RESUMO
BACKGROUND: Four international study groups undertook a large study in resectable osteosarcoma, which included two randomised controlled trials, to determine the effect on survival of changing post-operative chemotherapy based on histological response. PATIENTS AND METHODS: Patients with resectable osteosarcoma aged ≤40 years were treated with the MAP regimen, comprising pre-operatively of two 5-week cycles of cisplatin 120 mg/m(2), doxorubicin 75 mg/m(2), methotrexate 12 g/m(2) × 2 (MAP) and post-operatively two further cycles of MAP and two cycles of just MA. Patients were randomised after surgery. Those with ≥10% viable tumour in the resected specimen received MAP or MAP with ifosfamide and etoposide. Those with <10% viable tumour were allocated to MAP or MAP followed by pegylated interferon. Longitudinal evaluation of quality of life was undertaken. RESULTS: Recruitment was completed to the largest osteosarcoma study to date in 75 months. Commencing March 2005, 2260 patients were registered from 326 centres across 17 countries. About 1334 of 2260 registered patients (59%) were randomised. Pre-operative chemotherapy was completed according to protocol in 94%. Grade 3-4 neutropenia affected 83% of cycles and 59% were complicated by infection. There were three (0.13%) deaths related to pre-operative chemotherapy. At definitive surgery, 50% of patients had at least 90% necrosis in the resected specimen. CONCLUSIONS: New models of collaboration are required to successfully conduct trials to improve outcomes of patients with rare cancers; EURAMOS-1 demonstrates achievability. Considerable regulatory, financial and operational challenges must be overcome to develop similar studies in the future. The trial is registered as NCT00134030 and ISRCTN 67613327.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Ósseas/tratamento farmacológico , Osteossarcoma/tratamento farmacológico , Adolescente , Neoplasias Ósseas/cirurgia , Criança , Cisplatino/administração & dosagem , Cisplatino/efeitos adversos , Terapia Combinada , Doxorrubicina/administração & dosagem , Doxorrubicina/efeitos adversos , Etoposídeo/administração & dosagem , Etoposídeo/efeitos adversos , Feminino , Humanos , Ifosfamida/administração & dosagem , Ifosfamida/efeitos adversos , Interferon-alfa/administração & dosagem , Interferon-alfa/efeitos adversos , Masculino , Metotrexato/administração & dosagem , Metotrexato/efeitos adversos , Terapia Neoadjuvante , Osteossarcoma/cirurgia , Polietilenoglicóis/administração & dosagem , Polietilenoglicóis/efeitos adversos , Qualidade de Vida , Projetos de Pesquisa , Adulto JovemRESUMO
BACKGROUND: We evaluated the therapeutic results in 44 patients (17 girls and 27 boys) with osteosarcoma from 1997 to 2006.Their average age was 12.8 years (2.5-20.2). 41 patients had localised disease and 3 had primary metastases. PATIENTS AND METHODS: We treated our 44 patients using CCG 7921 POG 9351 INT 0133, the therapeutic protocol of the North American cooperative Children's Oncology Group.The median of the follow up was 5.5 years (2-11 years). RESULTS: 40 patients went into complete remission. 19 patients suffered relapses. Of these, 17 patients died - 15 progressed, 1 died due to treatment-related toxicity, 1 died due to secondary acute myeloid leukaemia. As a whole, the patients had a 5-year overall survival rate (OS) of 58.4% and a 5-year event free survival rate (EFS) of 46.7%. The patients with localised extremity osteosarcoma (n = 40) had a 5-year EFS rate of 51%. The patients with good histological response (n = 22) had a 5-year EFS rate of 63.6%, while patients with poor histological response (n = 18) achieved a 5-year EFS rate of 30.5% (p = 0.009). CONCLUSION: The results of treatment of patients with localised extremity osteosarcoma and patients with good histological response to preoperative treatment were very good. The prognosis of patients with axial localisation and metastatic involvement was poor.
Assuntos
Neoplasias Ósseas/terapia , Osteossarcoma/terapia , Adolescente , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/radioterapia , Criança , Pré-Escolar , Terapia Combinada , Feminino , Humanos , Masculino , Osteossarcoma/tratamento farmacológico , Osteossarcoma/radioterapia , Adulto JovemRESUMO
BACKGROUND: The aim of study was to evaluate outcome of international treatment protocol LCH II for children with Langerhans cell histiocytosis treated in FN Motol. METHODS AND RESULTS: Between November 1995 and December 2003, 46 children were treated, sex ratio M:F 29:17 and median age at diagnosis 6 years 8 months. 28 children (60.9%) suffered from monosystem disease with majority of bone lesions (23 times) with skull predominance (16 times). Surgery was primary treatment modality for monosystem disease. Five children with recurrence were successfully treated by protocol LCH II - LR (3x) and LCH III - LR /G2/, respectively. Eighteen children (39.1%) suffered from multisystem disease. 6 out of 18 patients were treated according to low-risk protocol LCH II - LR and 12 children by high-risk scheme LCH II - HR at the non-randomized branch included etoposide. Recurrence was revealed in 11 patients and 10 of them reached 2nd or 3rd complete remission (CR) by 2 - chlorodeoxyadenosine (CDA) monotherapy, and 1 child reached 2nd CR by LCH II - HR scheme. Two children underwent irradiation after bone lesion excision as well as 1 child as supplemental treatment. Totally, 29 children (63.0%) achieved 1st CR, 14 (30.4%) 2nd CR, 2 (4.4%) 3rd CR, and 1 child died because of LCH progression. There were no severe side effects of chemotherapy. Follow-up median time was 5 years 8 months (range 9 months - 9 years 6 months). CONCLUSIONS: LCH II protocol is safe and effective. Results revealed that treatment of patients with multisystem disease might demand some treatment modification.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Histiocitose de Células de Langerhans/tratamento farmacológico , Adolescente , Criança , Pré-Escolar , Quimioterapia Combinada , Etoposídeo/administração & dosagem , Feminino , Histiocitose de Células de Langerhans/patologia , Humanos , Lactente , Masculino , Mercaptopurina/administração & dosagem , Metotrexato/administração & dosagem , Prednisona/administração & dosagem , Indução de Remissão , Vimblastina/administração & dosagemRESUMO
The nucleotide sequence of a 1884 bp DNA fragment of E. coli, carrying the gene dacB, was determined. The DNA codes for penicillin-binding protein 4 (PBP4), an enzyme of 477 amino acids, being involved as a DD-carboxypeptidase-endopeptidase in murein metabolism. The enzyme is translated with a cleavable signal peptide of 20 amino acids, which was verified by sequencing the amino-terminus of the isolated protein. The characteristic active-site fingerprints SXXK, SXN and KTG of class A beta-lactamases and penicillin-binding proteins were located in the sequence. On the basis of amino acid alignments we propose, that PBP4 and class A beta-lactamases share a common evolutionary origin but PBP4 has acquired an additional domain of 188 amino acids in the region between the SXXK and SXN elements.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Transporte/genética , Proteínas de Escherichia coli , Escherichia coli/genética , Hexosiltransferases , Muramilpentapeptídeo Carboxipeptidase/genética , Penicilinas/metabolismo , Peptidil Transferases , D-Ala-D-Ala Carboxipeptidase Tipo Serina , Sequência de Aminoácidos , Sequência de Bases , Evolução Biológica , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , DNA Bacteriano , Dados de Sequência Molecular , Muramilpentapeptídeo Carboxipeptidase/química , Muramilpentapeptídeo Carboxipeptidase/metabolismo , Mapeamento de Nucleotídeos , Proteínas de Ligação às Penicilinas , Alinhamento de Sequência , beta-Lactamases/genéticaRESUMO
Three cases of congenital histiocytic disorders--generalized Langerhans cell histiocytosis, generalized juvenile xanthogranuloma and so-called congenital self-healing histiocytosis are compared using histiochemical, immunohistochemical and ultrastructural methods. The results showed a typical morphological pattern of Langerhans cell histiocytosis (S 100+, CD 1+, alpha-mannosidase +) with an unusual self-healing cutaneous phenomenon. The congenital self-healing histiocytosis showed a non-Langerhans cell immunophenotype (CD 14+, CD 1-, S 100-) and morphological appearance resembling the evolutive "early" stage of juvenile xanthogranuloma. A diffuse cellular positivity of alpha-mannosidase in juvenile xanthogranuloma and congenital self-healing histiocytosis differed from a typical perinuclear globular positivity of this enzyme in Langerhans cell histiocytosis. It is concluded that congenital self-healing histiocytosis may in some cases be of non-Langerhans cell type and under this term a clinically characteristic syndrome of histiocytic proliferation of Langerhans cells or tissue histiocytes may be included.
Assuntos
Histiocitose/congênito , Histiocitose/patologia , Biomarcadores , Histiócitos/enzimologia , Histiócitos/ultraestrutura , Histiocitose/enzimologia , Histiocitose de Células de Langerhans/congênito , Histiocitose de Células de Langerhans/patologia , Histocitoquímica , Humanos , Técnicas Imunoenzimáticas , Recém-Nascido , Masculino , Xantogranuloma Juvenil/congênito , Xantogranuloma Juvenil/patologiaRESUMO
BACKGROUND: Modern treatment of oncological diseases increases markedly the chance of long-term survival and permanent recovery. Due to frequently highly aggressive treatment it is however associated with the risk of late sequelae in the surviving patients. Comprehensive care of patients includes therefore not only control of the neoplastic disease but also efforts of maximal improvement of the quality of life of the patients. In young subjects, in view of their long-term perspective, this problem is particularly important. METHODS AND RESULTS: In 32 patients (25 boys and 7 girls) with extracranial solid tumours without primary endocrinological symptomatology (m. Hodgkin, neuroblastoma, ganglioneuroblastoma, nephroblastoma, Ewings sarcoma and others) a single examination was made assessing height, body weight, grade of sexual maturation according to Tanner, in boys testicular volume by means of a orchidometer and 20 other anthropometric dimensions. The mean age at the time of examination was 16.5 +/- 4.1 years, the mean age at the onset of treatment 6.1 +/- 4.8 years. The patients height, -0.4 +/- 0.9 SD, differs from the Czech national standard (p = 0.025). Impaired growth was recorded in 12.5% patients and had heterogenous causes. The authors proved a negative effect of radiotherapy on the growth of the spine, most markedly in children subjected to irradiation of the abdomen and chest and a highly significant reduction of the testicular volume in boys after cytostatic treatment of m. Hodgkin. CONCLUSIONS: The results are consistent with studies made abroad and indicate the necessity of comprehensive long-term follow-up of somatic growth and development of the gonads in oncological child patients.
Assuntos
Gônadas/efeitos da radiação , Crescimento/efeitos da radiação , Neoplasias/radioterapia , Puberdade/efeitos da radiação , Adolescente , Antineoplásicos/efeitos adversos , Criança , Feminino , Gônadas/efeitos dos fármacos , Crescimento/efeitos dos fármacos , Humanos , Masculino , Neoplasias/tratamento farmacológico , Puberdade/efeitos dos fármacos , Radioterapia/efeitos adversosRESUMO
The authors give an account of contemporary problems of child ophthalmological oncology from the paediatrician's aspect. The most serious intraocular tumors are retinoblastomas, in the orbitopalpebral area rhabdomyosarcomas. The authors draw attention to the five main alarming symptoms typical for tumorous processes at these sites: red painful eye, leukokoria, acute visual failure, acute strabism and various rapidly developing protrusions of the bulbus. Subsequently they inform on possible ophthalmological complications of comprehensive oncological treatment.
Assuntos
Neoplasias Oculares , Criança , Diagnóstico Diferencial , Neoplasias Oculares/diagnóstico , Neoplasias Oculares/terapia , HumanosRESUMO
Histiocytosis from Langerhans cells is a new term for a group of diseases formerly called histiocytosis X. In The Faculty Hospital Motol the authors treated between July 1974 and December 1980 84 children with this disease. Twenty one suffered from the malignant form (formerly Letterer-Siwe and Hand-Schüller-Christian disease) and in 63 patients the diagnosis of eosinophil granuloma was established. In 21 children with unequivocally malignant disease the authors started chemotherapy immediately after establishment of the diagnosis and in six they indicated in addition radiotherapy. Of these 21 children 16 are in complete remission and 5 children died from progression of the disease during treatment. In the group of 63 children with eosinophil granuloma in 44 only excochleation of the focus was performed. Chemotherapy was administered to 12 children, incl. 5 where it was combined with radiotherapy. A relapse of the disease was recorded in 8 children. At present all patients suffering from the disease are in complete remission.
Assuntos
Histiocitose de Células de Langerhans , Adolescente , Criança , Pré-Escolar , Feminino , Histiocitose de Células de Langerhans/diagnóstico , Histiocitose de Células de Langerhans/tratamento farmacológico , Humanos , Lactente , MasculinoAssuntos
Neoplasias Faciais , Hemangioma , Neoplasias Intestinais , Mesentério , Neoplasias Primárias Múltiplas , Neoplasias Peritoneais , Neoplasias Faciais/diagnóstico por imagem , Feminino , Hemangioma/diagnóstico por imagem , Humanos , Lactente , Neoplasias Intestinais/diagnóstico por imagem , Intestino Delgado/diagnóstico por imagem , Mesentério/diagnóstico por imagem , Neoplasias Primárias Múltiplas/diagnóstico por imagem , Neoplasias Peritoneais/diagnóstico por imagem , CintilografiaRESUMO
Langerhans cell histiocytosis (LCH) affecting child vulva alone is a very rare disease. Only 13 cases of primary vulvar LCH have been previously reported in the medical literature. We describe an additional case in which the LCH was confined to the vulva, with review of the literature. A 16.5-year-old girl presented with papulous and ulcerative lesions on her labia majora and minora. The biopsy revealed a typical histopathologic finding consistent with LCH. A metastatic work-up did not reveal any evidence of the disease except on the vulva. Treatment was carried out according to LCH II protocol. The patient was diagnosed with a recurrent disorder in the vulva 8 months after the completion of primary chemotherapy. For this reason, she underwent second line treatment with 2-chlorodeoxyadenosine. Eighteen months after the second line chemotherapy, the patient has no signs of a local or systemic recurrence. Primary LCH of vulva is very unusual, but we have to keep in mind this possibility when an adolescent girl presents with an atypical chronic lesion on the vulva. This patient appears to be the first case of adolescent 16.5 year old having a solely cutaneous lesion of the vulva.
Assuntos
Histiocitose de Células de Langerhans/diagnóstico , Doenças da Vulva/diagnóstico , Adolescente , Feminino , Histiocitose de Células de Langerhans/patologia , Histiocitose de Células de Langerhans/terapia , Humanos , Doenças da Vulva/patologia , Doenças da Vulva/terapiaRESUMO
A rapid method for screening the affinity of proteins to dye-modified resins is described. Performing the binding and elution of the protein extracts in a batch-wise manner and eluting the bound proteins with SDS-PAGE denaturation buffer speed up the screening process and allow the analysis of large collections of dyes. Penicillin-binding protein 4 of Escherichia coli was used as a model enzyme to determine the influences of pH, metal ions, and ionic strength (0 to 500 mM NaCl) on its binding behavior using a collection of 98 dye-affinity resins.
Assuntos
Proteínas de Bactérias/isolamento & purificação , Proteínas de Transporte/isolamento & purificação , Cromatografia de Afinidade , Corantes , Proteínas de Escherichia coli , Escherichia coli/química , Hexosiltransferases , Muramilpentapeptídeo Carboxipeptidase/isolamento & purificação , Peptidil Transferases , D-Ala-D-Ala Carboxipeptidase Tipo Serina , Proteínas de Bactérias/efeitos dos fármacos , Proteínas de Transporte/efeitos dos fármacos , Cromatografia de Afinidade/métodos , Concentração de Íons de Hidrogênio , Modelos Moleculares , Muramilpentapeptídeo Carboxipeptidase/efeitos dos fármacos , Proteínas de Ligação às Penicilinas , Ligação Proteica/efeitos dos fármacos , Sais/farmacologia , Triazinas , Zinco/farmacologiaRESUMO
The dacB gene of Escherichia coli, coding for penicillin-binding protein 4 (PBP4) was cloned under the control of the phage lambda pR promoter and cro gene translation signals. Depression of the phage lambda promoter for 2 h at 42 degrees C in E. coli led to the maximum over-production of PBP4 to 3.8% of the total soluble protein. Expression at 42 degrees C but not at 40 degrees C or 37 degrees C led to incomplete processing and aggregation of the preform of PBP4. Cibacron navyblue 2G-E was selected from a collection of triazine dyes as having a high affinity for PBP4. The immobilised dye was used in a two-step procedure to isolated 374 mg PBP4 from the soluble fraction of 125 g (wet mass) cells of the over-producing strain, with a recovery of 63.2% and a final purity of 99% as determined by active-site titration with radiolabelled penicillin. Saturation of PBP4 with various beta-lactam derivatives did not abolish binding to the dye material, nor was PBP4 eluted by addition of beta-lactams from the dye matrix. PBP4 behaved as a soluble protein throughout the purification, that was performed in the complete absence of detergents. Furthermore, in flotation experiments on sucrose density gradients and in Triton X-114 fractionation experiments, it showed the characteristics of a soluble protein. Cibacron navyblue 2G-E showed class specificity for all E. coli PBP except PBP3 and could be used for the isolation of these PBP from membrane extracts.
Assuntos
Proteínas de Bactérias , Proteínas de Transporte/isolamento & purificação , Cromatografia de Afinidade , Proteínas de Escherichia coli , Escherichia coli/genética , Hexosiltransferases , Muramilpentapeptídeo Carboxipeptidase/isolamento & purificação , Peptidil Transferases , D-Ala-D-Ala Carboxipeptidase Tipo Serina , Triazinas , Bacteriófago lambda/genética , Sequência de Bases , Proteínas de Transporte/genética , Centrifugação com Gradiente de Concentração , Clonagem Molecular , Corantes , Expressão Gênica , Dados de Sequência Molecular , Muramilpentapeptídeo Carboxipeptidase/genética , Proteínas de Ligação às Penicilinas , Plasmídeos , Regiões Promotoras Genéticas , SolubilidadeRESUMO
BACKGROUND: A retrospective review was carried out of nine children under 17 years of age with a diagnosis of intramedullary tumor seen during the period 1989-1995. Six had astrocytomas; one each had an ependymoma, a PNET, and a choroid plexus papilloma. Five patients had back pain, 3 others had mild pareses and the ninth had incapacitating defects. Seven of the 9 were treated by subtotal extirpation of the lesion, and biopsy alone was performed in the other two. All tumors were low grade (grade I or II) and therefore radiation therapy (RT) was performed as the only postoperative treatment in 8 of the 9 children. RESULTS: In February 1996, seven (77.8%) children were alive and two (22.2%) died of recurrent tumor (7 months and 5 years after diagnosis, respectively). Median follow-up was 3 years 4 months (range: 1 year 6 months to 7 years 3 months). CONCLUSION: Surgical removal of intraspinal tumors provides the best hope of control, but spinal column deformity after laminectomy and irradiation is a serious long-term problem in children. Orthopedic supervision for the prevention of these deformities; e.g., by external immobilization, is mandatory.
Assuntos
Neoplasias da Medula Espinal/terapia , Adolescente , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Estudos Retrospectivos , Neoplasias da Medula Espinal/mortalidade , Taxa de SobrevidaRESUMO
The penicillin-binding protein 4 (PBP4), from Escherichia coli, a DD-carboxypeptidase/DD-endopeptidase, was purified in an enzymatically active form to homogeneity by affinity chromatography on 6-aminopenicillanic acid/Sepharose and heparin/Sepharose. Polyclonal antibodies raised against the pure protein were used to identify and isolate PBP4 overproducing clones from an E. coli expression library, which was established on the basis of a temperature-inducible runaway replication plasmid. Three positive clones were isolated, one of which carried the intact structural gene dacB that codes for PBP4, on a 1.9kb SmaI-EcoRI fragment, whereas the other two carried truncated forms of this gene. The direction of transcription was determined. The PBP4 overproducing strain, when grown in rich medium, tolerated 160-fold overexpression. After disrupting cells by sonication, the majority (80%) of the overproduced PBP4 was detected in the 100,000 X g supernatant. Southern blotting analysis using the cloned dacB gene as a probe revealed that, in contrast to that described by Takeda et al. (1981), the plasmid pLC18-38 of the Clarke-Carbon collection does not code for PBP4. The overall composition of murein, synthesized in vitro or in vivo by the PBP4 overproducing strain, as determined by high-performance liquid chromatography analysis, suggests that PBP4 is not involved in transpeptidation but exclusively catalyses a DD-carboxypeptidase and DD-endopeptidase reaction.
Assuntos
Proteínas de Bactérias , Proteínas de Transporte/genética , Proteínas de Escherichia coli , Escherichia coli/genética , Hexosiltransferases , Muramilpentapeptídeo Carboxipeptidase/genética , Peptidoglicano/química , Peptidil Transferases , D-Ala-D-Ala Carboxipeptidase Tipo Serina , Southern Blotting , Proteínas de Transporte/isolamento & purificação , Proteínas de Transporte/metabolismo , Mapeamento Cromossômico , Cromossomos Bacterianos , Clonagem Molecular , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Muramilpentapeptídeo Carboxipeptidase/isolamento & purificação , Muramilpentapeptídeo Carboxipeptidase/metabolismo , Proteínas de Ligação às Penicilinas , Peptidoglicano/biossíntese , Plasmídeos , SolubilidadeRESUMO
A vector encoding the Staphylococcal protein A was modified by cloning the spa gene, including its signal peptide-encoding sequence, downstream of the translation initiation sites of the phage lambda cro gene and under the control of the temperature-inducible phage lambda pR promoter. The expression from this construct was studied using the Escherichia coli phoA gene as a reporter gene after fusion to the spa gene. Determination of alkaline phosphatase activity, 1 h after temperature induction of expression at 42 degrees C, revealed an 800-fold increase over host strain background level. The presence of the alternating selectable markers on the described vector, pHEMa153, which are essential for efficient oligonucleotide-directed construction of mutations by the gapped duplex DNA method, allows the construction of recombinant and mutated forms of Staphylococcal protein A fusion proteins and efficient expression of spa gene fusions without changing the vector system.
Assuntos
Clonagem Molecular/métodos , Vetores Genéticos , Proteínas Recombinantes de Fusão/biossíntese , Proteína Estafilocócica A/biossíntese , Sequência de Aminoácidos , Sequência de Bases , Cromatografia de Afinidade , Escherichia coli/metabolismo , Genes Bacterianos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Plasmídeos , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteína Estafilocócica A/isolamento & purificaçãoRESUMO
High-level expression of a soluble form of penicillin-binding protein 5 (PBP5), called PBP5s, and translocation across the cytoplasmic membrane results in lysis of Escherichia coli cells. The detrimental effect of increased amounts of this D,D-carboxypeptidase on the stability of murein polymer can be avoided by accumulation of the overexpressed protein in the cytoplasm. The signal peptide of the structural gene dacAs, coding for PBP5s was deleted by creating a BamHI site at the site of processing and the truncated gene dacAsc was cloned under the control of the lambda PR promoter. Temperature induction resulted in a 200-fold overproduction of the mature PBP5s in the cytosol (PBP5sc) which is no longer harmful to the cells. PBP5sc could quantitatively be recovered in the soluble fraction after disrupting the cells. The protein retained full enzymatic activity as measured by the release of D-alanine from bisacetyl-L-Lys-D-Ala-D-Ala and formation of [14C]penicillin-protein complex at a 1:1 stoichiometry. A one-step purification procedure using the immobilized dye Procion rubine MX-B resulted in homogeneous preparations of both wild-type and mutated forms of PBP5sc.
Assuntos
Proteínas de Bactérias , Proteínas de Transporte/genética , Citoplasma/enzimologia , Escherichia coli/enzimologia , Expressão Gênica , Hexosiltransferases , Muramilpentapeptídeo Carboxipeptidase/genética , Peptidil Transferases , Alanina/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Proteínas de Transporte/isolamento & purificação , Proteínas de Transporte/metabolismo , Membrana Celular/metabolismo , Cromatografia , Clonagem Molecular , Escherichia coli/genética , Dados de Sequência Molecular , Muramilpentapeptídeo Carboxipeptidase/isolamento & purificação , Muramilpentapeptídeo Carboxipeptidase/metabolismo , Mutagênese Sítio-Dirigida , Proteínas de Ligação às Penicilinas , Plasmídeos , Regiões Promotoras Genéticas/genética , Sinais Direcionadores de Proteínas/genética , Serina/genéticaRESUMO
Nephroblastomatosis means persisting embryonic, undifferentiated cells of renal blastoma which may give rise to a nephroblastoma. The foci of nephroblastomatosis create intrarenal nodi or a subcapsular border. The present scanning methods make it possible to correctly differentiate nephroblastomatosis in most cases and to differentiate it from most lesions previously hard to recognize from each other, while CT and arteriography represent the most sensitive methods. The main problem of differential diagnosis is still to differentiate nephroblastomatosis from nephroblastoma. For solving this problem it is frequently necessary to use more diagnostic methods including invasive arteriography and biopsy. It is the only way to prevent damage of the child by incorrect and most by too radical therapy. For the danger of the development of nephroblastoma even after regression of nephroblastomatosis in the X-ray picture a long-term observation is necessary, the ultrasound examination being the method of choice.
Assuntos
Neoplasias Renais/diagnóstico , Tumor de Wilms/diagnóstico , Angiografia , Pré-Escolar , Feminino , Humanos , Lactente , Neoplasias Renais/diagnóstico por imagem , Imageamento por Ressonância Magnética , Masculino , Tomografia Computadorizada por Raios X , Ultrassonografia , Tumor de Wilms/diagnóstico por imagemRESUMO
It was suggested previously that the primary structure of penicillin-binding protein 4 (PBP4) is new and unique among proteins that interact with penicillin. Our proposal that PBP4 carries an additional domain, located between the active-site fingerprints SXXK and SXN, was investigated by mutational deletion analysis. A clustered set of internal deletions was created in this region by exonuclease treatment of the dacB coding DNA, starting from two internal restriction sites. PBP4 mutants carrying internal deletions were selected by screening for immunoreactive forms of PBP4 with reduced molecular weight that were still active with respect to penicillin binding. DNA sequencing revealed 24 distinct PBP4 mutants with internal deletions ranging from 37 to 113 amino acids. The amino- and carboxy-terminal end points of the deletions were not randomly distributed but tended to cluster in certain areas. Overproduction of the individual mutated forms of PBP4 resulted in accumulation of the major portion of the proteins in the particulate cell fraction. The yield of soluble and active mutated forms of the protein was reduced from below 1% to 79% of the level obtained for the native protein. The deletions that were introduced had minor effects on the deacylation rate of bound benzylpenicillin. Two pairs of cysteine residues (Cys-139-Cys-153 and Cys-197-Cys-214) that are located in the deletable region may form disulfide bridges.
Assuntos
Proteínas de Bactérias , Proteínas de Transporte/genética , Proteínas de Escherichia coli , Escherichia coli/genética , Hexosiltransferases , Muramilpentapeptídeo Carboxipeptidase/genética , Peptidil Transferases , D-Ala-D-Ala Carboxipeptidase Tipo Serina , Sequência de Bases , Sítios de Ligação , Proteínas de Transporte/biossíntese , Mapeamento Cromossômico , Análise Mutacional de DNA , Engenharia Genética , Dados de Sequência Molecular , Muramilpentapeptídeo Carboxipeptidase/biossíntese , Mutagênese , Penicilina G/metabolismo , Proteínas de Ligação às Penicilinas , Conformação Proteica , Proteínas Recombinantes/biossíntese , Relação Estrutura-Atividade , Frações Subcelulares/químicaRESUMO
Streptococcus pneumoniae is the major cause of bacterial pneumonia, and it is also responsible for otitis media and meningitis in children. Apart from the capsule, the virulence factors of this pathogen are not completely understood. Recent technical advances in the field of bacterial pathogenesis (in vivo expression technology and signature-tagged mutagenesis [STM]) have allowed a large-scale identification of virulence genes. We have adapted to S. pneumoniae the STM technique, originally used for the discovery of Salmonella genes involved in pathogenicity. A library of pneumococcal chromosomal fragments (400 to 600 bp) was constructed in a suicide plasmid vector carrying unique DNA sequence tags and a chloramphenicol resistance marker. The recent clinical isolate G54 was transformed with this library. Chloramphenicol-resistant mutants were obtained by homologous recombination, resulting in genes inactivated by insertion of the suicide vector carrying a unique tag. In a mouse pneumonia model, 1.250 candidate clones were screened; 200 of these were not recovered from the lungs were therefore considered virulence-attenuated mutants. The regions flanking the chloramphenicol gene of the attenuated mutants were amplified by inverse PCR and sequenced. The sequence analysis showed that the 200 mutants had insertions in 126 different genes that could be grouped in six classes: (i) known pneumococcal virulence genes; (ii) genes involved in metabolic pathways; (iii) genes encoding proteases; (iv) genes coding for ATP binding cassette transporters; (v) genes encoding proteins involved in DNA recombination/repair; and (vi) DNA sequences that showed similarity to hypothetical genes with unknown function. To evaluate the virulence attenuation for each mutant, all 126 clones were individually analyzed in a mouse septicemia model. Not all mutants selected in the pneumonia model were confirmed in septicemia, thus indicating the existence of virulence factors specific for pneumonia.
Assuntos
Genes Bacterianos , Streptococcus pneumoniae/patogenicidade , Animais , Bacteriemia , Bases de Dados Factuais , Biblioteca Genômica , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Mutagênese Insercional , Análise de Sequência de DNA , Homologia de Sequência , Streptococcus pneumoniae/genética , Virulência/genéticaRESUMO
A 2.5-kb DNA fragment including the structural gene coding for the penicillin-binding protein 2x (PBP 2x) of Streptococcus pneumoniae has been cloned into the vector pJDC9 and expressed in Escherichia coli. Mapping of RNA polymerase binding sites by electron microscopy indicated that the pbpX promoter is well recognized by the E. coli enzyme. However, high-level expression occurred mainly under the control of the lac promoter upstream of the pJDC9 multiple cloning site. After induction with isopropyl beta-d-thiogalactopyranoside, PBP 2x was expressed as one of the major cellular proteins. PBP 2x produced in E. coli corresponded to the pneumococcal PBP 2x in terms of electrophoretic mobility, fractionation with the cytoplasmic membrane, and penicillin-binding capacity. Deletion of 30 hydrophobic N-terminal amino acid residues at positions 19-48 resulted in high-level expression of a cytoplasmic, soluble PBP 2x derivative (PBP 2x*) which still retained full beta-lactam-binding activity. A two-step procedure involving dye affinity chromatography was established for obtaining large amounts of highly purified enzymatically active PBP 2x*.