RESUMO
A variety of growth factors including transforming growth factor-alpha (TGF-alpha) are synthesized as transmembrane precursors. The short cytoplasmic domain of the transmembrane TGF-alpha precursor lacks any apparent motif associated with signal transduction. However, the sequence conservation of this cytoplasmic domain and its abundance of cysteine residues, reminiscent of the cytoplasmic domains of CD4 and CD8, suggest a biological function. In this study, we showed that transmembrane TGF-alpha was rapidly internalized after interaction with a specific antibody and that this internalization was greatly decreased when the COOH-terminal 31 amino acids were removed. Chemical cross-linking experiments revealed two associated proteins of 86 and 106 kD which coimmunoprecipitated with the TGF-alpha precursor. The association of p86 was dependent on the presence of the COOH-terminal cytoplasmic 31 amino acids of the TGF-alpha precursor, whereas p106 still remained associated when this segment was deleted. In addition, p106 was tyrosine-phosphorylated and exposed on the cell surface. The protein complex associated with transmembrane TGF-alpha displayed kinase activities towards tyrosine, serine, and threonine residues. These activities were not associated with transmembrane TGF-alpha when the COOH-terminal segment was truncated. The association of a protein kinase complex with transmembrane TGF-alpha may provide the basic elements for a "reverse" mode of signaling through the cytoplasmic domain of this growth factor, which may lead to two-directional communication during ligand-receptor interaction.
Assuntos
Substâncias de Crescimento/metabolismo , Proteínas Quinases/metabolismo , Precursores de Proteínas/metabolismo , Fator de Crescimento Transformador alfa/metabolismo , Animais , Células CHO , Sequência Conservada , Cricetinae , Citoplasma/metabolismo , Imunofluorescência , Proteínas de Membrana/metabolismo , Inibidores de Proteínas Quinases , Transdução de SinaisRESUMO
The gene defective in Huntington's disease encodes a protein, huntingtin, with unknown function. Antisera generated against three separate regions of huntingtin identified a single high molecular weight protein of approximately 320 kDa on immunoblots of human neuroblastoma extracts. The same protein species was detected in human and rat cortex synaptosomes and in sucrose density gradients of vesicle-enriched fractions, where huntingtin immunoreactivity overlapped with the distribution of vesicle membrane proteins (SV2, transferrin receptor, and synaptophysin). Immunohistochemistry in human and rat brain revealed widespread cytoplasmic labeling of huntingtin within neurons, particularly cell bodies and dendrites, rather than the more selective pattern of axon terminal labeling characteristic of many vesicle-associated proteins. At the ultrastructural level, immunoreactivity in cortical neurons was detected in the matrix of the cytoplasm and around the membranes of the vesicles. The ubiquitous cytoplasmic distribution of huntingtin in neurons and its association with vesicles suggest that huntingtin may have a role in vesicle trafficking.
Assuntos
Química Encefálica , Encéfalo/ultraestrutura , Citoplasma/química , Doença de Huntington/metabolismo , Proteínas do Tecido Nervoso/análise , Neurônios/ultraestrutura , Proteínas Nucleares/análise , Animais , Especificidade de Anticorpos , Fracionamento Celular , Centrifugação com Gradiente de Concentração , Dendritos/química , Humanos , Proteína Huntingtina , Soros Imunes/imunologia , Imunoquímica , Mutação , Proteínas do Tecido Nervoso/genética , Neuroblastoma , Proteínas Nucleares/genética , Ratos , Células Tumorais CultivadasRESUMO
Neuropoletic cytokines such as ciliary neurotrophic factor (CNTF) can activate multiple signaling pathways in parallel, including those involving Janus kinase (JAK)-signal transducers and activators of transcription (STATs), mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI 3-kinase) and mammalian target of rapamydn (mTOR)-p70 S6 kinase . Crosstalk occurs between these pathways, because studies have shown that STAT3 requires phosphorylation on tyrosine and serine residues by independent protein kinase activities for maximal activation of target gene transcription. Members of the JAK/Tyk family of tyrosine kinases mediate phosphorylation of STAT3 at Tyr705 during CNTF signaling; however, the kinase responsible for phosphorylation at STAT3 Tyr727 appears to depend on both the extracellular stimulus and the cellular context. Here we investigate the kinase activity responsible for phosphorylation of STAT3 on Ser727 in CNTF-stimulated neuroblastoma cells. We found that CNTF-induced phosphorylation of Ser727 was inhibited by the mTOR inhibitor rapamycin, but not by inhibitors of MAPK and protein kinase C (PKC) activation. A STAT3 peptide was efficiently phosphorylated on Ser727 in a CNTF-dependent manner by mTOR, but not by a kinase-inactive mTOR mutant or by p70 S6 kinase. In agreement with these biochemical studies, rapamycin treatment of cells transfected with a STAT-responsive promoter reporter decreased activation of the reporter to the same degree as a STAT3 Ser727Ala mutant The ability of mTOR to contribute to activation of STAT3 extends the function of mTOR in mammalian cells to include transcriptional regulation.
Assuntos
Fator Neurotrófico Ciliar/farmacologia , Proteínas de Ligação a DNA/metabolismo , Fosfosserina/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/fisiologia , Proteínas Quinases , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Serina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sirolimo/farmacologia , Transativadores/metabolismo , Linhagem Celular , Humanos , Sistema de Sinalização das MAP Quinases , Fragmentos de Peptídeos/farmacologia , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Fosfotransferases (Aceptor do Grupo Álcool)/efeitos dos fármacos , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Quinases S6 Ribossômicas/metabolismo , Fator de Transcrição STAT3 , Serina-Treonina Quinases TORRESUMO
Ciliary neurotrophic factor, along with other neuropoietic cytokines, signals through the shared receptor subunit gp130 [1-3], leading to the tyrosine phosphorylation of a number of substrates [4,5], including the transcription factors STAT1 and STAT3 and the protein tyrosine phosphatase SHP-2 [6,7] [8]. SHP-2 (also known as PTP1D, SHPTP2, Syp and PTP2C) is a positive regulatory molecule required for the activation of the mitogen-activated protein kinase pathway and the stimulation of gene expression in response to epidermal growth factor, insulin and platelet-derived growth factor stimulation [9-11]. We have previously shown that cytokines that signal via the gp130 receptor subunit activate transcription of the vasoactive intestinal peptide (VIP) gene through a 180 bp cytokine response element (CyRE) [12,13]. To characterize the role of SHP-2 in the regulation of gp130-stimulated gene expression, we examined the regulation of the VIP CyRE in two systems that prevented ligand-dependent SHP-2 phosphorylation. Inhibition of SHP-2, either by mutating the tyrosine residue in gp130 that mediates the SHP-2 interaction, or by expression of dominant-negative SHP-2, resulted in dramatic increases in gp130-dependent gene expression, through the VIP CyRE and more specifically through multimerized STAT-binding sites. These data suggest that SHP-2 has a negative role in gp130 signaling by modulating STAT-mediated transcriptional activation.
Assuntos
Regulação para Baixo , Fatores de Crescimento Neural/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Animais , Antígenos CD/metabolismo , Linhagem Celular , Fator Neurotrófico Ciliar , Receptor gp130 de Citocina , Proteínas de Ligação a DNA/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Glicoproteínas de Membrana/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Sequências Reguladoras de Ácido Nucleico , Fator de Transcrição STAT1 , Fator de Transcrição STAT3 , Transdução de Sinais , Transativadores/metabolismo , Ativação Transcricional , Peptídeo Intestinal Vasoativo/genéticaRESUMO
In experiments to identify molecules that might be important in the pathogenesis of glioblastoma multiforme, the most common malignant brain tumor, we found that annexin II (Lipocortin 2, p36), a likely second messenger in several different mitogenic pathways, was highly expressed in tumor tissue of glioblastoma multiforme (9 of 9) and highly anaplastic astrocytoma (2 of 6), but not in astrocytomas of lower pathological grade (0 of 6). We also detected high levels of annexin II expression in fetal brain during the period when radial glia proliferate, although annexin II expression was not detected in normal adult brain. These data demonstrate that annexin II expression is developmentally regulated in the human central nervous system and suggest that the early progenitor radial glia share important characteristics with highly malignant glial tumors.
Assuntos
Anexina A2/análise , Astrocitoma/metabolismo , Neoplasias Encefálicas/metabolismo , Encéfalo/metabolismo , Glioblastoma/metabolismo , Sequência de Aminoácidos , Anexina A2/genética , Sequência de Bases , Receptores ErbB/análise , Receptores ErbB/genética , Feto/metabolismo , Proteína Glial Fibrilar Ácida/análise , Proteína Glial Fibrilar Ácida/genética , Humanos , Dados de Sequência Molecular , RNA Mensageiro/análiseRESUMO
Interleukin 1beta-converting enzyme (ICE) is a member of a growing family of cysteine proteases shown to be a crucial component in the activation of a genetic program that leads to autonomous cell death in mammalian cells. In this study, a murine ICE-lacZ fusion gene was introduced into a novel retroviral vector designed to achieve regulated ectopic expression of a foreign gene in mammalian cells. By delivering the ICE-lacZ gene within a retroviral vector and under the control of a tetracycline-regulated promoter, we were able to utilize the intrinsic cell death program of ICE as a means for tumoricidal therapy in a rat brain tumor model. Both in culture and in vivo suppression of ICE-lacZ expression was extremely tight in the presence of tetracycline, as determined by the lack of X-galactosidase-positive tumor cells and by cell viability. When tetracycline was withdrawn, ICE-lacZ gene expression was rapidly turned on and apoptosis-mediated cell death occurred in essentially all tumor cells.
Assuntos
Apoptose/fisiologia , Neoplasias Encefálicas/terapia , Cisteína Endopeptidases/fisiologia , Terapia Genética , Vetores Genéticos/genética , Gliossarcoma/terapia , Vírus da Leucemia Murina de Moloney/genética , Animais , Apoptose/efeitos dos fármacos , Neoplasias Encefálicas/patologia , Caspase 1 , Corpo Estriado , Cisteína Endopeptidases/biossíntese , Cisteína Endopeptidases/genética , Indução Enzimática/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Gliossarcoma/patologia , Masculino , Transplante de Neoplasias , Regiões Promotoras Genéticas/efeitos dos fármacos , Ratos , Tetraciclina/farmacologia , Tetraciclina/uso terapêutico , TransfecçãoRESUMO
The control of cell cycle progression is orchestrated by an extraordinary diverse and dynamic in function group of proteins. Critical in the progression are the actions of the E2F family of transcription factors which regulate the expression of genes necessary for the G1/S transition and the WAF/CIP/KIP family of cdk inhibitors which can inhibit cell cycle progression. In this report, we have identified E2F binding sites in both the human and mouse p21 promoters that bind E2F protein complexes from nuclear extracts in a cell cycle-dependent manner. In ectopic expression experiments we determined that E2F1, but not E2F4, can strongly transactivate the human p21 gene through these E2F binding sites which are located in the -215/+1 region of the p21 gene. The transactivation of the p21 gene through regulatory elements within the -215/+1 region of the promoter was correlated with increased levels of endogenous E2F1 and p21 proteins at the G1/S boundary. The significance of transactivation of the p21 gene by E2F is that p21 function is important in cell cycle progression as well as for cell cycle arrest. Indeed, E2F-induced levels of p21 protein during the G1/ S transition is consistent with the recent findings demonstrating that p21 acts as an assembly factor for kinase active cyclin/cdk/p21 complexes.
Assuntos
Proteínas de Transporte , Proteínas de Ciclo Celular , Ciclo Celular/genética , Ciclinas/genética , Proteínas de Ligação a DNA , Inibidores Enzimáticos/metabolismo , Fatores de Transcrição/fisiologia , Animais , Sítios de Ligação/genética , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/metabolismo , Fatores de Transcrição E2F , Fator de Transcrição E2F1 , Fator de Transcrição E2F4 , Fase G1/genética , Regulação Neoplásica da Expressão Gênica , Glioma , Humanos , Camundongos , Regiões Promotoras Genéticas/genética , Proteína 1 de Ligação ao Retinoblastoma , Fase S/genética , Fator de Transcrição DP1 , Fatores de Transcrição/metabolismo , Células Tumorais CultivadasRESUMO
Cyclin D1 plays a key regulatory role during the G1 phase of the cell cycle and its gene is amplified and overexpressed in many cancers. To address the relationship between cyclin D1 and other cell cycle regulatory proteins, we established human glioma and rodent fibroblast cell lines in which cyclin D1 expression could be regulated ectopically with tetracycline. In both of these cell lines, we found that ectopic expression of cyclin D1 in asynchronously growing cells was accompanied by increased levels of the p53 tumor suppressor protein and the cyclin/cdk inhibitor p21. Despite the induction of these cell cycle inhibitory proteins, cyclin D1-associated cdk kinase remained activated and the cells grew essentially like that of the parent cells. Although growth parameters were unchanged in these cells, morphological changes were clearly identifiable and anchorage independent growth was observed in NIH3T3 cells. In a first step toward elaborating the mechanism for cyclin D1-mediated induction of p21 gene expression we show that co-expression of E2F-1 and DP-1 can specifically transactivate the p21 promoter. In support of these findings and a direct effect of E2F on induction of p21 gene expression a putative E2F binding site was identified within the p21 promoter. In summary, our results demonstrate that ectopic expression of cyclin D1 can induce gene expression of the cdk inhibitor p21 through an E2F mechanism the consequences of which are not to growth arrest cells but possibly to stabilize cyclin D1/cdk function.
Assuntos
Proteínas de Transporte , Ciclo Celular , Quinases Ciclina-Dependentes/antagonistas & inibidores , Ciclinas/genética , Ciclinas/metabolismo , Proteínas de Ligação a DNA , Regulação da Expressão Gênica , Proteínas Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas , Células 3T3 , Animais , Sequência de Bases , Western Blotting , Proteínas de Ciclo Celular/metabolismo , Divisão Celular/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Ciclina D1 , Quinase 4 Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p21 , Quinases Ciclina-Dependentes/metabolismo , Fatores de Transcrição E2F , Fator de Transcrição E2F1 , Inibidores Enzimáticos/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Glioma/metabolismo , Glioma/patologia , Humanos , Imuno-Histoquímica , Camundongos , Modelos Biológicos , Proteína 1 de Ligação ao Retinoblastoma , Tetraciclina/farmacologia , Fatores de Tempo , Fator de Transcrição DP1 , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologia , Ativação Transcricional , Proteína Supressora de Tumor p53/metabolismoRESUMO
DNA damaging agents such as ultraviolet (UV) induce cell cycle arrest followed by apoptosis in cells where irreparable damage has occurred. Here we show that during early phase G1 arrest which occurs in UV-irradiated human U343 glioblastoma cells, there are (1) decreases in cyclin D1 and cdk4 levels which parallel a loss of S-phase promoting cyclin D1/cdk4 complexes, and (2) increases in p53 and p21 protein levels. We also show that the late phase UV-induced apoptosis of U343 cells occurs after cell cycle re-entry and parallels the reappearance of cyclin D1 and cdk4 and cyclin D1/cdk4 complexes. These findings suggest that cyclin D1 can abrogate UV-induced G1 arrest and that the p53-mediated apoptosis that occurs in these cells is dependent on cyclin D1 levels. We examined these possibilities using U343 cells that ectopically express cyclin D1 and found that indeed cyclin D1 can overcome the cell cycle arrest caused by UV. Moreover, the appearance of p53 protein and the induction of apoptosis in UV-irradiated cells was found to be dependent on the level of ectopically expressed cyclin D1. These findings, therefore, indicate that expression of cyclin D1 following DNA damage is essential for cell cycle re-entry and p53-mediated apoptosis.
Assuntos
Apoptose/efeitos da radiação , Ciclina D1/fisiologia , Proteínas Proto-Oncogênicas , Proteína Supressora de Tumor p53/metabolismo , Ciclo Celular , Ciclina D1/biossíntese , Quinase 4 Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p21 , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Humanos , Células Tumorais Cultivadas , Raios UltravioletaRESUMO
Specific immune tolerance to fully allogeneic kidney grafts can be achieved in a miniature swine transplantation model by retrovirus-mediated transfer of allogeneic MHC class II genes into bone marrow cells (BMCs) of recipient animals. Graft survival correlated with transient expression of the somatic transgene (Tg) in the induction phase of tolerance. With the aim of investigating the effects of timing and threshold levels of Tg expression on induction of hyporesponsiveness to the grafted tissues, two recombinant retrovirus constructs containing the tetracycline binary regulatory system were used to achieve conditional expression of either the green fluorescent protein (tetGFP) as a control, or the porcine MHC class II DRbeta chain (tetDRB). Effective downregulation of GFP gene transcription was demonstrated in transduced murine fibroblasts after doxycycline treatment, leading to a > 90% reduction of GFP fluorescence. Similar diminution of the DRB gene transcription was achieved in transduced pig endothelial cells (ECs). Drug-dependent downregulation of DRBc gene expression in SLAd pig ECs coincided with complete inhibition of allogeneic activation of anti-class IIc-primed SLAd T cells. These in vitro results suggest that the binary tetracycline retrovirus system may also be adequate to regulate MHC class II Tg expression in vivo.
Assuntos
Transplante de Medula Óssea/métodos , Regulação da Expressão Gênica , Técnicas de Transferência de Genes , Genes MHC da Classe II/genética , Regiões Promotoras Genéticas , Retroviridae/genética , Transplante Homólogo/métodos , Animais , Antibacterianos/farmacologia , Northern Blotting , Southern Blotting , Linhagem Celular , Células Cultivadas , Regulação para Baixo , Doxiciclina/farmacologia , Endotélio/metabolismo , Fibroblastos/efeitos dos fármacos , Citometria de Fluxo , Proteínas de Fluorescência Verde , Proteínas Luminescentes/genética , Linfócitos/metabolismo , Camundongos , Modelos Genéticos , Suínos , Tetraciclina/farmacologia , Fatores de Tempo , Transcrição Gênica , Transdução Genética , TransfecçãoRESUMO
In this study we examined the distribution and developmental profile of the src homology 2 (SH2) domain-containing protein tyrosine phosphatase SHP-2 in the mouse brain. We found that SHP-2 is present in both mitotically active and postmitotic cells in the forebrains of embryonic day 12 (E12) mice. In a developmental study extending from embryonic day 12 to adulthood, Western blotting analysis demonstrated equivalent levels of SHP-2 protein at all of the ages examined. Expression of SHP-2 paralleled the level of enzymatic activity at the different developmental periods. In the adult brain SHP-2 was restricted to diverse classes of neurons, while the majority of glial cells did not express detectable levels of protein. However, reactive astrocytes in response to an ischemic brain injury showed SHP-2 immunolabelling. Our data suggest that SHP-2 may play a role in pathways of neuronal and glial progenitor cells, in a broad spectrum of neuronal responses in the adult brain and in the gliotic response to the injury.
Assuntos
Astrócitos/enzimologia , Neuroglia/enzimologia , Neurônios/enzimologia , Proteínas Tirosina Fosfatases/biossíntese , Animais , Antimetabólitos/farmacologia , Western Blotting , Encéfalo/citologia , Encéfalo/crescimento & desenvolvimento , Isquemia Encefálica/enzimologia , Bromodesoxiuridina/farmacologia , Feminino , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Camundongos , Mitose , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Proteínas Tirosina Fosfatases Contendo o Domínio SH2 , Células-Tronco/enzimologiaRESUMO
The protein tyrosine phosphatase SH-PTP2 has been implicated in a variety of cell signaling cascades, including those mediating neuronal survival. We therefore investigated the expression of SH-PTP2 in the adult human nervous system using Western blotting, immunohistochemistry and immunoelectron microscopy. SH-PTP2 immunoreactivity was noted only in neurons, but was not restricted to a specific neuronal type or location. Immunohistochemistry showed perikaryal staining, whereas Western blotting and ultrastructural analysis suggested that SH-PTP2 is present in axons as well. While immunohistochemistry showed a Nissl-like pattern in large motor neurons, immunoelectron microscopy demonstrated a diffuse pattern of cytoplasmic staining, without apparent preferential localization. The presence of the SH2 domain-containing tyrosine-specific phosphatase SH-PTP2 in diverse neurons in the adult nervous system suggests that SH-PTP2 may play a role in a broad spectrum of neuronal responses.
Assuntos
Encéfalo/enzimologia , Proteínas Tirosina Fosfatases/análise , Medula Espinal/enzimologia , Adulto , Western Blotting , Encéfalo/citologia , Humanos , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intracelular , Microscopia Eletrônica , Neurônios/enzimologia , Neurônios/ultraestrutura , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Proteínas Tirosina Fosfatases Contendo o Domínio SH2 , Medula Espinal/citologiaRESUMO
We have cloned a cDNA that encodes the sodium pump alpha-subunit of the cat flea Ctenocephalides felis. The deduced amino acid sequence of 1037 residues is 90% homologous with the Drosophila alpha-subunit sequence and shows 75% similarity to the sheep kidney alpha-subunit. One characteristic unique to the insect polypeptides is an extended sequence at the N-terminus. Northern blot analysis of C. felis RNA detects two alpha-subunit transcripts with lengths of 3.8 and 4.4 kb. Sodium pump alpha-subunit sequences were readily detected in homogenates of flea midguts and in cDNA synthesized from midgut RNA.
Assuntos
Sifonápteros/genética , ATPase Trocadora de Sódio-Potássio/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA/genética , Sondas de DNA , Sistema Digestório/metabolismo , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Sifonápteros/metabolismoRESUMO
Annual routine chest x-ray examinations have been recommended for many years for tuberculin skin test-positive hospital employees who have not received chemoprophylaxis. To examine the case-finding effectiveness of this policy in a university teaching hospital, employee health records covering an 18-year period were reviewed. Pulmonary tuberculosis was diagnosed in four employees during that time period. None of the patients were identified through the routine annual chest x-ray examination. In the study institution the policy requiring routine annual chest x-ray examinations for tuberculin-positive hospital employees who had not received preventive therapy was rescinded. Currently, the first chest x-ray examination is required at the time of skin test conversion, the second 1 year after conversion, and the third 2 years after conversion. Health teaching and counseling regarding the symptoms of tuberculosis have been initiated for tuberculin-positive hospital employees.
Assuntos
Testes Diagnósticos de Rotina , Recursos Humanos em Hospital , Teste Tuberculínico , Tuberculose Pulmonar/diagnóstico por imagem , Adulto , Análise Custo-Benefício , Feminino , Educação em Saúde , Humanos , Masculino , Pessoa de Meia-Idade , Radiografia , Fatores de TempoRESUMO
BACKGROUND: Bone marrow transplantation for the treatment of malignancies is on the increase. Unfortunately, there are no well-validated infection control guidelines for this highly susceptible population. METHODS: Literature was reviewed concerning infection risks and interventions to decrease risks for bone marrow transplant recipients. RESULTS: Definitive information was generally lacking. However, basic "common sense" infection control recommendations for bone marrow transplantation were made in the following areas: air ventilation systems, design issues, environmental services, patient care issues, barrier precautions, nosocomial surveillance, and discharge planning. Recommendations must be tailored to each facility or setting. CONCLUSION: We conclude that validation of many of these recommendations is necessary to provide optimum care for bone marrow transplant recipients.
Assuntos
Transplante de Medula Óssea , Ambiente de Instituições de Saúde , Controle de Infecções/métodos , Adulto , Microbiologia do Ar , Ambiente Controlado , Filtração , Humanos , Isolamento de Pacientes , Recursos Humanos em Hospital , Fatores de Risco , Esterilização , Ventilação , Visitas a PacientesRESUMO
Because accurate regulation of toxin gene expression is critical for safe and effective gene therapy applications, the authors have examined the regulation of diphtheria toxin A (DTA) fragment expression in human glioma cell lines using two transcriptional control systems derived from Escherichia coli: the tetracycline (Tet) system and the lactose (Lac) system. The Tet system includes a tetracycline-controlled transactivator (tTA), a tTA-responsive minimum human cytomegalovirus (hCMV) promoter controlling the expression of the DTA gene, and tetracycline as an allosteric inhibitor. The Lac system includes the lac repressor (lacR), a lacR-regulated Rous sarcoma virus-long terminal repeat (RSV-LTR) promoter controlling the expression of the DTA gene, and isopropyl-thio-beta-D-galactoside (IPTG) as an allosteric inducer. Expression plasmids encoding either tTA or lacR were transfected into U-87MG and U-343MG glioma cells along with the responsive DTA plasmid. Cell killing was monitored by the ability of the toxin to abolish protein synthesis and was quantitated using a luciferase reporter gene. In the Tet system, tumor cell killing could be regulated by tetracycline up to 120-fold. In contrast, only a twofold IPTG-dependent regulation was obtained using the Lac system because of an incomplete repression of DTA expression in the uninduced state. Replacement of the RSV-LTR promoter with the heavy metal-inducible mouse metallothionein-1 promoter in the lacR-responsive unit, as well as the generation of a clonal glioma cell line expressing lacR, did not significantly enhance regulation of DTA in the Lac system. In conclusion, this study demonstrates that the Tet system is of potential use in gene therapy applications in which regulated expression of a therapeutic gene is an important issue.
Assuntos
Neoplasias Encefálicas/genética , Toxina Diftérica/genética , Regulação da Expressão Gênica , Glioma/genética , Células Procarióticas/fisiologia , Transcrição Gênica , Animais , Linhagem Celular , Expressão Gênica , Técnicas Genéticas , Humanos , Óperon Lac , Camundongos , Óperon , Resistência a Tetraciclina/genéticaRESUMO
To assess the importance of TonB-dependent iron transport systems to growth of Shigella in vivo, a tonB mutant of Shigella dysenteriae was isolated and tested in cultured cells. The tonB mutant invaded epithelial cells, but did not form plaques in confluent monolayers of Henle cells, indicating an inability of this mutant to spread from cell to cell. The rate of intracellular multiplication of the tonB mutant was reduced significantly compared to that of the wild type. The loss of virulence in the tonB mutant was not due to loss of either Shu or Ent, the TonB-dependent systems which allow for transport of heme and ferrienterobactin, respectively. A shuA mutant lacking the outer membrane receptor for heme, an entB mutant defective in enterobactin synthesis, and a shuA entB double mutant each were able to invade cultured cells, multiply intracellularly, and form wild-type plaques. The ability of S. dysenteriae to access iron during intracellular growth was assessed by flow cytometric analysis of an iron- and Fur-regulated shuA-gfp reporter construct. Low levels of green fluorescent protein expression in the intracellular environment were observed in all strains, indicating that iron is available to intracellular bacteria, even in the absence of TonB-dependent iron transport. The failure of the tonB mutant to grow well in an iron-replete intracellular environment suggests that TonB plays a role in addition to heme- and siderophore-mediated iron acquisition in vivo, and this function is required for the intracellular growth and intercellular spread of S. dysenteriae.
Assuntos
Proteínas de Bactérias/fisiologia , Proteínas de Membrana/fisiologia , Shigella dysenteriae/crescimento & desenvolvimento , Transporte Biológico , Células Cultivadas , Humanos , Ferro/metabolismo , Regiões Promotoras Genéticas , Shigella dysenteriae/genética , Shigella dysenteriae/patogenicidade , VirulênciaRESUMO
Glial fibrillary acidic protein (GFAP) is an intermediate-filament (IF) protein that is highly specific for cells of astroglial lineage, although its tissue-specific role is speculative. Determination of the primary structure of this protein should be of importance for understanding the functional role it plays in astroglia. Therefore, we isolated a cDNA clone encoding this protein and determined its nucleotide sequence. The predicted amino acid sequence indicates that GFAP shares structural similarities--particularly in the central rod domain and to a lesser degree in the carboxyl-terminal domain--with other IF proteins found in nonepithelial cell types. Considerable sequence divergence in the amino-terminal region of GFAP suggests that the tissue-specific functions of this IF protein might be mediated through this region of the molecule. In contrast, conservation of structural characteristics and a moderate degree of sequence conservation in the carboxyl-terminal region suggest functional similarities. Blot hybridization analysis using the GFAP cDNA as a probe failed to detect GFAP mRNA in both normal and neoplastic human tissues in which IF proteins other than GFAP are known to be expressed.
Assuntos
Clonagem Molecular , Proteína Glial Fibrilar Ácida/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Southern Blotting , Linhagem Celular , Humanos , Dados de Sequência Molecular , Homologia de Sequência do Ácido Nucleico , Células Tumorais CultivadasRESUMO
Shigella flexneri possesses multiple iron acquisition systems, including proteins involved in the synthesis and uptake of siderophores and the Feo system for ferrous iron utilization. We identified an additional S. flexneri putative iron transport gene, sitA, in a screen for S. flexneri genes that are induced in the eukaryotic intracellular environment. sitA was present in all Shigella species and in most enteroinvasive Escherichia coli strains but not in any other E. coli isolates tested. The sit locus consists of four genes encoding a potential ABC transport system. The deduced amino acid sequence of the S. flexneri sit locus was homologous to the Salmonella enterica serovar Typhimurium Sit and Yersinia pestis Yfe systems, which mediate both manganese and iron transport. The S. flexneri sit promoter was repressed by either iron or manganese, and the iron repression was partially dependent upon Fur. A sitA::cam mutation was constructed in S. flexneri. The sitA mutant showed reduced growth, relative to the wild type, in Luria broth containing an iron chelator but formed wild-type plaques on Henle cell monolayers, indicating that the sitA mutant was able to acquire iron and/or manganese in the host cell. However, mutants defective in two of these iron acquisition systems (sitA iucD, sitA feoB, and feoB iucD) formed slightly smaller plaques on Henle cell monolayers. A strain carrying mutations in sitA, feoB, and iucD did not form plaques on Henle cell monolayers.