RESUMO
Massive present-day early-type (elliptical and lenticular) galaxies probably gained the bulk of their stellar mass and heavy elements through intense, dust-enshrouded starbursts--that is, increased rates of star formation--in the most massive dark-matter haloes at early epochs. However, it remains unknown how soon after the Big Bang massive starburst progenitors exist. The measured redshift (z) distribution of dusty, massive starbursts has long been suspected to be biased low in z owing to selection effects, as confirmed by recent findings of systems with redshifts as high as ~5 (refs 2-4). Here we report the identification of a massive starburst galaxy at z = 6.34 through a submillimetre colour-selection technique. We unambiguously determined the redshift from a suite of molecular and atomic fine-structure cooling lines. These measurements reveal a hundred billion solar masses of highly excited, chemically evolved interstellar medium in this galaxy, which constitutes at least 40 per cent of the baryonic mass. A 'maximum starburst' converts the gas into stars at a rate more than 2,000 times that of the Milky Way, a rate among the highest observed at any epoch. Despite the overall downturn in cosmic star formation towards the highest redshifts, it seems that environments mature enough to form the most massive, intense starbursts existed at least as early as 880 million years after the Big Bang.
RESUMO
Recent experiments in bacterial systems have established an extended database of sequences broadly relevant to all membrane transporters, allowing serious study of evolutionary relationships. The database will be especially useful in integrating conclusions derived from work with proteins in the major facilitator superfamily, because this kinship includes both eukaryotic and prokaryotic model systems. Even among carriers not linked by evolution, clear hints of functional homology have been note. Advances are also evident in the structural analysis of membrane carriers. Site-directed mutagenesis in a bacterial antiporter has shown how the translocation pathway might be identified; this should complement recent progress in preparing two-dimensional crystals of the eukaryotic anion-exchange protein, band 3. Together, these studies could soon verify or reject the idea that the transport pathway lies at the interface between the amino-terminal and carboxy-terminal helical bundles found in the hydrophobic core of most carrier proteins. If verified, the argument might allow construction of informed three-dimensional models in the absence of crystallographic evidence.
Assuntos
Antiporters/fisiologia , Proteínas de Bactérias/fisiologia , Evolução BiológicaRESUMO
DNA mismatch repair genes have been implicated in the pathogenesis and predisposition of certain malignancies through a mutator phenotype. In this study, we investigated, in 150 non-small cell lung carcinomas, the expression levels of hMLH1 and hMSH2 proteins in relation to loss of heterozygosity on chromosomes 3p and 2p, the mutational status of these genes' promoters and the hot spot exons. We have demonstrated that 88 of 150 (58.6%) tumor specimens had reduced expression levels of the hMLH1 protein, whereas 85 of 147 (57.8%) specimens had reduced expression levels of the hMSH2 protein. Reduced expression levels of both proteins were observed in 51 of 150 (34%) specimens. In adenocarcinomas, the reduction of hMSH2 expression was more frequently observed than that of hMLH1 (P<0.003), whereas in squamous cell carcinoma of the lung hMLH1 expression was more frequently reduced than hMSH2 (P<0.006). Reduced expression of hMLH1correlated with allelic imbalance on loci D3S1289 (P<0.0002) and D2S391 (P<0.05). It is of note that an inverse correlation was found between hMSH2 reduced expression and loss of heterozygosity at locus D3S1300 (P = 0.016). In addition, hMLH1 reduced expression was more frequently associated with heavy smokers, assessed by daily tobacco uptake (P = 0.018) and total smoking exposure (pack-years; P<0.05). In addition, a correlation between hMLH1 reduced expression and nodal metastasis in squamous cell carcinoma of the lung was observed (P = 0.015). No mutations were identified in the promoters or exons examined in these two genes. These findings indicate that hMLH1 and hMSH2 gene inactivation is a common event in the development of non-small cell lung carcinoma and allelic loss seems to be a major genetic event involved in hMLH1 silencing. In addition, we propose that a putative negative regulator of hMSH2 gene may be located at the locus 3p14.
Assuntos
Alelos , Carcinoma Pulmonar de Células não Pequenas/genética , Cromossomos Humanos Par 3 , Proteínas de Ligação a DNA , Neoplasias Pulmonares/genética , Proteínas de Neoplasias/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Adaptadoras de Transdução de Sinal , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Proteínas de Transporte , Cromossomos Humanos Par 2 , Análise Mutacional de DNA , Éxons/genética , Feminino , Humanos , Perda de Heterozigosidade , Neoplasias Pulmonares/metabolismo , Masculino , Pessoa de Meia-Idade , Proteína 1 Homóloga a MutL , Proteína 2 Homóloga a MutS , Mutação , Proteínas de Neoplasias/biossíntese , Proteínas Nucleares , Polimorfismo Conformacional de Fita Simples , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas/biossínteseRESUMO
We examined genomic instability in DNA from 80 bronchial lavage samples from patients with lung cancer and individuals with no malignant lung disease. We used a multiplex assay of eight fluorescent-tagged microsatellite markers that have a very high incidence of allelic imbalance in lung tumors. When genomic instability at individual loci was analyzed statistically against diagnosis, markers D3S1289 (P = 0.033), D3S1300 (P = 0.001), D13S171 (P = 0.009), and D17S2179E (P = 0.017) demonstrated significantly higher frequency of instability in bronchial lavage specimens from lung cancer cases than those with nonmalignant conditions. In contrast, markers D9S157, D9S161, D13S153, and D5S644 demonstrated lower specificity (P > 0.05) for lung tumors. These results suggest that genomic instability in some loci may be related to high proliferation rates but not necessarily to cell commitment to malignancy. When genomic instability was scored with only the four cancer-specific markers, the assay produced a sensitivity of 73.9% and a specificity of 76.5%. On combining the results from the cytological examination and the molecular assay, the sensitivity reached 82.6%. These results indicate that in our efforts to investigate genomic instability as a potential marker for the early detection of lung cancer, we need to identify cancer-specific genomic instability markers. This paper has shown that these first four markers may be considered to form an individual set of cancer-specific genomic instability markers.
Assuntos
Lavagem Broncoalveolar , Perda de Heterozigosidade , Neoplasias Pulmonares/genética , Repetições de Microssatélites/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Líquido da Lavagem Broncoalveolar/química , DNA de Neoplasias/sangue , DNA de Neoplasias/genética , Feminino , Fluorescência , Humanos , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase/métodos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
General principles of membrane function have been elucidated by the study of lactic acid bacteria. In this review, the operation and function of ion pumps, secondary transport systems and solute ATPases will be discussed. Despite their differences in kinetics and mechanisms between the transport systems, structural similarities can be recognized among these proteins irrespective of whether they originate from prokaryotes, lower or higher eukaryotes.
Assuntos
Membrana Celular/metabolismo , Lactobacillus/metabolismo , Adenosina Trifosfatases/metabolismo , Transporte Biológico Ativo , Membrana Celular/enzimologia , Canais Iônicos/metabolismo , Cinética , Lactobacillus/enzimologiaRESUMO
A general method has been developed for determining the rate of entry of lactose into cells of Escherichia coli that contain beta-galactosidase. Lactose entry is measured by either the glucose or galactose released after lactose hydrolysis. Since lactose is hydrolyzed by beta-galactosidase as soon as it enters the cell, this assay measures the activity of the lactose transport system with respect to the translocation step. Using assays of glucose release, lactose entry was studied in strain GN2, which does not phosphorylate glucose. Lactose entry was stimulated 3-fold when cells were also presented with readily metabolizable substrates. Entry of omicron-nitrophenyl-beta-D-galactopyranoside (ONPG) was only slightly elevated (1.5-fold) under the same conditions. The effects of arsenate treatment and anaerobiosis suggest that lactose entry may be limited by the need for reextrusion of protons which enter during H+/sugar cotransport. Entry of omicron-nitrophenyl-beta-D-galactopyranoside is less dependent on the need for proton reextrusion, probably because the stoichiometry of H+/substrate cotransport is greater for lactose than for ONPG.
Assuntos
Escherichia coli/metabolismo , Lactose/metabolismo , Arseniatos/farmacologia , Etilmaleimida/farmacologia , Frutosefosfatos/farmacologia , Galactose/metabolismo , Glucose/metabolismo , Glucofosfatos/farmacologia , Mutação , Nitrofenilgalactosídeos/metabolismo , Óperon , Consumo de Oxigênio , beta-Galactosidase/metabolismoRESUMO
Understanding the selectivity of aquaporin water channels will require structural and functional studies of wild-type and modified proteins; however, expression systems have not previously yielded aquaporins in the necessary milligram quantities. Here we report expression of a histidine-tagged form of Escherichia coli aquaporin-Z (AqpZ) in its homologous expression system. 10-His-AqpZ is solubilized and purified to near homogeneity in a single step with a final yield of approximately 2.5 mg/l of culture. The histidine tag is removed by trypsin, yielding the native protein with the addition of three N-terminal residues, as confirmed by microsequencing. Sucrose gradient sedimentation analysis showed that the native, solubilized AqpZ protein is a trypsin-resistant tetramer. Unlike other known aquaporins, AqpZ tetramers are not readily dissociated by 1% SDS at neutral pH. Hydrophilic reducing agents have a limited effect on the stability of the tetramer in 1% SDS, whereas incubations for more than 24 hours, pH values below 5.6, or exposure to the hydrophobic reducing agent ethanedithiol cause dissociation into monomers. Cys20, but not Cys9, is necessary for the stability of the AqpZ tetramer in SDS. Upon reconstitution into proteoliposomes, AqpZ displays very high osmotic water permeability (pf > or = 10 x 10(-14) cm3 s-1 subunit-1) and low Arrhenius activation energy (Ea = 3.7 kcal/mol), similar to mammalian aquaporin-1 (AQP1). No permeation by glycerol, urea or sorbitol was detected. Expression of native and modified AqpZ in milligram quantities has permitted biophysical characterization of this remarkably stable aquaporin tetramer, which is being utilized for high-resolution structural studies.
Assuntos
Aquaporinas/química , Aquaporinas/isolamento & purificação , Proteínas de Escherichia coli , Escherichia coli/química , Proteínas de Membrana , Sequência de Aminoácidos , Aquaporinas/genética , Aquaporinas/metabolismo , Cisteína/genética , Cisteína/metabolismo , Escherichia coli/genética , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Peso Molecular , Mutação , Concentração Osmolar , Permeabilidade , Conformação Proteica/efeitos dos fármacos , Proteolipídeos/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Substâncias Redutoras/farmacologia , Dodecilsulfato de Sódio/farmacologia , Solubilidade , Relação Estrutura-Atividade , Tripsina/metabolismo , Água/metabolismoRESUMO
Molecular water channels (aquaporins) allow living cells to adapt to osmotic variations by rapid and specific diffusion of water molecules. Aquaporins are present in animals, plants, algae, fungi and bacteria. Here we present an electron microscopic analysis of the most ancient water channel described so far: the aquaporin Z (AqpZ) of Escherichia coli. A recombinant AqpZ with a poly(histidine) tag at the N terminus has been constructed, overexpressed and purified to homogeneity. Solubilized with octylglucoside, the purified AqpZ remains associated as a homotetramer, and assembles into highly ordered two-dimensional tetragonal crystals with unit cell dimensions a = b = 95 A, gamma = 90 degrees when reconstituted by dialysis in the presence of lipids. Three-dimensional reconstruction of negatively stained lattices revealed the p42(1)2 packing arrangement that is also observed with the human erythrocyte water channel (AQP1). The 8 A projection map of the AqpZ tetramer in frozen hydrated samples is similar to that of AQP1, consistent with the high sequence homology between these proteins.
Assuntos
Aquaporinas/química , Proteínas de Escherichia coli , Escherichia coli/química , Proteínas de Membrana , Aquaporina 1 , Aquaporinas/genética , Aquaporinas/ultraestrutura , Antígenos de Grupos Sanguíneos , Cristalização , Cristalografia , Glucosídeos , Humanos , Processamento de Imagem Assistida por Computador , Bicamadas Lipídicas , Microscopia Eletrônica , Peso Molecular , Conformação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/ultraestrutura , Homologia de Sequência de Aminoácidos , Solubilidade , ÁguaRESUMO
Several [(1H-imidazol-1-yl)methyl]- and [(3-pyridinyl)methyl] pyrroles were prepared and evaluated in vitro as thromboxane synthetase inhibitors in human platelet aggregation studies. A number of structures, e.g. 10b,f,g,i (respective IC50 values: 1 microM, 50 nM, 42 nM, 44 nM) showed superior in vitro inhibition of TXA2 synthetase when compared to the standard dazoxiben (1). However, it was found that in vitro potency did not translate into nor correlate with in vivo activity when these compounds were evaluated in mice in a collagen-epinephrine-induced pulmonary thromboembolism model. (E)-1-Methyl-2-[(1H-imidazol-1-yl)methyl]-5-(2-carboxyprop-1-enyl) pyrrole (10b) was found to offer protection against collagen-epinephrine-induced mortality in mice, thereby demonstrating that oral administration is an effective route for absorption of this drug. Additional evidence for the oral effectiveness of 10b in lowering serum TXB2 levels was obtained by performing ex vivo radioimmunoassay experiments with rats. A 13-week study of 10b in rats with reduced renal mass was conducted in order to evaluate the role of TXA2 production in hypertension and renal dysfunction. Although serum and urinary TXB2 levels in rats were found to be lowered during this study by 10b, the levels of urinary protein excretion remained comparable to that of the control group.
Assuntos
Imidazóis/farmacologia , Piridinas/farmacologia , Pirróis/farmacologia , Tromboxano-A Sintase/antagonistas & inibidores , Animais , Aorta/ultraestrutura , Disponibilidade Biológica , Plaquetas/enzimologia , Fenômenos Químicos , Química , Humanos , Imidazóis/síntese química , Imidazóis/farmacocinética , Inflamação/enzimologia , Nefropatias/metabolismo , Masculino , Camundongos , Microssomos/enzimologia , Inibidores da Agregação Plaquetária , Endoperóxidos Sintéticos de Prostaglandinas/sangue , Prostaglandina H2 , Prostaglandinas H/sangue , Piridinas/síntese química , Piridinas/farmacocinética , Pirróis/síntese química , Pirróis/farmacocinética , Ratos , Relação Estrutura-Atividade , Suínos , Tromboxano A2/sangue , Tromboxano A2/metabolismo , Tromboxano B2/metabolismoRESUMO
A series of 1H-imidazol-1-yl- and 3-pyridyl-substituted 3,4-dihydroquinolin-2(1H)-ones was designed and synthesized as combined inhibitors of thromboxane (TXA2) synthase and cAMP phosphodiesterase (PDE) in human blood platelets. A number of structures, e.g. 4b, 7a, 7e, 13a, and 21-25, were superior to dazoxiben 26 as inhibitors of TXA2 synthase in in vitro ADP-induced aggregation experiments with human blood platelets. The TXA2 synthase inhibitory activity was confirmed by measurement of the prostanoid metabolites derived from 14C-labeled arachidonic acid. Three compounds (7a, 7e, and 25) demonstrated in vitro inhibition of human platelet cAMP PDE at micromolar concentrations in conjunction with their TXA2 synthase inhibitory activity. Synergistic enhancement of antiaggregatory and antithrombotic actions was expected when simultaneous stimulation of adenylate cyclase (through increased PGI2 production) and inhibition of platelet cAMP PDE were possible from the same compound. Ex vivo and in vivo experiments were conducted in rats and mice, respectively, to evaluate the effects of compounds 7e and 23 on platelet aggregation and thrombotic events within these animals. Compound 7e, which has a comparable level of TXA2 synthase (IC50 1.2 microM) and human platelet cAMP PDE (IC50 6.4 microM) inhibitory activities, was found to be orally bioavailable with a long duration of action and offered effective protection against mortality in a collagen-epinephrine-induced pulmonary thromboembolism model in mice. Significant blood pressure and heart rate effects were observed for several compounds, e.g. 7e, 9e, 13a, 13d, 18, 20, 21, and 23, when dosed orally in conscious spontaneously hypertensive rats.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/antagonistas & inibidores , Fibrinolíticos/síntese química , Quinolonas/síntese química , Tromboxano-A Sintase/antagonistas & inibidores , 6-Cetoprostaglandina F1 alfa/sangue , Difosfato de Adenosina/farmacologia , Animais , Aorta/enzimologia , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Fibrinolíticos/farmacologia , Fibrinolíticos/uso terapêutico , Humanos , Hipertensão/tratamento farmacológico , Masculino , Microssomos/enzimologia , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacologia , Quinolonas/química , Quinolonas/farmacologia , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos , Suínos , Tromboembolia/tratamento farmacológico , Tromboxano B2/sangueRESUMO
On the basis of previous observations that both 2,3-dihydro-2,2,3,3-tetrahydroxy-1,4-naphthoquinone (oxoline, 1) and 6-chloroisonaphthazarin (2) had demonstrated antipsoriatic activity in vivo, a series of structural derivatives of 2 were prepared and examined in the Scholtz-Dumas topical psoriasis bioassay. Of these six (5, 6, 9a, 10, 11a, 11b), the most effective compound was found to be 6-chloro-1,4-diacetoxy-2,3-dimethoxynaphthalene (RS-43179, lonapalene, 11a). An extensive series of 1,2,3,4-tetraoxygenated naphthalenes (16-74) incorporating variations of the ester, ether, and aryl substituents were prepared as analogues of 11a to examine the structural requirements for activity and were screened in vivo as inhibitors of arachidonic acid induced mouse ear edema, a topical bioassay capable of detecting 5-lipoxygenase inhibitors. Net lipophilicity, hydrolytic stability, and ring substitution play significant roles in determining the observed in vivo activity. Lonapalene (11a) is currently in clinical development as a topically applied nonsteroidal antipsoriatic agent.
Assuntos
Naftalenos/uso terapêutico , Psoríase/tratamento farmacológico , 3',5'-AMP Cíclico Fosfodiesterases/antagonistas & inibidores , Administração Tópica , Animais , Araquidonato Lipoxigenases , Ácido Araquidônico , Ácidos Araquidônicos , Bioensaio , Edema/induzido quimicamente , Edema/tratamento farmacológico , Feminino , Humanos , Inibidores de Lipoxigenase , Camundongos , Naftalenos/síntese química , Neutrófilos/enzimologia , Inibidores da Ornitina Descarboxilase , Oxigênio , Relação Estrutura-Atividade , Tetra-Hidronaftalenos/síntese química , Tetra-Hidronaftalenos/uso terapêuticoRESUMO
6-Azaandrost-4-en-3-ones were synthesized and tested versus human type 1 and 2 steroid 5 alpha-reductase (5AR) and human adrenal 3 beta-hydroxy-delta 5-steroid dehydrogenase/3-keto-delta 5-steroid isomerase (3BHSD) to explore the structure-activity relationship of this novel series in order to optimize potency versus both isozymes of 5AR and selectivity versus 3BHSD. Compounds with picomolar IC50's versus human type 2 5AR and low nanomolar Ki's versus human type 1 5AR with 100-fold selectivity versus 3BHSD were identified (70). Preliminary in vivo evaluation of some optimal compounds from this series in a chronic castrated rat model of 5AR inhibitor-induced prostate involution and dog pharmacokinetic measurements identified a series of 17 beta-[N-(diphenylmethyl)carbamoyl]-6-azaandrost-4-en-3-ones (compounds 54, 66, and 67) with good in vivo efficacy and half-life in the dog. Inhibitors with, at the minimum, low nanomolar potency toward both human 5AR's and selectivity versus 3BHSD may show advantages over previously known 5AR inhibitors in the treatment of disease states which depend upon dihydrotestosterone, such as benign prostatic hyperplasia.
Assuntos
3-Hidroxiesteroide Desidrogenases/antagonistas & inibidores , Inibidores de 5-alfa Redutase , Glândulas Suprarrenais/enzimologia , Azasteroides/farmacologia , Isoenzimas/antagonistas & inibidores , Esteroide Isomerases/antagonistas & inibidores , Animais , Azasteroides/química , Cães , Humanos , Ratos , Ratos Sprague-Dawley , Relação Estrutura-AtividadeRESUMO
A variety of C17 amide-substituted 6-azaandrost-4-en-3-ones were prepared and tested versus human type 1 and 2 steroid 5 alpha-reductase (5AR) and human adrenal 3 beta-hydroxy-delta 5-steroid dehydrogenase/3-keto-delta 5-steroid isomerase (3BHSD) in order to optimize potency versus both isozymes of 5AR and selectivity versus 3BHSD. Two series of potent and selective C17 amides were discovered, 2,5-disubstituted anilides and (arylcycloalkyl)amides. Compounds from each series with picomolar IC50's versus human type 2 5AR and low nanomolar to picomolar IC50's versus human type 1 5AR possessing 100-500-fold selectivity versus 3BHSD were identified. A conformational model to predict 3BHSD potency was developed which could rationalize 3BHSD potency within three different series of compounds. Evaluation of some optimal compounds from this series in a chronic castrated rat model of 5AR inhibitor induced prostate involution, and pharmacokinetic measurements identified compounds (9, 12, 16, and 29) with good in vivo efficacy and half-life in the dog. An intact rat model of in vivo selectivity for 5AR versus 3BHSD inhibition was also developed. Dual inhibitors of both human 5AR's may show advantages over type 2 selective 5AR inhibitors, such as finasteride (1), in the treatment of disease states which depend upon dihydrotestosterone.
Assuntos
3-Hidroxiesteroide Desidrogenases/antagonistas & inibidores , Inibidores de 5-alfa Redutase , Glândulas Suprarrenais/enzimologia , Azasteroides/farmacologia , Esteroide Isomerases/antagonistas & inibidores , Animais , Azasteroides/química , Azasteroides/farmacocinética , Cães , Humanos , Masculino , Modelos Moleculares , Ratos , Ratos Sprague-Dawley , Relação Estrutura-AtividadeRESUMO
A series of substituted phenethyl derivatives of 3-benzisothiazolylpiperazine incorporating potent D2 and 5-HT2A antagonist activity was investigated as an approach to a novel atypical antipsychotic agent. The in vitro profile of 8e from this series is a combination of D2 receptor affinity comparable to the typical antipsychotic agent haloperidol and a 5-HT2A/D2 ratio comparable to the atypical agent clozapine. In vivo 8e possesses activity consistent with an efficacious antipsychotic agent with less tendency to induce extrapyramidal side effects in man.
Assuntos
Antipsicóticos/farmacologia , Piperazinas/farmacologia , Antagonistas da Serotonina/farmacologia , Tiazóis/farmacologia , Anfetamina/farmacologia , Animais , Antipsicóticos/química , Apomorfina/farmacologia , Aprendizagem da Esquiva/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Catalepsia/metabolismo , Clozapina/farmacologia , Dopamina/metabolismo , Dopamina/farmacologia , Desenho de Fármacos , Humanos , Estrutura Molecular , Fosfatidilinositóis/antagonistas & inibidores , Fosfatidilinositóis/metabolismo , Piperazinas/química , Prazosina/antagonistas & inibidores , Prazosina/metabolismo , Ratos , Receptores Adrenérgicos/metabolismo , Receptores Dopaminérgicos/metabolismo , Antagonistas da Serotonina/química , Tiazóis/químicaRESUMO
Hydroxylapatite high performance liquid chromatography was used to prepare two fractions from 125I- Fab 96.5. One fraction (peak 1) had relatively low immunoreactivity (25-38%) and the second fraction (peak 2) had high immunoreactivity (70-81%). Scatchard analysis showed similar affinity constants for the two preparations (2.9 x 10(9) M-1 for peak 1; 3.4 x 10(9) M-1 for peak 2). In biodistribution and imaging studies in athymic mice with human melanoma (FEMX-II) xenografts the high immunoreactivity preparation rapidly cleared from the blood and nontumor organs while retention of radioactivity in the tumor was prolonged. The low immunoreactivity preparation, had slower blood and nontumor organ clearance but faster tumor clearance than the high immunoreactivity fraction. Thus, in these studies highly immunoreactive antibody gave higher tumor to nontumor ratios and enhanced the target to nontarget image contrast.
Assuntos
Anticorpos Monoclonais/imunologia , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos de Imunoglobulinas/imunologia , Melanoma/diagnóstico por imagem , Proteínas de Neoplasias/imunologia , Animais , Anticorpos Monoclonais/farmacocinética , Antígenos de Neoplasias , Feminino , Fragmentos Fab das Imunoglobulinas/farmacocinética , Fragmentos de Imunoglobulinas/farmacocinética , Técnicas In Vitro , Radioisótopos do Iodo , Melanoma/metabolismo , Antígenos Específicos de Melanoma , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transplante de Neoplasias , Cintilografia , Distribuição Tecidual , Transplante HeterólogoRESUMO
The serum clearance and biodistribution of a murine monoclonal antibody were compared to the in vitro complex formation of the antibody with patients' sera. Iodine-125-labeled 9.2.27, an anti-melanoma antibody, was incubated with sera from ten melanoma patients who had received 9.2.27 in an earlier study. Complexes were observed in all patients using size exclusion high performance liquid chromatography and complex formation was partially blocked by nonspecific murine antibody, suggesting the presence of human anti-murine antibody in serum. All patients subsequently underwent imaging studies with [131I] 9.2.27 given intravenously. The serum levels of the antibody obtained after the second administration were inversely correlated with the level of in vitro complex formation. Patients whose serum formed high levels of complex showed a rapid serum clearance, high hepatic uptake, and accelerated whole body clearance and urinary excretion of 131I. This suggests that in patients who receive repetitive administration of murine antibody the serum clearance rate and biodistribution of intravenously injected antibody are altered by antibody complex formation in the serum.
Assuntos
Anticorpos Monoclonais/imunologia , Formação de Anticorpos , Melanoma/imunologia , Camundongos/imunologia , Adulto , Animais , Anticorpos Monoclonais/metabolismo , Feminino , Humanos , Técnicas In Vitro , Radioisótopos do Iodo , Masculino , Melanoma/metabolismo , Pessoa de Meia-Idade , Distribuição TecidualRESUMO
A diester linkage was added between monoclonal anti-melanoma antibody 96.5 and a diethylenetriaminepentaacetic acid derivative to test if a tumor-to-blood and -to-organ ratio of the injected antibody in nude mice with human melanoma FEM XII xenografts could be increased by the addition of the readily cleavable linkage. Compared to the 111In-labeled antibody DTPA with a peptide linkage, the diester conjugate cleared much faster from the blood and was retained much less in muscle and normal organs such as liver, spleen and kidney over a 48-hr period. On the other hand, the activity retained in the tumor was larger than or similar to that of the peptide conjugate for this time period. This resulted in a 2.5, 2.1, and 2.6 fold increase in a tumor to blood, to liver and to kidney ratio at 48 hr for the diester conjugate as compared to the peptide conjugate. The whole-body biologic half life of the antibody was 36 hr, three times shorter than the peptide conjugate. The external imaging demonstrated a clearly visible tumor at 4 hr and a lower pool activity at 72 hr for the diester conjugate. The peptide conjugate, however, showed a persistant blood-pool activity at 72 hr. The addition of the diester linkage, therefore may be beneficial for imaging tumors in patients at early time intervals after injection.
Assuntos
Anticorpos Monoclonais , Radioisótopos de Índio , Melanoma/diagnóstico por imagem , Ácido Pentético , Animais , Ligação Competitiva , Fenômenos Químicos , Química , Humanos , Radioisótopos de Índio/farmacocinética , Masculino , Camundongos , Camundongos Nus , Ácido Pentético/farmacocinética , Cintilografia , Distribuição TecidualRESUMO
PURPOSE: To identify tumor-suppressor loci that may contribute to the pathogenesis of uveal melanoma. METHODS: Multiplex fluorescence microsatellite assays were performed on 27 uveal melanomas using markers at 3p25-p26, 3p14.2, 9p21-p23, 13q14, 13q12.3-q13, and 17p13, close to or within the von Hippel Lindau (VHL), fragile histidine triad (FHIT), p16/cyclin-dependent kinase inhibitor 2 (CDKN2A), retinoblastoma (RB1), breast cancer 2 (BRCA2), and p53 tumor suppressor loci, respectively. Further markers on chromosomes 3 and 9 were analyzed individually. RESULTS: Loss of heterozygosity (LOH) was identified in 63% of tumors, most frequently on chromosome 3 (52%), in association with epithelioid cells (P = 0.0002) and microvascular loops (P = 0.0008). In the majority of cases, LOH on chromosome 3 was detected at all informative markers. The second most common alteration was LOH at an RB1 intragenic marker (21% tumors), with retention of a more centromeric 13q marker (near BRCA2). The pattern of LOH on chromosome 9p was consistent with the involvement of a region telomeric to CDKN2A. LOH at TP53 was infrequent. CONCLUSIONS: In the majority of cases, chromosome 3 LOH involves an entire chromosome homologue, which hampers identification of the relevant suppressor loci. This LOH correlates with the presence of microvascular loops and epithelioid cells, two of the recognized histologic indicators of poor prognosis. Data for chromosomes 13 and 9 support a role for RB1 in the pathogenesis of uveal melanoma but also raise the possibility of the involvement of additional loci close to RB1 and CDKN2A.
Assuntos
Hidrolases Anidrido Ácido , Cromossomos Humanos Par 17 , Cromossomos Humanos Par 3 , Cromossomos Humanos Par 9 , Perda de Heterozigosidade , Melanoma/genética , Proteína do Retinoblastoma/genética , Neoplasias Uveais/genética , Proteína BRCA2 , Cromossomos Humanos Par 13 , Inibidor p16 de Quinase Dependente de Ciclina/genética , Análise Mutacional de DNA , DNA de Neoplasias/análise , Humanos , Melanoma/patologia , Repetições de Microssatélites , Proteínas de Neoplasias , Proteínas/genética , Fatores de Transcrição , Proteína Supressora de Tumor p53/genética , Neoplasias Uveais/patologiaRESUMO
Combined information from biochemical and molecular biological experiments reveals a consistent structural rhythm that underlies the construction of all membrane carriers and perhaps all transport systems. Biochemical work shows that while some carrier proteins function as monomers, others operate as dimers. But despite this variation, all examples can be modelled as having a pair of membrane-embedded domains, each of which contains an array of (about) six transmembrane helical elements. This pattern is best documented among membrane carriers, where the minimal functional unit is known in a reasonable number of cases. Nevertheless, the same conclusion is likely to characterize other solute transporters. These unexpected correlations suggest that all membrane carriers, including those that take part in "energy coupling", have a uniform structural design on which is superimposed a variety of kinetic and biochemical mechanisms.