RESUMO
Neurofibromas of the urinary bladder occur as isolated phenomena or as part of generalized neurofibromatosis or von Recklinghausen's disease. This benign mesenchymal tumor can be the cause of subvesical obstruction, hematuria or chronic cystitis. The lesion is rare. Our case of isolated extended neurofibromatosis of the urinary bladder was diagnosed in a 19-year-old girl with an 8-year history of chronic cystitis. Two transurethral resections were successful in relieving symptoms. Possible further therapy was delayed because of the patient's desire for children.
Assuntos
Cistite/diagnóstico , Cistite/etiologia , Neurofibromatoses/complicações , Neurofibromatoses/diagnóstico , Neoplasias da Bexiga Urinária/complicações , Neoplasias da Bexiga Urinária/diagnóstico , Adulto , Cistite/cirurgia , Feminino , Humanos , Neurofibromatoses/cirurgia , Prevenção Secundária , Resultado do Tratamento , Neoplasias da Bexiga Urinária/cirurgiaRESUMO
We describe a case of testicular duplication presenting as torsion of the supernumerary distal testis in a 14-year-old boy. Clinical examination showed an unclear mass above the left testis; intraoperatively, the supernumerary testis was linked to the regular testis by a common epididymis and shared a common vas with it. The distal testis was resected due to severe hemorrhagic damage. This condition represents the result of transversal division of the wolffian duct during organogenesis. Review of the literature revealed that testicular torsion, testicular tumor, unclear scrotal masses, or continuing potentia generandi after bilateral vasectomy were the reasons that polyorchidism was detected.
Assuntos
Torção do Cordão Espermático/etiologia , Testículo/anormalidades , Adolescente , Diagnóstico Diferencial , Humanos , Masculino , Fluxo Sanguíneo Regional/fisiologia , Torção do Cordão Espermático/diagnóstico por imagem , Torção do Cordão Espermático/patologia , Torção do Cordão Espermático/cirurgia , Testículo/irrigação sanguínea , Testículo/patologia , Ultrassonografia Doppler Dupla , Ductos Mesonéfricos/anormalidades , Ductos Mesonéfricos/patologiaRESUMO
We have reinvestigated the long form of cyclin-dependent kinase (CDK)2 that is expressed in many rodent cells. We show that the mRNA encoding CDK2L arises by alternative splicing and that the encoded protein can bind to, and be activated by, cyclins A and E. The complex of CDK2L with cyclin A has about half the specific activity of the equivalent CDK2-cyclin A complex. Also, CDK2L--cyclin A is inhibited to the same extent and by the same concentrations of p21(CIP1) as CDK2--cyclin A. The nucleotide sequences of intron V in the human and murine CDK2 genes, where the sequences encoding the 48-residue insert in CDK2L are located, show very high conservation in the position of the alternatively spliced exon and its surroundings. Despite this, we were not able to detect significant expression of CDK2L in human cell lines, although a low level is expressed in COS-1 cells from monkeys.
Assuntos
Processamento Alternativo , Quinases relacionadas a CDC2 e CDC28 , Ciclina A/metabolismo , Ciclina E/metabolismo , Quinases Ciclina-Dependentes/genética , Proteínas Serina-Treonina Quinases/genética , Sequência de Aminoácidos , Animais , Células COS , Ciclo Celular , Linhagem Celular , Chlorocebus aethiops , Quinase 2 Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p21 , Quinases Ciclina-Dependentes/metabolismo , Ciclinas , Ativação Enzimática , Inibidores Enzimáticos , Humanos , Isoenzimas , Camundongos , Dados de Sequência Molecular , Proteínas Serina-Treonina Quinases/metabolismo , Coelhos , Distribuição TecidualRESUMO
HIV-1 expresses a multifunctional protein called TAT (trans-acting transcriptional activator), the function of which in vivo is tightly correlated with the incidence of Kaposi's sarcoma in AIDS patients. TAT is angiogenic and apparently binds to receptors specific for vascular endothelial growth factor (VEGF). Amino acids 46-60 of HIV-TAT, known as the basic peptide, have been shown to be responsible for its functional interaction with VEGF receptors. To characterize further the binding properties of this peptide, its coding sequence was fused to the reading frame of bacterial thioredoxin, allowing the production of large amounts of chimaeric polypeptides in bacteria in a biologically active form. Binding of chimaeric proteins to VEGF receptors was studied in vitro in endothelial cell cultures expressing either of the two receptors. Chimaeric thioredoxin proteins carrying the basic domain of TAT bound to both VEGF receptors with affinities similar to those of HIV-TAT or VEGF. Interestingly, these polypeptides competed only partially with VEGF for receptor binding, implying different binding sites for the TAT peptide and VEGF. This suggests that TAT binds VEGF receptors at new sites that might be useful targets for pharmacological intervention during pathological angiogenesis. The thioredoxin/basic-peptide chimaeras are functional agonists that mediate VEGF receptor signalling: (1) they stimulate the growth of endothelial cells; (2) together with basic fibroblast growth factor they cause tube formation of endothelial cells in collagen gels; (3) they induce blood vessel formation on the chicken chorioallantoic membrane; and (4) they activate VEGF receptor kinase and mitogen-activated protein kinase activity.
Assuntos
Fatores de Crescimento Endotelial/fisiologia , Produtos do Gene tat/fisiologia , HIV/genética , Linfocinas/fisiologia , Transdução de Sinais , Animais , Sequência de Bases , Linhagem Celular , Galinhas , Clonagem Molecular , Primers do DNA , Cães , Fatores de Crescimento Endotelial/genética , Humanos , Linfocinas/genética , Mimetismo Molecular , Neovascularização Fisiológica , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio Vascular , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
BACKGROUND/AIMS: The occurrence of myeloid leukaemia in patients with systemic mastocytosis is a well recognised phenomenon. However, the pathophysiological basis of such a coevolution has not been clarified. Recent data have shown that the c-kit mutation Asp 816 to Val is detectable in neoplastic mast cells in most patients with systemic mastocytosis, including those who have associated haematological disorders. The aim of this study was to study clonal disease evolution by analysing bone marrow cells from a patient with systemic mastocytosis and associated chronic myelomonocytic leukaemia (CMML) for the presence of this mutation. METHODS: The DNA of microdissected bone marrow cells from a patient with systemic mastocytosis and associated CMML was analysed for the presence of the c-kit mutation Asp 816 to Val by means of HinfI digestion and direct sequencing of semi-nested polymerase chain reaction (PCR) products. RESULTS: The two neoplasms could easily be identified and discriminated in paraffin wax embedded bone marrow sections by tryptase and chloroacetate esterase staining. A total number of 10 tryptase positive systemic mastocytosis infiltrates and 10 tryptase negative CMML infiltrates were removed by microdissection. As assessed by HinfI digestion and direct sequencing of semi-nested PCR products, the c-kit mutation Asp 816 to Val was detected in five of seven systemic mastocytosis infiltrates and four of six CMML infiltrates. By contrast, no c-kit mutation Asp 816 to Val was found in bone marrow infiltrates in patients with CMML without associated systemic mastocytosis (n = 20). CONCLUSION: These data support a monoclonal evolution of systemic mastocytosis and concurrent CMML in the patient studied.
Assuntos
Leucemia Mielomonocítica Crônica/genética , Mastocitose/genética , Proteínas de Neoplasias/genética , Mutação Puntual , Proteínas Proto-Oncogênicas c-kit/genética , Idoso , Células da Medula Óssea/patologia , DNA de Neoplasias/genética , Humanos , Leucemia Mielomonocítica Crônica/complicações , Leucemia Mielomonocítica Crônica/patologia , Masculino , Mastocitose/complicações , Mastocitose/patologiaRESUMO
Immunoprecipitation after cell surface labeling of human neuroblastoma cells showed that the anti-neuroblastoma monoclonal antibody (mAb) chCE7 binds to a 200,000 M(r) cell surface protein. The protein was partially purified by immuno-affinity chromatography from a human renal carcinoma and a human neuroblastoma cell line, which both showed high levels of binding of MAb chCE7. NH(2)-terminal sequences of 18 and 15 amino acid residues were determined. Both sequences isolated from the renal carcinoma and the neuroblastoma cells showed strong homology to human cell adhesion molecule L1 (L1-CAM), and both were characterized by the NH(2)-terminal deletion of 5 amino acids, comprising exon 2 of L1-CAM. Reverse trancription-polymerase chain reaction (RT-PCR) analysis of the regions spanning exon 2 and exon 27 of L1-CAM indicated that in neuroblastoma cells both transcripts for the full-length and exon-deleted forms are present, whereas in the renal carcinoma cell lines only the exon-deleted L1-CAM isoform were detected. Western blot analysis showed that 6 of 7 tested renal carcinoma cell lines and 5 of 15 renal carcinoma tissues expressed L1-CAM. In normal adult kidney tissue, very low levels of protein expression were found. Northern blot analysis confirmed that in renal carcinoma and neuroblastoma cell lines L1-CAM mRNA levels are correlated with protein expression.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antineoplásicos/imunologia , Neoplasias Renais/química , Glicoproteínas de Membrana/análise , Proteínas de Neoplasias/análise , Moléculas de Adesão de Célula Nervosa/análise , Neuroblastoma/química , Animais , Células CHO , Cricetinae , Humanos , Rim/química , Complexo Antígeno L1 Leucocitário , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Moléculas de Adesão de Célula Nervosa/genética , Moléculas de Adesão de Célula Nervosa/imunologia , Testes de Precipitina , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais CultivadasRESUMO
A colorimetric enzyme-linked immunosorbent assay (ELISA) for the measurement of kinase activity of c-Jun N-terminal kinases (JNKs) in cell extracts is described. The assay involves passive immobilisation of the substrate GST-cJun on the surface of a microtiter plate, selection of JNK protein kinases directly in substrate-coated wells, kinase reaction, and detection of substrate phosphorylation by a phosphoepitope-specific antibody. The ability of this assay to selectively measure JNK activity relies on the high-affinity interaction between JNKs and c-Jun. Accordingly, we found that JNKs could be captured on the microtiter plate surface through binding to the immobilised GST-cJun. Moreover, JNKs retained the specificity of their interaction with and phosphorylation of c-Jun with respect to the dependence on both intact docking domain and the dimerisation state of c-Jun. This novel procedure represents a marked improvement on conventional radioactive assays in terms of sensitivity, accuracy of evaluation, low time consumption, high throughput and amenability to automation. It is expected to be useful forthe acceleration and facilitation of JNK activity measurement in cell extracts, in particular for large-scale screening of clinical samples.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Extratos Celulares , Ensaio de Imunoadsorção Enzimática/métodos , Proteínas Quinases Ativadas por Mitógeno , Células 3T3 , Trifosfato de Adenosina/metabolismo , Animais , Glutationa Transferase/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno , Cinética , Camundongos , Fosforilação , Especificidade por SubstratoRESUMO
The molecular biology of the angiogenic growth factor, vascular endothelial growth factor (VEGF), has been studied in the dog. All major isoforms of VEGF are present in the dog. The amino acid sequences are identical between human and dog in the loop regions that are responsible for receptor binding. Accordingly, the VEGF receptors of dogs and humans are very similar and permit functional exchange of the growth factor. Here we show that canine VEGF activates human endothelial cells to the same extent as human VEGF. Similarly, the two proteins display identical cell binding properties. The VEGF receptor 1 (Flt-1) shows the same alternative splicing in humans and dogs and is overexpressed in the majority of tumors in both species. VEGF occurs also in canine tumors in similar relative quantities as in human malignancies. Based on the literature and our study we suggest that the molecular biology and the function of the VEGF signaling system are virtually identical in humans and canines and in healthy as well as in disease conditions.
Assuntos
Fatores de Crescimento Endotelial/metabolismo , Linfocinas/metabolismo , Neoplasias Experimentais/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Processamento Alternativo , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA Complementar , Cães , Fatores de Crescimento Endotelial/fisiologia , Humanos , Linfocinas/fisiologia , Dados de Sequência Molecular , Neoplasias Experimentais/irrigação sanguínea , Neovascularização Patológica , RNA Mensageiro/genética , Homologia de Sequência de Aminoácidos , Fator A de Crescimento do Endotélio Vascular , Receptor 1 de Fatores de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
The bacteriophage Lambda head protein D (gpD) is a small major capsid protein (110aa; 11.6kDa; pI=5.68, devoid of cysteine residues) that is essential for stable head morphogenesis. We found that a His-tagged derivative of gpD (gpHD) is a monomeric protein with efficient expression properties and high resistance towards thermally induced irreversible aggregation. In addition, gpHD can be used as a fusion partner for high-level expression of soluble heterologous proteins in the cytoplasm of Escherichia coli. Its broad utility is illustrated by the production of various mammalian proteins by fusion to its C-terminus. As a fusion partner, gpHD is thought to mediate optimal translation initiation while reducing inclusion body formation and protein degradation. In addition, it provides a His-tag for simple purification. gpHD may act as a 'cytoplasmic anchor' by keeping its unfolded fusion partner in solution, thereby providing more time for proper folding. An ever-increasing number of open reading frames (ORFs) are being identified in the various genome sequencing programs. gpHD has the potential to be harnessed for the development of highly efficient cytoplasmic expression systems that might contribute to the production and characterization of these novel polypeptides. Protein D is also an established fusion partner for phage display. It thus presents the attractive opportunity of coupling the selection of heterologous proteins from a phage library to their subsequent high-level expression.
Assuntos
Bacteriófago lambda/genética , Escherichia coli/genética , Proteínas Virais/genética , Sítios de Ligação , Proteínas do Capsídeo , Colagenases/genética , Colagenases/metabolismo , Regulação Bacteriana da Expressão Gênica , Regulação Viral da Expressão Gênica , Hidrólise , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Solubilidade , Temperatura , Proteínas Virais/química , Proteínas Virais/metabolismoAssuntos
Encefalopatias/etiologia , Equinococose Hepática/complicações , Equinococose Pulmonar/etiologia , Doenças Linfáticas/etiologia , Adulto , Encefalopatias/diagnóstico , Broncoscopia , Equinococose Hepática/diagnóstico , Equinococose Pulmonar/diagnóstico , Humanos , Doenças Linfáticas/diagnóstico , Imageamento por Ressonância Magnética , Masculino , Tomografia Computadorizada por Raios XRESUMO
Exponentially growing V79 Chinese hamster lung fibroblasts irradiated with 7 Gy X-rays undergo cell cycle arrest in the S and G2 phases. These arrests are released, probably on completion of DNA repair. A premature release occurs after treatment of irradiated cells with caffeine. This release is accompanied by increased activity of the p34cdc2 serine/threonine protein kinase complex [Hain et al. (1993) Cancer Res. 53, 1507-1510]. We have investigated in V79 cells whether the association of p34cdc2 with its regulatory subunits cyclin A and B is affected by irradiation and subsequent caffeine treatment and found that this was not the case. The phosphorylation of p34cdc2 as assayed by mobility shift on SDS polyacrylamide gels was increased as early as 0.5 h after irradiation and decreased after subsequent caffeine treatment. A novel protein p40, detected with anti-PSTAIRE antibodies, appeared several fold more abundant than p34cdc2. Its phosphorylation state also changed after irradiation and after subsequent caffeine treatment.
Assuntos
Proteína Quinase CDC2/metabolismo , Cafeína/farmacologia , Ciclinas/metabolismo , Pulmão/efeitos dos fármacos , Pulmão/efeitos da radiação , Animais , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/efeitos da radiação , Células Cultivadas , Cricetinae , Cricetulus , Eletroforese em Gel de Poliacrilamida , Fibroblastos/efeitos dos fármacos , Fibroblastos/efeitos da radiação , Citometria de Fluxo , Pulmão/citologia , Fosforilação , Radiação Ionizante , Transdução de SinaisRESUMO
We have been investigating radiation-induced neoplastic transformants of C3H 10T1/2 mouse fibroblasts for evidence of heritable changes. C3H 10T1/2 cells were treated with 8 Gy X rays. After approximately 8 weeks of culture, type II/III foci were isolated from the monolayer using cloning rings. Cell lines developed from these foci, and clones established from these cell lines, were examined for DNA content. The isolated focus-derived populations and derived clones often display aneuploidy and/or polyploidization. In one instance a clone (derived from a single cell) displayed multiple polyploidies. During passage the ploidy of many of the anomalous populations gradually reverted to the ploidy of the non-neoplastically transformed state. The morphological features associated with the neoplastic transformation event were nevertheless retained. The results demonstrate that exposure to radiation can induce, in association with morphological neoplastic transformation, a heritable, genomically labile state.
Assuntos
Transformação Celular Neoplásica/efeitos da radiação , Genoma , Animais , Transformação Celular Neoplásica/genética , Células Cultivadas , Camundongos , Camundongos Endogâmicos C3H , Fenótipo , PloidiasRESUMO
We show here that the arrests of cells in G2 phase of the cell cycle induced by either staurosporine or ionizing radiation are closely related phenomena governed by a common kinase signaling pathway. The protein kinase inhibitor staurosporine induces a complete G2-phase arrest in exponentially growing TK6 human lymphoblastoid and V79 Chinese hamster fibroblast cells. Both cell types are equally sensitive to the kinase inhibitor and the arrest is dependent on its continued presence. Caffeine completely abrogates this arrest at concentrations comparable to those which abrogate radiation-induced G2-phase arrest. The kinetics of caffeine-induced release of both kinds of arrest are essentially identical. The activity of p34cdc2 kinase was also found to increase in a parallel fashion after caffeine-induced release of both kinds of arrest. As opposed to those transformed cell types which arrest only in G2 phase in response to staurosporine, immortalized C3H 10T1/2 fibroblasts and Muntjak skin fibroblasts display both G1- and G2-phase arrests. The results suggest that staurosporine and radiation interact with regulatory pathways in the cell cycle, and specifically with a caffeine-sensitive signal transduction pathway which recognizes DNA damage, regulates the G2/M-phase transition, and attenuates the biological consequences of radiation exposure.
Assuntos
Alcaloides/farmacologia , Cafeína/farmacologia , Fase G2/efeitos dos fármacos , Fase G2/efeitos da radiação , Animais , Proteína Quinase CDC2/análise , Células CHO , Células Cultivadas , Cricetinae , Cervos , Relação Dose-Resposta a Droga , Fibroblastos , Fase G1/efeitos dos fármacos , Fase G1/efeitos da radiação , Humanos , Estaurosporina , Fatores de TempoRESUMO
Both enzyme activity and mRNA concentration of beta-glucuronidase were measured in kidneys of mice treated with testosterone and the synthetic estrogen, diethylstilbestrol. Six congenic strains, all having a C57BL6/J genetic background but each having a different haplotype of the beta-glucuronidase gene complex, were compared. In each strain the induction caused by androgen was partially repressed by estrogen. The extent of this antagonism varied among the six haplotypes and was not coordinate with the extent of induction by androgen alone. Antagonism appears to be regulated by at least two alleles of a new locus, Gus-e, within the beta-glucuronidase gene complex. Repression by estrogen, like induction by androgen, appears to take place primarily at the transcriptional level. Kinetic studies revealed that estrogen causes the androgen response curve to plateau earlier and at a lower level. This suggests that estrogen increases the rate of gene deactivation rather than decreasing the rate of gene activation. Isoelectric focusing of beta-glucuronidase from Gus-ea and Gus-eb mice and their F1 progeny revealed that the genes are regulated in cis. Together, these findings support a model in which both sex hormones exert their effects on separate DNA response elements located in close proximity to the gene or within the gene itself.
Assuntos
Androgênios/farmacologia , Estrogênios/farmacologia , Glucuronidase/genética , Rim/enzimologia , Animais , Feminino , Regulação Enzimológica da Expressão Gênica , Glucuronidase/biossíntese , Haplótipos , Cinética , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/metabolismo , Receptores Androgênicos/metabolismo , Ativação TranscricionalRESUMO
The serine/threonine protein kinase p34cdc2 activity in V79 hamster cells 4 h after treatment with 7-Gy X-rays is similar to that of unirradiated cells. Nevertheless, the irradiated cells are arrested in the S and G2 phases of the cell cycle. The mRNA concentrations of histones H1 and H4 are reduced by a factor of about 2 in irradiated cells compared to unirradiated cells, as opposed to the mRNAs of high-mobility group I(Y) and 17 proteins which appear unchanged. Both the p34cdc2 activity and the mRNA concentrations of the histones rise within 30 min after the release of the radiation induced cell cycle block by caffeine. During this time span the p34cdc2 activity increases about 4-fold and the histone mRNA levels recover approximately to those of an exponentially growing cell population. Regulatory pathways influenced in irradiated and in subsequently caffeine treated cells apparently interact with basic cell cycle control mechanisms.
Assuntos
Proteína Quinase CDC2/biossíntese , Cafeína/farmacologia , Fase G2/efeitos dos fármacos , Protamina Quinase/metabolismo , RNA Mensageiro/metabolismo , Fase S/efeitos dos fármacos , Animais , Divisão Celular/efeitos da radiação , Células Cultivadas , Cricetinae , Fibroblastos/citologia , Fibroblastos/metabolismo , Fase G2/efeitos da radiação , Proteínas de Grupo de Alta Mobilidade/metabolismo , Fase S/efeitos da radiação , Fatores de TempoRESUMO
If the pyridoxal-phosphate-binding lysine residue 258 of aspartate aminotransferase is exchanged for a histidine residue, the enzyme retains partial catalytic competence [Ziak, M., Jaussi, R., Gehring, H. and Christen, P. (1990) Eur. J. Biochem. 187, 329-333]. The three-dimensional structures of the mutant enzymes of both chicken mitochondria and Escherichia coli were determined at high resolution. The folding patterns of the polypeptide chains proved to be identical to those of the wild-type enzymes, small conformational differences being restricted to parts of the active site. If aspartate or glutamate was added to the pyridoxal form of the mutant enzyme [lambda max 392 nm and 330 nm (weak); negative CD at 420 nm, positive CD at 370 nm and 330 nm], the external aldimine (lambda max = 430 nm; negative CD at 360 nm and 430 nm) transiently accumulated. Upon addition of 2-oxoglutarate to the pyridoxamine form (lambda max 330 nm, positive CD), a putative ketamine intermediate could be detected; however, with oxalacetate, an equilibrium between external aldimine and the pyridoxal form, which was strongly in favour of the former, was established within seconds. The transamination cycle with glutamate and oxalacetate proceeds only three orders of magnitude more slowly than the overall reaction of the wild-type enzyme. The specific activity of the mutant enzyme is 0.1 U/mg at 25 degrees C and constant from pH 6.0 to 8.5. Reconstitution of the mutant apoenzyme with [4'-3H]pyridoxamine 5'-phosphate resulted in rapid release of 3H with a first-order rate constant kappa' = 5 x 10(-4) s-1 similar to that of the wild-type enzyme. Apparently, in aspartate aminotransferase, histidine can to some extent substitute for the active-site lysine residue. The imidazole ring of H258, however, seems too distant from C alpha and C4' to act efficiently as proton donor/acceptor in the aldimine-ketamine tautomerization, suggesting that the prototropic shift might be mediated by an intervening water molecule. Transmination of the internal to the external aldimine apparently can be replaced by de novo formation of the latter, and by its hydrolysis in the reverse direction.
Assuntos
Aspartato Aminotransferases/metabolismo , Lisina/metabolismo , Mutagênese Sítio-Dirigida , Fosfato de Piridoxal/metabolismo , Animais , Aspartato Aminotransferases/química , Aspartato Aminotransferases/genética , Sítios de Ligação , Catálise , Galinhas , Dicroísmo Circular , Cristalização , Escherichia coli/enzimologia , Histidina/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Mitocôndrias/enzimologia , Estrutura Molecular , Conformação Proteica , Dobramento de Proteína , Espectrofotometria , Difração de Raios XRESUMO
Various investigators reported a reduced yield of chromosome and chromatid aberrations in short-term cultures of human lymphocytes if a 'challenge' exposure to ionizing radiation was preceded by an 'adaptive' exposure. In order to examine the cell cycle dependence of the 'adaptive response', chromosome and chromatid aberration yields were estimated after challenge doses in the G1, S or G2 phase of lymphocytes which had been adapted in the early G1 phase. On testing two donors no protective adaptive response was found. Blood samples of four donors were tested for their capability to evoke the adaptive response in a standard experiment with the adaptive dose in the S phase and the challenge dose in the G2 phase. A synergistic response occurred in one out of two similar experiments performed with the same blood sample. The three other blood samples tested did not respond. Apparently these data indicate a high frequency of human lymphocyte cultures that do not display an adaptive response.
Assuntos
Adaptação Fisiológica , Divisão Celular/efeitos da radiação , Linfócitos/efeitos da radiação , Tolerância a Radiação , Adulto , Aberrações Cromossômicas , Feminino , Humanos , Interfase/efeitos da radiação , Linfócitos/fisiologia , Masculino , Metáfase/efeitos da radiaçãoRESUMO
Studies on hormonal action frequently focus on a single hormone. In intact animals, however, genes may respond to the balance of multiple hormones. Therefore, we have studied the mutual influence of sex steroids on eight genes previously known to be testosterone-responsive in kidneys of mice. A variety of responses to estrogen were recorded. Effects occurred primarily at the transcriptional level although in several cases there was also evidence of decreased mRNA stability. Estrogen did not affect the nuclear location of the androgen receptor. Apparently each gene interacts with both androgen-receptor complex and estrogen-receptor complex, and the ultimate outcome depends on each gene's detailed regulatory structure.
Assuntos
Estrogênios/fisiologia , RNA Mensageiro , Testosterona/fisiologia , Animais , Dietilestilbestrol/farmacologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Rim/química , Rim/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptores Androgênicos/efeitos dos fármacos , Receptores Androgênicos/metabolismoRESUMO
Mitochondrial and cytosolic aspartate aminotransferase (AspAT) are homologous proteins with identically folded polypeptide chains. The cDNAs of the two isoenzymes of chicken were used to express the following proteins in yeast: the precursor of mitochondrial AspAT, mature mitochondrial AspAT, and two chimeric proteins in one of which (pc) the presequence of the precursor was attached to the entire cytosolic isoenzyme and in the other one (pmc) the N-terminal segment (amino acid residues -22 to 23) of the precursor was linked to the slightly truncated cytosolic isoenzyme (residues 34 to 412). All presequence containing proteins were imported into the mitochondria and processed to the mature form whereas mature mitochondrial AspAT remained in the cytosol. The rate of import of the authentic precursor was four times faster than that of the chimeric proteins pc and pmc, t1/2 for importation at 29 degrees C being 3, 13 and 14 min, respectively. Apparently, the mature moiety of the precursor of mitochondrial AspAT promotes importation.
Assuntos
Aspartato Aminotransferases/genética , Precursores Enzimáticos/genética , Mitocôndrias/enzimologia , Processamento de Proteína Pós-Traducional , Saccharomyces cerevisiae/genética , Sequência de Bases , Quimera , Citosol/enzimologia , Cinética , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Saccharomyces cerevisiae/enzimologiaRESUMO
Both the precursor and the mature form of chicken mitochondrial aspartate aminotransferase were synthesized in Escherichia coli. The precursor was found to sediment quantitatively together with insoluble cell material. In contrast, mature mitochondrial aspartate aminotransferase could be readily extracted from the cells and was indistinguishable from the enzyme isolated from chicken heart in all respects tested: specific activity 230 units mg-1; Mr 2 X 45,000; pI greater than 9; NH2-terminal sequence SSWWSHVEMG, the initiator methionine having been removed by the bacteria. Thus, the polypeptide chain representing mature mitochondrial aspartate aminotransferase is an autonomous folding unit which attains its functional spatial structure independently of the presence of the prepiece, trans-membrane passage, and proteolytic processing.