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1.
Methods ; 96: 75-84, 2016 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-26521976

RESUMO

Understanding the properties and functions of complex biological systems depends upon knowing the proteins present and the interactions between them. Recent advances in mass spectrometry have given us greater insights into the participating proteomes, however, monoclonal antibodies remain key to understanding the structures, functions, locations and macromolecular interactions of the involved proteins. The traditional single immunogen method to produce monoclonal antibodies using hybridoma technology are time, resource and cost intensive, limiting the number of reagents that are available. Using a high content analysis screening approach, we have developed a method in which a complex mixture of proteins (e.g., subproteome) is used to generate a panel of monoclonal antibodies specific to a subproteome located in a defined subcellular compartment such as the nucleus. The immunofluorescent images in the primary hybridoma screen are analyzed using an automated processing approach and classified using a recursive partitioning forest classification model derived from images obtained from the Human Protein Atlas. Using an ammonium sulfate purified nuclear matrix fraction as an example of reverse proteomics, we identified 866 hybridoma supernatants with a positive immunofluorescent signal. Of those, 402 produced a nuclear signal from which patterns similar to known nuclear matrix associated proteins were identified. Detailed here is our method, the analysis techniques, and a discussion of the application to further in vivo antibody production.


Assuntos
Anticorpos Monoclonais/química , Ensaios de Triagem em Larga Escala , Matriz Nuclear/química , Proteoma/administração & dosagem , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/isolamento & purificação , Afinidade de Anticorpos , Especificidade de Anticorpos , Atlas como Assunto , Células HeLa , Humanos , Hibridomas/química , Hibridomas/imunologia , Imunização , Aprendizado de Máquina , Camundongos , Camundongos Endogâmicos BALB C , Matriz Nuclear/imunologia , Análise de Componente Principal , Proteoma/química , Proteoma/imunologia , Vacinação
2.
Gen Physiol Biophys ; 22(2): 279-85, 2003 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-14661739

RESUMO

Interactions of nuclear extract and nuclear matrix proteins from rat hepatocytes with the hormone response element of the alpha2-macroglobulin gene were studied. By Western and South-Western blot analysis we have shown the presence of C/EBPbeta in the examined nuclear fractions as well as its increased binding affinity to the examined gene sequence during the acute-phase response. The results suggest that both nuclear protein fractions could participate in the transcriptional regulation of the alpha2-macroglobulin gene in the rat hepatocytes.


Assuntos
Reação de Fase Aguda/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Núcleo Celular/metabolismo , Hepatócitos/metabolismo , Fatores de Transcrição/metabolismo , alfa-Macroglobulinas/metabolismo , Reação de Fase Aguda/induzido quimicamente , Reação de Fase Aguda/imunologia , Animais , Proteínas Estimuladoras de Ligação a CCAAT/imunologia , Núcleo Celular/imunologia , Células Cultivadas , Hepatócitos/imunologia , Masculino , Matriz Nuclear/imunologia , Matriz Nuclear/metabolismo , Ratos , Ratos Wistar , Fator de Transcrição CHOP , Fatores de Transcrição/imunologia , Terebintina , alfa-Macroglobulinas/imunologia
3.
Cell Biol Int ; 24(7): 483-92, 2000.
Artigo em Inglês | MEDLINE | ID: mdl-10875896

RESUMO

A complex of three proteins (of 80, 70, 58 kDa-p80, p70, and p58, respectively) with the ability to bind alphoid DNA (alpha-satDNA) was revealed by gel mobility shift assay (GMSA) in human nuclear matrix. The probes of the alpha-satDNA bound in the GMSA with the greatest specificity, but the complex was capable of binding human satellite 3 fragment. According to ion exchange and affinity chromatography, the complex includes two DNA-binding proteins, p70 and p80, and a non-DNA-binding one, p58, which enhances the specificity of binding to the alpha-satDNA. GMSA, SDS-PAGE and immunoblotting showed that the lamins, as well as constitutive centromeric proteins (CENP-A, CENP-B, CENP-C, CENP-G), were not incorporated into the complex. It was demonstrated by immunoprecipitation assay that p70 and, probably p58, share a common antigen determinant with the rod domain of intermediate filaments (IF) proteins. The results obtained indicate that the nuclear matrix contains at least one IF-related protein that is able to bind specifically to alpha-satDNA in vitro and that this protein is distinct from the lamins.


Assuntos
DNA Satélite/metabolismo , Proteínas de Ligação a DNA/análise , Matriz Nuclear/química , Proteínas Nucleares/análise , Animais , Ciclo Celular , Fracionamento Celular , Cromatografia , Proteínas de Ligação a DNA/imunologia , Proteínas de Ligação a DNA/metabolismo , Eletroforese em Gel de Poliacrilamida , Cobaias , Humanos , Immunoblotting , Proteínas de Filamentos Intermediários/análise , Peso Molecular , Matriz Nuclear/imunologia , Proteínas Nucleares/imunologia , Proteínas Nucleares/metabolismo
4.
Biochim Biophys Acta ; 1501(2-3): 162-70, 2000 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-10838189

RESUMO

By using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting assays in the presence of polyclonal antiserum raised against electrophoretically specific polypeptides of colorectal cancer nuclear polypeptides with M(r) of 35-40 kDa, we have identified p36 protein whose expression accompanies tumorigenesis of large intestine. Immunological analysis of 35 nuclear protein preparations has indicated expression of p36 antigen in nine of 11 right-sided (81.8%) and 21 of 24 (87.5%) left-sided colorectal tumor cases, but not in any control tissue samples. In this study, we have identified p36 antigen in two colon tumor cell lines, i.e., SW620 and HT29 as well. Fractionation experiments based on selective extraction of nuclei isolated from cancerous specimens, which enables their separation into chromatin, nuclear matrix and its subfraction, i.e., internal and peripheral matrix have revealed the concentration of this particular antigen in the internal matrix.


Assuntos
Antígenos de Neoplasias/análise , Biomarcadores Tumorais/análise , Neoplasias Colorretais/imunologia , Proteínas Nucleares/análise , Antígenos Nucleares , Fracionamento Celular , Eletroforese em Gel de Poliacrilamida , Feminino , Humanos , Immunoblotting , Masculino , Pessoa de Meia-Idade , Matriz Nuclear/imunologia , Proteínas Nucleares/imunologia , Células Tumorais Cultivadas
5.
Appl Biochem Biotechnol ; 83(1-3): 255-68; discussion 268-9, 297-313, 2000.
Artigo em Inglês | MEDLINE | ID: mdl-10826965

RESUMO

The cytotoxicity of DNA-specific autoantibodies from sera of patients with systemic lupus erythematosis (SLE) and with lymphoproliferative diseases, and from blood of healthy donors was examined on tumor-cell lines L929 and HL-60. DNA-binding IgG fractions from SLE and chronic lymphocytic leukemia (CLL) sera were cytotoxic at concentrations of up to 10(-10) M. No detectable changes in cell viability were observed after incubation with antibodies devoid of DNA-binding activity and DNA-specific antibodies isolated from blood of healthy donors and patients with T-cell lymphoma, B-cell lymphosarcoma, and acute B-cell leukemia. There was good correlation between the cytotoxic activity and DNA-hydrolyzing activity of anti-DNA antibodies. The cytotoxic effect of DNA-binding antibodies presumably was complement-independent, because it was attributed only to the Fab fragment. The cytotoxic effect was completely inhibited by preincubation with double-stranded DNA (dsDNA). Both the cytotoxic effect and the DNA-hydrolyzing activity of anti-DNA antibodies were significantly increased in the antibody fraction that displayed cross-reactivity with nuclear matrix proteins. Possible mechanisms for the formation and pathogenicity of cytotoxic anti-DNA antibodies are discussed in this article.


Assuntos
Anticorpos Antinucleares/metabolismo , Anticorpos Catalíticos/metabolismo , Citotoxicidade Imunológica , DNA/imunologia , DNA/metabolismo , Anticorpos Antinucleares/sangue , Anticorpos Catalíticos/sangue , Reações Cruzadas , Células HL-60 , Humanos , Hidrólise , Fragmentos de Imunoglobulinas/sangue , Fragmentos de Imunoglobulinas/metabolismo , Imunoglobulina G/sangue , Imunoglobulina G/metabolismo , Técnicas In Vitro , Lúpus Eritematoso Sistêmico/imunologia , Transtornos Linfoproliferativos/imunologia , Membrana Nuclear/imunologia , Matriz Nuclear/imunologia , Células Tumorais Cultivadas
6.
Arch Dermatol Res ; 292(2-3): 55-9, 2000.
Artigo em Inglês | MEDLINE | ID: mdl-10749556

RESUMO

Serum containing anti-U1RNP antibodies reacts with the nuclear matrix, the relatively insoluble component of the cell nucleus, in addition to U1RNP. In this study, we determine the serum titer and clinical correlations of antinuclear matrix antibodies in samples from patients with anti-U1RNP antibodies. The patients with anti-U1RNP antibodies were classified as having mixed connective tissue disease (MCTD, 15 patients), systemic sclerosis (SSc, 12 patients), systemic lupus erythematosus (SLE, 7 patients), and undifferentiated CTD (UCTD, 9 patients). Antinuclear matrix antibodies were detected using indirect immunofluorescence staining on HCl-treated HEp-2 cells. The antinuclear matrix antibody titer was significantly higher in serum from patients with MCTD or SSc than in serum from patients with SLE or UCTD. The antinuclear matrix antibody titer was significantly increased in serum from patients with sclerodactyly, pitting scars, contracture of the phalanges, and decreased carbon monoxide diffusion capacity. Thus, a higher titer of antinuclear matrix antibodies in serum from patients with anti-U1RNP antibodies may be associated with a clinical diagnosis of MCTD or SSc rather than a diagnosis of SLE or UCTD.


Assuntos
Anticorpos Antinucleares/sangue , Doença Mista do Tecido Conjuntivo/imunologia , Matriz Nuclear/imunologia , Ribonucleoproteínas/imunologia , Adolescente , Adulto , Idoso , Feminino , Genes MHC da Classe II , Humanos , Masculino , Pessoa de Meia-Idade
7.
Acta Cytol ; 43(6): 1015-22, 1999.
Artigo em Inglês | MEDLINE | ID: mdl-10578972

RESUMO

OBJECTIVE: To evaluate, in a preclinical feasibility study, the efficacy of NMP179, a monoclonal antibody recognizing a cervical tumor-associated nuclear matrix antigen, for the early detection of high and low grade cervical intraepithelial neoplasia. STUDY DESIGN: In a blind study involving two clinical sites, NMP179 immunocytochemical staining data from 261 cervicovaginal Thin-Prep specimens were evaluated. Assay sensitivity and specificity were calculated based upon a positive threshold of > 10 immunostained cells per case, using cytologic diagnosis as an end point. RESULTS: Based upon the examination of squamous epithelial cells, NMP179 detected 96.7% of cases with cytologically diagnosed high grade squamous intraepithelial lesions (HSIL) and 70.5% of low grade squamous intraepithelial lesions. The antibody also reacted with 29.6% of normal (within normal limits or benign cellular changes) smears. CONCLUSION: The NMP179 assay detected HSIL with very high accuracy (96.7%). The assay was 79.3% sensitive for the detection of low and high grade cervical intraepithelial neoplasia (grades 1-3), with a specificity of 70.4%. NMP179 may be an effective marker for the early detection of preneoplastic squamous intraepithelial lesions of the cervix and may be useful as an adjunctive tool for better management of cervical intraepithelial neoplasia.


Assuntos
Biomarcadores Tumorais , Carcinoma in Situ/diagnóstico , Carcinoma de Células Escamosas/diagnóstico , Proteínas de Neoplasias/análise , Matriz Nuclear/imunologia , Displasia do Colo do Útero/diagnóstico , Anticorpos Monoclonais , Método Duplo-Cego , Estudos de Viabilidade , Feminino , Humanos , Imuno-Histoquímica , Sensibilidade e Especificidade
8.
Anal Cell Pathol ; 18(4): 175-81, 1999.
Artigo em Inglês | MEDLINE | ID: mdl-10609561

RESUMO

In tumour cell lines, the resistance of cancer cells to a variety of structurally unrelated chemotherapeutic drugs is termed multidrug-resistance or MDR. We reported previously [6] that MDR leukemic cells displayed nuclear texture changes, as assessed by image cytometry. The nature of these changes remained uncertain but they could be associated with alterations of the nuclear matrix which could serve an important role in DNA organization and chromatin structure. Therefore, we have compared the textural features observed in G0/G1 nuclei from human leukemic CEM cells and their MDR variant CEM-VLB, after staining of either DNA by Feulgen method or nuclear matrix by immunodetection of NuMA antigen on DNase treated samples. Chromatin or NuMA distributions within the nucleus were evaluated by image cytometry. Changes in textural parameters indicate that modifications of NuMA distribution observed in MDR cells are parallel to those observed at the whole chromatin level (i.e., a more decondensed and coarse texture with increase of Energy and Long-run sections and decrease of Contrast and Short-run sections). Moreover, Optical Densities measurements indicate that MDR cells seem to contain less NuMA, a datum confirmed by immunoblotting of nuclear proteins. In conclusion, chromatin changes observed by image cytometry in drug-resistant human leukemic CEM cells appear associated with modifications of the nuclear matrix structure.


Assuntos
Resistência a Múltiplos Medicamentos , Resistência a Múltiplos Medicamentos/fisiologia , Leucemia Linfoide/metabolismo , Proteínas Nucleares/metabolismo , Fuso Acromático/metabolismo , Antígenos Nucleares , Autoantígenos/metabolismo , Proteínas de Ciclo Celular , DNA de Neoplasias/metabolismo , Resistência a Múltiplos Medicamentos/imunologia , Humanos , Interfase , Leucemia Linfoide/tratamento farmacológico , Leucemia Linfoide/imunologia , Matriz Nuclear/imunologia , Matriz Nuclear/metabolismo , Proteínas Associadas à Matriz Nuclear , Proteínas Nucleares/imunologia , Fuso Acromático/imunologia , Células Tumorais Cultivadas
9.
Proc Natl Acad Sci U S A ; 96(21): 11970-5, 1999 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-10518560

RESUMO

Previous studies have identified nuclear matrix attachment regions (MARs) that are closely associated with transcriptional enhancers in the IgH, Igkappa, and T cell receptor (TCR) beta loci, but have yielded conflicting information regarding their functional significance. In this report, a combination of in vitro and in situ mapping approaches was used to localize three MARs associated with the human TCR delta gene. Two of these are located within the Jdelta3-Cdelta intron, flanking the core TCR delta enhancer (Edelta) both 5' and 3' in a fashion reminiscent of the Ig heavy chain intronic enhancer-associated MARs. The third is located about 20 kb upstream, tightly linked to Ddelta1 and Ddelta2. We have previously used a transgenic minilocus V(D)J recombination reporter to establish that Edelta functions as a developmental regulator of V(D)J recombination, and that it does so by modulating substrate accessibility to the V(D)J recombinase. We show here that the Edelta-associated MARs function synergistically with the core Edelta to promote V(D)J recombination in this system, as they are required for enhancer-dependent transgene rearrangement in single-copy transgene integrants.


Assuntos
DNA Nucleotidiltransferases/genética , Elementos Facilitadores Genéticos/genética , Matriz Nuclear/genética , Matriz Nuclear/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/genética , Recombinação Genética/fisiologia , Animais , Sequência de Bases , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Genéticos , Dados de Sequência Molecular , Plasmídeos/metabolismo , VDJ Recombinases
10.
J Korean Med Sci ; 14(1): 27-33, 1999 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-10102520

RESUMO

Autoimmune sera have been used in the diagnosis of autoimmune diseases as well as the analysis of nuclear substructures. In an attempt to study the biological characteristics of the nuclear matrix, we screened human sera using immunofluorescent staining and immunoblot. We detected antibodies against nuclear matrix (NM), a remnant nonchromatin protein compartment after the treatment of detergent, salt and nuclease, in 212 out of 284 tested sera (74.6%) by immunoblot. Peptides with molecular weights of 70 kDa, 50 kDa and 25 kDa were detected in the order of frequency. Clinical informations of 198 out of 212 cases were available and went as follows: 38 cases were autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis; 132 non-autoimmune and non-neoplastic diseases; 16 neoplastic diseases and 12 cases unclassified. The immunofluorescent staining intensity by anti-nuclear matrix protein (NMP) antibodies decreased variably, but fibrillogranular, speckled and nucleolar immunolocalization patterns were retained after in situ fractionation. Ku70 and La protein were detected by anti-NMP antibodies. Immunolocalization by anti-NMP antibodies indicates that the NMPs constitute a variety of characteristic nuclear substructures and may serve as autoantigens in diverse human diseases. In addition, the presence of Ku70 and La protein as NMPs suggests that the NM can be functionally active in association with DNA or RNA.


Assuntos
Antígenos Nucleares , Autoantígenos/análise , DNA Helicases , Proteínas de Ligação a DNA/análise , Proteínas Nucleares/análise , Ribonucleoproteínas/análise , Doenças Autoimunes/sangue , Doenças Autoimunes/imunologia , Sequência de Bases , DNA Complementar , Técnica Indireta de Fluorescência para Anticorpo , Células HeLa , Humanos , Immunoblotting , Autoantígeno Ku , Dados de Sequência Molecular , Matriz Nuclear/imunologia , Células Tumorais Cultivadas , Antígeno SS-B
11.
J Immunol ; 162(1): 292-8, 1999 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-9886398

RESUMO

The X-linked lymphocyte-regulated (Xlr) protein is a 30,000 Mr nuclear protein bearing homology with meiosis-specific proteins and expressed in late stage B lymphoid cell lines. In the present study we investigated its expression in the T lymphoid lineage. In adults, a high level of expression was detected in CD4-CD8- thymocytes. Most remarkably, the peak of Xlr expression occurred early during thymus cell ontogeny, precisely on days 14-15 of gestation, and was associated with the first wave of pre-T cell differentiation. Its onset preceded the rearrangement of TCR genes, as Xlr expression was conserved in thymus cells from RAG1(0/0) mice. The lower expression of Xlr on day 13 of fetal development, the bright Thy1+ phenotype of Xlr-positive cells, their large size, and their absence from subcapsular areas suggest that Xlr expression must be turned on within the thymus and not in prethymic precursors. From day 16 of gestation, Xlr expression decreased markedly. At birth and later, Xlr(high) cells were mostly large cells scattered throughout the cortical area. As shown by confocal microscopy, expression of Xlr closely overlapped that of SATB1, which binds special AT-rich DNA sequences associated with the nuclear matrix and plays an important regulatory role for many genes. The remarkably regulated expression of Xlr in the lymphoid cell lineage and of its homologue Xmr in the germ cell lineage suggests that they might play an important role in chromatin metabolism at critical stages of differentiation during which the genome undergoes irreversible rearrangements.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação à Região de Interação com a Matriz , Matriz Nuclear/metabolismo , Proteínas Nucleares/biossíntese , Subpopulações de Linfócitos T/metabolismo , Timo/metabolismo , Animais , Western Blotting , Diferenciação Celular/imunologia , Desenvolvimento Embrionário e Fetal/imunologia , Feminino , Masculino , Meiose , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , Microscopia de Fluorescência , Matriz Nuclear/imunologia , Proteínas Nucleares/química , Proteínas Nucleares/isolamento & purificação , Ligação Proteica/imunologia , Timo/citologia , Cromossomo X
13.
Artigo em Inglês | WPRIM (Pacífico Ocidental) | ID: wpr-96717

RESUMO

Autoimmune sera have been used in the diagnosis of autoimmune diseases as well as the analysis of nuclear substructures. In an attempt to study the biological characteristics of the nuclear matrix, we screened human sera using immunofluorescent staining and immunoblot. We detected antibodies against nuclear matrix (NM), a remnant nonchromatin protein compartment after the treatment of detergent, salt and nuclease, in 212 out of 284 tested sera (74.6%) by immunoblot. Peptides with molecular weights of 70 kDa, 50 kDa and 25 kDa were detected in the order of frequency. Clinical informations of 198 out of 212 cases were available and went as follows: 38 cases were autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis; 132 non-autoimmune and non-neoplastic diseases; 16 neoplastic diseases and 12 cases unclassified. The immunofluorescent staining intensity by anti-nuclear matrix protein (NMP) antibodies decreased variably, but fibrillogranular, speckled and nucleolar immunolocalization patterns were retained after in situ fractionation. Ku70 and La protein were detected by anti-NMP antibodies. Immunolocalization by anti-NMP antibodies indicates that the NMPs constitute a variety of characteristic nuclear substructures and may serve as autoantigens in diverse human diseases. In addition, the presence of Ku70 and La protein as NMPs suggests that the NM can be functionally active in association with DNA or RNA.


Assuntos
Humanos , Autoantígenos/análise , Doenças Autoimunes/imunologia , Doenças Autoimunes/sangue , Sequência de Bases , DNA Complementar , Proteínas de Ligação a DNA/análise , Técnica Indireta de Fluorescência para Anticorpo , Células HeLa , Immunoblotting , Dados de Sequência Molecular , Matriz Nuclear/imunologia , Proteínas Nucleares/análise , Ribonucleoproteínas/análise , Células Tumorais Cultivadas
14.
Genes Dev ; 12(7): 996-1009, 1998 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-9531537

RESUMO

The nuclear matrix antigen recognized by the monoclonal antibody (mAb) B1C8 is a novel serine (S) and arginine (R)-rich protein associated with splicing complexes and is named here SRm160 (SR-related matrix protein of 160 kD). SRm160 contains multiple SR repeats, but unlike proteins of the SR family of splicing factors, lacks an RNA recognition motif. SRm160 and a related protein SRm300 (the 300-kD nuclear matrix antigen recognized by mAb B4A11) form a complex that is required for the splicing of specific pre-mRNAs. The SRm160/300 complex associates with splicing complexes and promotes splicing through interactions with SR family proteins. Binding of SRm160/300 to pre-mRNA is normally also dependent on U1 snRNP and is stabilized by U2 snRNP. Thus, SRm160/300 forms multiple interactions with components bound directly to important sites within pre-mRNA. The results suggest that a complex of the nuclear matrix proteins SRm160 and SRm300 functions as a coactivator of pre-mRNA splicing.


Assuntos
Antígenos Nucleares , Proteínas Associadas à Matriz Nuclear , Proteínas Nucleares/genética , Precursores de RNA/metabolismo , Proteínas de Ligação a RNA/genética , Spliceossomos/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Células HeLa/metabolismo , Humanos , Interfase/fisiologia , Linfoma Difuso de Grandes Células B/patologia , Metáfase/fisiologia , Dados de Sequência Molecular , Matriz Nuclear/imunologia , Proteínas Nucleares/imunologia , Precursores de RNA/genética , Proteínas de Ligação a RNA/imunologia , Ribonucleoproteína Nuclear Pequena U1/metabolismo , Ribonucleoproteína Nuclear Pequena U2/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Spliceossomos/genética , Células Tumorais Cultivadas
15.
Hybridoma ; 17(5): 413-20, 1998 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-9873986

RESUMO

Two hybridoma clones, NMB1 and NML90, were established using nuclear matrix proteins from normal human thymi or malignant lymphoma as immunogens. They reacted with human Ku70 and Ku80, respectively, by immunoblotting. When HeLa cell nuclear proteins were fractionated and applied to immunoblotting, both Ku70 and Ku80 were detected in the nuclear matrix as well as the soluble nuclear protein fractions. By confocal scanning microscopy, the immunoreactivity of Ku70 and Ku80 was localized to distinct nucleoplasmic fibrillar network and fine granules in the interphase cell nuclei. When HeLa cells were fractionated in situ using DNase I and buffers containing 0.25 M (NH4)2SO4 and 2 M NaCl, the nucleoplasmic reticular structure was largely preserved, but granules disappeared. The nucleoplasmic distribution of Ku in the tissue and in cultured cells was distinct from each other. In the adult tissue, it consisted mostly of either distinct curvilinear lines along the nuclear periphery or of tangled, beaded lines throughout the nuclei. When xenotransplants of HeLa cell in Scid mice were examined, the "tissue type" immunolocalization pattern was reproduced consistently. In most fetal tissues, "tissue type" and "cell type" patterns were admixed. Monoclonal antibodies described here are useful tools for studying the structure and function of the nuclear matrix.


Assuntos
Antígenos Nucleares , DNA Helicases , Proteínas de Ligação a DNA/análise , Hibridomas , Matriz Nuclear/química , Proteínas Nucleares/análise , Animais , Anticorpos Monoclonais , Células Cultivadas , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/imunologia , Células HeLa , Humanos , Immunoblotting , Autoantígeno Ku , Fígado/embriologia , Fígado/ultraestrutura , Neoplasias Hepáticas/ultraestrutura , Masculino , Camundongos , Camundongos SCID , Microscopia Confocal , Microscopia de Fluorescência , Matriz Nuclear/imunologia , Proteínas Nucleares/química , Proteínas Nucleares/imunologia , Testes de Precipitina , Espermatogônias/ultraestrutura , Espermatozoides/ultraestrutura , Testículo/ultraestrutura , Distribuição Tecidual
16.
Cell Biol Int ; 21(6): 367-73, 1997 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-9268490

RESUMO

While much evidence indicates a high degree of spatial organization in the nucleus, the underlying molecular structures that support it remain poorly characterized. By extracting with high concentrations of RNase A in a modification of the sequential extraction protocol of Penman, we have identified a novel intranuclear network in the mouse lymphoma cell line, EL-4. Micrographs of embedment-free sections of extracted cells reveal anastomosing filaments of two different diameters: 3-5 nm and 8-10 nm. The 3-5-nm filaments are interconnected in many junctions and appear to blend smoothly into each other. The 8-10-nm fibers frequently split into two 3-5-nm filaments. Some 3-5-nm fibers appear to be connected at 90 degrees angles with the 8-10-nm fibers. All junctions are smooth with no apparent junction protein. Flow cytometric analysis of RNase A- (and DNase I-) extracted nuclear matrices indicates that they do not contain significant amounts of protein that react with anti-actin and anti-vimentin monoclonal antibodies. Extraction of EL-4 nuclear matrices with high salt does not reveal 8-10-nm core filaments described after similar treatment of tumor cell lines of cervical and mammary origin. The novel characteristics of the core filaments in EL-4 lymphoma cells may reflect cell-type specificity of the nuclear matrix.


Assuntos
Linfoma , Matriz Nuclear/química , Matriz Nuclear/ultraestrutura , Actinas/análise , Actinas/imunologia , Animais , Especificidade de Anticorpos , Citometria de Fluxo , Proteínas de Filamentos Intermediários/análise , Proteínas de Filamentos Intermediários/imunologia , Camundongos , Microscopia Eletrônica , Matriz Nuclear/imunologia , RNA/fisiologia , Células Tumorais Cultivadas/ultraestrutura
17.
Acta Urol Belg ; 65(1): 55-8, 1997 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-9175284

RESUMO

The early diagnosis of bladder cancer is central to the effective treatment of the disease. Presently, the detection of bladder tumors relies on cystoscopy and there are no methods available to easily and specifically identify the presence of bladder cancer cells. A variety of new technologies and potential tumor markers are being studied in bladder cancer and some are being translated into clinical use. It is important to realise that all available results on the diagnostic value of tumor markers do not allow firm clinical recommendations, but tests based on biomarkers will undoubtedly influence the management of bladder cancer in the near future.


Assuntos
Biomarcadores Tumorais/sangue , Neoplasias da Bexiga Urinária/diagnóstico , Antígenos de Neoplasias/isolamento & purificação , Antígenos de Grupos Sanguíneos/imunologia , Fibronectinas/sangue , Humanos , Isoantígenos/isolamento & purificação , Matriz Nuclear/imunologia , Antígeno Polipeptídico Tecidual/isolamento & purificação , Neoplasias da Bexiga Urinária/imunologia
18.
J Cell Sci ; 110 ( Pt 24): 3031-41, 1997 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-9365273

RESUMO

A hnRNP-free nuclear matrix prepared from chicken MSB-1 cells was used to raise monoclonal antibodies. The monoclonal antibodies 2H3 and 3B7 showed identical non-homogeneous immunofluorescence staining patterns of nuclei in MSB-1 cells and chicken embryonic fibroblasts. In a synchronized culture of MSB-1 cells, the immunoreactivity of nuclei with 2H3, but not with 3B7, antibody decreased markedly during the progression of S phase, but returned to the normal level at the next G1 phase. When cells were treated with Triton X-100 prior to fixation with paraformaldehyde or cells were fixed in methanol, nuclei were reactive with 2H3 antibody throughout the S phase. Both 2H3 and 3B7 antibodies recognized a high molecular mass nuclear antigen (HMNA) of approximately 550 kDa, which was associated with the nuclear matrix. HMNA was resistant to extraction with 0.5 M NaCl from the nuclei at the G1/S boundary but became extractable by the end of S phase. A cDNA clone, pBHB36, containing a partial sequence for HMNA was isolated by immunoscreening as a double positive clone with 2H3 and 3B7 antibodies. The deduced 1,150 residue-long sequence of pBHB36 shows no homology with any molecules in the nucleotide and protein sequence databases, and contains different epitope regions for 2H3 and 3B7 antibodies. A possibility of hydrophobic association of HMNA with nuclear protein(s) during the progression of S phase is discussed.


Assuntos
Matriz Nuclear/imunologia , Proteínas Nucleares/imunologia , Fase S , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Antígenos Nucleares , Sequência de Bases , Western Blotting , Linhagem Celular , Galinhas , DNA Complementar , Epitopos/imunologia , Imunofluorescência , Dados de Sequência Molecular , Peso Molecular , Proteínas Nucleares/química , Proteínas Nucleares/genética , RNA Mensageiro/genética , Fase S/imunologia
19.
Cell Biol Int ; 20(11): 769-75, 1996 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-8979370

RESUMO

The anti-desmin monoclonal antibodies (MAbs) DSB389, AC54, and DSB860 recognize intermediate filaments (IFs) and nuclear antigens that appear granular, locate around chromosomes, and are insoluble following 0.5% Triton X-100 and cetyltrimethylammonium bromide (CTAB) extraction. Nuclear antigens of the MAbs were searched for in an IFs-deficient clone of SW13 cells. Reactive materials specific to DSB389, AC54, and DSB860 MAbs were trapped at the top of the gel of SDS-agarose-PAGE. The reactivity of the materials disappeared after treatment with DNase I. The reactivity, or trapping of this material at the top of the gel, required previous heat treatment of the sample before application to the gel. The MAbs recognized both single-stranded and double-stranded DNA in ELISA. These results indicate that at least the main nuclear antigens of DSB389, DSB860 and AC54 MAbs are DNA.


Assuntos
Anticorpos Monoclonais/imunologia , DNA de Cadeia Simples/imunologia , DNA/imunologia , Desmina/imunologia , Matriz Nuclear/imunologia , Proteínas Nucleares/imunologia , Adenocarcinoma/imunologia , Adenocarcinoma/patologia , Especificidade de Anticorpos , Antígenos de Neoplasias/imunologia , Antígenos Nucleares , Cetrimônio , Compostos de Cetrimônio/farmacologia , DNA/isolamento & purificação , DNA de Neoplasias/imunologia , DNA de Cadeia Simples/isolamento & purificação , Detergentes/farmacologia , Ensaio de Imunoadsorção Enzimática , Técnica Indireta de Fluorescência para Anticorpo , Células HeLa/efeitos dos fármacos , Células HeLa/imunologia , Humanos , Immunoblotting , Filamentos Intermediários/imunologia , Filamentos Intermediários/ultraestrutura , Proteínas Nucleares/isolamento & purificação , Octoxinol/farmacologia , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/imunologia
20.
Histochem Cell Biol ; 105(1): 17-25, 1996 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-8824902

RESUMO

Previous studies have characterized pp125FAK as a focal adhesion (FA)-associated non-receptor tyrosine kinase. However, there are few data available on the expression and localization of this kinase in tissues. In this study we show that in human tissues the highest expression of pp125FAK is found in some developing epithelia, where pp125FAK is associated with either intercellular junctions or with sites of adhesion to the basement membrane, whereas the same adult tissues show only a faint reactivity. Connective tissue cells do not show any reactivity for pp125FAK in vivo, but developing arterial smooth muscle expresses pp125FAK at high levels. The expression pattern in malignant tissues is variable, but most carcinomas do not express this kinase. In primary cultures of human amnion epithelial cells pp125FAK first becomes associated with the polarized adhesion lamellae, but is subsequently translocated to the forming adherens junctions (AJs). Later upon culturing pp125FAK becomes associated with prominent FAs, as in cultured cell lines. Taken together, our results suggest that the association of pp125FAK with FAs in cultured cells is principally due to a process of adaptation, whereas in vivo pp125FAK mainly functions as a regulatory component of intercellular AJs and cell-matrix adhesions of developing epithelia and also in developing arterial smooth muscle.


Assuntos
Âmnio/imunologia , Moléculas de Adesão Celular/imunologia , Junções Intercelulares/imunologia , Matriz Nuclear/imunologia , Proteínas Tirosina Quinases/imunologia , Adulto , Âmnio/citologia , Âmnio/embriologia , Âmnio/metabolismo , Carcinoma/imunologia , Carcinoma/metabolismo , Carcinoma/patologia , Moléculas de Adesão Celular/metabolismo , Células Cultivadas , Neoplasias do Colo/imunologia , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Epitélio , Feminino , Quinase 1 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Neoplasias Gastrointestinais/imunologia , Neoplasias Gastrointestinais/metabolismo , Neoplasias Gastrointestinais/patologia , Humanos , Junções Intercelulares/metabolismo , Matriz Nuclear/metabolismo , Gravidez , Proteínas Tirosina Quinases/metabolismo
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