RESUMO
BACKGROUND: Metastatic colorectal cancer (mCRC) is commonly treated with 5-fluorouracil, folinic acid, and oxaliplatin or irinotecan. The multitargeted kinase inhibitor, regorafenib, was combined with chemotherapy as first- or second-line treatment of mCRC to assess safety and pharmacokinetics (primary objectives) and tumor response (secondary objective). PATIENTS AND METHODS: Forty-five patients were treated every 2 weeks with 5-fluorouracil 400 mg/m(2) bolus then 2400 mg/m(2) over 46 h, folinic acid 400 mg/m(2), and either oxaliplatin 85 mg/m(2) or irinotecan 180 mg/m(2). On days 4-10, patients received regorafenib 160 mg orally once daily. RESULTS: The median duration of treatment was 108 (range 2-345 days). Treatment was stopped for adverse events or death (17 patients), disease progression (11 patients), and consent withdrawal or investigator decision (11 patients). Six patients remained on regorafenib at data cutoff (two without chemotherapy). Drug-related adverse events occurred in 44 patients [grade ≥ 3 in 32 patients: mostly neutropenia (17 patients) and leukopenia, hand-foot skin reaction, and hypophosphatemia (four patients each)]. Thirty-three patients achieved disease control (partial response or stable disease) for a median of 126 (range 42-281 days). CONCLUSION: Regorafenib had acceptable tolerability in combination with chemotherapy, with increased exposure of irinotecan and SN-38 but no significant effect on 5-fluorouracil or oxaliplatin pharmacokinetics.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/patologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinética , Camptotecina/administração & dosagem , Camptotecina/análogos & derivados , Camptotecina/farmacocinética , Neoplasias Colorretais/metabolismo , Feminino , Fluoruracila/administração & dosagem , Fluoruracila/farmacocinética , Humanos , Leucovorina/administração & dosagem , Leucovorina/farmacocinética , Masculino , Pessoa de Meia-Idade , Compostos Organoplatínicos/administração & dosagem , Compostos Organoplatínicos/farmacocinética , Compostos de Fenilureia/administração & dosagem , Compostos de Fenilureia/farmacocinética , Piridinas/administração & dosagem , Piridinas/farmacocinética , Resultado do Tratamento , Adulto JovemRESUMO
BACKGROUND: Pattern and outcome of disease recurrence after autologous stem-cell transplantation (autoSCT) for follicular lymphoma (FL) is not well known. PATIENTS AND METHODS: Relapse cases were identified from 241 consecutive patients autografted for disseminated untransformed FL from 1990 to 2002 in three institutions. Prognostic factors for relapse and outcome after relapse were analyzed by log-rank comparisons and Cox regression analyses. RESULTS: One hundred and three relapses occurred. The 10-year relapse probability was 47%. Median time from autoSCT to relapse was 20 (2-128) months. Only three relapses were observed later than 6 years posttransplant. Median survival after relapse was 8.3 years. Patients with disease recurrence within 1 year from transplant and those who had received autoSCT as second-line treatment had significantly reduced survival by multivariate analysis, whereas Follicular Lymphoma International Prognostic Index score, age, remission status at autoSCT, high-dose regimen, and ex vivo purging had no impact. CONCLUSIONS: FL recurrence after autoSCT follows a biphasic pattern with continuing relapse during the first 6 years and only few events thereafter. The prognosis after relapse is relatively good and appears to be comparable to that of disease recurrence after standard treatment. The situation is less favorable for patients who relapse within the first posttransplant year.
Assuntos
Linfoma Folicular/cirurgia , Transplante de Células-Tronco , Seguimentos , Humanos , Linfoma Folicular/patologia , Recidiva , Condicionamento Pré-Transplante , Transplante AutólogoRESUMO
Translocation of chromosomes 14 and 18 [t(14;18)] for detection of minimal residual disease in follicular lymphoma patients can be analyzed by nested polymerase chain reaction (PCR) or by quantitative PCR like LightCycler-based assays. We have compared both methods in blood and bone marrow samples of 28 patients enrolled in a clinical study on immunochemotherapy. In 42% of samples, the bcl2-IgH rearrangement was detectable by nested PCR, but not by LightCycler PCR. Nested PCR was able to reveal a significant drop in positive bone marrow or peripheral blood samples after therapy. In contrast, with LightCycler PCR, the detected drop in t(14;18)-positive cells did not reach statistical significance. The majority of patients showed positive results with nested PCR of peripheral blood or bone marrow without any associations to presence or absence of histological bone marrow (BM) infiltration by lymphoma cells. With LightCycler PCR, the numbers of positive cells were higher in samples from patients with BM infiltration of lymphoma cells (1.9 x 10(-2)) compared to samples from patients without involvement (4.08 x 10(-5)). A similar trend was seen in samples derived from the peripheral blood. Positivity for t(14;18) after therapy in two patients correlated with clinical relapse 6 months later. The data shown here demonstrate a lower sensitivity of LightCycler vs. nested PCR for detection of t(14;18). The usefulness of nested PCR for t(14;18) for risk stratification after primary therapy has to be validated in larger trials.
Assuntos
Rearranjo Gênico , Cadeias Pesadas de Imunoglobulinas/genética , Linfoma Folicular/genética , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas c-bcl-2/genética , Adulto , Idoso , Medula Óssea/metabolismo , Ensaios Clínicos Fase II como Assunto , Feminino , Humanos , Linfoma Folicular/patologia , Masculino , Pessoa de Meia-Idade , Estudos Multicêntricos como Assunto , Neoplasia Residual , Reação em Cadeia da Polimerase/instrumentação , Reação em Cadeia da Polimerase/métodos , Ensaios Clínicos Controlados Aleatórios como Assunto , Sensibilidade e EspecificidadeRESUMO
The prevention of apoptosis may be critical for immunological function. Survivin is a recently cloned member of the inhibitor of apoptosis protein family. We analyzed survivin expression before and after lymphocyte activation in isolated cell populations. Prior to activation, survivin was undetectable. After activation with IL-2 and OKT-3, CD3(+) cells expressed survivin. Next, we correlated survivin expression with Fas, FasL and the amount of apoptosis over time in culture. After activation, survivin was readily detected by day 2 and decreased thereafter. Prior to activation (day 0), Fas was present on 60% of the cells and on 100% by days 2-6. Peak FasL mRNA expression was at day 2. During peak survivin expression (days 2-4) the apoptotic fraction was low, but when survivin expression decreased the apoptotic fraction increased rapidly. Finally, we found that CD45RO(+) memory T cells showed a higher expression of survivin than did CD45RA(+) naive T cells after activation. These results suggest that survivin may contribute to T-cell survival early in T-cell responses as well as in memory immune responses.
Assuntos
Apoptose/imunologia , Memória Imunológica/imunologia , Ativação Linfocitária/imunologia , Proteínas Associadas aos Microtúbulos , Proteínas/genética , Linfócitos T/imunologia , Expressão Gênica , Humanos , Proteínas Inibidoras de Apoptose , Proteínas de Neoplasias , Survivina , Fatores de TempoRESUMO
PURPOSE: Sorafenib (BAY 43-9006), a multikinase inhibitor, has been shown to inhibit tumor growth and tumor angiogenesis by targeting Raf kinase, vascular endothelial growth factor receptor, and platelet-derived growth factor receptor. This study investigated the safety, pharmacokinetics, and preliminary efficacy of sorafenib in combination with gemcitabine and cisplatin. METHODS: Patients with advanced solid tumors were treated with 75 mg/m(2) cisplatin on day 1 and 1,250 mg/m(2) gemcitabine on days 1 and 8 of each 21-day cycle. On day 5 of cycle 1, sorafenib 400 mg twice daily was started and continued throughout the complete treatment cycles without interruption. RESULTS: Nineteen patients were valid for safety analysis. The most frequent toxicities related to the cytotoxic agents were hematological disorders. Sorafenib-related toxicities were skin-related, gastrointestinal, and constitutional symptoms. No clinically relevant pharmacokinetic drug-drug interaction between sorafenib, cisplatin, and gemcitabine was detected. AUC(0-72) and C (max) of total and unbound platinum were only marginally changed by concomitant sorafenib. Concomitant sorafenib increased mean AUC and C (max) of gemcitabine by 12 and 21%. CONCLUSIONS: Sorafenib as continuous oral treatment in combination with gemcitabine and cisplatin demonstrated an acceptable safety profile. No clinically relevant pharmacokinetic interaction was detected. Preliminary antitumor activity, pharmacokinetic, and safety data support the recommendation of 400 mg sorafenib twice daily in combination with cisplatin and gemcitabine to be further evaluated in clinical studies.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias/tratamento farmacológico , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinética , Área Sob a Curva , Benzenossulfonatos/administração & dosagem , Benzenossulfonatos/efeitos adversos , Benzenossulfonatos/farmacocinética , Cisplatino/administração & dosagem , Cisplatino/efeitos adversos , Cisplatino/farmacocinética , Desoxicitidina/administração & dosagem , Desoxicitidina/efeitos adversos , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacocinética , Esquema de Medicação , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Gastroenteropatias/induzido quimicamente , Doenças Hematológicas/induzido quimicamente , Humanos , Taxa de Depuração Metabólica , Pessoa de Meia-Idade , Neoplasias/metabolismo , Neoplasias/patologia , Niacinamida/análogos & derivados , Compostos de Fenilureia , Piridinas/administração & dosagem , Piridinas/efeitos adversos , Piridinas/farmacocinética , Dermatopatias/induzido quimicamente , Sorafenibe , Resultado do Tratamento , GencitabinaRESUMO
BACKGROUND: Cytokine-induced killer (CIK) cells are ex vivo expanded T cells with co-expression of CD3 and CD56 and NK activity. They have recently been evaluated in a phase I/II clinical trial against malignant lymphoma. Bispecific Ab (bsAb) redirect CIK cells to tumor targets, thus enhancing their cytotoxicity. While bsAb may improve T-cell mediated anti-tumor activity, little is known about the fate of effector cells upon redirection to tumor targets using a bsAb. METHODS: Using ex vivo-activated CIK cells, Her2/neu expressing breast and ovarian cell lines and a F(ab')2 Her2/neu x CD3 bsAb, we investigated the anti-tumor activity and the proliferative and apoptotic outcome of CIK cells. RESULTS: When redirected to tumor targets with bsAb, there was a significant increase in anti-tumor activity as well as an increase in both CIK cell proliferation and apoptosis. The addition of agonistic Ab against CD28 did not significantly increase proliferation or apoptosis of CIK cells redirected to CD80- and CD86- tumor targets. To attempt to reduce T-cell apoptosis, we incubated CIK cells in the presence of the pan-caspase inhibitor z-VAD-fmk, which led to a partial reduction in T-cell apoptosis without increasing cellular cytotoxicity. DISCUSSION: bsAb are effective in redirecting activated T cells to tumor targets and such redirection leads to both T-cell proliferation and apoptosis that are not altered by co-stimulation through CD28. Effector cell apoptosis can be reduced by using a caspase inhibitor but this does not increase CIK cell cytotoxicity.
Assuntos
Anticorpos Biespecíficos/farmacologia , Apoptose/efeitos dos fármacos , Citocinas/imunologia , Células Matadoras Naturais/citologia , Células Matadoras Naturais/imunologia , Neoplasias/imunologia , Anticorpos Biespecíficos/imunologia , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Sítios de Ligação de Anticorpos , Proliferação de Células/efeitos dos fármacos , Testes Imunológicos de Citotoxicidade , Citotoxicidade Imunológica , Feminino , Citometria de Fluxo , Humanos , Células Matadoras Naturais/efeitos dos fármacos , Pessoa de Meia-Idade , Neoplasias/patologia , Receptor ErbB-2/metabolismo , Receptores de Superfície Celular/metabolismo , Células Tumorais CultivadasRESUMO
Thrombotic microangiopathy (TMA) has been described as a complication of bone marrow or stem cell transplantation. It is usually associated with high dose therapy for autologous transplantation or myeloablative conditioning in the allogeneic setting. Here we report three cases of TMA after reduced intensity conditioning and allogeneic peripheral blood stem cell transplantation. All three patients had high dose Melphalan therapy with autologous stem cell support preceding the allogeneic transplantation for several weeks, which may have contributed to endothelial damage and subsequent development of TMA.
Assuntos
Síndrome Hemolítico-Urêmica/etiologia , Mieloma Múltiplo/complicações , Transplante de Células-Tronco de Sangue Periférico/efeitos adversos , Púrpura Trombocitopênica Trombótica/etiologia , Antineoplásicos/efeitos adversos , Endotélio Vascular/patologia , Humanos , Mieloma Múltiplo/terapia , Transplante de Células-Tronco de Sangue Periférico/métodos , Radioterapia Adjuvante/efeitos adversos , Condicionamento Pré-Transplante/efeitos adversos , Condicionamento Pré-Transplante/métodos , Transplante Autólogo , Transplante HomólogoRESUMO
Hybrid proteins were constructed in which C-terminal regions of the bacterial cell surface and extracellular protein pullulanase were replaced by the mature forms of the normally periplasmic Escherichia coli proteins beta-lactamase or alkaline phosphatase. In E. coli strains expressing all pullulanase secretion genes, pullulanase-beta-lactamase hybrid protein molecules containing an N-terminal 834-amino-acid pullulanase segment were efficiently and completely transported to the cell surface. This hybrid protein remained temporarily anchored to the cell surface, presumably via fatty acids attached to the N-terminal cysteine of the pullulanase segment, and was subsequently specifically released into the medium in a manner indistinguishable from that of pullulanase itself. These results suggest that the C-terminal extremity of pullulanase lacks signal(s) required for export to the cell surface. When beta-lactamase was replaced by alkaline phosphatase, the resulting hybrid also became exposed at the cell surface, but exposition was less efficient and specific release into the medium was not observed. We conclude that proteins that do not normally cross the outer membrane can be induced to do so when fused to a permissive site near the C-terminus of pullulanase.
Assuntos
Escherichia coli/genética , Glicosídeo Hidrolases/genética , beta-Lactamases/genética , Transporte Biológico , Membrana Celular/enzimologia , Membrana Celular/metabolismo , Clonagem Molecular , Escherichia coli/enzimologia , Imunofluorescência , Glicosídeo Hidrolases/análise , Glicosídeo Hidrolases/metabolismo , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/metabolismo , Mapeamento por Restrição , beta-Lactamases/análise , beta-Lactamases/metabolismoRESUMO
The determined nucleotide sequence of the Klebsiella pneumoniae UNF5023 gene pulA comprises a single open reading frame coding for a 1090-residue precursor of the secreted protein pullulanase. The predicted sequence of this protein is highly homologous to that of pullulanase of Klebsiella aerogenes strain W70. However, the UNF5023 pullulanase lacks a collagen-like sequence present at the N-terminus of the mature W70 enzyme and differs further from the W70 pullulanase around residue 300 and at the C-terminus. Pullulanases with or without the collagen-like sequence could not be separated by gel electrophoresis under denaturing or non-denaturing conditions, and were unaffected by collagenase. A large central domain which is highly conserved in both UNF5023 and W70 polypeptides contains eight short sequences that are also found in amylases and iso-amylases. Linker mutations in the region of the UNF5023 pulA gene coding for this domain abolished catalytic activity without affecting transport of the polypeptide across the outer membrane. Hybrid proteins comprising at least the amino-terminal 656 residues of prepullulanase fused to alkaline phosphatase were partially localized to the cell surface, as judged by their accessibility to anti-pullulanase serum in immuno-fluorescence tests. On the basis of these results, we tentatively propose that secretion signals required for recognition and translocation across the outer membrane via the pullulanase-specific extension of the secretion pathway are located near the N-terminus of the pullulanase polypeptide.
Assuntos
Genes Bacterianos , Glicosídeo Hidrolases/genética , Klebsiella pneumoniae/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Colágeno , Escherichia coli/genética , Glicosídeo Hidrolases/biossíntese , Klebsiella pneumoniae/enzimologia , Dados de Sequência Molecular , Mutação , Plasmídeos , Mapeamento por RestriçãoRESUMO
Recent findings suggest that axial flagellar proteins and virulence proteins of Gram-negative bacteria are exported from the cytoplasm via conserved translocation systems. To identify residues essential for secretion of flagellar axial proteins we examined the 591-residue Caulobacter crescentus flagellar hook protein. Western blot assays of the culture media of strains producing mutant hook proteins show that only residues 38-58 are essential for its secretion to the cell surface. We discuss the observation that this unprocessed 21-residue sequence is not conserved in other axial proteins and does not correspond to the SGL-, ANNLAN- and heptad repeat motifs that are located just upstream of the essential secretion information in the hook protein and are conserved near the N-termini of other axial proteins. These motifs, for which an essential role in export or assembly has been proposed, are required for motility. However, we also demonstrate that hook protein can only be secreted when the flagellar basal body is present in the cell envelope. The cell-cycle regulation of hook protein secretion confirms the specificity of the assay used in these studies and suggests that the basal body itself may serve as a secretion channel for the hook protein.
Assuntos
Proteínas de Bactérias/biossíntese , Caulobacter crescentus/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sequência de Bases , Caulobacter crescentus/genética , Caulobacter crescentus/crescimento & desenvolvimento , Ciclo Celular , Bactérias Gram-Negativas/genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos , Sinais Direcionadores de Proteínas/biossíntese , Sinais Direcionadores de Proteínas/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Homologia de Sequência de AminoácidosRESUMO
The fatty acid-acylated enzyme pullulanase is normally found in either of two locations in Escherichia coli, depending on whether or not the producing strains also express the genes specifically required for the second step in pullulanase secretion. When they are expressed, the enzyme is localized to the cell surface, while in their absence, it is directed to an unidentified location in the cell envelope which, upon lysis, forms vesicles whose density is intermediate between those of outer and cytoplasmic membrane vesicles. In order to test the role of the putative lipoprotein sorting signal, Asp2, in pullulanase sorting and secretion, the structural gene (pulA) was subjected to site-directed mutagenesis. Replacement of the Asp2 residue by Asn, Glu, or Ser caused the enzyme to fractionate with outer membrane-derived vesicles rather than with intermediate density vesicles from E. coli cells devoid of pullulanase secretion genes. A pronounced secretion defect was observed in a two-step secretion assay in which the first (sec gene-dependent) and second (pul gene-dependent) secretion steps were uncoupled. We propose that the Asp residue increases the efficiency of pullulanase secretion by allowing the enzyme to be initially sorted to a region of the cell envelope wherein most of the pullulase-specific secretion factors are located.
Assuntos
Escherichia coli/enzimologia , Glicosídeo Hidrolases/metabolismo , Lipoproteínas/metabolismo , Sequência de Bases , Compartimento Celular , Membrana Celular/enzimologia , Análise Mutacional de DNA , Escherichia coli/metabolismo , Oligonucleotídeos/química , Processamento de Proteína Pós-Traducional , Sinais Direcionadores de Proteínas/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Relação Estrutura-AtividadeRESUMO
Three different techniques, protease accessibility, cell fractionation and in situ immunocytochemistry, were used to study the location of the lipoprotein pullulanase produced by Escherichia coli K12 carrying the cloned pullulanase structural gene (pulA) from Klebsiella pneumoniae, with or without the K. pneumoniae genes required to transport pullulanase to the cell surface (secretion-competent and secretion-incompetent, respectively). Pullulanase produced by secretion-competent strains could be slowly but quantitatively released into the medium by growing the cells in medium containing pronase. The released pullulanase lacked the N-terminal fatty-acylated cysteine residue (and probably also a short N-terminal segment of the pullulanase polypeptide), confirming that the N-terminus is the sole membrane anchor in the protein. Pullulanase produced by secretion-incompetent strains was not affected by proteases, confirming that it is not exposed on the cell surface. Pullulanase cofractionated with both outer and inner membrane vesicles upon isopycnic sucrose gradient centrifugation, irrespective of the secretion competence of the strain. Examination by electronmicroscopy of vesicles labelled with antipullulanase serum and protein A-gold confirmed that pullulanase was associated with both types of vesicles. When thin-sectioned cells were examined by the same technique, pullulanase was found to be located mainly on the cell surface of the secretion-competent cells and mainly in the proximity of the inner membrane in the secretion-incompetent cells. Thus, while the results from three independent techniques (substrate accessibility, protease accessibility and in situ immunocytochemistry) show that pullulanase is transported to the cell surface of secretion-competent cells, this could not be confirmed by cell-fractionation techniques. Possible explanations for this discrepancy are discussed.
Assuntos
Escherichia coli/genética , Genes Bacterianos , Glicosídeo Hidrolases/genética , Klebsiella pneumoniae/genética , Proteínas de Bactérias/análise , Membrana Celular/enzimologia , Eletroforese em Gel de Poliacrilamida , Escherichia coli/ultraestrutura , Glicosídeo Hidrolases/biossíntese , Klebsiella pneumoniae/enzimologia , Lipoproteínas/análise , Proteínas de Membrana/análise , Peptídeo HidrolasesRESUMO
The analyses of hybrid proteins and of deletion and insertion mutations reveal that the only amino acid at the amino-proximal end of the cell surface lipoprotein pullulanase that is specifically required for its extracellular secretion is an aspartate at position +2, immediately after the fatty acylated amino-terminal cysteine. To see whether the requirement for this amino acid is related to its proposed role as a cytoplasmic membrane lipoprotein sorting signal, we used sucrose gradient floatation analysis to determine the subcellular location of pullulanase variants (with or without the aspartate residue) that accumulated in cells lacking the pullulanase-specific secretion genes. A non-secretable pullulanase variant with a serine at position +2 cofractionated mainly with the major peak of outer membrane porin. In contrast, most (55%) of a pullulanase variant with an aspartate at position +2 cofractionated with slightly lighter fractions that contained small proportions of both outer membrane porin and the cytoplasmic membrane marker NADH oxidase. Only 5% of this pullulanase variant cofractionated with the major NADH oxidase peak, while the rest (c. 40%) remained at the bottom of the gradient in fractions totally devoid of porin and NADH oxidase. When analysed by sedimentation through sucrose gradients, however, a large proportion of this variant was recovered from fractions near the top of the gradient that also contained the major NADH oxidase peak. When this peak fraction was applied to a floatation gradient the pullulanase activity remained at the bottom while the NADH oxidase floated to the top.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Ácido Aspártico , Escherichia coli/genética , Glicosídeo Hidrolases/biossíntese , Lipoproteínas/biossíntese , Sinais Direcionadores de Proteínas/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular/métodos , Escherichia coli/enzimologia , Genes Bacterianos , Variação Genética , Glicosídeo Hidrolases/genética , Dados de Sequência Molecular , Mutagênese , Mutagênese Insercional , Oligodesoxirribonucleotídeos , Deleção de Sequência , Homologia de Sequência de AminoácidosRESUMO
Two distinct steps in the secretion of the extracellular, cell-surface-anchored lipoprotein pullulanase by Escherichia coli were uncoupled by allowing export of the enzyme to the cytoplasmic membrane via the signal peptide/sec-gene-dependent general export pathway, and then inducing the pulC-O operon of genes required for translocation to the cell surface. The secretion intermediate cofractionated mainly with intermediate-density vesicles when cells were gently lysed and the resulting vesicles were separated by isopycnic sucrose density centrifugation. Cytoplasmic forms of pullulanase (which are not exported because they lack a functional signal peptide) are more sensitive to heat inactivation, denaturation by sodium dodecyl sulphate and carboxymethylation than the intermediate and cell-surface forms. The latter are distinguished only by the fact that the secretion intermediate is less susceptible to proteinase K and trypsin, and is partially inaccessible to substrate or in an inactive conformation in sphaeroplasts. These and other results indicate that the secretion intermediate can acquire considerable higher-ordered structure, including disulphide bridges, before it is transported to the cell surface; this seems to rule out the possibility that it is threaded through this membrane as a locally unfolded polypeptide.
Assuntos
Proteínas de Bactérias/metabolismo , Escherichia coli/enzimologia , Glicosídeo Hidrolases/metabolismo , Fracionamento Celular , Centrifugação com Gradiente de Concentração , Dissulfetos/metabolismo , Ditiotreitol/farmacologia , Eletroforese em Gel de Poliacrilamida , Endopeptidase K , Temperatura Alta , Iodoacetamida/farmacologia , Cinética , Metilação , Conformação Proteica , Proteínas Recombinantes de Fusão/metabolismo , Serina Endopeptidases/metabolismo , Dodecilsulfato de Sódio/metabolismo , Esferoplastos/enzimologia , Tripsina/metabolismoRESUMO
Site-directed mutagenesis was used to construct three mutant derivatives of the extracellular, cell surface lipoprotein pullulanase (PulA) in which the normally fatty acylated cysteine of the signal peptide-bearing precursor was replaced by other amino acids. When produced in Escherichia coli expressing all genes required for pullulanase secretion, approximately 90% of the PulA derivatives persisted as cell-associated precursors, indicating inefficient signal peptide processing. Processed (intermediate-sized) forms of the two derivatives that were studied in detail were found to result from proteolytic cleavage at different sites within the signal peptide. Both were further processed to smaller polypeptides by cleavage at an undetermined site that is presumably close to their C termini. The intermediate-sized pullulanase derived from prepullulanase in which Cys+1 had been replaced by Leu and Gly-1 by Glu (PulA:C1L/G-1E) appeared rapidly, was apparently entirely extracellular, and accounted for approximately 10% of synthesized PulA. Prolonged incubation did not result in further conversion of the precursor to the intermediate form, and the precursor remained anchored to the cytoplasmic membrane. The smaller processed form was also found extracellularly. The active form of the extracellular enzyme was monomeric, which is again in contrast to the fatty acylated, wild-type enzyme. Taken together, these results indicate that replacement of Cys+1 of prePulA eliminates processing by lipoprotein signal peptidase and does not permit processing by leader peptidase, but allows inefficient, aberrant processing by an unknown peptidase and immediate secretion of the resulting polypeptide, which retains most of its signal peptide. Processing and secretion only occur when the pullulanase secretion functions are expressed.
Assuntos
Escherichia coli/enzimologia , Glicosídeo Hidrolases/metabolismo , Acilação , Sequência de Aminoácidos , Sequência de Bases , Compartimento Celular , Membrana Celular/metabolismo , Cisteína/química , Análise Mutacional de DNA , Escherichia coli/metabolismo , Glicosídeo Hidrolases/química , Dados de Sequência Molecular , Oligonucleotídeos/química , Processamento de Proteína Pós-Traducional , Relação Estrutura-AtividadeRESUMO
Pullulanase is an extracellular, cell surface-anchored lipoprotein produced by Gram-negative bacteria belonging to the genus Klebsiella. Its correct localization in recombinant Escherichia coli requires the products of 14 genes that are linked to the enzyme structural gene in the Klebsiella chromosome. In addition, we show here that six sec genes (secA, secB, secD, secE, secF and secY) are all required for processing of the prepullulanase signal peptide to occur. This implies that pullulanase crosses the cytoplasmic membrane via the general export pathway of which the sec gene products are essential components. Removal or drastic alteration of the prepullulanase signal peptide cause the enzyme to remain cytoplasmic. We propose that pullulanase secretion occurs in two steps, the first of which is common to all signal peptide-bearing precursors of exported and secreted proteins, whereas the second is specifically involved in translocating pullulanase to the cell surface.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli , Escherichia coli/genética , Glicosídeo Hidrolases/metabolismo , Proteínas de Membrana , Proteínas de Transporte de Monossacarídeos , Serina Endopeptidases , Sequência de Aminoácidos , Sequência de Bases , Transporte Biológico , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Clonagem Molecular , DNA Bacteriano , Endopeptidases/metabolismo , Escherichia coli/enzimologia , Escherichia coli/metabolismo , Genes Bacterianos , Proteínas Ligantes de Maltose , Dados de Sequência Molecular , Mutação , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Sinais Direcionadores de Proteínas/metabolismoRESUMO
Genetic methods based on fusion proteins allow the power of a genetic approach to be applied to the self-assembly of proteins or protein fragments, regardless of whether or not the normal function of the fused assembly domains is either known or amenable to selection or screening. The widespread adoption of variations of the yeast two-hybrid system originally described by S. Fields and O. Song (1989, Nature 340, 245-246) demonstrates the usefulness of these kinds of assays. This review describes some of the many systems used to select or screen for protein-protein interactions based on the regulation of reporter constructs by hybrid proteins expressed in bacteria, including recent implementations of generalizable two-hybrid systems for Escherichia coli.
Assuntos
Proteínas de Bactérias/química , Escherichia coli/genética , Hibridização Genética , Sequência de Aminoácidos , Sequência de Bases , Escherichia coli/química , Dados de Sequência Molecular , Ativação TranscricionalRESUMO
Sarcoidosis-lymphoma syndrome is a well-established syndrome where sarcoidosis is followed by the development of a lymphoproliferative disease such as non-Hodgkin's lymphoma (NHL). Here we report two patients with NHL who developed sarcoidosis subsequent to the diagnosis of lymphoproliferative disease. In both cases, chemotherapeutic treatment had already been initiated or was completed when sarcoidosis occurred. In these patients, sarcoidosis may have been triggered by immunologic aberrations induced by antineoplastic therapy or as a consequence of an underlying immunologic disturbance associated with the lymphoma. When a suspected relapse of lymphoma presents with signs and symptoms compatible with sarcoidosis, this rare immunologic disorder has to be ruled out by careful clinical and histopathologic analysis to prevent mistreatment.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Pneumopatias/induzido quimicamente , Linfoma Folicular/tratamento farmacológico , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Sarcoidose/induzido quimicamente , Adulto , Antibacterianos/uso terapêutico , Anti-Infecciosos/uso terapêutico , Anti-Inflamatórios/uso terapêutico , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/efeitos adversos , Anticorpos Monoclonais Murinos , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Claritromicina/uso terapêutico , Ciclofosfamida/administração & dosagem , Ciclofosfamida/efeitos adversos , Doxorrubicina/administração & dosagem , Doxorrubicina/efeitos adversos , Etoposídeo/administração & dosagem , Etoposídeo/efeitos adversos , Humanos , Pneumopatias/tratamento farmacológico , Masculino , Prednisolona/administração & dosagem , Prednisolona/efeitos adversos , Prednisona/administração & dosagem , Prednisona/efeitos adversos , Prednisona/uso terapêutico , Rituximab , Sarcoidose/tratamento farmacológico , Combinação Trimetoprima e Sulfametoxazol/uso terapêutico , Vincristina/administração & dosagem , Vincristina/efeitos adversosRESUMO
The Escherichia coli activator and repressor proteins AraC and LexA bind DNA as homodimers. Here we show that their heterodimerization through fused cognate dimerization domains results in repression of AraC-dependent gene activation by LexA. Repression also requires a LexA operator half-site located several helical turns downstream of the AraC operator. This requirement for a specific spatial organization of the operators suggests the formation of a DNA loop between operator-bound Ara/LexA heterodimers, and we propose that heterodimerization with the AraC hybrid provides co-operativity for operator binding and repression by the LexA hybrid. Consistent with a mechanism that involves DNA looping, repression increases when the E. coli DNA looping and transcriptional effector protein IHF binds between the AraC and LexA operators. Thus, we have combined the functions of three distinct transcriptional effector proteins to achieve a new mode of gene regulation by DNA looping, in which the activator protein is an essential part of the repressor complex. The flexibility of the DNA loop may facilitate this novel combinatorial arrangement of those proteins on the DNA. The requirement for protein interactions between the AraC and LexA hybrids for gene regulation suggests that this regulatory circuit may prove useful as an E. coli-based two-hybrid system.