RESUMO
The extracellular matrix (ECM) is an important regulator of the differentiated phenotype of mammary epithelial cells in culture. Despite the fact that ECM-degrading enzymes have been implicated in morphogenesis and tissue remodeling, there is little evidence for a direct role for such regulation in vivo. We generated transgenic mice that express autoactivated isoforms of the matrix metalloproteinase stromelysin-1, under the control of the whey acidic protein gene promoter, to examine the effect of inappropriate expression of this enzyme. Stromelysin-1 is implicated as the primary player in the loss of basement membrane and loss of function in the mammary gland during involution. The transgene was expressed at low levels in mammary glands of virgin female mice, leading to an unexpected phenotype: The primary ducts had supernumerary branches and showed precocious development of alveoli that expressed beta-casein at levels similar to that of an early- to mid-pregnant gland. Lactating glands showed high levels of transgene expression, with accumulation at the basement membrane, and a decrease in laminin and collagen IV, resulting in a loss of basement membrane integrity; this was accompanied by a dramatic alteration of alveolar morphology, with decreased size and shrunken lumina containing little beta-casein. During pregnancy, expression of endogenous whey acidic protein and beta-casein was reduced in transgenic glands, confirming the observed dependence of milk protein transcription of ECM in mammary epithelial cells in culture. These data provide direct evidence that stromelysin-1 activity can be morphogenic for mammary epithelial cells, inducing hyperproliferation and differentiation in virgin animals, and that its lytic activity can, indeed, disrupt membrane integrity and reduce mammary-specific function. We conclude that the balance of ECM-degrading enzymes with their inhibitors, and the associated regulation of ECM structure, is crucial for tissue-specific gene expression and morphogenesis in vivo.
Assuntos
Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/enzimologia , Metaloendopeptidases/metabolismo , Animais , Sequência de Bases , Membrana Basal/ultraestrutura , Caseínas/genética , Diferenciação Celular , Colágeno/genética , Primers do DNA/química , Feminino , Regulação da Expressão Gênica , Genes , Lactação , Laminina/genética , Metaloproteinase 3 da Matriz , Camundongos , Camundongos Transgênicos , Proteínas do Leite/genética , Dados de Sequência Molecular , Morfogênese , Gravidez , Regiões Promotoras Genéticas , Mapeamento por Restrição , Proteínas do Soro do LeiteRESUMO
To develop an animal model for sickle cell anemia, we have created transgenic mice that express a severe naturally occurring human sickling hemoglobin, Hb S Antilles. Due to its low solubility and oxygen affinity, Hb S Antilles has a greater propensity to cause red cell sickling than Hb S. To make transgenic animals that express a high level of Hb S Antilles, the erythroid-specific DNAse I hypersensitive site II from the human beta-globin cluster was linked independently to the human alpha 2-globin gene and to the beta S Antilles gene. Embryos were injected with both constructs simultaneously and seven transgenic mice were obtained, three of which contained both the human alpha and the human beta S Antilles transgene. After crossing the human transgenes into the mouse beta-thalassemic background a transgenic mouse line was derived in which approximately half the beta-globin chains in the murine red cells were human beta S Antilles. Deoxygenation of the transgenic red cells in vitro resulted in extensive sickling. An increase of in vivo sickling was achieved by placing these transgenic mice in a low oxygen environment. This murine model for red cell sickling should help to advance our understanding of sickle cell disease and may provide a model to test therapeutic interventions.
Assuntos
Eritrócitos/ultraestrutura , Hemoglobina Falciforme/metabolismo , Hipóxia/fisiopatologia , Animais , Sequência de Bases , Southern Blotting , Cromatografia Líquida de Alta Pressão , DNA/análise , Modelos Animais de Doenças , Globinas/genética , Globinas/isolamento & purificação , Espectrometria de Massas , Camundongos , Camundongos Transgênicos , Dados de Sequência MolecularRESUMO
Circulating alpha 1-antitrypsin is synthesized primarily in the liver and secreted into the bloodstream, where it serves as the major protease inhibitor. The PiZ variant of alpha 1-antitrypsin is associated with decreased levels of the protein in sera as a result of its retention within hepatocytes. Homozygosity for the variant allele predisposes individuals to the development of pulmonary emphysema and an increased risk for liver disease. We and others have previously demonstrated that the normal PiM human alpha 1-antitrypsin gene can be properly expressed in the livers of transgenic mice. The PiZ variant of the human alpha 1-antitrypsin gene was introduced into the germline of mice to determine whether the mutant protein would accumulate in mouse hepatocytes and if such accumulation would result in the development of liver damage in an animal model. As expected, the mutant human protein was abundantly synthesized in the livers of the transgenic animals and accumulated within the rough endoplasmic reticulum of hepatocytes as it does in human patients. PiZ mice developed significantly more liver necrosis and inflammation than PiM transgenic mice or control littermates. The degree of liver damage was correlated with the amount of PiZ alpha 1-antitrypsin accumulated in the liver of the different pedigrees of mice. Although 40% of PiZ mice tested were seropositive for mouse hepatitis virus (MHV), the degree of liver damage was not influenced by the MHV seropositivity; rather, it was related only to the presence of accumulated PiZ protein.
Assuntos
Fígado/patologia , alfa 1-Antitripsina/metabolismo , Animais , Humanos , Fígado/efeitos dos fármacos , Fígado/ultraestrutura , Cirrose Hepática Experimental/etiologia , Cirrose Hepática Experimental/metabolismo , Cirrose Hepática Experimental/patologia , Camundongos , Camundongos Transgênicos , Necrose , Fenótipo , Especificidade da Espécie , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/fisiologiaRESUMO
Vaccination with irradiated granulocyte-macrophage colony-stimulating factor (GM-CSF)-secreting gene-transduced cancer vaccines induces tumoricidal immune responses. In a Phase I human gene therapy trial, eight immunocompetent prostate cancer (PCA) patients were treated with autologous, GM-CSF-secreting, irradiated tumor vaccines prepared from ex vivo retroviral transduction of surgically harvested cells. Expansion of primary cultures of autologous vaccine cells was successful to meet trial specifications in 8 of 11 cases (73%); the yields of the primary culture cell limited the number of courses of vaccination. Side effects were pruritus, erythema, and swelling at vaccination sites. Vaccine site biopsies manifested infiltrates of dendritic cells and macrophages among prostate tumor vaccine cells. Vaccination activated new T-cell and B-cell immune responses against PCA antigens. T-cell responses, evaluated by assessing delayed-type hypersensitivity (DTH) reactions against untransduced autologous tumor cells, were evident in two of eight patients before vaccination and in seven of eight patients after treatment. Reactive DTH site biopsies manifested infiltrates of effector cells consisting of CD45RO+ T-cells, and degranulating eosinophils consistent with activation of both Th1 and Th2 T-cell responses. A distinctive eosinophilic vasculitis was evident near autologous tumor cells at vaccine sites, and at DTH sites. B-cell responses were also induced. Sera from three of eight vaccinated men contained new antibodies recognizing polypeptides of 26, 31, and 150 kDa in protein extracts from prostate cells. The 150-kDa polypeptide was expressed by LNCaP and PC-3 PCA cells, as well as by normal prostate epithelial cells, but not by prostate stromal cells. No antibodies against prostate-specific antigen were detected. These data suggest that both T-cell and B-cell immune responses to human PCA can be generated by treatment with irradiated, GM-CSF gene-transduced PCA vaccines.
Assuntos
Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Neoplasias da Próstata/terapia , Vacinas Sintéticas/imunologia , Linfócitos B/imunologia , Técnicas de Transferência de Genes , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Humanos , Hipersensibilidade Tardia/etiologia , Masculino , Pessoa de Meia-Idade , Neoplasias da Próstata/imunologia , Neoplasias da Próstata/patologia , Linfócitos T/imunologia , VacinaçãoRESUMO
Granulocyte-macrophage colony-stimulating factor (GM-CSF) gene-transduced, irradiated tumor vaccines induce potent, T-cell-mediated antitumor immune responses in preclinical models. We report the initial results of a Phase I trial evaluating this strategy for safety and the induction of immune responses in patients with metastatic renal cell carcinoma (RCC). Patients were treated in a randomized, double-blind dose-escalation study with equivalent doses of autologous, irradiated RCC vaccine cells with or without ex vivo human GM-CSF gene transfer. The replication-defective retroviral vector MFG was used for GM-CSF gene transfer. No dose-limiting toxicities were encountered in 16 fully evaluable patients. GM-CSF gene-transduced vaccines were equivalent in toxicity to nontransduced vaccines up to the feasible limits of autologous tumor vaccine yield. No evidence of autoimmune disease was observed. Biopsies of intradermal sites of injection with GM-CSF gene-transduced vaccines contained distinctive macrophage, dendritic cell, eosinophil, neutrophil, and T-cell infiltrates similar to those observed in preclinical models of efficacy. Histological analysis of delayed-type hypersensitivity responses in patients vaccinated with GM-CSF-transduced vaccines demonstrated an intense eosinophil infiltrate that was not observed in patients who received nontransduced vaccines. An objective partial response was observed in a patient treated with GM-CSF gene-transduced vaccine who displayed the largest delayed-type hypersensitivity conversion. No replication-competent retrovirus was detected in vaccinated patients. This Phase I study demonstrated the feasibility, safety, and bioactivity of an autologous GM-CSF gene-transduced tumor vaccine for RCC patients.
Assuntos
Vacinas Anticâncer/uso terapêutico , Carcinoma de Células Renais/terapia , Técnicas de Transferência de Genes , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Neoplasias Renais/terapia , Adulto , Idoso , Vacinas Anticâncer/efeitos adversos , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/efeitos da radiação , Carcinoma de Células Renais/imunologia , Vírus Defeituosos/genética , Método Duplo-Cego , Toxidermias/etiologia , Toxidermias/imunologia , Toxidermias/patologia , Feminino , Vetores Genéticos/genética , Humanos , Hipersensibilidade Tardia/patologia , Neoplasias Renais/imunologia , Masculino , Pessoa de Meia-Idade , Vacinação/efeitos adversosRESUMO
We report the close correlation between changes in serum immunoreactive vascular endothelial growth factor 165 (iVEGF165) levels and metastatic tumor burden measured by computed tomography scan before treatment, during the antitumor response, and during early progression in a patient treated with ex vivo gene therapy for renal cell carcinoma. With the researcher blinded to outcome, iVEGF levels were measured in archived serum samples from a patient with metastatic renal cell carcinoma who demonstrated a 7-month partial remission to treatment with autologous, irradiated human GM-CSF gene transduced tumor vaccine. Although a spontaneous regression could not be formally excluded in this patient, the appearance of 20 new pulmonary metastases on computed tomography scan after nephrectomy and before vaccination indicates that if spontaneous regression occurred, it took place at the start of vaccine treatment.
Assuntos
Biomarcadores Tumorais/sangue , Carcinoma de Células Renais/sangue , Carcinoma de Células Renais/terapia , Fatores de Crescimento Endotelial/sangue , Terapia Genética , Neoplasias Renais/patologia , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/terapia , Linfocinas/sangue , Carcinoma de Células Renais/secundário , Humanos , Neoplasias Pulmonares/secundário , Masculino , Pessoa de Meia-Idade , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
When irradiated and administered intradermally as vaccines, cancer cells engineered to secrete high levels of granulocyte-macrophage colony-stimulating factor (GM-CSF) by gene transfer elicit potent anticancer immune responses in a variety of animal tumor models. Upon vaccination, antigens present in the cancer cells are phagocytosed and processed by skin dendritic cells. These dendritic cells then prime anticancer immune responses by presenting antigenic peptides to T cells. The immune responses generated are capable of eradicating small but lethal cancer cell inocula with minimal toxicity in preclinical animal tumor studies. To develop this vaccination strategy for the treatment of human genitourinary cancers, we have conducted phase I clinical trials using human genitourinary cancer cells as sources of cancer cell antigens. In the first human clinical trial of genetically engineered cancer cell vaccines, a phase I clinical trial of kidney cancer cell vaccines (n = 18), kidney cancer cells were removed at surgery, propagated briefly in vitro, and then genetically modified to secrete high levels of GM-CSF via ex vivo transduction with the retrovirus MFG-GM-CSF. After irradiation, the kidney cancer cells were administered as vaccines to 18 patients with advanced kidney cancers. Vaccine treatment, which caused few side effects, nonetheless appeared to trigger anticancer immune responses manifest as conversion of delayed-type hypersensitivity (DTH) skin responses against irradiated autologous cancer cells after vaccination. Biopsies of vaccine sites yielded findings reminiscent of biopsies from preclinical animal model studies, with evidence of vaccine cell recruitment of dendritic cells, T cells, and eosinophils. One patient with measurable kidney cancer metastases treated at the highest vaccine dose level experienced a partial treatment response. The bioactivity of GM-CSF-secreting autologous cancer cell vaccines was confirmed in a phase I clinical trial for prostate cancer (n = 8). Vaccine cells were prepared from surgically harvested prostate tumors by ex vivo transduction with MFG-GM-CSF in a manner similar to that used for the kidney cancer trial. Vaccine treatment was well tolerated and associated with induction of anticancer immunity as assessed using DTH skin testing. In addition, new antiprostate cancer cell antibodies were detected in serum samples from treated men as a consequence of vaccination. These first clinical trials of GM-CSF-secreting cancer cell vaccines for the treatment of genitourinary cancers have demonstrated both safety and bioactivity, in that very few side effects have been seen and anticancer immune responses have been detected. Future clinical studies will be required to assess vaccine treatment efficacy, refine vaccination dose and schedule, define the appropriate clinical context for the use of such vaccines, and ascertain optimal combinations involving vaccines and other local or systemic anticancer treatments.
Assuntos
Vacinas Anticâncer/imunologia , Carcinoma de Células Renais/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Neoplasias Renais/imunologia , Neoplasias da Próstata/imunologia , Adulto , Idoso , Antígenos de Neoplasias/sangue , Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/genética , Vacinas Anticâncer/uso terapêutico , Carcinoma de Células Renais/terapia , Relação Dose-Resposta Imunológica , Feminino , Técnicas de Transferência de Genes , Engenharia Genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Humanos , Hipersensibilidade Tardia/imunologia , Neoplasias Renais/terapia , Masculino , Pessoa de Meia-Idade , Neoplasias da Próstata/terapia , Retroviridae/genéticaRESUMO
The isolation and characterization of a mutant mouse T-cell lymphoma (S49) with altered purine metabolism is described. This mutant, AU-100, was isolated from a mutagenized population of S49 cells by virtue of its resistance to 0.1 mM 6-azauridine in semisolid agarose. The AU-100 cells are resistant to adenosine mediated cytotoxicity but are extraordinarily sensitive to killing by guanosine. High performance liquid chromatography of AU-100 cell extracts has demonstrated that intracellular levels of GTP, IMP, and GMP are all elevated about 3-fold over those levels found in wild type cells. The AU-100 cells also contain an elevated intracellular level of pyrophosphoribosylphosphate (PPriboseP), which accounts for its resistance to adenosine. However AU-100 cells synthesize purines de novo at a rate less than 35% of that found in wild type cells. Furthermore, the intact cells of this mutant S49 cell line cannot efficiently incorporate labeled hypoxanthine into nucleotides since the salvage enzyme HGPRTase is inhibited in situ. The AU-100 cell line was found to be 80% deficient in adenylosuccinate synthetase, but these cells are not auxotrophic for adenosine or other purines. The significant alterations in the control of purine de novo and salvage metabolism caused by the defect in adenylosuccinate synthetase are mediated by the resulting increased levels of guanosine nucleotides.
Assuntos
Adenilossuccinato Sintase/deficiência , Ligases/deficiência , Linfoma/metabolismo , Purinas/metabolismo , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Camundongos , Mutação , Neoplasias Experimentais/metabolismo , Fosforribosil Pirofosfato/metabolismo , Purinas/farmacologia , Ribonucleotídeos/metabolismo , Linfócitos T/metabolismoRESUMO
Two exploratory studies are reported on the perceived benefits associated with active participation in choral singing. In the first study, 84 members of a university college choral society completed a brief questionnaire that asked whether they had benefited personally from their involvement in the choir and whether there were ways in which participation could benefit their health. A large majority of respondents agreed they had benefited socially (87%) and emotionally (75%), with 58% agreeing they had benefited in some physical way, and 49% spiritually. A content analysis of written comments served to elaborate the ways in which choir members felt they had benefited. Common themes expressed were: meeting new people, feeling more positive, increased control over breathing, feeling more alert and feeling spiritually uplifted. With respect to health benefits, 84% of participants gave answers, the main themes of which related to improved lung function and breathing, improved mood and stress reduction. In the second study, 91 members of the choir completed a structured questionnaire consisting of 32 statements about singing reflecting the ideas expressed in the first study. Over 40% of respondents strongly agreed that 'singing helps to make my mood more positive', 'singing is a moving experience for me sometimes', 'singing makes me feel a lot happier' and 'singing is good for my soul'. A principal components analysis followed by Oblimin rotation identified six dimensions of benefit associated with singing. These were labelled as: benefits for well-being and relaxation, benefits for breathing and posture, social benefits, spiritual benefits, emotional benefits, and benefits for heart and immune system. Cronbach alpha coefficients were satisfactory for all components except the third, social benefits, due primarily to the small number of items loading on this component. Women were significantly more likely to experience benefits for well-being and relaxation, younger people were more likely to report social benefits, and those professing religious beliefs were more likely to experience spiritual benefits. The present studies have a number of limitations, but they provide a useful foundation for future larger scale surveys, more sophisticated qualitative studies, and experimental investigations of the impact of singing on psycho-physiological functioning.
Assuntos
Música , Satisfação Pessoal , Qualidade de Vida , Adulto , Idoso , Feminino , Humanos , Estilo de Vida , Masculino , Pessoa de Meia-Idade , Fatores Sexuais , Inquéritos e Questionários , UniversidadesRESUMO
Findings are presented from a questionnaire survey of 562 gay men resident in southern England on their sexual behaviour in the context of holidays. Data were gathered during summer 1996 by means of a self-completion questionnaire; 395 men reported at least one holiday undertaken in 1996 and the analysis focuses on the first or only holiday described. Of 391 men giving details of sexual activity, 187 reported sex with a new partner, 113 men reported penetrative sex and 17 reported penetration without consistent use of condoms. Sexual activity on holiday was predicted by: being on holiday alone or with friends, taking condoms, being motivated by 'gay social life and sex' in planning a holiday, higher expectations of sexual activity/risk and a higher number of new partners at home. Penetrative sex was predicted by: more sexual partners on holiday, taking condoms and higher expectations of sexual activity/risk. Unprotected sex was predicted by: not taking condoms, higher expectations of sexual activity/risk and positive HIV status. Survey findings are discussed in relation to previous research, and implications for travel-related HIV prevention initiatives targeting gay men are explored.
Assuntos
Homossexualidade Masculina , Assunção de Riscos , Síndrome da Imunodeficiência Adquirida/psicologia , Adulto , Inglaterra , Infecções por HIV/psicologia , Férias e Feriados , Humanos , Masculino , Análise de Regressão , Fatores de Risco , Inquéritos e QuestionáriosRESUMO
In Western societies high density lipoprotein (HDL) levels correlate inversely with the risk for coronary heart disease. The primary protein component of both human and mouse HDL is apolipoprotein A-I (apoAI), which comprises greater than 70% of HDL protein and 30% of HDL mass. Human HDLs include particles of several distinct size subpopulations, whereas HDLs from inbred C57BL/6 mice contain a single population of particles. To study the regulation of apoAI expression and its role in HDL assembly, we created transgenic C57BL/6 mice containing the human apoAI gene. Two independent lines of transgenic mice with approximately twice the normal plasma levels of total apoAI were studied. The level of mouse apoAI is reduced greater than 4-fold in both transgenic lines, comprising only 4% of total plasma apoAI levels in one transgenic line and 13% in the other. We demonstrate that the mechanism responsible for the decrease in mouse apoAI is posttranscriptional. Parallel to the replacement of mouse with human apoAI, the single HDL species normally present in the plasma of C57BL/6 is replaced by two HDL subclasses similar in size to human HDL2b and HDL3a. The changes in murine apolipoprotein levels and HDL subclass size are inherited by all transgenic offspring of the two founder animals. These results suggest a dominant role of apoAI in determining the HDL particle size distribution and a mechanism involving expression of human apoAI transgenes that alters the plasma levels of mouse apoAI.
Assuntos
Apolipoproteínas A/genética , Lipoproteínas HDL/genética , Animais , Apolipoproteína A-I , Apolipoproteínas A/sangue , Northern Blotting , DNA/genética , Humanos , Lipoproteínas HDL/sangue , Lipoproteínas HDL/classificação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Peso Molecular , RNA/genética , Mapeamento por RestriçãoRESUMO
The inherited deficiency of purine-nucleoside phosphorylase (PNPase; purine-nucleoside:orthophosphate ribosyltransferase, EC 2.4.2.1) in humans is associated with a severe deficiency of the T lymphocytes of the immune system. Because of the unsatisfactory nature of previously described model systems, we have selected, cloned, and characterized a mutant mouse T cell lymphoma (S49) completely deficient in PNPase. Of the four substrates of PNPase, only deoxyguanosine at low concentrations is toxic to the PNPase-deficient (NSU-1) cells. In order to delineate the biochemical processes necessary for the sensitivity of the NSU-1 cells to deoxyguanosine, we have isolated a series of secondary mutants resistant to deoxyguanosine from the PNPase-deficient line. One of these mutants is defective in its ability to transport deoxyguanosine into the cell. A second type of mutant cannot phosphorylate the deoxyguanosine and is totally deficient in deoxycytidine kinase activity. A third type of mutant (NSU-1-dGuo-L) can both transport and phosphorylate deoxyguanosine and accumulates dGTP. However, unlike its parent, NSU-1-dGuo-L does not become depleted of dCTP and TTP when exposed to exogenous deoxyguanosine. This observation is accounted for by the fact that the reduction of CDP to dCDP by the ribonucleotide reductase (ribonucleoside-diphosphate reductase, 2'-deoxyribonucleoside-diphosphate:oxidized-thioredoxin 2'-oxidoreductase, EC 1.17.4.1) of NSU-1-dGuo-L cells is not normally sensitive to feedback inhibition by dGTP.Thus, in order to exert its toxicity deoxyguanosine must be transported into the cell, be phosphorylated by deoxycytidine kinase, and be accumulated as dGTP. By inhibiting ribonucleotide reductase, dGTP depletes the cell of dCTP and to some extent TTP, thus preventing the synthesis of DNA, a process necessary for any proliferation-dependent function of T cells.
Assuntos
Síndromes de Imunodeficiência/enzimologia , Linfoma/enzimologia , Pentosiltransferases/deficiência , Purina-Núcleosídeo Fosforilase/deficiência , Ribonucleotídeo Redutases/metabolismo , Animais , Linhagem Celular , Desoxirribonucleotídeos/metabolismo , Cinética , Camundongos , Mutação , Neoplasias Experimentais/enzimologia , FenótipoRESUMO
Ribonucleotide reductase is responsible for the reduction of the 2'-hydroxy moiety of all four ribonucleoside diphosphates to the corresponding deoxyribonucleotides. The overall activity of the enzyme is regulated by the allosteric effectors ATP (activator) and dATP (inhibitor), and the enzyme's substrate specificity is also controlled by nucleotide effectors. For instance, wild type ribonucleotide reductase from mouse T-lymphoma (S49) cells requires dGTP as a positive effector for ADP reduction. This effect of dGTP causes a reciprocal inhibition of CDP reduction. The dGuo-L mutant cell line, resistant to growth inhibition by exogenous deoxyguanosine, contains a nucleotide-binding subunit, protein M1, that conveys to its CDP reductase an insensitivity to dGTP (and dTTP) inhibition. The dGuo-L protein M1 also shows a decreased capacity to use ADP as a substrate, and therefore, the regulation of the substrate specificity is altered in the mutant protein M1. Another mutant cell line, dGuo-200-1, is resistant to deoxyadenosine and its ribonucleotide reductase is abnormally resistant to inhibition by dATP. The isolated mutant protein M1 from dGuo-200-1 cells has a CDP reductase activity which is stimulated by dATP, unlike the wild type enzyme which is inhibited by dATP. It appears that this mutant enzyme has lost the capacity to distinguish between dATP and ATP, but is still sensitive to regulation by dGTP and dTTP. Thus, the site of protein M1 regulating overall activity is altered in the dGuo-200-1 mutant, while the site regulating substrate specificity is normal. These characteristics of the mutants provide genetic evidence for two independent allosteric domains of protein M1, each responsible for a different aspect of nucleotide sensitivity of ribonucleotide reductase.
Assuntos
Linfoma/enzimologia , Ribonucleotídeo Redutases/genética , Trifosfato de Adenosina/farmacologia , Regulação Alostérica , Animais , Linhagem Celular , Nucleotídeos de Desoxiadenina/farmacologia , Cinética , Substâncias Macromoleculares , Camundongos , Neoplasias Experimentais/enzimologia , Ribonucleotídeo Redutases/metabolismoRESUMO
Eight overlapping phage clones, spanning 34.4 kilobase pairs of genomic DNA, containing the 7.2-kilobase pair rat beta-casein gene have been isolated and characterized. The first 510 base pairs (bp) of 5' flanking, 110 bp of 3' flanking, and all the exon/intron junctions have been sequenced. The beta-casein gene contains 9 exons ranging in size from 21 to 525 bp. We have attempted to identify potential regulatory elements by searching for regions of sequence homology shared between milk protein genes which respond similarly to lactogenic hormones and by searching for previously reported hormone receptor-binding sites. Within the conserved first 200 bp of 5' flanking sequences 3 regions of greater than 70% homology were observed between the rat beta- and gamma-casein genes. One of these contains a region 90% homologous to the chicken progesterone receptor-binding site. The conserved 5' noncoding region, the highly conserved signal peptide, and the hydrophobic carboxyl-terminal region of the protein are each encoded by a separate exon. In contrast the evolutionarily conserved phosphorylation site of beta-casein is formed by an RNA-splicing event. The exons which encode the phosphorylation sites of beta-casein appear to have resulted from an intragenic duplication. Based upon the exon structure of the casein genes, an evolutionary model of intragenic and intergenic exon duplications for this gene family is proposed.
Assuntos
Caseínas/genética , DNA/análise , Animais , Sequência de Bases , Sítios de Ligação , Fosforilação , Splicing de RNA , RatosRESUMO
Normal and mutant human alpha-1-antitrypsin genes were cloned from a PiMZ heterozygous individual. Nucleotide sequence comparison demonstrated a T to C transition in exon III and an G to A transition in exon V of the PiZ gene. A 14.4 kb DNA fragment containing the entire PiM gene plus 2 kb of 5' and 3' flanking genomic DNA sequences was introduced into the germ line of mice and five F0 transgenic lines were established. Transgenic F1 progeny from F0 parents exhibited high levels of human alpha-1-antitrypsin protein in their plasma. The human gene was expressed primarily in liver of the transgenic mice as it is in man. However, expression of the human alpha-1-antitrypsin gene was also observed in kidneys of the transgenic mice, which led to the observation that the endogenous mouse gene is also expressed in the kidney. These data indicate that cis-acting elements within or proximal to the human alpha-1-antitrypsin gene are able to direct its in vivo transcription with a high degree of tissue specificity.
Assuntos
Clonagem Molecular , Genes , alfa 1-Antitripsina/genética , Animais , Sequência de Bases , Enzimas de Restrição do DNA , Heterozigoto , Humanos , Rim/metabolismo , Fígado/metabolismo , Camundongos , PlasmídeosRESUMO
Apolipoproteins A-I and A-II comprise approximately 70 and 20%, respectively, of the total protein content of HDL. Evidence suggests that apoA-I plays a central role in determining the structure and plasma concentration of HDL, while the role of apoA-II is uncertain. To help define the function of apoA-II and determine what effect increasing its plasma concentration has on HDL, transgenic mice expressing human apoA-II and both human apoA-I and human apoA-II were produced. Human apoA-II mRNA is expressed exclusively in the livers of transgenic animals, and the protein exists as a dimer as it does in humans. High level expression of human apoA-II did not increase HDL concentrations or decrease plasma concentrations of murine apoA-I and apoA-II in contrast to what was observed in mice overexpressing human apoA-I. The primary effect of overexpressing human apoA-II was the appearance of small HDL particles composed exclusively of human apoA-II. HDL from mice transgenic for both human apoA-I and human apoA-II displayed a unique size distribution when compared with either apoA-I or apoA-II transgenic mice and contain particles with both these human apolipoproteins. These results in mice, indicating that human apoA-II participates in determining HDL size, parallel results from human studies.
Assuntos
Apolipoproteína A-II/biossíntese , Lipoproteínas HDL/sangue , Animais , Apolipoproteína A-I/metabolismo , Apolipoproteína A-II/fisiologia , Northern Blotting , Eletroforese em Gel de Poliacrilamida , Expressão Gênica , Humanos , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , PlasmídeosRESUMO
The 5'-flanking sequence of the human alpha 1-antitrypsin (AAT) gene contains multiple cis-regulatory elements, including a distal enhancer and proximal sequences essential for its transcription in cultured hepatoma cells. To understand better the promoter specificity of the AAT gene in vivo, transgenic mice harboring the AAT-SV40 hybrid promoter or the natural AAT promoter fused to a reporter gene (CAT) were generated. Examination of CAT activity in various tissues indicated that the CAT gene was expressed primarily in the liver and also, to a lesser extent, in tissues known to express the AAT gene. In addition, the cis-acting elements of the human AAT gene were utilized to drive the transcription of the SV40 T antigen gene in transgenic mice. Hepatocellular malignancy was found in all founder animals examined, while sporadic occurrence of malignancy was also observed in stomach, pancreas, and kidney. These results verify that the 5'-flanking region of the human AAT gene contains cis-regulatory elements sufficient to confer tissue specificity in vivo.
Assuntos
Regulação da Expressão Gênica , Fígado/metabolismo , Camundongos Transgênicos/metabolismo , Regiões Promotoras Genéticas , alfa 1-Antitripsina/genética , Animais , Neoplasias Hepáticas/genética , Camundongos , Neoplasias Experimentais/genéticaRESUMO
Epidemiological surveys have identified a strong inverse relationship between the amount in the plasma of high density lipoproteins (HDL), apolipoprotein AI (ApoA-I), the major protein component of HDL, and the risk for atherosclerosis in humans. It is not known if this relationship arises from a direct antiatherogenic effect of these plasma components or if it is the result of other factors also associated with increases in ApoA-I and HDL levels. Because some strains of mice are susceptible to diet-induced formation of preatherosclerotic fatty streak lesions, and because of available techniques for the genetic manipulation of this organism, the murine system offers a unique setting in which to investigate the process of early atherogenesis. To test the hypothesis that induction of a high plasma concentration of ApoA-I and HDL would inhibit this process, we studied the effects of atherogenic diets on transgenic mice expressing high amounts of human ApoA-I. We report that transgenic mice with high plasma ApoA-I and HDL levels were significantly protected from the development of fatty streak lesions.
Assuntos
Aorta/patologia , Apolipoproteínas A/genética , Arteriosclerose/genética , Manteiga , Colesterol na Dieta , Dieta Aterogênica , Lipoproteínas HDL/genética , Animais , Apolipoproteína A-I , Apolipoproteínas A/sangue , Arteriosclerose/patologia , Arteriosclerose/fisiopatologia , Predisposição Genética para Doença , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Músculo Liso Vascular/patologiaRESUMO
The isolation and characterization of a mutant murine T-cell lymphoma (S49) with altered purine metabolism is described. This mutant, AU-100, was isolated from a mutagenized population of S49 cells by virtue of its resistance to 0.1 mM 6-azauridine in semisolid agarose. The AU-100 cells are resistant to adenosine mediated cytotoxicity but are extraordinarily sensitive to killing by guanosine. High performance liquid chromatography of AU-100 cell extracts has demonstrated that intracellular levels of GTP, IMP, and GMP are all elevated about 3-fold over those levels found in wild type cells. The AU-100 cells also contain an elevated intracellular level of pyrophosphoribosylphosphate (PPriboseP), which as in wild type cells is diminished by incubation of AU-100 cells with adenosine. However AU-100 cells synthesize purines de novo at a rate less than 35% of that found in wild type cells. In other growth rate experiments, the AU-100 cell line was shown to be resistant to 6-thioguanine and 6-mercaptopurine. Levels of hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) measured in AU-100 cell extracts, however, are 50-66% greater than those levels of HGPRTase found in wild type cell extracts. Nevertheless this mutant S49 cell line cannot efficiently incorporate labeled hypoxanthine into nucleotides since the salvage enzyme HGPRTase is inhibited in vivo. The AU-100 cell line was found to be 80% deficient in adenylosuccinate synthetase, but these cells are not auxotrophic for adenosine or other purines. The significant alterations in the control of purine de novo and salvage metabolism caused by the defect in adenylosuccinate synthetase are mediated by the resulting increased levels of guanosine nucleotides.