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1.
J Clin Invest ; 82(1): 354-9, 1988 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-2839551

RESUMEN

The effect of insulin-like growth factor I (IGF-I) on growth of small cell lung cancer (SCLC) cell lines was studied. Western blot analysis of whole cell lysates of cell lines NCI-H345 and NCI-N417 demonstrated the presence of a 16-kD band consistent with an IGF-I precursor molecule. Scatchard plot analysis of cell line NCI-H345 using 125I-labeled IGF-I demonstrated two high affinity specific binding sites (Kd 1.3 and 4.0 nM with maximal rate (Bmax) 200 and 500 fmol/mg protein, respectively). The exogenous addition of IGF-I, IGF-II, or insulin resulted in marked proliferation of human SCLC cells as evaluated using an in vitro growth assay. These peptides stimulated the growth of SCLC cell lines NCI-H82, NCI-H209, NCI-H345, and NCI-N417. The concentration of IGF-I producing maximal SCLC cell growth was 10-100-fold less than that of insulin or IGF-II, whereas the maximal growth stimulated by the optimal concentration of these peptides were similar. An MAb that specifically binds to the IGF-I receptor (but not to the insulin receptor) mediates a dose-dependent inhibition of cell growth in basal media as well as IGF-I, IGF-II, or insulin-supplemented media. The IGF-I receptor thus appears to be the common pathway for the mitogenic activity by IGF-I, IGF-II, and insulin for human SCLC cell lines. The demonstration of an IGF-I precursor molecule, specific IGF-I receptor binding, IGF-I-mediated growth stimulation, and inhibition of basal cell growth by an MAb to the IGF-I receptor suggests that an IGF-I-like molecule can function in vitro as an autocrine growth factor for human SCLC cell lines.


Asunto(s)
Carcinoma de Células Pequeñas/patología , División Celular/efectos de los fármacos , Factor I del Crecimiento Similar a la Insulina/farmacología , Neoplasias Pulmonares/patología , Somatomedinas/farmacología , Animales , Anticuerpos Monoclonales/fisiología , Carcinoma de Células Pequeñas/metabolismo , Línea Celular , Inhibidores de Crecimiento/fisiología , Humanos , Insulina/farmacología , Factor I del Crecimiento Similar a la Insulina/aislamiento & purificación , Factor II del Crecimiento Similar a la Insulina/farmacología , Neoplasias Pulmonares/metabolismo , Ratones , Precursores de Proteínas/aislamiento & purificación , Receptor de Insulina/análisis , Receptor de Insulina/inmunología , Receptores de Somatomedina , Células Tumorales Cultivadas
2.
Mol Cell Biol ; 8(8): 3129-35, 1988 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-3211139

RESUMEN

The 27-amino-acid gastrin-releasing peptide (GRP1-27) is a neuropeptide and growth factor that is synthesized by various neural and neuroendocrine cells. The major pro-GRP hormone (isoform I) contains both GRP1-27 and a novel C-terminal extension peptide termed pro-GRP31-125. In order to define potentially active neuropeptides that could be generated from this novel protein domain, we analyzed the posttranslational processing of endogenous human pro-GRP1-125 in a small-cell lung cancer cell line. Because such studies are much easier in an overexpression system, we investigated at the same time the posttranslational processing of baculovirus-expressed human pro-GRP1-125 in an insect ovary cell line. In the small-cell lung cancer cell line, GRP1-27 was cleaved as expected from the endogenous prohormone at a pair of basic amino acids (29 and 30) and alpha-amidated at its C-terminal methionine; however, a number of novel peptides were generated by additional cleavages in the pro-GRP31-125 domain. In the insect ovary cell line, GRP1-27 was cleaved from the expressed prohormone by a different mechanism, as were a number of other peptides that appeared to be similar in size to those produced by the human neuroendocrine tumor cell line. These data show for the first time that an insect ovary cell line that is widely used to overexpress proteins can process a human neuropeptide precursor. They also reveal the existence of novel pro-GRP-derived peptides that are candidates for biologically active ligands.


Asunto(s)
Hormonas Gastrointestinales/genética , Péptidos/genética , Precursores de Proteínas/genética , Procesamiento Proteico-Postraduccional , Secuencia de Aminoácidos , Animales , Línea Celular , Clonación Molecular , Genes , Humanos , Virus de Insectos/genética , Datos de Secuencia Molecular , Mariposas Nocturnas
3.
J Natl Cancer Inst ; 83(20): 1470-6, 1991 Oct 16.
Artículo en Inglés | MEDLINE | ID: mdl-1656058

RESUMEN

We have evaluated an anti-autocrine growth factor monoclonal antibody for potential use in the treatment of patients with small-cell lung cancer. The monoclonal antibody, designated 2A11, binds to the C-terminal region of the autocrine growth factor gastrin-releasing peptide and neutralizes its growth-promoting effects in vitro and in vivo. Equilibrium-binding analysis demonstrated that the peptide binds to the antibody (dissociation constant = 1.5 x 10(-10) at least as avidly as it binds to the tumor peptide receptor. Pharmacokinetic studies in normal BALB/c mice demonstrated an initial clearance half-life (alpha t1/2) of 24.3 +/- 4 hours and a secondary clearance half-life (beta t1/2) of 1039.6 +/- 309 hours, and biodistribution studies revealed a distribution pattern which generally reflected blood flow. Single intravenous infusions of 2A11 (20 mg/20-25-kg dogs) into normal mongrel dogs with surgically created gastric fistulas antagonized the stimulatory effects of exogenously infused gastrin-releasing peptide or bombesin on plasma gastrin release and gastric acid secretion. Toxicology studies in normal dogs (with gastric fistulas) infused with 50 mg 2A11 intravenously three times a week for 4 weeks failed to reveal any adverse behavioral, clinical, or pathological effects. Four of six dogs developed an immune response to 2A11. Anti-idiotypic antibodies elicited in two cases did not mimic the functional effects of the peptide. We conclude that the concept of immunoblockade of an autocrine growth factor appears feasible in vivo.


Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Bombesina/inmunología , Carcinoma de Células Pequeñas/terapia , Sustancias de Crecimiento/inmunología , Neoplasias Pulmonares/terapia , Péptidos/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Perros , Ácido Gástrico/metabolismo , Péptido Liberador de Gastrina , Gastrinas/metabolismo , Ratones , Ratones Endogámicos BALB C , Distribución Tisular
4.
Cancer Res ; 52(10): 2771-6, 1992 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-1349849

RESUMEN

Amplification and/or overexpression of the erbB-2 gene have been demonstrated in 20-30% of adenocarcinomas of the breast, ovary, lung, and stomach and are associated with aggressive clinical course and poor prognosis. Interference with erbB-2 function by the use of monoclonal antibodies is a promising approach to the treatment of these diseases. In this study we demonstrate that a combination of two anti-erbB-2-specific antibodies inhibited the growth of human gastric tumor cells in vitro. This combination antibody therapy also inhibited the growth of human tumor cell lines growing as xenografts in nude mice and was able to dramatically reduce established tumors. This is the first reported observation of tumor regression induced by anti-erbB-2 monoclonal antibodies. Treatment was not curative in that tumors regrew after 6 weeks. Treatment with either single antibody alone did not inhibit cell growth or tumor formation. Pulse chase and tyrosine kinase activity experiments were used to investigate the activity of the erbB-2 gene product (gp185erbB-2). The formation of complexes by two antibodies was found to interfere with receptor function and mimic some properties of a typical receptor ligand. Selective interference of the erbB-2 receptor by combination antibody therapy may be advantageous for the treatment of human cancers.


Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Proteínas Proto-Oncogénicas/inmunología , Neoplasias Gástricas/terapia , Células 3T3/fisiología , Animales , Anticuerpos Monoclonales/metabolismo , División Celular/fisiología , Colorimetría , Modelos Animales de Enfermedad , Expresión Génica/genética , Humanos , Inmunización , Inmunoterapia , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Receptor ErbB-2 , Neoplasias Gástricas/genética , Neoplasias Gástricas/inmunología , Sales de Tetrazolio , Tiazoles , Trasplante Heterólogo , Células Tumorales Cultivadas
5.
Cancer Res ; 59(12): 2939-43, 1999 Jun 15.
Artículo en Inglés | MEDLINE | ID: mdl-10383158

RESUMEN

Homocamptothecin (hCPT) is a semisynthetic analogue of camptothecin (CPT) with a seven-membered beta-hydroxylactone resulting from the insertion of a methylene spacer between the alcohol moiety and the carboxyl function of the naturally occurring six-membered alpha-hydroxylactone of CPT. This E-ring modification provides a less reactive lactone with enhanced stability and decreased protein binding in human plasma. Biological testing against CPT revealed that, instead of being detrimental, the modified lactone of hCPT has a positive impact on topoisomerase I (Topo I) poisoning properties. In vitro tests showed hCPT to fully conserve the ability to stabilize Topo I-DNA cleavage complexes and to stimulate a higher level of DNA cleavage than CPT. A similar trend toward improvement was also observed in antiproliferative assays with human tumor cell lines (including cells overexpressing P-glycoprotein). In two distinct in vivo models, using L1210 murine leukemia or human colon carcinoma HT29, hCPT was found to be more efficacious than CPT. The slow, but irreversible, hydrolysis of hCPT, instead of the fast equilibrium of CPT, may account for its good in vivo activity. Overall, these results provide evidence that a highly reactive lactone is not a requisite for the Topo I-mediated antitumor activity of CPT analogues, and that hCPT is an interesting pharmacological tool with improved solution behavior as well as a promising new template for the preparation of more efficacious Topo I poisons.


Asunto(s)
Antineoplásicos/uso terapéutico , Camptotecina/análogos & derivados , Lactonas/metabolismo , Inhibidores de Topoisomerasa I , Animales , Antineoplásicos/química , Antineoplásicos/metabolismo , Camptotecina/química , Camptotecina/farmacología , División Celular/efectos de los fármacos , ADN/efectos de los fármacos , ADN/metabolismo , Modelos Animales de Enfermedad , Ensayos de Selección de Medicamentos Antitumorales , Estabilidad de Medicamentos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/uso terapéutico , Células HT29 , Humanos , Células K562 , Leucemia L1210/tratamiento farmacológico , Leucemia Experimental/tratamiento farmacológico , Trasplante de Neoplasias , Trasplante Heterólogo , Células Tumorales Cultivadas
6.
Clin Cancer Res ; 2(1): 75-80, 1996 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-9816093

RESUMEN

The recombinant oncotoxin OLX-209 [e23(Fv)PE38KDEL] has been developed to target cancers with erbB-2 expression and is nearing a clinical trial. Important in clinical planning is the selection of patients on the basis of tumor expression of erbB-2. ErbB-2 gene amplification occurs in cancers of the breast, stomach, and ovary. Patients with these diseases and evident overexpression are candidates for OLX-209 therapy. In lung cancer, overexpression of erbB-2 is also frequent, but in most cases, it is not caused by gene amplification. This study demonstrates that OLX-209 has activity on lung cancer cells with varying levels of erbB-2 expression in the presence and absence of gene amplification. In vitro sensitivity of cell lines to OLX-209 is related to erbB-2 expression level. Normal bronchial epithelial cells were not sensitive. Effective treatment of lung cancer cell lines growing as xenografts in nude mice was shown with Calu-3 (a lung adenocarcinoma line with high levels of p185(erbB-2) caused by gene amplification) and three other lung adenocarcinomas (A549, NCI-H1466, and 201T) with lower levels of p185(erbB-2) and no gene amplification. The 201T cell line was isolated recently from a lung tumor with erbB-2 expression in the original tumor. The results of this study indicate that patients with erbB-2-positive, non-small cell lung cancer should be included in clinical trials of OLX-209.


Asunto(s)
Amplificación de Genes , Genes erbB-2 , Inmunotoxinas/uso terapéutico , Neoplasias Pulmonares/tratamiento farmacológico , Adenocarcinoma/tratamiento farmacológico , Animales , Anticuerpos , Exotoxinas , Humanos , Inmunotoxinas/farmacología , Neoplasias Pulmonares/genética , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Proteínas Recombinantes de Fusión/farmacología , Proteínas Recombinantes de Fusión/uso terapéutico , Anticuerpos de Cadena Única , Trasplante Heterólogo , Células Tumorales Cultivadas
7.
J Med Chem ; 41(27): 5410-9, 1998 Dec 31.
Artículo en Inglés | MEDLINE | ID: mdl-9876111

RESUMEN

Homocamptothecin (hCPT), a camptothecin (CPT) analogue with a seven membered beta-hydroxylactone which combines enhanced plasma stability and potent topoisomerase I (Topo I)-mediated activity, is an attractive template for the elaboration of new anticancer agents. Like CPT, hCPT carries an asymmetric tertiary alcohol and displays stereoselective inhibition of Topo I. The preparation and biological screening of racemic hCPT analogues are described. The 10 hCPTs tested were better Topo I inhibitors than CPT. Fluorinated hCPTs 23c, d,f,g were found to have potent cytotoxic activity on A427 and PC-3 tumor cell lines. Their cytotoxicity remained high on the K562adr and MCF7mdr cell lines, which overexpress a functionally active P-glycoprotein. Fluorinated hCPTs were more efficacious in vivo than CPT on HT-29 xenografts. In this model, a tumor growth delay of 25 days was reached with hCPT 23g at a daily dose of 0.32 mg/kg, compared to 4 days with CPT at 0.625 mg/kg. Thus difluorinated hCPT 23g warrants further investigation as a novel Topo I inhibitor with high cytotoxicity toward tumor cells and promising in vivo efficacy.


Asunto(s)
Antineoplásicos/síntesis química , Benzoxepinas/síntesis química , Camptotecina/análogos & derivados , Camptotecina/síntesis química , Inhibidores Enzimáticos/síntesis química , Inhibidores de Topoisomerasa I , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Benzoxepinas/química , Benzoxepinas/farmacología , Camptotecina/química , Camptotecina/farmacología , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Femenino , Humanos , Concentración 50 Inhibidora , Masculino , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Estereoisomerismo , Relación Estructura-Actividad , Trasplante Heterólogo , Células Tumorales Cultivadas
8.
Ann N Y Acad Sci ; 922: 100-11, 2000.
Artículo en Inglés | MEDLINE | ID: mdl-11193885

RESUMEN

Homocamptothecins (hCPT) are modified camptothecins (CPT) with a seven-membered beta-hydroxylactone instead of the naturally occurring six-membered alpha-hydroxylactone. This E-ring modification fully conserves the ability to stabilize topo I-DNA single-strand breaks and stimulates high levels of DNA cleavage. A key feature is the irreversibility of E-ring opening, which should give reduced toxicity. Substituted hCPTs have been selected for their high antiproliferative activity on a panel of tumor cell lines, including those with cross resistance, and were found to be active at very low doses in a variety of human tumor xenografts when administered orally. BN 80915, a difluoro-hCPT, has entered clinical trials.


Asunto(s)
Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Camptotecina/análogos & derivados , Camptotecina/síntesis química , Camptotecina/farmacología , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Animales , Humanos , Ensayos Antitumor por Modelo de Xenoinjerto
10.
Anticancer Res ; 20(5A): 2835-47, 2000.
Artículo en Inglés | MEDLINE | ID: mdl-11062691

RESUMEN

The peripheral-type benzodiazepine receptor (PBR) expression and localization correlate with human breast cancer cell proliferation and aggressive phenotype expression. The standardized extract of Ginkgo biloba leaves (EGb 761) and isolated ginkgolide B (GKB) were shown to decrease PBR mRNA expression in adrenal cells. We examined the effect of EGb 761 and GKB on PBR expression and cell proliferation in human breast cancer cells. EGb 761 and GKB decreased in a time- and dose-dependent manner PBR expression and cell proliferation in the highly aggressive, rich in PBR, human breast cancer cell line MDA-231 whereas they did not affect the proliferation of the non-aggressive human breast cancer cell line MCF-7, which contains very low PBR levels. This effect was reversible and not due to the antioxidant properties of the compounds tested. Using a human cDNA expression array we determined that EGb 761 treatment altered, in addition to PBR, the expression of 36 gene products involved in various pathways regulating cell proliferation. These in vitro data were further validated in an in vivo model where EGb 761 and GKB significantly inhibited the nuclear PBR expression and growth of MDA-231 cell xenografts in nude mice. Taken together, these data suggest that the manipulation of PBR expression could be used to control tumor growth and that EGb 761 and GKB, under the conditions used, exert cytostatic properties.


Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Antioxidantes/farmacología , Diterpenos , Flavonoides/farmacología , Lactonas/farmacología , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Receptores de GABA-A/biosíntesis , Animales , Antineoplásicos Fitogénicos/uso terapéutico , Antioxidantes/uso terapéutico , División Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Femenino , Flavonoides/uso terapéutico , Regulación de la Expresión Génica/efectos de los fármacos , Ginkgo biloba , Ginkgólidos , Humanos , Lactonas/uso terapéutico , Ligandos , Ratones , Ratones Desnudos , Fitoterapia , Extractos Vegetales , Plantas Medicinales , ARN Mensajero , Receptores de GABA-A/genética , Transcripción Genética/efectos de los fármacos , Células Tumorales Cultivadas
11.
Oncology (Williston Park) ; 5(5): 25-32; discussion 32-3, 37, 1991 May.
Artículo en Inglés | MEDLINE | ID: mdl-1832003

RESUMEN

The current mortality rate for lung cancer, which approaches 90% at two years, is not decreasing despite intensive clinical trials attempting to improve systemic therapy. New drug discoveries and dose intensification approaches have not resulted in more effective anti-tumor control. An alternative approach using sputum immunocytology for early lung cancer detection was recently reported. The proposed basis for this early detection reflects the underlying biology of immunodominant carbohydrate tumor-associated antigens, which are recognized as a class of oncofetal antigens. The process of bronchial epithelial carcinogenesis is associated with an evolution of carbohydrate display which reverses the sequence originally associated with fetal organ development. By elucidating this pattern of fetal carbohydrate display, a precise map of stage of bronchial carcinogenesis may emerge.


Asunto(s)
Neoplasias Pulmonares/diagnóstico , Antígenos de Neoplasias/análisis , Antígenos de Carbohidratos Asociados a Tumores/análisis , Biomarcadores de Tumor/análisis , Sustancias de Crecimiento/análisis , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Factores de Tiempo , Tretinoina/uso terapéutico
16.
Anticancer Agents Med Chem ; 8(8): 857-62, 2008 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-19075568

RESUMEN

The identification of a CDC25 inhibitor to arrest the cell cycle closely followed the discovery of CDC25 by Russell and Nurse in 1986. Recent advances at the preclinical and clinical stages reinforce the rationale to consider CDC25 as a relevant target for a cancer treatment. Here, in order to exemplify recent drug discovery efforts, we present our own experience with various chemical series of CDC25 inhibitors. We discuss how we have progressed and how we are considering the next steps to define the clinical entry points and hopefully complete this target validation to generate a new class of therapeutic agents.


Asunto(s)
Antineoplásicos/farmacología , Inhibidores Enzimáticos/farmacología , Fosfatasas cdc25/antagonistas & inhibidores , Animales , Humanos
17.
Br J Cancer ; 97(12): 1673-82, 2007 Dec 17.
Artículo en Inglés | MEDLINE | ID: mdl-18026194

RESUMEN

Therapies for hormone-independent prostate and breast cancer are limited, with the effectiveness of the taxanes compromised by toxicity, lack of oral bioavailability and drug resistance. This study aims to identify and characterise new microtubule disruptors, which may have improved efficacy relative to the taxanes in hormone-independent cancer. 2-Methoxy-3-O-sulphamoyl-17beta-cyanomethyl-oestra-1,3,5(10)-triene (STX641), 2-methoxy-3-hydroxy-17beta-cyanomethyl-oestra-1,3,5(10)-triene (STX640) and 2-methoxyoestradiol-3,17-O,O-bis-sulphamate (STX140) were all potent inhibitors of cell proliferation in a panel of prostate and breast cancer cell lines. STX641 and STX640 significantly inhibited tumour growth in the MDA-MB-231 xenograft model. STX641 inhibited both in vitro and in vivo angiogenesis. Despite good in vivo activity, STX641 was not as potent in vivo as STX140. Therefore, STX140 was evaluated in the prostate hormone-independent PC-3 xenograft model. STX140 had superior efficacy to docetaxel, 2-MeOE2 and bevacizumab. In contrast to vinorelbine, no significant toxicity was observed. Furthermore, STX140 could be dosed daily over a 60-day period leading to tumour regression and complete responses, which were maintained after the cessation of dosing. This study demonstrates that STX641 and STX140 have considerable potential for the treatment of hormone-independent breast and prostate cancer. In contrast to the taxanes, STX140 can be dosed orally, with no toxicity being observed even after prolonged daily dosing.


Asunto(s)
Inhibidores de la Angiogénesis/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Estrenos/uso terapéutico , Neoplasias de la Próstata/tratamiento farmacológico , Moduladores de Tubulina/uso terapéutico , Animales , Apoptosis , Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de la radiación , Células Cultivadas , Resistencia a Antineoplásicos , Células Endoteliales/efectos de los fármacos , Femenino , Humanos , Masculino , Ratones , Ratones Desnudos , Neoplasias Hormono-Dependientes
18.
Biochemistry ; 25(1): 141-51, 1986 Jan 14.
Artículo en Inglés | MEDLINE | ID: mdl-2869781

RESUMEN

Probes for fluorescence energy transfer measurements were introduced into active sites of dodecameric glutamine synthetase from Escherichia coli by substituting appropriate ATP analogues for ATP in the autoinactivation reaction of this enzyme with L-Met-(S)-sulfoximine and Mn2+ [Maurizi, M. R., & Ginsburg, A. (1986) Biochemistry (preceding paper in this issue)]. Two fluorescent donors, 8-mercapto-ATP alkylated with either 5-[[[(iodoacetyl)amino]ethyl]amino]naphthalene-1-sulfonic acid (AEDANS-ATP) or 1,N6-etheno-2-aza-ATP (aza-epsilon-ATP), and two acceptors, 6-mercaptopurine ribonucleotide triphosphate or 8-mercapto-ATP alkylated with the chromophore 4'-[[4-(dimethylamino)-phenyl]azo]-2-iodoacetanilide (6-Y- or 8-Y-ATP), were used. Fluorescence emissions of enzyme derivatives with 1 or 2 equiv of fluorescent donor per dodecamer and either an acceptor (Y) or ADP at the remaining active sites were compared at pH 7.0. The results, together with the known geometry of the enzyme, indicate that active site probes in the dodecamer are widely separated and that energy transfer occurs from a single donor to two or three acceptors on adjacent subunits. The calculated distance between equidistant active site probes on heterologously bonded subunits within the same hexagonal ring is 56-60 A. Probes on isologously bonded subunits are no closer than 60 A and may be as far apart as 78 A. Thus, active sites are away from the 6-fold axis of symmetry toward the outer edges of the dodecamer and are located greater than or equal to 30 A from the plane separating the hexagonal rings. During Zn2+-induced stacking of the same enzyme derivatives along the 6-fold axes of symmetry, additional quenches of fluorescent probes were dependent on the presence of acceptors on separate dodecamers. The Zn2+-induced face to face aggregation of dodecamers in the presence of 46 microM ZnCl2 and 9 mM MgCl2 at pH 7.0 had an Arrhenius activation energy of 22.3 +/- 0.2 kcal/mol and a second-order rate constant at 25 degrees C of approximately 10(5) M-1 s-1 at early stages. Time-dependent fluorescence quenches correlated well with the degree of linear polymer formation and reached maximum values of 47-70% quench when the average n-mer was six dodecamers. After correction for unquenched polymer ends, a fluorescent donor and an acceptor probe in layered dodecamers were estimated to be approximately 36 A apart--an average value if there is some twisting of single strands.(ABSTRACT TRUNCATED AT 400 WORDS)


Asunto(s)
Adenosina Trifosfato/análogos & derivados , Escherichia coli/enzimología , Glutamato-Amoníaco Ligasa/metabolismo , Adenosina Trifosfato/farmacología , Sitios de Unión , Transferencia de Energía , Colorantes Fluorescentes , Cinética , Sustancias Macromoleculares , Matemática , Modelos Moleculares , Conformación Proteica , Espectrometría de Fluorescencia/métodos , Relación Estructura-Actividad
19.
Biochemistry ; 22(8): 1877-82, 1983 Apr 12.
Artículo en Inglés | MEDLINE | ID: mdl-6342671

RESUMEN

Fluorescence energy transfer experiments were used to measure distances between three fluorescently labeled sulfhydryl sites on Escherichia coli carbamoyl-phosphate synthetase, an unsymmetrical dimer. When five different combinations of fluorescent donor-acceptor pairs are used, the distance between site 1, located on the large subunit, and site 2, located on the small subunit, is in the range of 27-33 A. Similarly, the distance between site 1 and site 3 (large subunit) was approximately 27 A and between site 2 and site 3 was approximately 21 A. A similar approach was employed to determine distances between each sulfhydryl group and the ATP site(s), and in all cases no fluorescence quenching was observed using Cr3+ATP or Co(NH3)4ATP as substrate analogues. A lower limit could be calculated from these data, resulting in a distance of greater than or equal to 21 A from each sulfhydryl site to the ATP site. Additional experiments were performed to evaluate if the substrates ATP, HCO3(-), or glutamine or the allosteric modifiers ornithine, IMP, and UMP altered the distance relationships among the sulfhydryl sites. IMP and UMP produced a slight decrease in fluorescence between sites while glutamine and ATP produced a slight increase in fluorescence.


Asunto(s)
Carbamoil-Fosfato Sintasa (Amoniaco)/metabolismo , Escherichia coli/enzimología , Ligasas/metabolismo , Transferencia de Energía , Cinética , Espectrometría de Fluorescencia , Compuestos de Sulfhidrilo/análisis , Reactivos de Sulfhidrilo/farmacología
20.
Proc Natl Acad Sci U S A ; 89(13): 5867-71, 1992 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-1352878

RESUMEN

Immunotoxins were made using five different murine monoclonal antibodies to the human erbB2 gene product and LysPE40, a 40-kDa recombinant form of Pseudomonas exotoxin (PE) lacking its cell-binding domain. All five conjugates were specifically cytotoxic to cancer cell lines overexpressing erbB2 protein. The most active conjugate was e23-LysPE40, generated by chemical crosslinking of anti-erbB2 monoclonal antibody e23 to LysPE40. In addition, a recombinant immunotoxin, e23(Fv)PE40, was constructed that consists of the light-chain variable domain of e23 connected through a peptide linker to its heavy-chain variable domain, which in turn is fused to PE40. The recombinant protein was made in Escherichia coli, purified to near homogeneity, and shown to selectively kill cells expressing the erbB2 protooncogene. To improve the cytotoxic activity of e23(Fv)PE40, PE40 was replaced with a variant, PE38KDEL, in which the carboxyl end of PE is changed from Arg-Glu-Asp-Leu-Lys to Lys-Asp-Glu-Leu and amino acids 365-380 of PE are deleted. The e23(Fv)PE38KDEL protein inhibits the growth of tumors formed by the human gastric cancer cell line N87 in immunodeficient mice.


Asunto(s)
ADP Ribosa Transferasas , Exotoxinas/química , Inmunotoxinas/química , Proteínas Proto-Oncogénicas/inmunología , Receptores de Superficie Celular/inmunología , Factores de Virulencia , Secuencia de Aminoácidos , Animales , Citotoxicidad Celular Dependiente de Anticuerpos , Toxinas Bacterianas/química , Toxinas Bacterianas/toxicidad , Secuencia de Bases , Citotoxicidad Inmunológica , Exotoxinas/toxicidad , Inmunoterapia , Técnicas In Vitro , Ratones , Ratones Desnudos , Datos de Secuencia Molecular , Trasplante de Neoplasias , Oligodesoxirribonucleótidos/química , Pseudomonas aeruginosa , Receptor ErbB-2 , Proteínas Recombinantes de Fusión/toxicidad , Neoplasias Gástricas/terapia , Relación Estructura-Actividad , Células Tumorales Cultivadas , Exotoxina A de Pseudomonas aeruginosa
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