RESUMO
Certainpolynuclear aromatic hydrocarbons (PAHs) such as benzo[a]pyrene (BaP) induce CYP1A-dependent enzyme activities. Because PAHs are ubiquitous environmental contaminants, and some are aryl hydrocarbon agonists, CYP1A has been used as a biomarker for PAH exposure. However, PAHs exist in the environment in complex mixtures that may confound biomarker results. In in vitro studies, the PAH fluoranthene (FL) failed to increase or enhance CYP1A1-dependent ethoxyresorufin-O-deethylase (EROD) activity in cells, but rather inhibited activities induced by AhR agonists such as 2,3,7,8-tetrachlorodibenzo-p-dioxin and benzo(k)fluoranthene. In order to determine the in vivo effects of FL on CYP1A and DNA adduct levels, Fundulus heteroclitus were given single ip injections of either BaP (5 mg/kg), BaP + FL (5 mg/kg each), BaP + FL (5 and 50 mg/kg, respectively), FL (5 mg/kg), FL (50 mg/kg), or corn oil control. BaP-treated fish had liver microsome EROD activities significantly higher than controls, whereas FL-treated fish were not different from controls. EROD activities in BaP + FL cotreatments were significantly lower compared to fish treated with BaP alone. When FL was incubated with BaP-induced microsomes, the IC50 for inhibition of EROD activity was 1.4 x 10(-5) M FL. Kinetic studies indicated a significant noncompetitive component to the FL inhibition. When fish were treated with [(14)C]FL, the concentration of radiolabel associated with microsomal preparations was four orders of magnitude lower than the IC50. Therefore, the presence of FL or a FL metabolite was insufficient to account for the inhibition by a kinetic mechanism. In contrast to cell studies, CYP1A immunoreactive protein was significantly decreased in vivo by FL cotreatment, indicating that FL may inhibit EROD activity by down-regulating the CYP1A protein. A covalent interaction of [(14)C]FL with CYP1A was not detected. Total (32)P-postlabeled DNA adducts were not significantly changed by cotreatment of FL and BaP; however, cotreatment with 50 mg/kg FL decreased one adduct and increased another significantly. Because FL and perhaps other inhibitory PAHs, co-occur in the environment with CYP1A inducers, CYP1A-dependent bioassays may cause an underestimation of PAH exposures.
Assuntos
Citocromo P-450 CYP1A1/metabolismo , Inibidores Enzimáticos/farmacologia , Fluorenos/farmacologia , Fundulidae/metabolismo , Hidrocarbonetos Policíclicos Aromáticos/farmacologia , Animais , Benzo(a)pireno/farmacologia , Ligação Competitiva/efeitos dos fármacos , Adutos de DNA/análise , Adutos de DNA/biossíntese , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Feminino , Fígado/química , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , Dibenzodioxinas Policloradas/farmacologiaRESUMO
The pgk-tpi gene locus of Thermotoga maritima encodes both phosphoglycerate kinase (PGK) and a bienzyme complex consisting of a fusion protein of PGK with triosephosphate isomerase (TIM). No separate tpi gene for TIM is present in T. maritima. A frame-shift at the end of the pgk gene has been previously proposed as a mechanism to regulate the expression of the two protein variants [Schurig et al., EMBO J. 14 (1995), 442-451]. Surprisingly, the complete T. maritima genome was found to contain a pgk-tpi sequence not requiring the proposed frameshift mechanism. To clarify the apparent discrepancy, a variety of DNA sequencing techniques were applied, disclosing an anomalous local variability in the pgk-tpi fusion region. The comparison of different DNA samples and the mass spectrometric analysis of the amino acid sequence of the natural fusion protein from T. maritima MSB8 confirmed the local variability of the DNA variants. Since not all peptide masses could be assigned, further variations are conceivable, suggesting an even higher heterogeneity of the T. maritima MSB8 strain.
Assuntos
Proteínas de Bactérias , Mutação da Fase de Leitura/genética , Variação Genética/genética , Complexos Multienzimáticos/genética , Fosfoglicerato Quinase/genética , Thermotoga maritima/genética , Triose-Fosfato Isomerase/genética , Sequência de Aminoácidos/genética , Sequência de Bases/genética , Dados de Sequência Molecular , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Taxon-specific epsilon-crystallin (epsilonC) from duck eye lens is identical to duck heart muscle lactate dehydrogenase. It forms a dimer of dimers with a dissociation constant of 2.2 x 10-7 M, far beyond the value observed for other vertebrate lactate dehydrogenases. Comparing the characteristics of wild-type epsilon-crystallin with those of three mutants, G115N, G119F and 115N/119F, representing the only significant peripheral sequence variations between duck epsilonC and chicken or pig heart muscle lactate dehydrogenase, no significant conformational differences are detectable. Regarding the catalytic properties, the Michaelis constant of the double mutant 115N/119F for pyruvate is found to be decreased; for wild-type enzyme, the effect is overcompensated by the high expression level of epsilonC in the eye lens. As taken from spectral analysis of the guanidine-induced and temperature-induced denaturation transitions, epsilonC in its dimeric state is relatively unstable, whereas the native tetramer exhibits the high intrinsic stability characteristic of common vertebrate heart and muscle lactate dehydrogenases. The denaturation mechanism of epsilonC is complex and only partially reversible. In the case of thermal unfolding, the predominant side reaction competing with the reconstitution of the native state is the kinetic partitioning between proper folding and aggregation. alpha-Crystallin, the major molecular chaperone in the eye lens, inhibits the aggregation of epsilonC by trapping the misfolded protein.
Assuntos
Cristalinas/química , L-Lactato Desidrogenase/química , Cristalino/química , Sequência de Aminoácidos , Animais , Catálise , Cromatografia em Gel , Clonagem Molecular , Cristalinas/genética , Cristalinas/metabolismo , Patos , Eletroforese em Gel de Poliacrilamida , Estabilidade Enzimática , Cinética , L-Lactato Desidrogenase/metabolismo , Dados de Sequência Molecular , Miocárdio/enzimologia , Estrutura Quaternária de Proteína , Homologia de Sequência de AminoácidosRESUMO
Thermotoga maritima XynA is an extremely thermostable modular enzyme with five domains (A1-A2-B-C1-C2). Its catalytic domain (-B-) is flanked by duplicated non-catalytic domains. The C-terminal repeated domains represent cellulose-binding domains (CBDs). Xylanase domains related to the N-terminal domains of XynA (A1-A2) are called thermostabilizing domains because their deletion normally leads to increased thermosensitivity of the enzymes. It was found that a glutathione-S-transferase (GST) hybrid protein (GST-A1A2) containing both A-domains of XynA can interact with various soluble xylan preparations and with mixed-linkage beta-1,3/beta-1,4-glucans. GST-A1A2 showed no affinity for insoluble microcrystalline cellulose, whereas, vice versa, GST-C2, which contains the C-terminal CBD of XynA, did not interact with soluble xylan. Another hybrid protein, GST-A2, displayed the same binding properties as GST-A1A2, indicating that A2 alone can also promote xylan binding. The dissociation constants for the binding of xylose, xylobiose, xylotriose, xylotetraose and xylopentaose by GST-A2, as determined at 20 degrees C by fluorescence quench experiments, were 8.1 x 10(-3) M, 2.3 x 10(-4) M, 2.3 x 10(-5) M, 2.5 x 10(-6)M and 1.1 x 10(-6) M respectively. The A-domains of XynA, which are designated as xylan binding domains (XBD), are, from the structural as well as the functional point of view, prototypes of a novel class of binding domains. More than 50 related protein segments with hitherto unknown function were detected in about 30 other multidomain beta-glycanases, among them putative plant (Arabidopsis thaliana) xylanases. It is argued that polysaccharide binding and not thermostabilization is the main function of A-like domains.
Assuntos
Glucanos/metabolismo , Thermotoga maritima/enzimologia , Xilanos/metabolismo , Xilosidases/metabolismo , beta-Glucanas , Sítios de Ligação , Celulose/metabolismo , Eletroforese em Gel de Poliacrilamida/métodos , Endo-1,4-beta-Xilanases , Estabilidade Enzimática , Escherichia coli , Expressão Gênica , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Oligossacarídeos/metabolismo , Polissacarídeos/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Solubilidade , Especificidade por Substrato , Thermotoga maritima/genética , Xilosidases/genética , Xilosidases/isolamento & purificaçãoRESUMO
Recombinant maltose-binding protein from Thermotoga maritima (TmMBP) was expressed in Escherichia coli and purified to homogeneity, applying heat incubation of the crude extract at 75 degrees C. As taken from the spectral, physicochemical and binding properties, the recombinant protein is indistinguishable from the natural protein isolated from the periplasm of Thermotoga maritima. At neutral pH, TmMBP exhibits extremely high intrinsic stability with a thermal transition >105 degrees C. Guanidinium chloride-induced equilibrium unfolding transitions at varying temperatures result in a stability maximum at approximately 40 degrees C. At room temperature, the thermodynamic analysis of the highly cooperative unfolding equilibrium transition yields DeltaG(N-->U)=100(+/-5) kJ mol(-1 )for the free energy of stabilization. Compared to mesophilic MBP from E. coli as a reference, this value is increased by about 60 kJ mol(-1). At temperatures around the optimal growth temperature of T. maritima (t(opt) approximately 80 degrees C), the yield of refolding does not exceed 80 %; the residual 20 % are misfolded, as indicated by a decrease in stability as well as loss of the maltose-binding capacity. TmMBP is able to bind maltose, maltotriose and trehalose with dissociation constants in the nanomolar to micromolar range, combining the substrate specificities of the homologs from the mesophilic bacterium E. coli and the hyperthermophilic archaeon Thermococcus litoralis. Fluorescence quench experiments allowed the dissociation constants of ligand binding to be quantified. Binding of maltose was found to be endothermic and entropy-driven, with DeltaH(b)=+47 kJ mol(-1) and DeltaS(b)=+257 J mol(-1) K(-1). Extrapolation of the linear vant'Hoff plot to t(opt) resulted in K(d) approximately 0.3 microM. This result is in agreement with data reported for the MBPs from E. coli and T. litoralis at their respective optimum growth temperatures, corroborating the general observation that proteins under their specific physiological conditions are in corresponding states.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Proteínas de Transporte/metabolismo , Proteínas de Escherichia coli , Proteínas de Transporte de Monossacarídeos , Thermotoga maritima/metabolismo , Sequência de Bases , Sítios de Ligação , Metabolismo dos Carboidratos , Proteínas de Transporte/genética , Proteínas de Transporte/isolamento & purificação , Clonagem Molecular , Primers do DNA , Concentração de Íons de Hidrogênio , Proteínas Ligantes de Maltose , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Espectrofotometria Ultravioleta , TermodinâmicaRESUMO
Proteins from (hyper-)thermophiles are known to exhibit high intrinsic stabilities. Commonly, their thermodynamic characterization is impeded by irreversible side reactions of the thermal analysis or calorimetrical problems. Small single-domain proteins are suitable candidates to overcome these obstacles. Here, the thermodynamics of the thermal denaturation of the recombinant cold-shock protein (Csp) from the hyperthermophilic bacterium Thermotoga maritima (Tm) was studied by differential scanning calorimetry. The unfolding transition can be described over a broad pH range (3.5-8.5) by a reversible two-state process. Maximum stability (DeltaG (25 degrees C)=6.5 kcal/mol) was observed at pH 5-6 where Tm Csp unfolds with a melting temperature at 95 degrees C. The heat capacity difference between the native and the denatured states is 1.1(+/-0.1) kcal/(mol K). At pH 7, thermal denaturation occurs at 82 degrees C. The corresponding free energy profile has its maximum at 30 degrees C with DeltaGN-->U=4.8(+/-0.5) kcal/mol. At the optimal growth temperature of T. maritima (80 degrees C), Tm Csp in the absence of ligands is only marginally stable, with a free energy of stabilization not far beyond the thermal energy. With the known stabilizing effect of nucleic acids in mind, this suggests a highly dynamical interaction of Tm Csp with its target molecules.
Assuntos
Proteínas de Bactérias/química , Proteínas de Choque Térmico/química , Dobramento de Proteína , Thermotoga maritima/metabolismo , Proteínas de Bactérias/metabolismo , Varredura Diferencial de Calorimetria , Clonagem Molecular , Proteínas de Choque Térmico/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Desnaturação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , TermodinâmicaRESUMO
Of all surgical interventions of intestinal non-Hodgkin's lymphomas 58% (15 or 26 patients) are performed in an emergency situation. In 42% of cases, examination by ultrasonography, endosonography, intestinoscopy. Sellink's enema, thoracic, abdominal/pelvic CT and bone marrow puncture could determine the stage preoperatively. This could also be done by examining the regional and juxtaregional lymph nodes or performing a liver biopsy intraoperatively. Crucial for the therapy is in all cases the adequate staging even in emergency situations. Only special knowledge of the intestinal non-Hodgkin's lymphoma can lead to the necessary stage-adapted multimodal therapy--operation/irradiation/chemotherapy.
Assuntos
Neoplasias Intestinais/terapia , Linfoma não Hodgkin/terapia , Adulto , Medula Óssea/patologia , Terapia Combinada , Feminino , Humanos , Neoplasias Intestinais/diagnóstico , Neoplasias Intestinais/patologia , Linfonodos/patologia , Linfoma de Zona Marginal Tipo Células B/diagnóstico , Linfoma de Zona Marginal Tipo Células B/patologia , Linfoma de Zona Marginal Tipo Células B/terapia , Linfoma não Hodgkin/diagnóstico , Linfoma não Hodgkin/patologia , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Equipe de Assistência ao Paciente , PrognósticoRESUMO
The hyperthermophilic bacterium Thermotoga maritima is capable of gaining metabolic energy utilizing xylan. XynA, one of the corresponding hydrolases required for its degradation, is a 120-kDa endo-1,4-D-xylanase exhibiting high intrinsic stability and a temperature optimum approximately 90 degrees C. Sequence alignments with other xylanases suggest the enzyme to consist of five domains. The C-terminal part of XynA was previously shown to be responsible for cellulose binding (Winterhalter C, Heinrich P, Candussio A, Wich G, Liebl W. 1995. Identification of a novel cellulose-binding domain within the multi-domain 120 kDa Xylanase XynA of the hyperthermophilic bacterium Thermotoga maritima. Mol Microbiol 15:431-444). In order to characterize the domain organization and the stability of XynA and its C-terminal cellulose-binding domain (CBD), the two separate proteins were expressed in Escherichia coli. CBD, because of its instability in its ligand-free form, was expressed as a glutathione S-transferase fusion protein with a specific thrombin cleavage site as linker. XynA and CBD were compared regarding their hydrodynamic and spectral properties. As taken from analytical ultracentrifugation and gel permeation chromatography, both are monomers with 116 and 22 kDa molecular masses, respectively. In the presence of glucose as a ligand, CBD shows high intrinsic stability. Denaturation/renaturation experiments with isolated CBD yield > 80% renaturation, indicating that the domain folds independently. Making use of fluorescence emission and far-UV circular dichroism in order to characterize protein stability, guanidine-induced unfolding of XynA leads to biphasic transitions, with half-concentrations c1/2 (GdmCl) approximately 4 M and > 5 M, in accordance with the extreme thermal stability. At acid pH, XynA exhibits increased stability, indicated by a shift of the second guanidine-transition from 5 to 7 M GdmCl. This can be tentatively attributed to the cellulose-binding domain. Differences in the transition profiles monitored by fluorescence emission and dichroic absorption indicate multi-state behavior of XynA. In the case of CBD, a temperature-induced increase in negative ellipticity at 217 nm is caused by alterations in the environment of aromatic residues that contribute to the far-UV CD in the native state.
Assuntos
Celulose/metabolismo , Bactérias Anaeróbias Gram-Negativas/enzimologia , Xilosidases/química , Sequência de Aminoácidos , Cristalização , Endo-1,4-beta-Xilanases , Estabilidade Enzimática , Guanidina , Guanidinas , Temperatura Alta , Dados de Sequência Molecular , Peso Molecular , Ligação Proteica , Desnaturação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Análise Espectral , Termodinâmica , Xilosidases/metabolismoRESUMO
Surgical therapy of lung metastases is now an established procedure. The operation's purpose is radical, and therefore potentially curative, resection. There are also diagnostic and palliative indications. Median sternotomy is the standard approach as it allows revision of both lungs, which is important even when disease seems to be unilateral. Preoperative staging does not give a reliable idea of the number and extent of metastases. From 1972 to 1991, a total of 843 operations for lung metastases were carried out in 729 patients in the surgical department of the Thorax Clinic in Heidelberg (Rohrbach). The 30-day mortality was 2.9%, and the overall 5-year survival from the date of resection of the metastases was 33%. The best results were achieved in testicular cancer, with a 5-year-survival rate of 67%, and the poorest were observed in melanomas, with only 12% 3-year survival. In addition to the primary tumour, and in some cases depending on it, several other prognostic factors were relevant: radicality, sarcoma vs. carcinoma (carcinoma involved a better prognosis), disease-free interval, type of resection, thoracic lymph node involvement. Multivariate analysis showed that the prognostic influence of the factors varies considerably with the kind of primary tumour. Surgical treatment of lung metastases is part of an interdisciplinary oncological therapeutic concept offering prolongation of survival to most of the patients concerned and even the possibility of cure to some. Even if prolongation of survival is not feasible, an improved quality of life and therefore good palliation are obtained.
Assuntos
Neoplasias Pulmonares/secundário , Equipe de Assistência ao Paciente , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma/diagnóstico por imagem , Carcinoma/mortalidade , Carcinoma/secundário , Carcinoma/cirurgia , Criança , Pré-Escolar , Intervalo Livre de Doença , Feminino , Seguimentos , Humanos , Neoplasias Pulmonares/diagnóstico por imagem , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/cirurgia , Excisão de Linfonodo , Masculino , Pessoa de Meia-Idade , Pneumonectomia , Complicações Pós-Operatórias/mortalidade , Radiografia , Estudos Retrospectivos , Sarcoma/diagnóstico por imagem , Sarcoma/mortalidade , Sarcoma/secundário , Sarcoma/cirurgia , Taxa de SobrevidaRESUMO
Surgical therapy of lung metastases nowadays is an established procedure. The operation's purpose is the radical and therefore potential curative resection. Beside there are diagnostic and palliative indications. Beside there are diagnostic and palliative indications. Median sternotomy is the standard approach for revision of both lungs even in unilateral seeming disease. Preoperative staging is not reliable concerning number and extension of metastases. From 1972 to 1991 843 operations for lung metastases were carried out in 729 patients in the surgical department of the "Thoraxklinik Heidelberg-Rohrbach". 30-day-mortality amounted to 2.9%, 5-year-survival-rate was 33% overall from date of metastases resection. The best results were achieved in testicular cancer with 67% 5-years-survival-rate, poorest survival was observed in melanomas with 12% 3-years-survival. Beside the primary tumor and partly dependent on it several prognostic factors were relevant: radicality, sarcoma vs carcinoma in favour of carcinomas, disease-free interval, type of resection, thoracic lymphnode involvement. As figured out by multivariate analysis the prognostic influence of the factors varies considerably due to the kind of primary tumor. Surgery of lung metastases is part of an interdisciplinary oncological therapeutical concept and offers a prolonged survival to most of the patients and the possibility of cure to some. Even if prolongation of life is not feasible an improved quality and therefore a good palliation is obtained.
Assuntos
Neoplasias Pulmonares/secundário , Equipe de Assistência ao Paciente , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Feminino , Seguimentos , Humanos , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/cirurgia , Masculino , Pessoa de Meia-Idade , Cuidados Paliativos , Pneumonectomia , Taxa de SobrevidaRESUMO
We have localized the transcription start site of the Drosophila melanogaster muscle myosin heavy chain (MHC) gene and find that all forms of the alternatively spliced MHC mRNA initiate at the same location. Therefore the alternative inclusion/exclusion of the 3' penultimate exon in transcripts from this gene (Bernstein, S.I., Hansen, C.J., Becker, K.D., Wassenberg, D.R., II, Roche, E.S., Donady, J.J., and Emerson, C. P., Jr. (1986) Mol. Cell. Biol. 6, 2511-2519; Rozek, C.E., and Davidson, N. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 2128-2134) does not result from the use of different 5' transcription initiation sites. This gene is the first invertebrate MHC gene shown to have TATA and CAAT box consensus sequences and a noncoding 5' exon, properties that are shared with some vertebrate and invertebrate contractile protein genes. The intron that splits the 5' noncoding region of the Drosophila MHC gene contains no major conserved elements relative to other Drosophila contractile protein genes. The introns within the coding region near the 5' end of the Drosophila MHC gene are located at the same sites as nematode and vertebrate MHC gene introns, indicating that these MHC genes are derived from a common ancestral sequence. The putative ATP binding domain encoded in the fourth exon of the Drosophila MHC gene is highly conserved relative to vertebrate, invertebrate, and non-muscle MHC genes suggesting that each of these myosins bind ATP by the same mechanism. Two divergent copies of the third exon are present within the 5' region of the Drosophila MHC gene, suggesting that alternative splicing produces MHC isoforms with different globular head regions.
Assuntos
Drosophila melanogaster/genética , Miosinas/genética , Splicing de RNA , Transcrição Gênica , Sequência de Aminoácidos , Sequência de Bases , Evolução Biológica , DNA/genética , Enzimas de Restrição do DNA , Éxons , Íntrons , Hibridização de Ácido Nucleico , RNA Mensageiro/genéticaRESUMO
Genomic and cDNA sequencing studies show that transcripts from the muscle myosin heavy-chain (MHC) gene of Drosophila melanogaster are alternatively spliced, producing RNAs that encode at least two MHC isoforms with different C termini. Transcripts encoding an MHC isoform with 27 unique C-terminal amino acids accumulate during both larval and adult muscle differentiation. Transcripts for the second isoform encode one unique C-terminal amino acid and accumulate almost exclusively in pupal and adult thoracic segments, the location of the indirect flight muscles. The 3' splice acceptor site preceding the thorax-specific exon is unusually purine rich and thus may serve as a thorax-specific splicing signal. We suggest that the alternative C termini of these two MHC isoforms control myofilament assembly and may play a role in generating the distinctive myofilament organizations of flight muscle and other muscle types.