RESUMO
The aim of the study was to evaluate the association of vascular endothelial growth factor (VEGF) genotypes with treatment efficacy in a randomized trial. This study compared two chemotherapy regimens (FOLFIRI versus XELIRI) combined with bevacizumab, as first-line treatment for metastatic colorectal cancer. DNA was extracted from blood samples of 173 patients participating in the trial. Genotyping was performed for selected SNPs (VEGF-1154, +936, -634, -2578 and -1498). All candidate genotypes were evaluated for associations with overall survival (OS), progression-free survival (PFS) and response rate (RR). There were no significant differences with respect to the distribution of genotypes in the treatment groups. The VEGF-1154 GG genotype was more frequent in patients not responding to treatment compared with responders (65.5 versus 39.8%, P = 0.032). Furthermore, the VEGF-1154 GG genotype was associated with inferior median OS compared with GA (hazards ratio = 1.68; 95% confidence interval: 1.10-2.57; P = 0.016) or with the alternative genotypes (GA and AA) combined (hazards ratio = 1.62; 95% confidence interval: 1.09-2.40; P = 0.017). In multivariate analysis, the VEGF-1154 GG genotype remained a significant adverse factor for OS. Our results support the potential predictive ability of VEGF genotypes in patients with metastatic colorectal cancer receiving irinotecan-based chemotherapy plus bevacizumab, in terms of RR and OS. However, current results should be validated prospectively, in larger cohorts.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Polimorfismo de Nucleotídeo Único , Fator A de Crescimento do Endotélio Vascular/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Monoclonais Humanizados/administração & dosagem , Bevacizumab , Camptotecina/administração & dosagem , Camptotecina/análogos & derivados , Neoplasias Colorretais/genética , Neoplasias Colorretais/mortalidade , Feminino , Genótipo , Humanos , Irinotecano , Desequilíbrio de Ligação , Masculino , Pessoa de Meia-Idade , Modelos de Riscos ProporcionaisRESUMO
Combination antimicrobial therapy represents common practice in the treatment of febrile neutropenia aiming to broaden the antimicrobial spectrum against Gram-negative pathogens. We did a prospective, non-randomized, comparative study to evaluate ceftazidime plus either levofloxacin or once-daily amikacin as empirical regimens for febrile neutropenia in patients with solid tumor or hematopoietic neoplasm in a region of high baseline resistance prevalence. We included 285 febrile neutropenic episodes in 235 individual patients. One hundred forty-eight cases received levofloxacin and 137 received amikacin, both in combination with ceftazidime. More cases in the levofloxacin than the amikacin group had underlying hematological malignancy; most other characteristics of the two groups were well balanced. Nephrotoxicity requiring treatment discontinuation occurred in one case in the amikacin group. No difference in clinical success (79.7% vs. 80.3%, p>0.99) or all-cause mortality (12.8% vs. 11.7%, p=0.86) was noted between the levofloxacin and the amikacin groups, even after adjustment for the independent predictor variables for each endpoint. Sepsis at presentation, presence of localizing symptoms/signs of infection, and isolation of a non-susceptible Gram-negative pathogen independently predicted both clinical success and all-cause mortality. Additionally, underlying solid tumor independently predicted clinical success, while poor prognosis of the underlying neoplasia and skin/soft tissue infection independently predicted mortality. Ceftazidime plus levofloxacin had similar effectiveness to ceftazidime plus amikacin as empirical regimens for febrile neutropenia. Nephrotoxicity with once-daily amikacin was minimal. Inappropriate empirical therapy was associated with worse prognosis.
Assuntos
Amicacina/administração & dosagem , Antibacterianos/administração & dosagem , Ceftazidima/administração & dosagem , Febre de Causa Desconhecida/tratamento farmacológico , Levofloxacino , Ofloxacino/administração & dosagem , Idoso , Amicacina/efeitos adversos , Antibacterianos/efeitos adversos , Ceftazidima/efeitos adversos , Quimioterapia Combinada/efeitos adversos , Quimioterapia Combinada/métodos , Feminino , Febre de Causa Desconhecida/complicações , Febre de Causa Desconhecida/mortalidade , Humanos , Nefropatias/induzido quimicamente , Masculino , Pessoa de Meia-Idade , Neoplasias/complicações , Neutropenia/complicações , Ofloxacino/efeitos adversos , Estudos Prospectivos , Análise de Sobrevida , Resultado do TratamentoRESUMO
Cellular DNA is subjected to continual attack, both by reactive species inside cells and by environmental agents. Toxic and mutagenic consequences are minimized by distinct pathways of repair, and 130 known human DNA repair genes are described here. Notable features presently include four enzymes that can remove uracil from DNA, seven recombination genes related to RAD51, and many recently discovered DNA polymerases that bypass damage, but only one system to remove the main DNA lesions induced by ultraviolet light. More human DNA repair genes will be found by comparison with model organisms and as common folds in three-dimensional protein structures are determined. Modulation of DNA repair should lead to clinical applications including improvement of radiotherapy and treatment with anticancer drugs and an advanced understanding of the cellular aging process.
Assuntos
Reparo do DNA/genética , Genes , Genoma Humano , DNA/metabolismo , Dano ao DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Bases de Dados Factuais , Desoxirribonucleases/genética , Desoxirribonucleases/metabolismo , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , N-Glicosil Hidrolases/genética , N-Glicosil Hidrolases/metabolismo , Polimorfismo Genético , Rad51 Recombinase , Recombinação Genética , Transdução de SinaisRESUMO
We have identified a >600-kb region at 16q23.2 that is homozygously deleted from malignant ovarian ascites using representational difference analysis. Overlapping homozygous deletions were also observed in the colon carcinoma cell line HCT116 and a xenograft established from the small cell lung cancer cell line WX330. This region coincides with that described previously by others as showing loss of heterozygosity in prostate and breast cancers (C. Li et al., Genes Chromosomes Cancer, 24: 175-182, 1999; A. Latil et al., Cancer Res., 57: 1058-1062, 1997; K. Driouch et al., Genes Chromosomes Cancer, 19: 185-191, 1997; A. Iida et al., Br. J. Cancer, 75: 264-267, 1997). In addition, the minimally deleted region spans the common fragile site FRA16D. We have constructed a 700-kb physical map encompassing the deleted region. By fluorescence in situ hybridization of aphidicolin-induced metaphase chromosomes, we have preliminary data to suggest that P1-derived bacterial artificial chromosome clones from the contig lie on both sides of FRA16D. This is confirmed by extensive fluorescence in situ hybridization analysis of the region reported in the accompanying article (M. Mangelsdorf et al., Cancer Res., 60: 1683-1689, 2000) and is consistent with an involvement of this common fragile site in the loss of 16q23.2 material in various cancer types. The minimally deleted region of approximately 210 kb has been characterized using our own markers and public domain markers. Eleven distinct expressed sequences mapped to the region, providing a basis for identifying the predicted tumor suppressor gene in this region.
Assuntos
Deleção Cromossômica , Fragilidade Cromossômica , Cromossomos Humanos Par 16/genética , Neoplasias/genética , Bacteriófago P1 , Bandeamento Cromossômico , Sítios Frágeis do Cromossomo , Cromossomos Artificiais de Levedura , Clonagem Molecular , Mapeamento de Sequências Contíguas , DNA de Neoplasias/genética , Predisposição Genética para Doença/genética , Homozigoto , Humanos , Hibridização in Situ Fluorescente , Mapeamento Físico do Cromossomo , Células Tumorais CultivadasRESUMO
BACKGROUND: The serum CA 125 marker is elevated in 80% of patients with ovarian adenocarcinoma. MDR 1 gene expression has been identified in a variety of tumor types and its expression has been correlated with multidrug resistance. Whether there is a correlation between CA 125 levels and MDR 1 expression has not been sufficiently investigated. Therefore, the aim of this study was to examine whether an association between serum CA 125 levels and MDR 1 expression exists. PATIENTS AND METHODS: Serum CA 125 levels were measured during the diagnosis of ovarian cancer. Fresh tumor specimens or ascitic fluid samples were studied for MDR 1 expression by the polymerase chain reaction method (PCR). RESULTS: Forty patients with ovarian cancer were studied, 34 (85%) of whom had elevated CA 125. Twenty-eight out of the 40 patients were tested for MDR 1 expression; 20 expressed the gene and 8 did not. The median level of CA 125 in specimens expressing the MDR1 gene was 327, and in specimens that did not it was 376. There was no correlation between the CA 125 levels and MDR 1 expression (p = 0.484). CONCLUSION: There does not seem to be an association between CA 125 levels and expression of the MDR1 gene in patients with ovarian cancer.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Adenocarcinoma/metabolismo , Antígeno Ca-125/sangue , Neoplasias Ovarianas/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Adenocarcinoma/genética , Adenocarcinoma/patologia , Adulto , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Reação em Cadeia da PolimeraseAssuntos
Carcinoma Embrionário/secundário , Histiocitose de Células de Langerhans/etiologia , Neoplasias Pulmonares/secundário , Fumar/efeitos adversos , Neoplasias Testiculares/patologia , Adulto , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma Embrionário/terapia , Quimioterapia Adjuvante , Histiocitose de Células de Langerhans/diagnóstico , Humanos , Neoplasias Pulmonares/terapia , Masculino , Orquiectomia , Abandono do Hábito de Fumar , Neoplasias Testiculares/terapia , Resultado do TratamentoRESUMO
A novel Drosophila melanogaster gene UBL3 was characterized and shown to be highly conserved in man and Caenorhabditis elegans (C. elegans). The human and mouse homologues were cloned and sequenced. UBL3 is a ubiquitin-like protein of unknown function with no conserved homologues in yeast. Mapping of the human and mouse UBL3 genes places them within a region of shared gene order between human and mouse chromosomes on human chromosome 13q12-13 and telomeric mouse chromosome 5 (MMU5).
Assuntos
Proteínas de Drosophila , Proteínas de Insetos/genética , Sequência de Aminoácidos , Animais , Mapeamento Cromossômico , Clonagem Molecular , DNA Complementar , Drosophila melanogaster/genética , Humanos , Dados de Sequência Molecular , Homologia de Sequência de AminoácidosRESUMO
PURPOSE: To evaluate the efficacy and safety of weekly paclitaxel and trastuzumab in patients with HER2-positive metastatic breast cancer, with trastuzumab administered beyond disease progression. PATIENTS AND METHODS: Twenty-six women with metastatic breast cancer, that was HER2-positive as determined by immunohistochemistry, were treated with weekly paclitaxel 70 or 90 mg/m2 and trastuzumab (4 mg/kg initial dose followed by 2 mg/kg weekly). RESULTS: The median duration of treatment was 28 (8-72) weeks for paclitaxel and 59 (14-150) weeks for trastuzumab. Two (8%) patients experienced complete and 14 (54%) partial responses, for an overall response rate of 62%. The median time to disease progression was 11 (2.89-36) months and median survival 34+ months. Grade 3/4 adverse events were alopecia (46%), neurotoxicity (15%), leukopenia (12%) and neutropenia (12%). Infusion-related reactions were mild to moderate. No symptomatic cardiac toxicity was observed. No patient discontinued trastuzumab due to toxicity. CONCLUSION: Prolonged administration of weekly paclitaxel and trastuzumab is effective and well-tolerated in women with HER2-positive metastatic breast cancer.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Antineoplásicos Fitogênicos/administração & dosagem , Neoplasias da Mama/terapia , Imunoterapia/métodos , Paclitaxel/administração & dosagem , Adulto , Idoso , Anticorpos Monoclonais/efeitos adversos , Anticorpos Monoclonais Humanizados , Antineoplásicos Fitogênicos/efeitos adversos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Terapia Combinada , Esquema de Medicação , Feminino , Humanos , Imunoterapia/efeitos adversos , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Paclitaxel/efeitos adversos , Receptor ErbB-2/biossíntese , TrastuzumabRESUMO
BACKGROUND: Annualised figures show an up to 7-fold higher incidence of vascular thromboembolism (VTE) in patients with advanced pancreatic cancer (APC) compared to other common malignancies. Concurrent VTE has been shown to confer a worse overall prognosis in APC. METHODS: One hundred and twenty three APC patients were randomised to receive either gemcitabine 1000 mg/m(2) or the same with weight-adjusted dalteparin (WAD) for 12 weeks. Primary end-point was the reduction of all-type VTE during the study period. NCT00462852, ISRCTN: 76464767. FINDINGS: The incidence of all-type VTE during the WAD treatment period (<100 days from randomisation) was reduced from 23% to 3.4% (p = 0.002), with a risk ratio (RR)of 0.145, 95% confidence interval (CI) (0.035-0.612) and an 85% risk reduction. All-type VTE throughout the whole follow-up period was reduced from 28% to 12% (p = 0.039), RR = 0.419, 95% CI (0.187-0.935) and a 58% risk reduction. Lethal VTE <100 days was seen only in the control arm, 8.3% compared to 0% (p = 0.057), RR = 0.092, 95% CI (0.005-1.635). INTERPRETATION: Weight adjusted dalteparin used as primary prophylaxis for 12 weeks is safe and produces a highly significant reduction of all-type VTE during the prophylaxis period. The benefit is maintained after dalteparin withdrawal although decreases with time.
Assuntos
Anticoagulantes/uso terapêutico , Antimetabólitos Antineoplásicos/uso terapêutico , Dalteparina/uso terapêutico , Desoxicitidina/análogos & derivados , Neoplasias Pancreáticas/sangue , Neoplasias Pancreáticas/tratamento farmacológico , Tromboembolia Venosa/tratamento farmacológico , Tromboembolia Venosa/prevenção & controle , Adulto , Idoso , Idoso de 80 Anos ou mais , Desoxicitidina/uso terapêutico , Feminino , Seguimentos , Humanos , Masculino , Adesão à Medicação , Pessoa de Meia-Idade , Análise Multivariada , Taxa de Sobrevida , Tromboembolia Venosa/etiologia , GencitabinaRESUMO
Conventional treatment for brain metastases (BM) is whole-brain radiotherapy (WBRT). Efficacy is poor. It might be increased by a potent radiosensitiser such as gemcitabine which is believed to cross the disrupted blood-brain barrier. Primary objective of this study was to determine the maximum tolerated dose (MTD) of twice weekly gemcitabine given concurrently with WBRT. Patients with BM from carcinoma were included. The dose of WBRT was 30 Gys (10 daily fractions). Gemcitabine was given 2-4 h prior to WBRT on days 1 and 8 for the first cohort of patients and then on days 1, 4, 8 and 11. Starting dose was 25 mg m(-2), escalated by 12.5 mg m(-2) increments. At least three patients were included per level. Dose limiting toxicity (DLT) was defined as grade 4 haematological or grade > or =3 nonhaematological toxicity. A total of 25 patients were included; 74% had a PS 1 (ECOG). In all, 23 had non-small-cell lung cancer, six colorectal, four breast, two renal cell and one oesophageal carcinoma. A total of 92% had concurrent extracranial disease. Six had single BM, 13 had two or three BM and six multiple. Up to 50 mg m(-2) (level 4) no DLT was observed. At 62.5 mg m(-2), one out of six patients developed DLT (thrombocytopenia-bleeding). The next dose level (75 mg m(-2)) was abandoned after grade 4 bone marrow toxicity (fatal neutropenic sepsis) was seen in one out of two patients. So that the dose of 50 mg m(-2) will be taken forward for further study.
Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/secundário , Desoxicitidina/análogos & derivados , Desoxicitidina/toxicidade , Radiossensibilizantes/toxicidade , Adulto , Idoso , Neoplasias Encefálicas/radioterapia , Relação Dose-Resposta a Droga , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Indução de Remissão/métodos , GencitabinaRESUMO
BACKGROUND: Cohesin is a macromolecular complex that links sister chromatids together at the metaphase plate during mitosis. The links are formed during DNA replication and destroyed during the metaphase-to-anaphase transition. In budding yeast, the 14S cohesin complex comprises at least two classes of SMC (structural maintenance of chromosomes) proteins - Smc1 and Smc3 - and two SCC (sister-chromatid cohesion) proteins - Scc1 and Scc3. The exact function of these proteins is unknown. RESULTS: Searches of protein sequence databases have revealed new homologs of cohesin proteins. In mouse, Mmip1 (Mad member interacting protein 1) and Smc3 share 99% sequence identity and are products of the same gene. A phylogenetic tree of SMC homologs reveals five families: Smc1, Smc2, Smc3, Smc4 and an ancestral family that includes the sequences from the Archaea and Eubacteria. This ancestral family also includes sequences from eukaryotes. A cohesion interaction network, comprising 17 proteins, has been constructed using two proteomic databases. Genes encoding six proteins in the cohesion network share a common upstream region that includes the MluI cell-cycle box (MCB) element. Pairs of the proteins in this network share common sequence motifs that could represent common structural features such as binding sites. Scc2 shares a motif with Chk1 (kinase checkpoint protein), that comprises part of the serine/threonine protein kinase motif, including the active-site residue. CONCLUSIONS: We have combined genomic and proteomic data into a comprehensive network of information to reach a better understanding of the function of the cohesin complex. We have identified new SMC homologs, created a new SMC phylogeny and identified shared DNA and protein motifs. The potential for Scc2 to function as a kinase - a hypothesis that needs to be verified experimentally - could provide further evidence for the regulation of sister-chromatid cohesion by phosphorylation mechanisms, which are currently poorly understood.
Assuntos
Proteoglicanas de Sulfatos de Condroitina , Cromátides/metabolismo , Biologia Computacional , Proteínas de Drosophila , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Filogenia , Proteínas de Saccharomyces cerevisiae , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinase 1 do Ponto de Checagem , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Sequência Consenso/genética , Bases de Dados como Assunto , Proteínas Fúngicas , Genômica , Humanos , Substâncias Macromoleculares , Camundongos , Dados de Sequência Molecular , Proteínas Nucleares/genética , Fosfoproteínas , Fosforilação , Ligação Proteica , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteoma , Elementos de Resposta/genética , Saccharomyces cerevisiae , Homologia de Sequência , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , CoesinasRESUMO
The hemoglobin of the Great Crested Newt (Triturus cristatus), an animal maintaining the gas exchange to about 85% through the skin, consists of a major (HbM = 65%) and a minor (Hbm = 35%) component. The primary structures of the four chains are presented. They could be separated by reversed-phase HPLC and were cleaved with trypsin and additionally by acid hydrolysis. Both the native chains and their peptides were sequenced by liquid and gas phase sequenators. At the N-terminus the alpha M-chains are by one amino-acid residue longer and the beta M-chains by one residue shorter, resulting in a chain length of 142 and 145, respectively. The alpha m-chains are of normal length whereas in the beta m-chains the C-terminal histidine in position 146 is missing. Both alpha-chains differ by 50 residues (35.2%) and the beta-chains by 63 (43.2%). The alpha-chains were compared with those of other salamandroid hemoglobins. The difference to human hemoglobin is marked by 61 (43.3%) amino-acid substitutions in both alpha-chains and by 78 (53.4%) in both beta-chains. Numerous heme contacts and positions involved in the subunit interface are affected by replacements. The most interesting of them were studied by molecular modeling. The importance of the missing beta m-146(HC3)His and of the substitution of several amino-acid residues involved in the binding of organic phosphates is discussed with respect to the reduced Bohr effect of Triturus cristatus hemoglobin.
Assuntos
Hemoglobinas , Triturus/sangue , Sequência de Aminoácidos , Animais , Gráficos por Computador , Globinas/isolamento & purificação , Hemoglobina A , Hemoglobinas/isolamento & purificação , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/isolamento & purificação , Conformação Proteica , Especificidade da Espécie , TripsinaRESUMO
The hemoglobin of the Indian flying fox Cynopterus sphinx contains only one component. In this work, we are presenting its primary structure. The globin chains were separated by high-performance liquid chromatography and the sequences determined by automatic liquid and gas-phase Edman degradation of the chains and their tryptic peptides, as well as of the peptide obtained by acid hydrolysis of the Asp-Pro bond in the beta-chains. The alpha-chains show 14 and the beta-chains 19 exchanges compared with the human alpha- and beta-chains, respectively. In the alpha-chains one amino-acid exchange involves an alpha 1/beta 1 contact. In the beta-chains one heme contact, three alpha 1/beta 1- and one alpha 1/beta 2-contacts are exchanged. The functional and evolutionary aspects of these findings are discussed.
Assuntos
Quirópteros/sangue , Eritrócitos/análise , Globinas/análise , Hemoglobinas/análise , Sequência de Aminoácidos , Animais , Cromatografia Líquida de Alta Pressão , Fragmentos de Peptídeos/análiseRESUMO
The hemoglobin of the European marmot Marmota marmota marmota has been found to consist of only one component. In this work, we are presenting its primary structure. The globin chains have been separated by high performance liquid chromatography and the sequences have been determined by automated Edman degradation of the chains and their tryptic peptides, as well as of the peptide obtained by acid hydrolysis of the Asp-Pro bond in the beta-chains. In the alpha-chains we have found 13 and in the beta-chains 34 exchanges compared with the human alpha- and beta-chains, respectively. The amino acids which are substituted in the alpha-chains are not involved in any contacts, whereas in the beta-chains, one exchange involves a heme contact, two alpha 1/beta 1- and one alpha 1/beta 2-contacts. The functional and evolutionary aspects of these findings are discussed.
Assuntos
Hemoglobinas/isolamento & purificação , Marmota/sangue , Sciuridae/sangue , Sequência de Aminoácidos , Animais , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Eletroforese em Gel de Poliacrilamida , Globinas/isolamento & purificação , Hemoglobina A , Humanos , Fragmentos de Peptídeos/análise , Especificidade da Espécie , TripsinaRESUMO
The hemoglobin of the Indian false vampire Megaderma lyra contains only one component. In this paper, we are presenting its primary structure. The globin chains were separated by high-performance liquid chromatography and the sequences determined by automatic liquid and gas phase Edman degradation of the chains and their tryptic peptides, as well as of the prolyl-peptides obtained by acid hydrolysis of the Asp-Pro bond in the alpha- and beta-chains. The alpha-chains show 23 and the beta-chains 20 exchanges compared with the human alpha- and beta-chains, respectively. In the alpha-chains, three exchanges involved alpha 1/beta 1 contacts. In the beta-chains one heme-and three alpha 1/beta 1 contacts are exchanged. The functional and systematic aspects of these replacements are discussed.
Assuntos
Quirópteros/sangue , Hemoglobinas/isolamento & purificação , Sequência de Aminoácidos , Animais , Globinas/análise , Dados de Sequência Molecular , Fragmentos de Peptídeos/isolamento & purificação , Conformação ProteicaRESUMO
The coelacanth (Latimeria chalumnae, Actinistia) has a single hemoglobin component. The primary structures of the alpha- and beta-chains are presented. They could be separated by reversed-phase HPLC. Peptides obtained by tryptic digestion of the native and oxidized chains were isolated by reversed-phase HPLC and sequenced in liquid and gas-phase sequenators. The alignment was achieved by employing the N-terminal sequences of the native chains and those of a beta-chain cyanogen bromide peptide as well as fragments obtained by acid hydrolysis. The Latimeria alpha-chains consist of 142 amino-acid residues, due to a fish-specific insertion between positions 46 and 47, whereas the beta-chains are of normal length (146 residues). Latimeria alpha- and beta-chains share 72 (51.1%) and 70 (47.9%) identical residues with human hemoglobin, respectively. Numerous heme contacts and positions involved in subunit interface contacts are replaced. The most interesting of them were studied by molecular modeling. The loss of an alpha 1/beta 2-contact by the exchanges alpha 92(FG4)Arg----Leu and beta 43(CD2)Glu----Lys might be responsible for the easy dissociation of the tetrameric hemoglobin molecule. A comparison of the residues replaced in contact positions with fishes and amphibians revealed the highest number of matches between Latimeria and tadpoles. The same result was obtained by the evaluation of other regions relevant for structure and function of the molecule, like exon-intron boundary regions, phosphate binding sites and salt bridges responsible for the Bohr effect.
Assuntos
Anfíbios/genética , Peixes/genética , Fósseis , Hemoglobinas/genética , Sequência de Aminoácidos , Animais , Evolução Biológica , Eletroforese em Gel de Poliacrilamida , Peixes/sangue , Hemoglobinas/química , Humanos , Substâncias Macromoleculares , Dados de Sequência Molecular , Filogenia , Conformação Proteica , Homologia de Sequência do Ácido NucleicoRESUMO
The primary structures of the hemoglobins of two Flying Foxes of the genus Pteropus are presented. Both comprise two components: in P. alecto hemoglobin two alpha-chains at a ratio of 1:1 and two beta-chains at a ratio of 4:1 were detected. The hemoglobin of P. poliocephalus comprises one alpha-chain and two beta-chains, the latter at a ratio of 1:1. The globin chains were separated by high-performance liquid chromatography and the sequences determined by automatic liquid and gas phase Edman degradation of the chains and their tryptic peptides. Compared with human hemoglobin, the alpha-chains of P. alecto and P. poliocephalus show 18 and 19 exchanges, respectively, whereas in the beta-chains 16/17 substitutions are found in both cases. In the alpha-chains of P. alecto, one exchange involves an alpha 1/beta 1-contact. In the beta-chains of both species one heme-, one alpha 1/beta 2- and two alpha 1/beta 1-contacts are exchanged. The relevant side chains are the same in both species. The functional and systematic aspects of these findings are discussed.
Assuntos
Raposas/sangue , Hemoglobinas , Sequência de Aminoácidos , Animais , Cromatografia Líquida de Alta Pressão , Globinas/isolamento & purificação , Hemoglobinas/isolamento & purificação , Substâncias Macromoleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/análise , Especificidade da Espécie , TripsinaRESUMO
Mammalian DNA polymerases alpha and beta lack 3' exonuclease activity and are unable to edit errors after DNA synthesis. However, editing exonucleases can be functions of separate polypeptides. We isolated a widely distributed DNA-specific 3' exonuclease from rabbit liver nuclei, sequenced tryptic peptides by mass spectrometry, and identified the corresponding human open reading frame. The protein expressed from the cloned human sequence exhibits 3' exonuclease activity. The human clone shares sequence homology with the editing function of the Escherichia coli DNA polymerase III holoenzyme, i.e., the DnaQ/MutD protein, and weakly with the editing 3' exonuclease domain of eukaryotic DNA polymerase epsilon. The gene maps to human chromosome 3p21.2-21.3. In a reconstituted human DNA repair system containing DNA polymerase beta and DNA ligase III-XRCC1, accurate rejoining of a 3' mismatched base residue at a single-strand break is dependent on addition of the exonuclease.