RESUMO
Secondary palate formation is a complex process that is frequently disturbed in mammals, resulting in the birth defect cleft palate. Gene targeting has identified components of cytokine/growth factor signalling systems such as Tgf-alpha/Egfr, Eph receptors B2 and B3 (Ephb2 and Ephb3, respectively), Tgf-beta2, Tgf-beta3 and activin-betaA (ref. 3) as regulators of secondary palate development. Here we demonstrate that the mouse orphan receptor 'related to tyrosine kinases' (Ryk) is essential for normal development and morphogenesis of craniofacial structures including the secondary palate. Ryk belongs to a subclass of catalytically inactive, but otherwise distantly related, receptor protein tyrosine kinases (RTKs). Mice homozygous for a null allele of Ryk have a distinctive craniofacial appearance, shortened limbs and postnatal mortality due to feeding and respiratory complications associated with a complete cleft of the secondary palate. Consistent with cleft palate phenocopy in Ephb2/Ephb3-deficient mice and the role of a Drosophila melanogaster Ryk orthologue, Derailed, in the transduction of repulsive axon pathfinding cues, our biochemical data implicate Ryk in signalling mediated by Eph receptors and the cell-junction-associated Af-6 (also known as Afadin). Our findings highlight the importance of signal crosstalk between members of different RTK subfamilies.
Assuntos
Anormalidades Craniofaciais/genética , Receptor Cross-Talk/fisiologia , Receptores Proteína Tirosina Quinases/metabolismo , Animais , Animais Recém-Nascidos , Anormalidades Craniofaciais/embriologia , Desenvolvimento Embrionário e Fetal/genética , Feminino , Genótipo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Camundongos Knockout , Mutação , Fenótipo , Receptores Proteína Tirosina Quinases/deficiência , Receptores Proteína Tirosina Quinases/genética , Receptor EphB2 , Transdução de SinaisRESUMO
Protein tyrosine kinases (PTKs) are integral components of the cellular machinery that mediates the transduction and/or processing of many extra- and intracellular signals. Members of the JAK family of intracellular PTKs (JAK1, JAK2 and TYK2) are characterized by the possession of a PTK-related domain and five additional homology domains, in addition to a classical PTK domain. An important breakthrough in the understanding of JAK kinases function(s) has come from the recent observations that many cytokine receptors compensate for their lack of a PTK domain by utilizing members of the JAK family for signal transduction.
RESUMO
Interleukin-6 (IL-6), leukemia inhibitory factor, oncostatin M, interleukin-11, and ciliary neurotrophic factor bind to receptor complexes that share the signal transducer gp130. Upon binding, the ligands rapidly activate DNA binding of acute-phase response factor (APRF), a protein antigenically related to the p91 subunit of the interferon-stimulated gene factor-3 alpha (ISGF-3 alpha). These cytokines caused tyrosine phosphorylation of APRF and ISGF-3 alpha p91. Protein kinases of the Jak family were also rapidly tyrosine phosphorylated, and both APRF and Jak1 associated with gp130. These data indicate that Jak family protein kinases may participate in IL-6 signaling and that APRF may be activated in a complex with gp130.
Assuntos
Antígenos CD , Proteínas de Ligação a DNA/metabolismo , Interleucina-6/farmacologia , Glicoproteínas de Membrana/metabolismo , Proteínas Tirosina Quinases/metabolismo , Transativadores , Sequência de Bases , Receptor gp130 de Citocina , Citocinas/farmacologia , Humanos , Fator Gênico 3 Estimulado por Interferon , Fator Gênico 3 Estimulado por Interferon, Subunidade gama , Interferon gama/farmacologia , Janus Quinase 1 , Dados de Sequência Molecular , Fosforilação , Fator de Transcrição STAT1 , Fator de Transcrição STAT3 , Transdução de Sinais , Fatores de Transcrição/metabolismo , Células Tumorais Cultivadas , Tirosina/metabolismoRESUMO
The protein-tyrosine kinases (PTKs) are a burgeoning family of proteins, each of which bears a conserved domain of 250 to 300 amino acids capable of phosphorylating substrate proteins on tyrosine residues. We recently exploited the existence of two highly conserved sequence elements within the catalytic domain to generate PTK-specific degenerate oligonucleotide primers (A. F. Wilks, Proc. Natl. Acad. Sci. USA 86:1603-1607, 1989). By application of the polymerase chain reaction, portions of the catalytic domains of several novel PTKs were amplified. We describe here the primary sequence of one of these new PTKs, JAK1 (from Janus kinase), a member of a new class of PTK characterized by the presence of a second phosphotransferase-related domain immediately N terminal to the PTK domain. The second phosphotransferase domain bears all the hallmarks of a protein kinase, although its structure differs significantly from that of the PTK and threonine/serine kinase family members. A second member of this family (JAK2) has been partially characterized and exhibits a similar array of kinase-related domains. JAK1 is a large, widely expressed membrane-associated phosphoprotein of approximately 130,000 Da. The PTK activity of JAK1 has been located in the C-terminal PTK-like domain. The role of the second kinaselike domain is unknown.
Assuntos
DNA/genética , Fosfotransferases/genética , Proteínas Quinases/genética , Proteínas Tirosina Quinases/genética , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Clonagem Molecular , DNA/isolamento & purificação , Expressão Gênica , Humanos , Dados de Sequência Molecular , Fosforilação , Fosfotransferases/metabolismo , Filogenia , Reação em Cadeia da Polimerase , Proteínas Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Homologia de Sequência do Ácido NucleicoRESUMO
The 84-, 91-, and 113-kDa proteins of the ISGF-3 alpha complex are phosphorylated on tyrosine residues upon alpha interferon (IFN-alpha) treatment and subsequently translocate to the nucleus together with a 48-kDa subunit. In this study, we investigated the presence and the functional status of ISGF-3 alpha subunits and Tyk-2 and JAK1 tyrosine kinases in mutant HeLa cells defective in the IFN-alpha/beta and -gamma response. Stable cell fusion analysis revealed a single complementation group among one class (class B) of mutants. The class B mutants contain detectable level of mRNA and proteins of the 84-, 91-, and 113-kDa proteins, but neither the protein nor mRNA is inducible by IFN-alpha or -gamma. The 91-kDa protein IFN-gamma-activated factor fails to be activated into a DNA-binding state after IFN-alpha or -gamma treatment. In addition, the 91-kDa protein is unable to localize in the nucleus after IFN-alpha and -gamma treatment, and the 113-kDa protein fails to translocate after IFN-alpha treatment. Immunoprecipitation studies document a failure of phosphorylation of the 84- or 91-kDa proteins after IFN-alpha or -gamma treatment. Similarly, no tyrosine-phosphorylated 113-kDa protein was detected after IFN-alpha treatment. The inability of class B mutants to phosphorylate the 84-, 91-, or 113-kDa protein on tyrosine residues correlated with the loss of biological response to IFN-alpha and -gamma. The genetic defect appears to be the absence of the tyrosine kinase JAK1. Our data therefore confirm a recent report that JAK1 plays a critical early signaling role for both IFN-alpha/beta and IFN-gamma systems.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Interferon Tipo I/farmacologia , Interferon gama/farmacologia , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogênicas , Fatores de Transcrição/metabolismo , Western Blotting , Compartimento Celular , Proteínas de Ligação a DNA/química , Imunofluorescência , Regulação da Expressão Gênica , Teste de Complementação Genética , Células HeLa , Humanos , Técnicas In Vitro , Fator Gênico 3 Estimulado por Interferon , Fator Gênico 3 Estimulado por Interferon, Subunidade gama , Janus Quinase 2 , Peso Molecular , Fosfoproteínas/química , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Proteínas Tirosina Quinases/fisiologia , Proteínas/metabolismo , RNA Mensageiro/genética , Transdução de Sinais , TYK2 Quinase , Fatores de Transcrição/químicaRESUMO
Gamma interferon (IFN-gamma), a macrophage-activating cytokine, modulates gene expression through the activity of a transcription factor designated IFN-gamma activation factor (GAF). GAF is formed after phosphorylation on tyrosine and dimerization of the 91-kDa protein STAT1. We have recently reported that differentiation of the promonocytic cell line U937 into monocytes increases the amount of cellular GAF after IFN-gamma treatment and at the same time increases the phosphorylation of STAT1. Here we show that activation of the JAK family kinases, which are instrumental in mediating STAT1 phosphorylation on tyrosine, did not increase upon monocytic U937 differentiation. Consistent with this finding, levels of STAT1 tyrosine phosphorylation were virtually identical in promonocytic and monocytic U937 cells. Analysis of STAT1 phosphoamino acids and mapping of phosphopeptides showed an IFN-gamma-dependent increase in Ser phosphorylation in differentiated cells. Analyses of STAT1 isoforms by two-dimensional gel electrophoresis demonstrated a differentiation-induced shift toward more acidic isoforms. All isoforms were equally sensitive to subsequent tyrosine phosphorylation, as indicated by a sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobility shift typical for tyrosine-phosphorylated STAT1. Consistent with the importance of Ser phosphorylation for high-affinity binding to the IFN-gamma activation site sequence, phosphatase 2A treatment strongly reduced the formation of IFN-gamma activation site-GAF complexes in an electrophoretic mobility shift assay. Our data indicate that the activity of GAF is modulated by STAT1 serine kinases/phosphatases and suggest that this mechanism is employed in the developmental control of macrophage responsiveness to IFN-gamma.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Macrófagos/fisiologia , Proteínas Proto-Oncogênicas , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Aminoácidos/análise , Diferenciação Celular , Regulação da Expressão Gênica , Fator Gênico 3 Estimulado por Interferon , Janus Quinase 1 , Janus Quinase 2 , Modelos Biológicos , Mapeamento de Peptídeos , Fosfopeptídeos/análise , Fosforilação , Fosfosserina/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/análise , Fator de Transcrição STAT1 , Serina/metabolismo , Tirosina/metabolismoRESUMO
Tumor angiogenesis involves a combination of events including the production of inhibitors, proteases, and angiogenic factors that have a chemotactic and mitogenic effect on endothelial cells. Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen that promotes angiogenesis in solid tumors, including brain tumors such as astrocytomas. As an approach to the development of new strategies for gene therapy of brain tumors, we have interrupted the VEGF/VEGF receptor paracrine pathway in an attempt to inhibit angiogenesis and thereby control tumor growth. Rat C6 glioma cells were transfected with a eukaryotic expression vector bearing an antisense-VEGF cDNA. Stable transfectants were observed to express reduced levels of VEGF in culture under hypoxic conditions. When implanted s.c. into nude (nu/nu) mice, growth of the antisense-VEGF cell lines was observed to be greatly inhibited compared to control cells, despite the fact that they have a faster division time in vitro. Analysis of these tumors revealed that they have fewer blood vessels and a higher degree of necrosis, which is a plausible explanation for the reduced tumor size. We believe antisense-VEGF can be successfully used to control tumor growth and may provide the basis for the development of antiangiogenic gene therapy.
Assuntos
Neoplasias Encefálicas/irrigação sanguínea , Neoplasias Encefálicas/terapia , Fatores de Crescimento Endotelial/genética , Terapia Genética , Glioma/irrigação sanguínea , Glioma/terapia , Linfocinas/genética , Neovascularização Patológica/terapia , Oligonucleotídeos Antissenso/genética , Animais , Sequência de Bases , Neoplasias Encefálicas/patologia , Divisão Celular/fisiologia , Hipóxia Celular , Clonagem Molecular , DNA Complementar/genética , Fatores de Crescimento Endotelial/biossíntese , Fatores de Crescimento Endotelial/fisiologia , Endotélio Vascular/crescimento & desenvolvimento , Endotélio Vascular/fisiologia , Feminino , Glioma/patologia , Linfocinas/biossíntese , Linfocinas/fisiologia , Masculino , Camundongos , Camundongos Nus , Dados de Sequência Molecular , Necrose , Ratos , Transfecção , Células Tumorais Cultivadas , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
The murine c-fes protein is encoded by an mRNA of 2.8 kb which is expressed predominantly in haemopoietic cell lines of the myeloid lineage. We have isolated over-lapping cDNA clones encompassing the entire coding region of murine c-fes. The predicted translation product of the c-fes mRNA is an 820 amino acid protein with 89% overall similarity to feline and human c-fes sequences, and 66% similarity to the chicken c-fps sequence. The murine c-fes proto-oncogene is related to other members of the protein tyrosine kinase family of oncogenes, although it is structurally distinct from both the receptor type, and the src family of tyrosine kinases. A comparison of the predicted protein sequence of murine, feline, human and chicken c-fps/fes proteins highlights several domains of possible functional significance in the molecule.
Assuntos
DNA/isolamento & purificação , Proteínas Tirosina Quinases , Proteínas Proto-Oncogênicas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Gatos , Linhagem Celular , DNA/genética , Células-Tronco Hematopoéticas/análise , Humanos , Camundongos , Dados de Sequência Molecular , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-fes , RNA Mensageiro/genética , Homologia de Sequência do Ácido NucleicoRESUMO
Loss of DCC gene expression has now been demonstrated in a wide variety of metastatic cancers. Here we present the nucleotide sequence and predicted amino acid sequence of mouse DCC. Mouse and human DCC share 96% identity at the amino acid level. Analysis of DCC mRNA expression throughout the mid and late stages of gestation in the mouse, demonstrated that DCC mRNA is expressed at significantly higher levels in the developing mouse embryo than in any adult tissue. In addition, we show that an embryo-specific, alternatively spliced, form of DCC is expressed in day 9.5 through day 18.5 embryos. The expression of both alternatively spliced forms of DCC is developmentally regulated such that the embryonic form of DCC predominates in day 9.5 and 10.5 embryos. In the later stage embryos the expression of this alternatively spliced form of DCC is down-regulated with respect to that of the adult form. Whole-mount in situ hybridization of day 11.5 mouse embryos revealed that DCC mRNA is expressed at high levels in the developing brain and the neural tube. However, no DCC mRNA could be detected in any other embryonic tissue at this stage of development. These observations suggest that during embryogenesis DCC may play a pivotal role in the development of the central nervous system.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Genes DCC , Processamento Alternativo , Sequência de Aminoácidos , Animais , Sequência de Bases , Encéfalo/embriologia , Encéfalo/metabolismo , Clonagem Molecular , DNA Complementar , Humanos , Hibridização In Situ , Camundongos , Dados de Sequência Molecular , Biossíntese de Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Especificidade da EspécieRESUMO
We have isolated cDNA clones encoding a third, widely expressed, member of the JAK family of protein tyrosine kinases (PTKs). The anticipated amino acid sequence of JAK2 predicts the presence of two kinase-related domains, a feature characteristic of this family of PTKs. The structural similarity of JAK2 to the other members of this family extends towards their N-termini, beyond the two kinase-related domains, and reveals five further domains of substantial amino acid similarity. The C-terminal portion of one of these domains, the JH4 domain, bears an intriguing, albeit tenuous, similarity to the core element of the SH2 domain, whereas the remaining JAK homology domains do not appear to be a feature of other known proteins.
Assuntos
Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas , Sequência de Aminoácidos , Animais , Sequência de Bases , Janus Quinase 1 , Janus Quinase 2 , Camundongos , Dados de Sequência Molecular , Proteínas Tirosina Quinases/química , Proteínas/química , Homologia de Sequência do Ácido Nucleico , TYK2 QuinaseRESUMO
A protein tyrosine kinase (NYK/FLK-1), bearing all the hallmarks of a growth factor receptor, has been isolated from a cDNA library generated from enriched populations of mouse day 10 embryonic neuroepithelium and from day 18 embryonic colon. Sequence analysis of cDNAs covering the entire coding region of this 5.7 kb mRNA predicted the presence of seven immunoglobulin-like domains in the extracellular region of this molecule. This feature, coupled with the detection of an insert domain bisecting the kinase domain of the predicted protein sequence, places NYK/FLK-1 firmly in the Platelet-derived Growth Factor Receptor-related class of molecules. NYK/FLK-1 is expressed at high levels in adult heart, lung, kidney, brain and skeletal muscle, but is also expressed at lower levels in most other adult tissues. In situ hybridization of day 12.5 embryo sections demonstrated NYK/FLK-1 mRNA expression in endothelial cells lining the dorsal aorta and intervertebral veins. In addition, expression was found in cells lining the capillary plexus which surrounds the developing neuroepithelium, and in the endothelial cells which are found within the embryonic lung, spleen, liver and metanephros.
Assuntos
Embrião de Mamíferos/enzimologia , Endotélio/enzimologia , Proteínas Tirosina Quinases/análise , Células 3T3 , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA/isolamento & purificação , Epitélio/enzimologia , Feminino , Hibridização In Situ , Camundongos , Dados de Sequência Molecular , Gravidez , Proteínas Tirosina Quinases/genéticaRESUMO
A cDNA encoding the human homologue of mouse RYK (related to receptor tyrosine kinases) has been cloned from an interleukin 1 (IL-1)-stimulated human hepatoma cDNA library by cross-species hybridization using the mouse RYK cDNA as a probe. The sequence of the 3067-bp cDNA clone encoding human RYK predicts a transmembrane protein with a cytoplasmic domain that contains the consensus sequences (subdomains I-XI) of the protein tyrosine kinase (PTK) family. The highly conserved motif -D-F-G- (subdomain VII) of the catalytic domain of other receptor-type tyrosine kinases is altered to -D-N-A- in human RYK. In addition, a number of other changes were found in the ATP binding site (subdomains I and II) and the motif [-I-H-R-D-L-A-A-R-N-] found in subdomain VI. Comparison of the human and mouse RYK sequences shows a 92% conservation at the nucleotide level and 97% at the amino acid level. There was no significant homology between the extracellular domain of RYK and the other families of receptor tyrosine kinases described to date. RYK therefore appears to define a new subclass of receptor-type tyrosine kinases whose structure has remained highly conserved across species.
Assuntos
Mapeamento Cromossômico , Clonagem Molecular , Proteínas Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases , Receptores de Superfície Celular/genética , Homologia de Sequência de Aminoácidos , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Cromossomos Humanos Par 17 , Cromossomos Humanos Par 3 , Sequência Conservada , Humanos , Hibridização In Situ , Camundongos , Dados de Sequência Molecular , Proteínas Tirosina Quinases/análise , Proteínas Tirosina Quinases/química , RNA Mensageiro/análise , Receptores de Superfície Celular/análise , Receptores de Superfície Celular/químicaRESUMO
An endogenous protein kinase activity of liver heterogeneous nuclear ribonucleoprotein particles has been characterized and the particle proteins which it phosphorylates in vitro have been fractionated on two-dimensional polyacrylamide gels. The activity is dependent on Mg2+ and is further stimulated by cyclic AMP, polyamines and Mn2+.
Assuntos
Fígado/metabolismo , Proteínas Quinases/metabolismo , RNA Nuclear Heterogêneo/metabolismo , Animais , AMP Cíclico/metabolismo , Magnésio/metabolismo , Masculino , Manganês/metabolismo , Fosforilação , Poliaminas/metabolismo , RatosRESUMO
In the Drosophila embryo, a subset of muscles require expression and function of the RYK subfamily RTK gene derailed (drl) for correct attachment. We have isolated a second RYK homolog, doughnut (dnt), from Drosophila. The DNT protein exhibits 60% amino acid identity to DRL, and is structurally as similar to the mammalian RYK proteins as is DRL, indicating an ancient duplication event. dnt is expressed in dynamic patterns in the embryonic epidermis, being found at high level in epithelia adjacent to cells that are invaginating into the interior of the embryo, including ventral furrow, cephalic furrow, fore- and hindgut, optic lobe and tracheal pits. dnt is capable of a partial rescue of the muscle attachment defect of drl-/- embryos, indicating that it encodes a receptor with a related and significantly overlapping biochemical function.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento , Genes de Insetos , Proteínas Musculares/biossíntese , Músculos/embriologia , Proteínas Tirosina Quinases/biossíntese , Sequência de Aminoácidos , Animais , Mapeamento Cromossômico , Clonagem Molecular , Drosophila melanogaster/embriologia , Epiderme/embriologia , Epiderme/metabolismo , Teste de Complementação Genética , Mamíferos/genética , Dados de Sequência Molecular , Morfogênese/genética , Proteínas Musculares/genética , Proteínas Musculares/fisiologia , Músculos/metabolismo , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/fisiologia , Receptores Proteína Tirosina Quinases/deficiência , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/fisiologia , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Degenerate oligodeoxyribonucleotide (oligo) primers derived from amino acid (aa) sequence motifs held in common between all members of the protein tyrosine kinase (PTK) family were used to prime the amplification of PTK-related sequences from a variety of murine cDNA sources, including the haemopoietic cell lines, FDC-P1 and WEHI-3B D+, peritoneal macrophages and whole brain. Several parameters, such as the length (short, i.e., less than 20 nucleotides (nt) vs. long, i.e., greater than 30 nt) and degeneracy (i.e., moderately degenerate vs. highly degenerate) of the oligo primers and the temperature of the extension phase of the reaction, were examined. The data from these analyses suggest that the most effective type of primer in this application of the polymerase chain reaction is a short, moderately degenerate oligo such as that which might be derived from the small patches of aa sequence homology that are frequently found to be held in common among members of protein families. In addition to a number of previously described PTK sequences, a novel mammalian PTK-related sequence was uncovered.
Assuntos
Clonagem Molecular , Família Multigênica , Técnicas de Amplificação de Ácido Nucleico , Reação em Cadeia da Polimerase/métodos , Proteínas Tirosina Quinases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Células Clonais , Biblioteca Gênica , Immunoblotting , Camundongos , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Poli A/genética , RNA/genética , RNA Mensageiro/genéticaRESUMO
The first natural MHC ligand to be sequenced directly was the nonapeptide SYFPEITHI eluted from H-2 Kd molecules of a mouse tumour line, P815 [1]. A GenBank search indicated high homology to a nonapeptide contained within the human tyrosine kinase JAK1: SFFPEITHI, residues 355-363 [2]. This high homology prompted us to look at whether the mouse JAK1 protein has a Tyr residue at position 356 instead of Phe as in the human sequence. Cloning and sequencing of the mouse homologue gene confirmed that this is indeed the case. Thus, the physiological MHC ligand SYFPEITHI is derived from the protein tyrosine kinase, JAK1. The mouse tumor line P815 expresses the 5.4-kb JAK1 mRNA, and the 130,000 kDa JAK1 protein can be readily detected.
Assuntos
Antígenos H-2/metabolismo , Proteínas Tirosina Quinases/metabolismo , Células 3T3 , Sequência de Aminoácidos , Animais , Western Blotting , Expressão Gênica , Humanos , Janus Quinase 1 , Ligantes , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Oligopeptídeos , RNA Mensageiro/metabolismo , Homologia de Sequência de Aminoácidos , Células Tumorais CultivadasRESUMO
The core proteins of heterogeneous ribonucleoprotein particles, which migrate as four major bands on one-dimensional sodium dodecyl sulphate-polyacrylamide gels, exhibit an complex pattern on two-dimensional gels in which each protein resolves into strings of polypeptides of differing charge. Tryptic peptide mapping was employed to analyse the structural relationship of their components. The 34,000-dalton protein (polypeptide-group B) resolves into a string of seven major components, of which four have been analysed. Tryptic peptide maps indicated that minor variants of at least two proteins occur in this group. The A group of polypeptides, comprising six major components of mol.wt. 32,000 and the D group, comprising three major polypeptides of mol.wt. 40,000, were also examined. Tryptic peptide maps indicate that the two protein types are fundamentally different from each other and from the B-group proteins, but they again exhibit minor variation consistent with post-translational modification.
Assuntos
Nucleoproteínas/análise , Peptídeos/análise , Ribonucleoproteínas/análise , Eletroforese em Gel de Poliacrilamida , Peso Molecular , Biossíntese de Proteínas , RNA Nuclear Heterogêneo , TripsinaRESUMO
Human gliomas, including astrocytomas, consist of highly heterogeneous populations of cells that represent different stages of malignancy. Glioblastoma multiforme is the most highly vascularised class of solid tumour. In order to develop efficacious adjuvant therapies for gliomas the growth pathway(s) targeted must be common to all of these tumours. As angiogenesis is a requirement for all solid tumour growth, we have targeted this process in order to suppress glioma growth in vivo. We have applied antisense VEGF gene expression to disrupt the VEGF/VEGF receptor paracrine pathway in C6 glioma cells and, thereby, inhibit tumour angiogenesis. C6 glioma cells which constitutively express antisense VEGF sequences were demonstrated to have significantly inhibited growth rates when implanted intracranially. Antisense VEGF expressing tumours had a markedly lower level of vascularisation which was accompanied by an increased level of necrosis compared to control tumours. Furthermore, these data support the notion that VEGF is the sole factor required for tumour angiogenesis as other potentially angiogenic factors could not compensate for the reduced level of VEGF expression in the antisense-VEGF expressing tumours. Our findings also suggest that the inhibition of angiogenesis is sufficient to significantly suppress tumour growth and is thus an effective point for therapeutic intervention for gliomas and all solid tumours.