RESUMEN

Two glioblastoma groups, which are distinguished from each other by expression level of 416 genes (P < 0.05), were determined using a mathematical model of linear Boolean programming on the basis of gene expression data, obtained by microarray analysis of the glioblastomas and available in Gene Expression Omnibus (GEO) data base. The expression level of 15 genes was more than two-fold higher in the first group of glioblastoma (80 samples) in comparison with the second group (144 samples) and 401 genes and--more than two-fold lower as compared to the second group. 10 of 15 genes, which expression level prevailed in the first group, encode the proteins involved in cell cycle regulation and cell proliferation. A significant percentage of 401 genes are the genes that encode proteins involved in the functioning of neural cells and participating in the processes such as synaptic transmission, neurogenesis, the formation of myelin sheath, axon formation. Kohonen map, built on the basis of the data of 15 genes with prevailed expression in the first group and 60 (of4 01) genes, whose expression level elevated in the second group, confirmed the existence of two glioblastoma groups with specific gene expression profiles. Distribution of the glioblastomas into two groups may reflect two pathways of astrocytic glioma development, one of which leads to the formation of tumors with higher levels of gene expression, which protein products are involved in cell cycle regulation and proliferation. On the other hand, the existence of two molecular variants may reflect different states of glioblastoma progression.

Asunto(s)

Neoplasias Encefálicas/clasificación , Neoplasias Encefálicas/genética , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Glioblastoma/clasificación , Glioblastoma/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Análisis por Conglomerados , Bases de Datos Genéticas , Biblioteca de Genes , Glioblastoma/metabolismo , Glioblastoma/patología , Humanos

RESUMEN

Cancer evolution is a stochastic process both at the genome and gene levels. Most of tumors contain multiple genetic subclones, evolving in either succession or in parallel, either in a linear or branching manner, with heterogeneous genome and gene alterations, extensively rewired signaling networks, and addicted to multiple oncogenes easily switching with each other during cancer progression and medical intervention. Hundreds of discovered cancer genes are classified according to whether they function in a dominant (oncogenes) or recessive (tumor suppressor genes) manner in a cancer cell. However, there are many cancer "gene-chameleons", which behave distinctly in opposite way in the different experimental settings showing antagonistic duality. In contrast to the widely accepted view that mutant NADP(+)-dependent isocitrate dehydrogenases 1/2 (IDH1/2) and associated metabolite 2-hydroxyglutarate (R)-enantiomer are intrinsically "the drivers" of tumourigenesis, mutant IDH1/2 inhibited, promoted or had no effect on cell proliferation, growth and tumorigenicity in diverse experiments. Similar behavior was evidenced for dozens of cancer genes. Gene function is dependent on genetic network, which is defined by the genome context. The overall changes in karyotype can result in alterations of the role and function of the same genes and pathways. The diverse cell lines and tumor samples have been used in experiments for proving gene tumor promoting/suppressive activity. They all display heterogeneous individual karyotypes and disturbed signaling networks. Consequently, the effect and function of gene under investigation can be opposite and versatile in cells with different genomes that may explain antagonistic duality of cancer genes and the cell type- or the cellular genetic/context-dependent response to the same protein. Antagonistic duality of cancer genes might contribute to failure of chemotherapy. Instructive examples of unexpected activity of cancer genes and "paradoxical" effects of different anticancer drugs depending on the cellular genetic context/signaling network are discussed.

Asunto(s)

Genes Supresores de Tumor , Oncogenes , Animales , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias/genética , Neoplasias/metabolismo

RESUMEN

The process of cellular transformation has been amply studied in vitro using immortalized cell lines. Immortalized cells never have the normal diploid karyotype, nevertheless, they cannot grow over one another in cell culture (contact inhibition), do not form colonies in soft agar (anchorage-dependent growth) and do not form tumors when injected into immunodeficient rodents. All these characteristics can be obtained with additional chromosome changes. Multiple genetic rearrangements, including whole chromosome and gene copy number gains and losses, chromosome translocations, gene mutations are necessary for establishing the malignant cell phenotype. Most of the experiments detecting transforming ability of genes overexpressed and/or mutated in tumors (oncogenes) were performed using mouse embryonic fibroblasts (MEFs), NIH3T3 mouse fibroblast cell line, human embryonic kidney 293 cell line (HEK293), and human mammary epithelial cell lines (mainly HMECs and MC-F10A). These cell lines have abnormal karyotypes and are prone to progress to malignantly transformed cells. This review is aimed at understanding the mechanisms of cell immortalization by different "immortalizing agents", oncogene-induced cell transformation of immortalized cells and moderate response of the advanced tumors to anticancer therapy in the light of tumor "oncogene and chromosome addiction", intra-/intertumor heterogeneity, and chromosome instability.

Asunto(s)

Transformación Celular Neoplásica/genética , Cromosomas/genética , Células Eucariotas , Oncogenes/genética , Cariotipo Anormal , Animales , Línea Celular Transformada , Línea Celular Tumoral , Aberraciones Cromosómicas , Células Eucariotas/metabolismo , Células Eucariotas/patología , Dosificación de Gen , Humanos , Cariotipificación , Ratones , Mutación

RESUMEN

Increased expression of the insulin-like growth factor (IGF) family members, IGF1, IGF2, their receptors and binding proteins, or combinations thereof has been documented in various malignancies including gliomas. The results of multiple investigations suggest that the IGFs can play a paracrine and/or autocrine role in promoting tumor growth in situ during tumor progression but that these roles may vary depending on the tissue of origin. Enhanced IGF1 expression was not found in glioblastomas and it was supposed that IGF1 participation in the development of glial tumors may be substituted by protein products of highly expressed other genes, also participating in PI3K and MAPK pathways. Increased expression of IGF-binding protein genes in brain tumors makes the picture even more complicated. As other binding proteins, IGFBPs regulate the activity of their ligands by prolonging their half-life. The discrepancies arising from conflicting evidence on the results obtained by different laboratories in human gliomas are discussed. Our data highlight the importance of viewing the IGF-related proteins as a complex multifactorial system and show that changes in the expression levels of any one component of the system, in a given malignancy, should be interpreted with caution. As IGF targeting for anticancer therapy is rapidly becoming clinical reality, an understanding of this complexity is very timely.

Asunto(s)

Astrocitoma/metabolismo , Neoplasias Encefálicas/metabolismo , Encéfalo/metabolismo , Regulación Neoplásica de la Expresión Génica , Glioblastoma/metabolismo , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Factor II del Crecimiento Similar a la Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Transducción de Señal/genética , Astrocitoma/genética , Encéfalo/patología , Neoplasias Encefálicas/genética , Bases de Datos Genéticas , Perfilación de la Expresión Génica , Glioblastoma/genética , Semivida , Humanos , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/genética , Factor I del Crecimiento Similar a la Insulina/genética , Factor II del Crecimiento Similar a la Insulina/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Unión Proteica , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Reacción en Cadena en Tiempo Real de la Polimerasa

RESUMEN

Human cartilage chitinase 3-like protein 2 (CHI3L2, YKL-39) is secreted by articular chondrocytes, also synoviocytes, lung, and heart. Increased levels of YKL-39 have been demonstrated in synovial fluids of patients with rheumatoid or osteoarthritis as well as in some other pathologies and in malignant tumors, particularly in glioblastomas. It belongs to glycosyl hydrolase family 18 and the most closely related to human cartilage glycoprotein 39 (HC gp-39 or chitinase 3-like protein 1, CHI3L1 or YKL-40), which as it was shown previously, promotes the growth of human synovial cells as well as skin and fetal lung fibroblasts. Dose-dependent growth stimulation was observed when the fibroblastic cell line was exposed to YKL-40 in a concentration range from 0.1 to 2 nM, which is similar to the effective dose of the well characterized mitogen, insulin-like growth factor 1. The use of selective inhibitors of the mitogen-activated protein kinase (MAP kinase) signaling pathway indicates that both, YKL-40 and IGF-I are involved in phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/ERK2). Thus YKL-40 initiates a signaling cascade which leads to increased cell proliferation, suggesting that this protein could play some role in the inhibition of apoptosis. We report here that YKL-39, which as YKL-40 has significantly increased expression in glioblastomas, also activates signal-regulated kinases ERK1/ERK2 in human embryonic kidney (HEK293) and human glioblastoma (U87 MG) cells.

Asunto(s)

Fibroblastos , Glioblastoma/enzimología , Glicoproteínas/fisiología , Lectinas/fisiología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Adipoquinas , Secuencia de Aminoácidos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proteína 1 Similar a Quitinasa-3 , Relación Dosis-Respuesta a Droga , Fibroblastos/enzimología , Fibroblastos/metabolismo , Glioblastoma/metabolismo , Glioblastoma/patología , Glicoproteínas/antagonistas & inhibidores , Glicoproteínas/química , Glicoproteínas/farmacología , Humanos , Lectinas/antagonistas & inhibidores , Lectinas/química , Lectinas/farmacología , Mitosis/efectos de los fármacos , Datos de Secuencia Molecular , Fosforilación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Transducción de Señal/efectos de los fármacos , Líquido Sinovial/enzimología , Líquido Sinovial/metabolismo

RESUMEN

Analysis of the expression of genes encoding myelin basic protein (MBP) and glial fibrillary acidic protein (GFAP) genes in human glial tumors was carried out for determination of the expression specificity of these genes according to tumor types and their malignancy. Low levels of MBP mRNA in astrocytoma specimens of malignancy grades II-IV and significantly higher levels in perifocal zone adjacent to them have been determined by Northern hybridization. Diffuse astrocytomas and anaplastic astrocytomas are characterized mostly by low level of MBP gene expression and high level of GFAP gene expression, but distinct subtypes of diffuse and anaplastic astrocytomas with high level of MBP gene and low level of GFAP gene expression can be also detected that may be the reflection of different oncogenic pathways. Very low levels or even absence of MBP mRNA were revealed in oligodendroglioma and all oligoastrocytomas. Thus, Northern hybridization data are correlated with Serial Analysis of Gene Expression (SAGE). Obtained results show that MBP is nonspecific marker for tumors of oligodendroglial origin, but determination of relative levels of MBP and GFAP mRNAs may be useful for glial tumors recognition. By such a way, these two genes together with previously found by us YKL-40 and TSC-22 can be included into the gene panel for the determination of so called "gene signatures" of brain tumors. However, severe requirements in relation to a clinical value of these "gene signatures" can not be formulated without their verification on plenty of clinical samples of tumors and valid control.

Asunto(s)

Biomarcadores de Tumor/genética , Neoplasias Encefálicas/genética , Expresión Génica , Proteína Ácida Fibrilar de la Glía/genética , Glioma/genética , Proteína Básica de Mielina/genética , Northern Blotting , Neoplasias Encefálicas/metabolismo , Estudios de Casos y Controles , Marcadores Genéticos , Glioma/metabolismo , Humanos , ARN Mensajero/genética

RESUMEN

A comparison of gene expression profiles in different types of human brain tumours and normal brain by Serial Analysis of Gene Expression (SAGE) revealed exceptionally high content of CTTGGGTTTT tag in meningioma and ependimoma SAGE-libraries. A search of the most relevant gene for this tag on the website "SAGE Anatomic Viewer" showed that it belonged to the nucleotide sequence of insulin-like growth factor II (IGF-II) gene as well as to the open reading frame 43 on a chromosome 11 (C11orf43). This nucleotide sequence encodes putative insulin-like growth factor II associated protein (IGF-IIA). mRNA for this protein is produced as a result of the processing of IGF-II gene primary transcript. Northern analysis of glial tumours and meningiomas showed the exceptionally high level of mRNA of IGF-II-associated protein in meningiomas. Protein, encoded by this mRNA, can play the important role in meningioma formation and may be used as their specific molecular marker.

Asunto(s)

Biomarcadores de Tumor/biosíntesis , Regulación Neoplásica de la Expresión Génica , Factor II del Crecimiento Similar a la Insulina/biosíntesis , Neoplasias Meníngeas/metabolismo , Meningioma/metabolismo , ARN Mensajero/biosíntesis , Secuencia de Aminoácidos , Secuencia de Bases , Biomarcadores de Tumor/genética , Northern Blotting , Encéfalo/metabolismo , Encéfalo/patología , Humanos , Factor II del Crecimiento Similar a la Insulina/genética , Neoplasias Meníngeas/patología , Meningioma/patología , Datos de Secuencia Molecular , ARN Mensajero/genética

RESUMEN

To enhance glioblastoma (GB) marker discovery we compared gene expression in GB with human normal brain (NB) by accessing SAGE Genie web site and compared obtained results with published data. Nine GB and five NB SAGE-libraries were analyzed using the Digital Gene Expression Displayer (DGED), the results of DGED were tested by Northern blot analysis and RT-PCR of arbitrary selected genes. Review of available data from the articles on gene expression profiling by microarray-based hybridization showed as few as 35 overlapped genes with increased expression in GB. Some of them were identified in four articles, but most genes in three or even in two investigations. There was found also some differences between SAGE results of GB analysis. Digital Gene Expression Displayer approach revealed 676 genes differentially expressed in GB vs. NB with cut-off ratio: twofold change and P < or = 0.05. Differential expression of selectedgenes obtained by DGED was confirmed by Northern analysis and RT-PCR. Altogether, only 105 of 955 genes presented in published investigations were among the genes obtained by DGED. Comparison of the results obtained by microarrays and SAGE is very complicated because authors present only the most prominent differentially expressed genes. However, even available data give quite poor overlapping of genes revealed by microarrays. Some differences between results obtained by SAGE in different investigations can be explained by high dependence on the statistical methods used. As for now, the best solution to search for molecular tumor markers is to compare all available results and to select only those genes, which significant expression in tumor combined with very low expression in normal tissues was reproduced in several articles. 105 differentially expressed genes, common to both methods, can be included in the list of candidates for the molecular typing of GBs. Some genes, encoded cell surface or extra-cellular proteins may be useful for targeting gliomas with antibody-based therapy.

Asunto(s)

Neoplasias Encefálicas/genética , Perfilación de la Expresión Génica , Expresión Génica/genética , Glioblastoma/genética , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , ADN Complementario/genética , Etiquetas de Secuencia Expresada , Biblioteca de Genes , Humanos , Proteínas de Neoplasias/genética , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

AIM: To evaluate expression patterns of protein product of putative tumor suppressor gene TSC-22 in human astrocytic tumors by immunohistochemical approach. METHODS: Plasmid pET-23d-TSC22 was constructed for the expression of human TSC-22 protein in bacterial system, and polyclonal rabbit antibodies against recombinant TSC-22 were produced. Immunohistochemical analysis of TSC-22 and GFAP expression with the use of anti-human-TSC-22- and anti-human-GFAP-antibodies was performed on histological slides of astrocytic tumors. RESULTS: Immunohistochemical analysis has shown that the number of cells expressing TSC-22 was significantly lower in glioblastoma tissues than that in diffuse astrocytoma. Double immunohistochemical staining of astrocytic tumors using anti-human-TSC-2- and anti-human-GFAP-antibodies showed that both TSC-22 and GFAP expression is co-localized in astrocytes. CONCLUSION: TSC-22 protein is expressed in astrocytes, but not in macrophage/microglial cells. In more aggressive forms of astrocytic tumors decreased expression of TSC-22 mRNA correlates with its lowered expression on protein level.

Asunto(s)

Astrocitoma/metabolismo , Neoplasias Encefálicas/metabolismo , Regulación Neoplásica de la Expresión Génica , Proteínas Represoras/biosíntesis , Secuencia de Aminoácidos , Astrocitos/metabolismo , Astrocitoma/patología , Secuencia de Bases , Biomarcadores de Tumor/análisis , Neoplasias Encefálicas/patología , Perfilación de la Expresión Génica , Proteína Ácida Fibrilar de la Glía/biosíntesis , Humanos , Inmunohistoquímica , Microglía/metabolismo , Datos de Secuencia Molecular , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Represoras/genética

RESUMEN

In the present study, we have used the gene expression data available in the SAGE database in an attempt to identify glioblastoma molecular markers. Of 129 genes with more than 5-fold difference found by comparison of nine glioblastoma with five normal brain SAGE libraries, 44 increased their expression in glioblastomas. Most corresponding proteins were involved in angiogenesis, host-tumor immune interplay, multidrug resistance, extracellular matrix (ECM) formation, IGF-signalling, or MAP-kinase pathway. Among them, 16 genes had a high expression both in glioblastomas and in glioblastoma cell lines suggesting their expression in transformed cells. Other 28 genes had an increased expression only in glioblastomas, not in glioblastoma cell lines suggesting an expression possibly originated from host cells. Many of these genes are among the top transcripts in activated macrophages, and involved in immune response and angiogenesis. This altered pattern of gene expression in both host and tumor cells, can be viewed as a molecular marker in the analysis of malignant progression of astrocytic tumors, and as possible clues for the mechanism of disease. Moreover, several genes overexpressed in glioblastomas produce extracellular proteins, thereby providing possible therapeutic targets. Further characterization of these genes will thus allow them to be exploited in molecular classification of glial tumors, diagnosis, prognosis, and anticancer therapy.

Asunto(s)

Biomarcadores de Tumor/metabolismo , Neoplasias Encefálicas/genética , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Glioblastoma/genética , Biomarcadores de Tumor/genética , Encéfalo/metabolismo , Neoplasias Encefálicas/metabolismo , Línea Celular Tumoral , Bases de Datos Genéticas , Biblioteca de Genes , Glioblastoma/metabolismo , Humanos

RESUMEN

The identification and characterization of genes either induced or repressed in human brain tumors are important in understanding the mechanism of tumor initiation and progression, stages of malignancy, in developing new approaches to the diagnosis and treatment of tumors. In this paper, differential hybridization of arrayed human fetal brain and postnatal brain and postnatal brain cDNA libraries revealed differences in the rate of hybridization signals with total cDNA probes of the human brain and glioblastoma multiforme for more than 150 cDNA clones. Sixteen nucleotide sequences with changed contents in tumors were identified by repeated differential hybridization of the cDNA clones selected by primary screening with the same total cDNA probes of the human brain and glioblastoma multiforme and by Northern-hybridization of RNA samples from the human and glial tumors. The results of an analysis of the increased expression of the gene encoding apolipoprotein E. DNA-binding protein B, mitochondrial (16S and 12S) and cytoplasmic 28S rRNAs, Alu-containing transcripts, inactivation of cullin 1 gene and potential tumor suppressor gene TSC-22 are described.

Asunto(s)

Neoplasias Encefálicas/genética , Proteínas Cullin , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Glioma/genética , Elementos Alu , Apolipoproteínas E/genética , Northern Blotting , Encéfalo/embriología , Encéfalo/crecimiento & desarrollo , Proteínas de Ciclo Celular/genética , ADN Complementario , Humanos , Hibridación in Situ , Valor Predictivo de las Pruebas , ARN Ribosómico , ARN Ribosómico 16S , ARN Ribosómico 28S

RESUMEN

The chum salmon insulin-like growth factor II (IGF-II) gene is highly expressed in liver tissue. In this study we demonstrate that two transcription factors, Sp1 and C/EBPbeta, are involved in the enhanced expression of the salmon IGF-II gene. The presence of the fish homolog for C/EBPbeta in salmon liver RNA was confirmed by Northern blotting. The sIGF-II promoter was activated up to 20-fold by co-transfection with C/EBPbeta. The functional importance of four out of the five putative C/EBPbeta binding sites was demonstrated with mutational analysis in transient transfection assays. The transcription factor Sp1 binds to two sites within the salmon IGF-II promoter. Interestingly, mutation of the Sp1 binding sites decreases not only the basal IGF-II promoter activity but also the C/EBPbeta-induced transactivation. These results demonstrate that liver-enriched C/EBPbeta and ubiquitously expressed Sp1 each activate the sIGF-II promoter and that Sp1 is required for full transactivation of the sIGF-II gene by C/EBPbeta. This suggests that C/EBPbeta and Sp1 act in synergy.

Asunto(s)

Proteína beta Potenciadora de Unión a CCAAT/farmacología , Factor I del Crecimiento Similar a la Insulina/genética , Oncorhynchus keta/genética , Regiones Promotoras Genéticas/efectos de los fármacos , Factor de Transcripción Sp1/farmacología , Animales , Secuencia de Bases , Sitios de Unión/genética , Northern Blotting , Sinergismo Farmacológico , Hígado/metabolismo , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , ARN Mensajero/análisis , Activación Transcripcional/efectos de los fármacos

RESUMEN

As far as a physiological state of immune system cells is of great importance, for the investigation of the growth hormone influence on spleen T-lymphocytes ability to divide, we utilised plasmid-mediated gene transfer of hGH cDNA regulated by a powerful human cytomegalovirus immediate-early (CMV-IE) enhancer-promoter. The femur muscles of mice were injected with 50 mg of the plasmid DNA and then electrostimulated. 7 days later muscle tissues were harvested, and hGH DNA and RNA presence were proved by Southern and Northern blot analysis, respectively. Splenocytic proliferative response after ConA stimulation was shown to be increased by 6.8 times in experimental mice comparing to controls. Besides that in mice treated with plasmid vector increased cellularity of spleen. These results demonstrate that gene transfer of hGH is one of possible methods to increase immunity in aging organisms.

Asunto(s)

División Celular/fisiología , Hormona del Crecimiento/genética , Hormona del Crecimiento/fisiología , Plásmidos , Bazo/química , Animales , Femenino , Ratones , Ratones Endogámicos BALB C

RESUMEN

One autapomorphic character restricted to all Metazoa including Porifera [sponges] is the existence of transmembrane receptor tyrosine kinases (RTKs). In this study we screened for molecules from one subfamily within the superfamily of the insulin receptors. The subfamily includes the insulin receptors (InsR), the insulin-like growth factor I receptors, and the InsR-related receptors--all found in vertebrates--as well as the InsR-homolog from Drosophila melanogaster. cDNAs encoding putative InsRs were isolated from the hexactinellid sponge Aphrocallistes vastus, the demosponge Suberites domuncula, and the calcareous sponge Sycon raphanus. Phylogenetic analyses of the catalytic domains of the putative RTKs showed that the sponge polypeptides must be grouped with the InsRs. The relationships revealed that all sponge sequences fall into one branch of this group, whereas related sequences from mammals (human, mouse, and rat), insects and molluscs, and polypeptides from one cephalochordate, fall together into a second branch. We have concluded that (i) the InsR-like molecules evolved in sponges prior to the "Cambrian Explosion" and contributed to the rapid appearance of the higher metazoan phyla; (ii) the sponges constitute a monophyletic taxon, and (iii) epidermal growth factor (EGF)-like domains are present in sponges, which allows the insertion of this domain into potential receptor and matrix molecules.

Asunto(s)

Poríferos/genética , Receptor de Insulina/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Dominio Catalítico , Clonación Molecular , ADN Complementario , Evolución Molecular , Humanos , Ratones , Datos de Secuencia Molecular , Filogenia , Ratas , Receptor de Insulina/clasificación , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido

RESUMEN

Mice immunized with plasmid DNA encoding Nef regulatory protein of human immunodeficiency virus type 1 developed high levels of anti-Nef antibodies. After 4 intramuscular injections of 100 microg plasmid DNA, anti-Nef antibodies reached titers up to 2 x 10(4). A significant specific antibody response was maintained for at least 16 months. Using a set of seven 31-66 mer synthetic peptides covering the entire sequence of Nef, we analysed the specificity of ant-Nef antibodies. Interestingly, specific antibodies produced in response to Nef expressing plasmid DNA did not recognize the linear peptides except the long C-terminal peptide (aa 141-205) for 3 of the 10 sera. With anti-Nef antibodies produced in mice immunized with the protein Nef without any adjuvant, the same restraint epitope binding was found. Only 3 of the 5 Nef positive sera reacted with the C-terminal peptide. This suggests that specific antibodies induced by plasmid DNA as well as by the non-denatured protein recognize conformation-dependent epitopes. On the contrary, anti-Nef antibodies from mice immunized with the protein in Freund's adjuvant showed a broader epitope reactivity pattern. Interestingly, the analysis of immunoglobulin isotype profiles of antibodies generated by the different protocols of immunization showed that plasmid DNA immunization induced predominantly IgG2a, whereas immunization with Nef protein, with or without adjuvant, yielded a preponderance of IgG1 antibodies.

Asunto(s)

ADN Viral/genética , Productos del Gen nef/inmunología , VIH-1/genética , Inmunización , Secuencia de Aminoácidos , Animales , Especificidad de Anticuerpos , Linfocitos B/inmunología , Mapeo Epitopo , Código Genético , Vectores Genéticos , Humanos , Isotipos de Inmunoglobulinas , Modelos Logísticos , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Proteínas Recombinantes/inmunología , Productos del Gen nef del Virus de la Inmunodeficiencia Humana

RESUMEN

IGF-II plays an important role in growth and development of vertebrates and is highly expressed in adult salmon liver. In the present study, we demonstrate that a liver-enriched transcription factor, hepatocyte nuclear factor 3beta (HNF-3beta), is an activator of the chum salmon IGF-II gene. Multiple binding sites for HNF-3beta were identified within the 5'-UTR using electrophoretic mobility shift assays and mutation of these sites prevents binding of HNF-3beta. In transient transfection assays it was shown that mutation of the HNF-3beta binding sites results in a substantial decrease of HNF-3beta-activated salmon IGF-II gene expression. This is the first identified transcription factor that is functionally involved in the regulation of fish IGF-II expression.

Asunto(s)

Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Factor II del Crecimiento Similar a la Insulina/genética , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas , Factores de Transcripción/metabolismo , Animales , Sitios de Unión , Proteínas de Unión al ADN/genética , Factor Nuclear 3-beta del Hepatocito , Proteínas Nucleares/genética , Oncorhynchus keta , Factores de Transcripción/genética , Activación Transcripcional

RESUMEN

The long arm of human chromosome 21 likely contains several hundred genes. To determine which of these are responsible for specific aspects of the Down Syndrome phenotype, protein functional analysis coupled to phenotypic analysis of transgenic mice will be required. Because such experiments are both time consuming and expensive, prioritizing 21q genes for further studies would be advantageous. Here, we discuss expression analysis, specifically the use of Northern analysis, cDNA array screening and RNA tissue in situ hybridization to assess place and time of expression of forty-two genes. For a subset of these, over expression in normal versus trisomy cell lines and mouse tissues is discussed. Lastly, several examples of alternative processing and their potential for generation of brain specific proteins are described. Together, these experiments give information on time, place and level of expression of a number of 21q genes and suggest some interesting candidates worth further investigation for relevance to Down Syndrome. These data also illustrate the complexities and ambiguities inherent in interpretation and use of expression information.

Asunto(s)

Encéfalo/metabolismo , Cromosomas Humanos Par 21 , Síndrome de Down/genética , Adulto , Animales , Mapeo Cromosómico , Expresión Génica , Humanos , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/genética

RESUMEN

In vitro experiments revealed that the scrapie prion protein, PrP(Sc), as well as the PrP fragment PrP106-126, and the HIV-1 coat protein gp120 induce apoptosis of rat cortical neurons. The toxic effect displayed by PrP and gp120 could be blocked by NMDA receptor antagonists. Treatment of neuronal cells with PrP106-126 resulted in a drop of intracellular glutathione level and changes in the level of Bcl-2. Evidence is presented that gp120 causes an activation of phospholipase A2, resulting in the increased release of arachidonic acid, which may in turn sensitize the NMDA receptor.

Asunto(s)

Proteína gp120 de Envoltorio del VIH/toxicidad , Neuronas/efectos de los fármacos , Proteínas PrPSc/toxicidad , Animales , Muerte Celular/efectos de los fármacos , Células Cultivadas , Humanos , Neuronas/metabolismo , Ratas

RESUMEN

Mice immunized with plasmid DNA encoding Nef accessory protein of human immunodeficiency virus type 1 developed high levels of anti-Nef antibodies which were maintained for at least 16 months. These antibodies produced in response to Nef-expressing plasmid DNA did not recognize the linear peptides except the long C-terminal peptide for three of the ten sera. With anti-Nef antibodies produced in mice immunized with the protein Nef without any adjuvant, the same restraint epitope binding was found. On the contrary, anti-Nef antibodies from mice immunized with the protein in Freund's adjuvant showed a broader epitope reactivity pattern. Interestingly, the analysis of immunoglobulin isotype profiles of antibodies generated by the different protocols of immunization showed that plasmid DNA immunization induced predominantly IgG2a, whereas immunization with Nef protein, with or without adjuvant, yielded a preponderance of IgG1 antibodies.

Asunto(s)

Productos del Gen nef/genética , Productos del Gen nef/inmunología , VIH-1/genética , VIH-1/inmunología , Vacunas de ADN/inmunología , Vacunas de ADN/farmacología , Secuencia de Aminoácidos , Animales , Formación de Anticuerpos/inmunología , Especificidad de Anticuerpos , Mapeo Epitopo , Epítopos de Linfocito B/inmunología , Femenino , Anticuerpos Anti-VIH/sangre , Anticuerpos Anti-VIH/inmunología , VIH-1/metabolismo , Humanos , Inmunoglobulina G/clasificación , Inmunoglobulina G/inmunología , Isotipos de Inmunoglobulinas/inmunología , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Plásmidos/genética , Vacunación , Productos del Gen nef del Virus de la Inmunodeficiencia Humana

RESUMEN

IGF-II plays an important role in growth and development of vertebrates. In the present study, the characterization of the first fish IGF-II gene, chum salmon IGF-II, is described. The sIGF-II gene consists of four exons, spanning a region of 9 kbp, that encode the 214 aa IGF-II precursor. While the amino acid sequences of fully processed IGF-II of salmon and mammalian species are very similar, the prepro-peptide sequence deviates extensively in the signal- and E-peptide domains. The transcription initiation site of the sIGF-II gene was localized within a 30 nt region employing RT-PCR. Using sIGF-II promoter-luciferase constructs it was demonstrated that the sIGF-II gene has a relatively strong promoter that contains tissue-specific regulatory elements.

Asunto(s)

Factor II del Crecimiento Similar a la Insulina/biosíntesis , Factor II del Crecimiento Similar a la Insulina/genética , Oncorhynchus keta/genética , Animales , Secuencia de Bases , Cartilla de ADN , Exones , Regulación de la Expresión Génica , Humanos , Datos de Secuencia Molecular , Oncorhynchus mykiss , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Precursores de Proteínas/biosíntesis , Precursores de Proteínas/genética , Proteínas Recombinantes/biosíntesis , Transfección , Células Tumorales Cultivadas