein in hepatic microsomes, and no longer recognized CYP2C19 and CYP2C8. Monospecific anti-CYP2C9 not only retained the ability to inhibit CYP2C9-catalyzed reactions, as evidenced by its marked (90%) inhibition of diclofenac 4'-hydroxylation by purified CYP2C9 and by human liver microsomes, but also exhibited metabolic specificity, as indicated by its negligible (<15%) inhibitory effect on S-mephenytoin 4'-hydroxylation by purified CYP2C19 or hepatic microsomes containing CYP2C19. Monospecific anti-CYP2C9 was also found to inhibit rates of tolbutamide hydroxylation by 93 +/- 4 and 78 +/- 6% in CYP2C19-deficient and CYP2C19-containing human liver microsomes, respectively. Taken together, our results indicate that both CYP2C9 and CYP2C19 are involved in tolbutamide hydroxylation by human liver microsomes, and that CYP2C19 underlies at least 14 to 22% of tolbutamide metabolism. Although expression of CYP2C19 in human liver is less than that of CYP2C9, it may play an important role in tolbutamide disposition in subjects expressing either high levels of CYP2C19 or a catalytically deficient CYP2C9 enzyme.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas , Sistema Enzimático del Citocromo P-450/metabolismo , Microsomas Hepáticos/metabolismo , Oxigenasas de Función Mixta/metabolismo , Esteroide 16-alfa-Hidroxilasa , Tolbutamida/metabolismo , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/aislamiento & purificación , Anticuerpos Monoclonales/farmacología , Reacciones Cruzadas , Citocromo P-450 CYP2C19 , Citocromo P-450 CYP2C8 , Citocromo P-450 CYP2C9 , Sistema Enzimático del Citocromo P-450/inmunología , Diclofenaco/metabolismo , Humanos , Hidroxilación/efectos de los fármacos , Masculino , Mefenitoína/metabolismo , Microsomas Hepáticos/efectos de los fármacos , Oxigenasas de Función Mixta/inmunología , Conejos , Esteroide Hidroxilasas/inmunología , Esteroide Hidroxilasas/metabolismoRESUMEN
Individuals with drug metabolism polymorphisms involving CYP2C enzymes exhibit deficient oxidation of important therapeutic agents, including S-mephenytoin, omeprazole, warfarin, tolbutamide, and nonsteroidal anti-inflammatory drugs. While recombinant CYP2C19 and CYP2C9 proteins expressed in yeast or Escherichia coli have been shown to oxidize these agents, the capacity of the corresponding native P450s isolated from human liver to do so is ill defined. To that end, we purified CYP2C19, CYP2C9, and CYP2C8 from human liver samples using conventional chromatographic techniques and examined their capacity to oxidize S-mephenytoin, omeprazole, and tolbutamide. Upon reconstitution, CYP2C19 metabolized S-mephenytoin and omeprazole at rates that were 11- and 8-fold higher, respectively, than those of intact liver microsomes, whereas neither CYP2C9 nor CYP2C8 displayed appreciable metabolic activity with these substrates. CYP2C19 also proved an efficient catalyst of tolbutamide metabolism, exhibiting a turnover rate similar to CYP2C9 preparations (2.0-6.4 vs 2.4-4.3 nmol hydroxytolbutamide formed/min/nmol P450). The kinetic parameters of CYP2C19-mediated tolbutamide hydroxylation (Km = 650 microM, Vmax = 3.71 min-1) somewhat resembled those of the CYP2C9-catalyzed reaction (Km = 178-407 microM, Vmax = 2.95-7.08 min-1). Polyclonal CYP2C19 antibodies markedly decreased S-mephenytoin 4'-hydroxylation (98% inhibition) and omeprazole 5-hydroxylation (85% inhibition) by human liver microsomes. CYP2C19 antibodies also potently inhibited (>90%) microsomal tolbutamide hydroxylation, which was similar to the inhibition (>85%) observed with antibodies to CYP2C9. Moreover, excellent correlations were found between immunoreactive CYP2C19 content, S-mephenytoin 4'-hydroxylase activity (r = 0.912; P < 0. 001), and omeprazole 5-hydroxylase activity (r = 0.906; P < 0.001) in liver samples from 13-17 different subjects. A significant relationship was likewise observed between microsomal tolbutamide hydroxylation and CYP2C9 content (r = 0.664; P < 0.02) but not with CYP2C19 content (r = 0.393; P = 0.184). Finally, immunoquantitation revealed that in these human liver samples, expression of CYP2C9 (88. 5 +/- 36 nmol/mg) was 5-fold higher than that of CYP2C19 (17.8 +/- 14 nmol/mg) and nearly 8-fold higher than that of CYP2C8 (11.5 +/- 12 nmol/mg). Our results, like those obtained with recombinant CYP2C enzymes, indicate that CYP2C19 is a primary determinant of S-mephenytoin 4'-hydroxylation and low-Km omeprazole 5-hydroxylation in human liver. Despite its tolbutamide hydroxylase activity, the low levels of hepatic CYP2C19 expression (relative to CYP2C9) may preclude an important role for this enzyme in hepatic tolbutamide metabolism and any polymorphisms thereof.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas , Sistema Enzimático del Citocromo P-450/metabolismo , Mefenitoína/farmacocinética , Microsomas Hepáticos/enzimología , Oxigenasas de Función Mixta/metabolismo , Omeprazol/farmacocinética , Esteroide 16-alfa-Hidroxilasa , Esteroide Hidroxilasas/metabolismo , Tolbutamida/farmacocinética , Antiinflamatorios no Esteroideos/metabolismo , Cromatografía DEAE-Celulosa , Cromatografía por Intercambio Iónico , Citocromo P-450 CYP2C19 , Citocromo P-450 CYP2C8 , Citocromo P-450 CYP2C9 , Sistema Enzimático del Citocromo P-450/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida , Humanos , Hidroxilación , Cinética , Oxigenasas de Función Mixta/aislamiento & purificación , Esteroide Hidroxilasas/aislamiento & purificación , Especificidad por Sustrato , UltrafiltraciónRESUMEN
BACKGROUND: Envelope mutant forms of hepatitis B virus (HBV), impairing HBV antibody recognition, have been reported with mutations in single or multiple sites of the hepatitis B surface antigen (HBsAg) group-specific "a" determinant. Blood donors infected with such an HBsAg mutant form of HBV may escape detection by HBsAg screening assays and therefore may affect the safety of the blood supply. CASE REPORT: A repeat blood donor became HBsAg-reactive in an enzyme immunoassay. Confirmatory testing yielded negative results for HBsAg in a radioimmunoassay and in four enzyme immunoassays used in blood donor screening. The specificity of the HBsAg reactivity in the first enzyme immunoassay was confirmed by HBsAg neutralization with antibody to HBsAg. Additional HBV confirmatory test results were positive for antibody to hepatitis B core antigen and antibody to hepatitis B e antigen; negative for antibody to HBsAg and for hepatitis B e antigen; and positive for HBV DNA. DNA sequence analysis of the "a" determinant region of HBsAg revealed amino acid substitutions from Q (Gln) to R (Arg) at codon 129 and from M (Met) to T (Thr) at codon 133. CONCLUSION: This case illustrates the presence of HBsAg mutant forms of HBV in a West European blood donor population that were undetected by several HBsAg screening assays. Adaptation of HBsAg screening is indicated to overcome deficiencies in sensitivity in detecting HBsAg mutant forms of HBV. Screening for antibody to hepatitis B core antigen or HBV DNA may also detect blood donors infected with HBsAg mutant forms of HBV
Asunto(s)
Donantes de Sangre , Antígenos de Superficie de la Hepatitis B/análisis , Virus de la Hepatitis B/aislamiento & purificación , Hepatitis B/diagnóstico , Adulto , Femenino , Antígenos de Superficie de la Hepatitis B/genética , Virus de la Hepatitis B/genética , Humanos , Técnicas para Inmunoenzimas , Mutación , Valor Predictivo de las Pruebas , Juego de Reactivos para Diagnóstico , Viremia/diagnósticoRESUMEN
Cytochrome P450 (CYP) 2E1 is implicated in a variety of chemically initiated hepatotoxicities, including alcoholic liver disease. These pathological conditions arise from increased production of reactive intermediates caused by elevated enzyme concentrations. Thus, the ability to detect enhanced CYP2E1 levels would aid in identifying individuals at high risk for xenobiotic-promoted liver injury. With this in mind, the present investigation assessed in vivo chlorzoxazone metabolism and compared pharmacokinetic parameters with CYP2E1 expression in blood. Twenty-two subjects were recruited and divided into two groups, control subjects and alcohol abusers, based on responses to two screening questionnaires. Those individuals with higher survey scores, i.e. those who consumed alcohol more frequently, exhibited higher rates of chlorzoxazone metabolism. Indeed, a correlation (r = 0.66, p < 0.01) was obtained when scores were compared with the pharmacokinetic parameter AUC for chlorzoxazone. Lymphocyte microsomes isolated from blood samples obtained from these same individuals were subjected to immunoblot analyses to detect CYP2E1 levels. That lymphocytes contained CYP2E1 was confirmed by reverse transcription-polymerase chain reaction and sequence analysis of the cDNA. Quantification of immunoreactive bands revealed that levels of this P450 were 2.3-fold higher in alcoholics than in control subjects. This increase in lymphocyte CYP2E1 content in alcoholic subjects coincided with a 2.1-fold increase in chlorzoxazone clearance and a 2-fold decrease in the AUC for chlorzoxazone. Importantly, a correlation (r = 0.62, p < 0.01) was observed between CYP2E1 content in lymphocytes and chlorzoxazone clearance rates. Thus, monitoring lymphocyte CYP2E1 expression may provide a substitute for estimating hepatic activity of this P450.
Asunto(s)
Clorzoxazona/metabolismo , Citocromo P-450 CYP2E1/efectos de los fármacos , Etanol/farmacología , Hígado/efectos de los fármacos , Hígado/enzimología , Linfocitos/efectos de los fármacos , Linfocitos/enzimología , Adulto , Biomarcadores/sangre , Citocromo P-450 CYP2E1/sangre , Femenino , Humanos , Masculino , Persona de Mediana Edad , Distribución AleatoriaRESUMEN
BACKGROUND: Paclitaxel is a novel agent with significant activity in several solid tumors. Preclinical data suggested that methotrexate prior to paclitaxel would be synergistic. To determine the qualitative and quantitative toxicity of this regimen we performed a phase I study in patients with solid tumors. PATIENTS AND METHODS: Patients with solid tumors previously treated with no more than two prior chemotherapy regimens were given methotrexate intravenously on day 1, followed by paclitaxel, as a 24-hour infusion on day 2. The starting dose (level '0') was 40 mg/m2 for methotrexate and 135 mg/m2 for paclitaxel. RESULTS: After achieving a maximum tolerated dose, additional patients were enrolled with the addition of G-CSF 5 mu g/kg/d on days 4-13. At the starting dose level, dose-limiting toxicity consisting of neutropenic fever occurred in 3 of 4 patients. At dose level -1, methotrexate 30 mg/m2 and paclitaxel 110 mg/m2, neutropenic fever occurred in 7 of 10 patients during the first course. At dose level -2, methotrexate 23 mg/m2 and paclitaxel 85 mg/m2, neutropenic fever occurred in 1 of 7 patients. To abrogate the neutropenia we explored the same combination with the addition of G-CSF. Neutropenic fever remained the only dose-limiting toxicity. At dose level '0' with G-CSF, 1 of 7 patients developed dose-limiting toxicity. At dose level 1 plus G-CSF, methotrexate 40 mg/m2 and paclitaxel 170 mg/m2, dose-limiting neutropenic fever occurred in 4 of 6 patients. Partial responses occurred in 4 of 41 patients entered on this study. Pharmacokinetic data suggested that methotrexate did not increase paclitaxel levels. CONCLUSION: The combination of methotrexate and paclitaxel is feasible, but neutropenic fever, even with the addition of G-CSF prevents further escalations of paclitaxel beyond 135 mg/m2 following methotrexate.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Esquema de Medicación , Sinergismo Farmacológico , Factor Estimulante de Colonias de Granulocitos/administración & dosificación , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Humanos , Infusiones Intravenosas , Neoplasias Pulmonares/tratamiento farmacológico , Metotrexato/administración & dosificación , Persona de Mediana Edad , Paclitaxel/administración & dosificaciónRESUMEN
Human cytochrome P450s 2C8, 2C9, 2C18, and 2C19 and rabbit cytochrome P450s 2C1, 2C2, 2C4, 2C5, and 2C16 were expressed from their respective cDNAs in Escherichia coli as chimeric enzymes in which a portion of the N-terminal membrane anchor sequence was replaced with a modified sequence derived from P450 17A. For 2C1 and 2C2 removal of the extraneous 3'-untranslated sequence allowed the successful expression of constructs that were unproductive in its presence. The levels of expression varied from 180 to 1500 nmol/liter of culture and the addition of delta-aminolevulinic acid to the culture media increased the amount of spectrally detectable P450 for several of these enzymes 2- to 10-fold. The catalytic properties of the modified human 2C P450s expressed in E. coli were concordant with previously published data for several marker substrates including (S)-mephenytoin for P450 2C19, tolbutamide and tetrahydrocannabinol (THC) for P450 2C9, and taxol for P450 2C8. Interestingly, P450 2C19 catalyzed the 21-hydroxylation of progesterone and, to a lesser extent, catalyzed the formation of 16 alpha-hydroxyprogesterone. The rabbit enzyme P450 2C16 catalyzed the formation of 17 alpha- and 16 alpha-hydroxyprogesterone in addition to 21-hydroxylation. P450 2C19 also catalyzed the methylhydroxylation of tolbutamide and the 7-hydroxylation of THC at rates that were similar to or greater than that of P450 2C9. This work has identified important factors required for the high-level expression of 2C subfamily P450s in E. coli. The availability of these enzymes will facilitate detailed kinetic measurements for known and yet to be identified substrates.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas , Sistema Enzimático del Citocromo P-450/genética , Escherichia coli/genética , Esteroide 16-alfa-Hidroxilasa , Esteroide Hidroxilasas/genética , Alelos , Animales , Secuencia de Bases , Clonación Molecular , Sistema Enzimático del Citocromo P-450/biosíntesis , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , Técnicas Genéticas , Humanos , Datos de Secuencia Molecular , Conejos , Proteínas Recombinantes de Fusión/genética , Esteroide Hidroxilasas/biosíntesisRESUMEN
To determine whether the ALL (acute lymphoblastic leukemia) CDR3 (third complementarity-determining region) repertoire represents the recombination repertoire, or shows evidence of selectional processes inherent to normal B cell differentiation or malignant transformation, we analyzed 68 ALL CDR3 regions and included 127 previously published sequences in the analyses. We found no evidence of selection prior to malignant transformation as recombination was random with 1/3 "in frame" and 2/3 "out of frame" joinings and usage of all three D reading frames was observed. D and JH gene segments were predominantly unmutated which allowed a detailed analysis of gene usage and rearrangement characteristics. JH4 and JH6 usage (both 32.2%) was significantly different (p = 0.005) from that observed in peripheral B lymphocytes. D gene family usage roughly represented D gene family size with the exception of the DXP and DA/K family which were over- and underrepresented (p = < or = 0.05), respectively. D-D fusions were found in 26.2% of CDR3 regions. If less stringent criteria were applied DIR homology was found in 40/65 sequences, suggesting the frequent involvement of DIR gene segments in human CDR3 formation. The rearranged D genes were evenly distributed over the D locus, suggesting that D recombination is a predominantly random process, independent of physical location at the locus. Also, there was no correlation between JH gene usge and physical location of the rearranged D gene segment, which excludes a major contribution of the DJH replacement recombination mechanism. In 36.1% of CDR3 regions N-nucleotides at the DJH junction were absent. This frequency is higher than observed for peripheral B lymphocytes. It is suggested that for a number of ALL the initial transformational event took place in early fetal life. We conclude that ALL CDR3 sequences show no evidence of selection prior to malignant transformation, nor of extensive changes subsequent to malignant transformation.
Asunto(s)
Transformación Celular Neoplásica , Feto/inmunología , Región Variable de Inmunoglobulina/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/inmunología , Leucemia-Linfoma Linfoblástico de Células Precursoras/inmunología , Recombinación Genética , Factores de Edad , Secuencia de Bases , Preescolar , Reordenamiento Génico , Humanos , Región de Unión de la Inmunoglobulina/genética , Datos de Secuencia Molecular , Nucleótidos/análisisRESUMEN
The TH line was established by bringing tumour cells from a multiple myeloma patient into suspension culture and subsequently cloning them by limiting dilution. The cultured cells show marked heterogeneity; there are ultrastructural differences between small and large TH cells, particularly with respect to the rough endoplasmatic reticulum (RER). Karyotyping revealed chromosome numbers in the triploid range, with many structural abnormalities, at the 14q32 region among others. A t(14;18) could not be demonstrated. TH was shown to have germline and a rearranged allele for kappa light chain, and only a single rearranged gene for heavy chain immunoglobulin. TH expressed PCA-1, CD9, CD28 and CD38 antigens, HLA class II, RER and kappa light chain, but few or no other antigens associated with the B-cell lineage. Light chain kappa and trace amounts of IgG3 were found intracellularly as well as in culture supernatant. The addition of IL-6 to cultures of TH increased proliferation, as well as the secretion of kappa light chain and the membrane expression of CD28 and CD38 antigens. Because TH has relatively few B cell markers on its membrane, it may be useful for the induction of monoclonal antibodies specific for human plasma cells. It also provides a model for the demonstration that IL-6 can act as a paracrine growth and differentiation factor for cells of myelomal origin.
Asunto(s)
Plasmacitoma/ultraestructura , Adulto , División Celular , Línea Celular , ADN/análisis , Humanos , Inmunofenotipificación , Cariotipificación , Masculino , Microscopía Electrónica , Plasmacitoma/genética , Plasmacitoma/inmunologíaRESUMEN
Strategies for diminishing the risk of blood transfusion-associated transmission of HIV-1 were evaluated. HIV-1-infected peripheral blood mononuclear cells were added to blood that was subsequently filtered by using different white cell (WBC) filters (cellulose acetate and polyester). The average log reduction of infected cells with polyester filters was at least 2.5 as measured by ID50 titration and polymerase chain reaction. In two WBC filtration experiments with blood from seropositive donors diluted 1:4 with seronegative blood, log reductions of 2.4 and greater than 2.5 were observed. No cell-free virus was retained by the filter used. A freeze-thaw procedure applied to HIV-1-contaminated blood resulted in a minimal log reduction. These results indicate that the reduction of HIV-1 infectivity as a result of filtration is mainly due to the removal of HIV-1-infected WBCs, and that complete removal of infected WBCs cannot be achieved by the current filtration or freeze-thaw procedures. However, the development of filters with enhanced ability to remove (possibly infected) WBCs may have the added benefit of improving the safety of donor blood, especially in multiply transfused patients.
Asunto(s)
Sangre/microbiología , Congelación , Infecciones por VIH/sangre , VIH/aislamiento & purificación , Leucocitos/citología , Filtración/instrumentación , Genes pol , VIH/genética , Seropositividad para VIH , Humanos , Reacción en Cadena de la PolimerasaRESUMEN
Several authors have reported the in vitro production of colony-stimulating factors (CSF) and interleukin-1 (IL-1) by the neoplastic cells from patients with acute myeloid leukemia (AML). Using a sensitive bioassay for IL-6, the capacity of the leukemic cells of 30 patients with AML to produce IL-6 was examined. IL-6 production was found to be specific for cells from patients with an AML with monocytic differentiation (12 of 15 M4 and M5 patients, 0 of 15 M1 and M2 patients). Moreover, IL-6 production was paralleled by IL-1 production. The IL-6- and IL-1-producing cells were mainly found in the more mature monocytic cell fractions, defined as CD14-positive and CD34-negative adherent cells. By limiting dilution experiments, it could be excluded that the production of IL-1 or IL-6 was due to contamination with normal monocytes.
Asunto(s)
Interleucina-1/biosíntesis , Interleucina-6/biosíntesis , Leucemia Monocítica Aguda/metabolismo , Leucemia Mielomonocítica Aguda/metabolismo , Antígenos CD34 , Antígenos de Diferenciación , Antígenos de Diferenciación Mielomonocítica , Northern Blotting , Adhesión Celular , Diferenciación Celular , Humanos , Técnicas In Vitro , Interleucina-6/genética , Receptores de Lipopolisacáridos , ARN Mensajero/genéticaRESUMEN
Neurodermatitis is a relatively common skin condition characterized by patches of scaly, pruritic skin in one or more of several classic locations. It can be treated with cryosurgery using Refrigerant 12 with successful clinical and aesthetic results.
Asunto(s)
Criocirugía , Neurodermatitis/cirugía , Adulto , Clorofluorocarburos de Metano , Humanos , Masculino , Cuero CabelludoRESUMEN
A recently developed radioimmunoprecipitation assay, using 125I-labeled human immunodeficiency virus (HIV) viral proteins enriched for glycoproteins gp120env, gp41env (GRIPA), was compared to the immunoblot assay with respect to sensitivity and specificity for the detection of antibodies to HIV. Longitudinal sets of serum samples of seroconverting homosexual men were studied, as were sera of six blood-bank donors likely to be false-positive in immunoblot. In addition, HIV isolation was attempted from white blood cells of these blood-bank donors and of seropositive and seronegative individuals. In sets of seroconversion samples, the GRIPA appeared at least as sensitive as the immunoblot. Some sera already were clearly positive in the GRIPA at a time when there was only weak reactivity in immunoblot. In contrast, sera from blood-bank donors that were regarded as false-positive in immunoblot were negative in GRIPA. Virus culture from these donors was also negative. It is concluded that reactivity in immunoblot to core proteins only may well be false-positive, whereas antibody reactivity in the radioimmunoprecipitation assay to p24gag solely suggests ongoing seroconversion. This feature, in addition to a sensitivity for anti-gp120env comparable to immunoblotting, makes the GRIPA a useful confirmatory assay in sera that yield conflicting results in other HIV-antibody assays.