PubMed Health⌕ Search

Biomedical subjects

R K Busch

Publications and source records attributed to R K Busch.

At least 37 records · Page 2Linked to original sources
PubMed1990

Phosphorylation of the human cell proliferation-associated nucleolar protein p120.

The human cell proliferation-associated nucleolar protein p120 was found in a variety of human cancer specimens but not in most normal resting cells. Polyclonal antibodies raised against bacterially expressed p120 were used to immunoprecipitate the p120 protein isolated from 32P-labeled HeLa cells. The p120 protein was phosphorylated at serine, threonine and tyrosine residues. A tryptic peptide map showed it contained three labeled peptides. One of these peptides comigrated with a p120 peptide phosphorylated in vitro by casein kinase II. This peptide was phosphorylated in vitro both at Ser-181 and Thr-185. This region is juxtaposed to the epitope site recognized by the anti-p120 monoclonal antibody.

Amino Acid Sequence↗
PubMed1990

Characterization of U6 small nuclear RNA cap-specific antibodies. Identification of gamma-monomethyl-GTP cap structure in 7SK and several other human small RNAs.

The cap structure in human U6 small nuclear (sn)RNA, gamma-monomethylguanosine triphosphate (meGTP), was conjugated to human serum albumin and used as antigen to raise polyclonal antibodies in rabbits. The resulting antibodies reacted specifically with meGTP but not with GTP, GDP, GMP, meGMP, meATP, meCTP, meUTP, or with methyl phosphate in enzyme-linked immunosorbent assay and/or in radioimmunoassays. Although less efficiently, meGDP was also recognized by these antibodies. Indirect immunofluorescence studies with anti-meGTP antibodies showed predominantly nuclear immunofluorescence. Anti-meGTP antibodies immunoprecipitated intact U6 snRNA from a mixture of HeLa cell RNAs. In addition to the U6 snRNA, anti-meGTP antibodies immunoprecipitated several additional small RNAs that varied in length from approximately 50 to 330 nucleotides. These RNAs contained the meGTP cap structure and are structurally distinct from U6 snRNA. One of these meGTP-containing RNAs was found to be previously characterized 7SK RNA; human 7SK RNA synthesized in vitro also contained the same cap structure. Results obtained in this study provide evidence for the presence of gamma-monomethyl-GTP cap structure in a wide spectrum of human cellular RNAs. These antibodies will be useful in studying the structure and function of this new family of small RNAs.

Antibodies↗
PubMed1990

Identification of an epitope region of the human proliferation-associated nucleolar antigen P120.

An epitope region, located at amino acid residues 173-180 (EAAA-GIQW), of a human cell proliferation-associated nucleolar antigen, P120, has been defined by mutational analysis and competition assays. A synthetic peptide corresponding to this epitope region completely blocks the binding of the anti-P120 antibody to Escherichia coli-expressed P120 and the HeLa nucleolar P120 protein. Adjacent peptides lack inhibitory effects. The antigenic site includes a hydrophilic residue and a hydrophobic stretch. The glutamyl and tryptophanyl residues in this region make major contributions to the binding of P120 to its antibody, since peptides lacking either the glutamyl or the tryptophanyl residue do not block the antibody binding to the P120 antigen. This study provides a basis for drug design for specific binding to the epitope region of the P120 protein.

Antigens, Nuclear↗
PubMed1989

Proliferation-related nucleolar antigens P145 and P120 associated with separate nucleolar elements and differences in tissue distribution.

Nucleolar antigens p145 and p120 are associated with proliferating cells (Freeman, J.W.; McRorie, D.K.; Busch, R.K.; Gyorkey, P.; Gyorkey, F.; Ross, B.E.; Spohn, W.H.; Busch, H. Cancer Res. 46:3593; 1986 and Freeman, J.W.; Busch, R.K.; Gyorkey, P.; Gyorkey, F.; Ross, B.E.; Busch, H. Cancer Res. 48:1244; 1988) and are not detectable in normal resting cells. Recent immunoelectron microscopic studies (Ochs, R.L.; Reilly, M.T.; Freeman, J.W.; Busch, H. Cancer Res. 48:6523; 1988) suggest that the two antigens have overlapping nucleolar localizations. In this study the nucleolus was physicochemically and biochemically studied to determine whether p145 and p120 were associated with a common nucleolar component. Antigen p145 was associated with 40-80 S ribonucleoprotein particles (RNPs), and the p145 antigen was not detected in HeLa cells following in situ RNAse digestion. P120 was found in a 40-80 S, RNAse resistant complex. Sequential extraction of HeLa nucleoli showed that most of antigen p145 was extractable in 10 mM Tris with 0.2% deoxycholate, whereas p120 was found in a nucleolar residue fraction requiring DNAse and high salt treatment for optimal extraction. Neither antigen p145 nor p120 was detectable in normal resting tissues. Antigen p145 was detected in all proliferating tissues examined, including a variety of malignant tumors (ten of ten), benign tissues including adenomas and hyperplasias (eight of eight), and in normal proliferating cells such as colonic epithelium and spermatogonia of the testes. Antigen p120 was not detected in all tumors, being absent in three of seven lymphomas and in one melanoma examined.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigens, Nuclear↗
PubMed1989

mRNA levels for human nucleolar protein P120 in tumor and nontumor cells.

A monoclonal antibody to a human tumor nucleolar 120 kD protein was developed by Freeman et al. (Cancer Res. 48: 1244-1251, 1988). Its complementary DNA (cDNA) has been isolated and sequenced (Fonagy et al., submitted). To determine the relative messenger RNA (mRNA) level for protein p120, cellular mRNA was extracted, slot-blotted onto nitrocellulose filters, and hybridized to radioactive p120 cDNA fragments. Human tumor cells contained 15-60 times more p120 mRNA than human term placenta. The rat Novikoff hepatoma ascites cell mRNA hybridized to the p120 cDNA probes, but the p120 monoclonal antibody did not react with the Novikoff hepatoma proteins. Novikoff hepatoma mRNA contained 8 times as much p120 mRNA as normal rat liver. As a control, a cDNA was used for protein B23, an abundant nucleolar protein; there were 3.5, 29, and 14 times more B23 mRNA than p120 mRNA in normal rat liver, Novikoff hepatoma ascites cells, and HeLa cells, respectively. Whereas the increased levels of the mRNA and protein B23 reflect increased activity of the nucleolus for any increment of nucleolar function, the increased levels of p120 mRNA and the p120 protein reflect the activity of the G1 phase of the cell cycle. The elevated level of p120 mRNA in tumors may reflect the heightened G1 cascade in transformed cells.

Animals↗
PubMed1989

Cloning of the cDNA and sequence of the human proliferating-cell nucleolar protein P120.

The 120 kDa proliferating-cell nucleolar antigen described by Freeman et al. (Cancer Res. 48:1244; 1988) is the most cancer specific of the proliferation-associated nucleolar proteins identified thus far. It is localized in a novel nucleolar microfibrillar structure recently described by Ochs et al. (Cancer Res. 48:6523; 1988). The amino acid sequence has been determined by a combination of cDNA and genomic DNA sequences. This molecule contains, consecutively, four major domains: a basic domain, an acidic domain, a hydrophobic and methionine-rich domain, and a domain rich in cysteine and proline residues. The isolated cDNA was shown to code for the HeLa P120 protein as shown by a similarity in immunoreactivity, mobility on sodium dodecylsulfate-polyacrylamide gel electrophoresis, and patterns of partial digestion of the Escherichia coli-expressed P120 and the HeLa nucleolar P120 protein. This protein is of special interest because it is expressed in early G1 and, in studies to date, it has not been detected in benign tumors and most normal resting tissues.

Amino Acid Sequence↗
PubMed1988

Identification and characterization of a human proliferation-associated nucleolar antigen with a molecular weight of 120,000 expressed in early G1 phase.

Tumor nucleoli were treated with polyclonal antisera to normal human tissue nucleoli to block some determinants common to tumor and normal tissue nucleoli. Immunization of mice with these immune complexes resulted in the development of a monoclonal antibody (FB2) to a novel Mr 120,000 nucleolar proliferation-associated antigen. By indirect immunofluorescence, antibody FB2 produced bright nucleolar staining in a variety of malignant tumors, including cancers of the breast, liver, gastrointestinal tract, genitourinary tract, blood, lymph system, lung, and brain. Although specific nucleolar immunofluorescence was not detectable in most normal tissues, it was detectable in some proliferating nonmalignant tissues including spermatogonia of the testes, ductal regions of hypertrophied prostates, and phytohemagglutinin-stimulated lymphocytes. The Mr 120,000 antigen was not detectable in 48-h serum-deprived HeLa cells but was readily detectable (within 30 min) following serum refeeding. The Mr 120,000 antigen was not detected in retinoic acid-treated HL-60 cells following morphological differentiation but was detectable in 48-h phytohemagglutinin-treated lymphocytes. These studies suggest that the Mr 120,000 antigen is a proliferation-associated antigen which plays a role in the early G1 phase of the cell cycle.

Antibodies, Monoclonal↗
PubMed1987

Studies on satellite nucleoli in rat hepatocytes and Novikoff hepatoma cells.

The present study was undertaken to provide information on the presence and frequency of satellite nucleoli in cells with increased nucleolar biosynthetic activity. The number of hepatocytes containing satellite nucleoli was analyzed in rat liver, regenerating liver 18 h after partial hepatectomy and in Novikoff hepatoma ascites cells. In comparison with hepatocytes of normal liver, the number of both stimulated hepatocytes and malignant hepatoma cells containing satellite nucleoli was significantly reduced. The results also indicated that whereas most satellite nucleoli contain protein C23, a smaller percentage contain protein B23.

Animals↗
PubMed1987

Novel nucleolar antigens in autoimmune disease.

Antinucleolar antibodies are of interest in 2 important areas, namely, autoimmune diseases and specific products involved in the "mitogenic cascade." The former have delineated a series of novel nuclear and nucleolar elements including the recently described proliferating cell nuclear antigens (PCNA), some of which are present in the nucleolus only at specific times in the G1-S phase of the cell cycle. Important new proteins such as "fibrillarin" (34 kD/pI 8.5) and a 125 kD nucleotide containing protein have been identified with antinucleolar antibodies. Protein p145 is a nucleolar PCNA that is present in growing and dividing cells but not in normal resting tissues. Antinucleolar antibodies offer powerful tools, not only for identification of specific nucleolar proteins important in the cell cycle, but also for purification of their genes and analysis of mechanisms of gene control that operate the "time windows" of the cell cycle.

Amino Acid Sequence↗
PubMed1986

Identification and partial characterization of a nucleolar antigen with a molecular weight of 145,000 found in a broad range of human cancers.

Previous studies in our laboratory have indicated the presence of nucleolar antigens in tumors which were not detected in normal tissues. Some of the polyclonal antisera produced in these studies were shown to identify a Mr, 145,000 nucleolar antigen on immunoblots of tumor nucleoli but not in normal human liver nucleoli. A monoclonal antibody to a Mr 145,000 nucleolar protein (p145) was produced by immunization of mice with a nucleolar extract of HeLa cells which is enriched with this antigen. The monoclonal antibody showed bright nucleolar immunofluorescence localization in a broad range of human tumors including cancers of the gastrointestinal tract, genitourinary tract, lung, liver, muscle, cartilage, and blood. The p145 nucleolar antigen was not detected in most normal human tissues or in benign tumors, with only weak nucleolar staining observed in spermatogonia of the testes and in ductal regions of some hypertrophied prostates. Nucleolar antigen p145 was extracted from HeLa cell nucleoli by homogenization in a 0.01 M Tris buffer containing 0.2% deoxycholate. On sucrose density gradient centrifugation, the antigen remained sedimented with the nucleolar ribonucleoprotein fraction. Nucleolar antigen p145 was released from ribonucleoproteins following treatment with 4 M guanidinium hydrochloride or RNase. Peptide mapping of nucleolar antigen p145 showed that it was distinct from other known nucleolar antigens. Although it remains to be determined if the p145 antigen plays a role in cell transformation, maintenance of the malignant phenotype, or in cell division, it may have value as a tumor marker or as a therapeutic target.

Adenofibroma↗
PubMed1985

Effects of actinomycin D analogs on nucleolar phosphoprotein B23 (37,000 daltons/pI 5.1).

Localization of protein B23 in HeLa cells after treatment with actinomycin D and its analogs was studied using indirect immunofluorescence. Bright nucleolar fluorescence was observed in control HeLa cells. After treatment with actinomycin D (250 ng/ml) for 2 hr, a uniform nucleoplasmic fluorescence was observed. Similar results were obtained with the actinomycin analogs, actinomycin Z5 and actinomycin K2T. Only after a much longer incubation (24 hr) with actinomycin 4-4'-gly was nucleoplasmic fluorescence observed. Actinomycin D, actinomycin Z5, and actinomycin K2T inhibited [3H]uridine incorporation into the trichloroacetic acid insoluble fraction of HeLa cells with IC50 values of 9.5 +/- 3.2, 59.1 +/- 19.6 and 1423.3 +/- 212.2 ng/ml respectively. No inhibition of [3H]uridine incorporation was observed using actinomycin 4-4'-gly (2000 ng/ml, 2-hr incubation). The order of potency for the loss of nucleolar fluorescence and the concurrent increase in nucleoplasmic fluorescence was actinomycin D greater than actinomycin Z5 greater than actinomycin K2T greater than actinomycin 4-4'-gly, which correlated with the order of their IC50 values for inhibition of [3H]uridine incorporation. Studies of the effects of actinomycin D and its analogs on RNA synthesis and localization of protein B23 indicated that there is a direct relationship between the B23 "translocation" from nucleolus to nucleoplasm and the inhibition of RNA synthesis. At 45-55% inhibition of RNA synthesis, both nuclear and nucleolar B23 immunofluorescence were observed. At 75-85% inhibition, only a uniform nucleoplasmic fluorescence was observed.

Cell Nucleolus↗
PubMed1985

Electrophoretic analysis of HeLa cell and human liver nucleolar proteins and antigens.

The major antigens in HeLa nucleolar extracts recognized by immunoblots with rabbit antisera had molecular weights of 145, 110, and 34 kd. The major antigens in the HeLa nucleolar residues had molecular weights of 145, 110, 86, 68, 55, 48, and 34 kd. In the liver, the major antigens in the nucleolar extracts had molecular weights of 86 and 76 kd. In the liver residues, the major antigens had molecular weights of 110, 86, 76, 65, and 55 kd. On two-dimensional gels stained with Coomassie blue, the HeLa nucleolar extract contained large amounts of protein B23 and lesser amounts of protein C23. In the liver nucleolar samples separated on two-dimensional (2-D) gels, protein 55/7.6 (Mr/pI) was the major protein in the extract. Lesser amounts of protein B23 were identified. Addition of protease inhibitors markedly improved the quality of the protein samples as shown in one-dimensional gel patterns for liver nucleolar proteins and to a lesser extent for HeLa nucleolar proteins. In the 2-D immunoblots of the HeLa extract and HeLa residues, the major stained band was protein C23 (110/5.2-5.6). In the liver extract, the major bands were 70/5.8-7 and 60/5.8-7. With a monoclonal antibody (MS-3) to protein C23 a 76/5.8 band was more notable in the residue which supported the results of the Coomassie blue and immunostains with rabbit antinucleolar antibodies. Some degradation products of protein C23 were observed in both the liver extract and the liver residue despite the use of protease inhibitors. Protein C23 along with other proteins are major antigens in HeLa nucleoli. In human liver nucleoli, a major protein 55/7.6 was identified which was not observed in the HeLa extracts. These studies show that a combination of protease inhibitors markedly reduced degradation of proteins in liver samples and provided a more satisfactory sample for comparison with HeLa cells. Qualitative and quantitative differences were found in the nucleolar proteins by Coomassie blue and immunostaining.

Antigens↗
PubMed1985

Masking of nontumorous antigens for development of human tumor nucleolar antibodies with improved specificity.

Nucleoli purified from HeLa cells were spread by homogenization in low ionic strength buffer and by chelation of divalent cations. The antigenic determinants of these tumor nucleoli that are shared by normal liver nucleoli were masked by addition of rabbit anti-liver nucleolar antisera to form immune complexes. New Zealand White rabbits were immunized with these antibody-nucleolar complexes. The resulting antisera produced nucleolar fluorescence in HeLa cells but not in normal human liver or human kidney cells. One- and two-dimensional immunoblots identified a major Mr 58,000/pl 5.8 antigen in HeLa cells, which was not detected on corresponding immunoblots of normal human liver nucleoli. This method offers an improved approach to selection of tumor-associated antigens.

Animals↗
PubMed1984

Actinomycin D inhibition of monoclonal antibody binding to nucleolar phosphoprotein 37/5.2 (B23).

Actinomycin D was found to block binding of a monoclonal antibody to nucleolar phosphoprotein 37/5.2 (B23) when incubated either simultaneously with or prior to addition of the antibody. Daunorubicin had no significant blocking activity of this antigen-antibody reaction. In comparative studies with a monoclonal antibody to nucleolar phosphoprotein 110/5.2 (C23), actinomycin D exhibited little blocking activity. When a 42-mer peptide containing the epitope of protein 37/5.2 (B23) was tested as the antigen, similar inhibition by actinomycin D of binding of the monoclonal antibody was found. These results provide evidence for binding of actinomycin D to a specific epitope of protein 37/5.2 (B23).

Amino Acid Sequence↗
PubMed1984

Further studies on satellite nucleoli in rat and mouse hepatocytes.

To provide more information on satellite nucleoli, these nuclear structures were studied by means of cytochemical and immunofluorescence procedures in rat and mouse hepatocytes without and following experimental inhibition of the RNA synthesis. The immuno-staining specific for nucleoli or B23 as well as C23 proteins demonstrated that satellite nucleoli and characteristic nucleoli exhibit the same fluorescence. The number of satellite nucleoli decreased after inhibition of nucleolar RNA synthesis in a similar way to the number of silver-stained granules (SSGs) of characteristic nucleoli. Inhibition of RNA synthesis also reduced the number of hepatocytes containing satellite nucleoli. Thus, satellite nucleoli seem to be real nucleoli from single NORs which did not fuse in the formation of a characteristic nucleolus.

Animals↗
PubMed1983

Identification and purification of Namalwa nuclear RNP antigen 52/5.3.

Using affinity purified rabbit antibodies to HeLa nucleoli and the Western blotting techniques, an antigen with an approximate molecular weight of 52,000 and pI of 5.3 was found in Namalwa cells (a Burkitt lymphoma), but not in normal liver cells. This antigen was purified from Namalwa RNP by column chromatography on Sephacryl S-200, hydroxylapatite and one-dimensional SDS gel electrophoresis. A liver protein with the same molecular weight and pI value was purified from RNP fraction by one-dimensional SDS gel electrophoresis. Both proteins had similar amino-acid compositions. The tryptic map of 125I-labeled protein 52/5.3 contained approximately nine major spots; spot 9 was present in the Namalwa protein but not in the liver protein. The similarity of the structures of these proteins and their differences in antigenicity are noteworthy and require further structural and functional analysis.

Amino Acids↗
PubMed1983

Ultrastructural and purification studies on human tumor nucleolar antigens and nucleolar particles.

The presence of common nucleolar antigens in a broad array of human malignant tumors has led to several lines of investigations. In addition to studies on an increasing number of benign and malignant neoplasms with a variety of antibodies designed to statistically evaluate the presence of nucleolar antigens, purification procedures and chemical analyses are being used to characterize the specific antigens. The localization of the nucleolar antigens in HeLa cells was studied by the postembedding immunoelectron microscopic procedure employing rabbit antibodies to nucleoli or nuclear Tris extracts of these cells. The products of the peroxidase-antiperoxidase complexes visualized by the reaction with diaminobenzidine in nucleoli were mainly found in the nucleolonemas which contain the dense nucleolar RNP components. When these nucleoli became compact after treatment of HeLa cells with adriamycin, the distribution of the immunoreactive products was altered along with distribution of the dense nucleolar components. The human nucleolar antigens were mainly localized to nucleolar regions containing the nucleolar RNP components. Improved purification of the antigens made it possible to provide a satisfactory amino acid analysis of one pI 6.3 antigen. Interestingly, some of the nucleolar antigen was found in miniparticle undescribed until now.

Amino Acids↗