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Biomedical subjects

H Busch

Publications and source records attributed to H Busch.

At least 127 records · Page 7Linked to original sources
PubMed1986

Studies on satellite nucleoli in human normal and leukemic lymphocytes.

Lymphocytes from normal and leukemic patients, and phytohemagglutinin-stimulated lymphocytes were investigated by means of immunofluorescence procedures and a silver reaction for the demonstration of proteins characteristic of nucleolus organizer regions in interphasic cells to provide basic information on the presence of satellite nucleoli in these cells. The results clearly indicated that satellite nucleoli are present in a limited but constant percentage of peripheral lymphocytes. An increased percentage of lymphocytes with satellite nucleoli was found only in leukemic patients and after silver staining. In contrast, a decreased percentage of satellite nucleoli was found 24 h after stimulation with phytohemagglutinin vitro. In leukemic patients, the discrepancy in the percentage of lymphocytes with satellite nucleoli between immunostained and silver-stained preparations may suggest that the silver reaction demonstrates the presence of an additional argyrophilic protein besides proteins B23 and C23, or altered forms of these proteins, which does not react with the specific antibodies.

Cell Nucleolus↗
PubMed1986

Pulsed Doppler echocardiography in patients with aorto-pulmonary connection.

The pulsed Doppler principle enables us to study the bloodflow velocity at specific points in the heart and great vessels. In such aorto-pulmonary connections as patent ductus arteriosus, aorto-pulmonary window and truncus arteriosus the bloodflow velocity patterns are typical of the malformation. Based on knowledge of these abnormal flow patterns the diagnoses of these malformations can be made.

Aortopulmonary Septal Defect↗
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↗
PubMed1986

Effects of luzopeptins on protein B23 translocation and ribosomal RNA synthesis in HeLa cells.

Localization of protein B23 in HeLa cells after treatment with luzopeptin A and its analogues was studied using indirect immunofluorescence. Bright nucleolar fluorescence was observed in control HeLa cells. After treatment with luzopeptin A (50 ng/ml), luzopeptin B (500 ng/ml), or luzopeptin D (10 ng/ml) for 2 h, uniform nucleoplasmic rather than specific nucleolar fluorescence was observed. Luzopeptin C had no effect on protein B23 translocation. Luzopeptin D, A, and B inhibited [3H]uridine incorporation into the trichloroacetic acid insoluble fraction of HeLa cells with 50% inhibitory concentration values of 3.7 +/- 1.1 (SD), 10.8 +/- 2.1, and 122.0 +/- 34.0 ng/ml, respectively. Less than 10% inhibition of [3H]uridine incorporation was found with luzopeptin C (500 ng/ml and 2 h incubation). Ribosomal RNAs (28 and 18S) were isolated from HeLa cells treated with luzopeptin D (50 ng/ml; 2 h). They were then separated and analyzed in 1% agarose gel electrophoresis. There were 90.1 +/- 1.38 and 95.0 +/- 1.04% inhibition of [3H]uridine incorporation into 28 and 18S ribosomal RNA, respectively. The order of potency for the loss of nucleolar fluorescence and the concurrent increase in nucleoplasmic fluorescence was luzopeptin D greater than luzopeptin A greater than luzopeptin B much greater than luzopeptin C, which correlates with the order of their 50% inhibitory concentration values for inhibition of [3H]uridine incorporation. With 34-55% inhibition of RNA synthesis, both nuclear and nucleolar B23 immunofluorescence were observed. With 70-85% inhibition of RNA synthesis, a uniform nucleoplasmic fluorescence was observed. These results indicate that translocation of protein B23 as observed by indirect immunofluorescence may be a rapid and simple screening test for the selection of antitumor agents which inhibit ribosomal RNA synthesis.

Biological Transport↗
PubMed1985

Translocation of nucleolar phosphoprotein B23 (37 kDa/pI 5.1) induced by selective inhibitors of ribosome synthesis.

To elucidate the possible role of nucleolar phosphoprotein B23 in ribosome synthesis, drugs which inhibit the processing of ribosomal RNA were employed. After treatment with actinomycin D, toyocamycin or high doses of alpha-amanitin, a uniform nucleoplasmic fluorescence was observed. Low doses of alpha-amanitin and the protein synthesis inhibitor puromycin and cycloheximide had no effect on protein B23 translocation. By ELISA immunoassay, there was a 60% decrease in the amount of protein B23 in the nucleoli of the actinomycin D-treated cells as compared with the control nucleoli. Conversely, the amount of protein B23 in the nucleoplasm (excluding nucleoli) was 3-fold higher in the actinomycin D-treated cells. Preribosomal ribonucleoprotein particles (pre-rRNPs) were extracted from isolated nucleoli of Novikoff hepatoma ascites cells and fractionated on sucrose density gradients. Protein B23 was found co-localized with the pre-rRNPs as determined by ELISA assays which agrees with previous studies. The proteins in these 80 S and 55 S pre-ribosomal ribonucleoprotein particles were fractionated by 10% gel electrophoresis. Immunoblots showed protein B23 was present in both pre-rRNPs.

Amanitins↗
PubMed1985

Isolation and characterization of a 125-kilodalton rapidly labeled nucleolar phosphoprotein.

A 125-kilodalton (kDa) phosphoprotein was isolated from nucleoli of Novikoff hepatoma cells in the presence of various inhibitors of proteases, alkaline phosphatase, and RNase. This protein was the most highly phosphorylated protein found thus far in the nucleolus. The half-life of [32P]phosphate in the 125-kDa phosphoprotein was approximately 60 min. Amino acid analysis of the protein showed it had a high serine content (15.5 mol %), a high glutamine plus glutamic acid content (15.5 mol %), and a high lysine content (10.3 mol %). Phosphoserine was the only phosphorylated amino acid identified. After alkaline hydrolysis of the 32P-labeled protein, ribonucleotides were found which accounted for approximately 8.5% of the [32P]phosphate. After cytidine 3',5'-[32P]diphosphate ([32P]pCp) labeling by RNA ligase, several oligoribonucleotide sequences were purified including GGGCOH and GGGGCOH. The binding of oligonucleotides to peptides was stable under denaturing fractionation conditions including 6 M urea treatment and incubation at 100 degrees C for 10 min in sodium dodecyl sulfate and beta-mercaptoethanol. Furthermore, when nucleotide-peptide complex was treated with ribonuclease T2 followed by snake venom phosphodiesterase, the junctional nucleotide pCp was released. These results suggest that one or more ribonucleotides are covalently bound to the 125-kDa phosphoprotein.

Animals↗
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

Purification and partial characterization of a nucleolar scleroderma antigen (Mr = 34,000; pI, 8.5) rich in NG,NG-dimethylarginine.

A new scleroderma antigen of Mr = 34,000; pI, 8.5 has been identified. This 34-kDa protein is a nucleolar protein as determined by immunostaining procedures with affinity-purified antibodies. The 34-kDa protein was shown to localize to the fibrillar regions of the nucleolus by immunoelectron microscopy. Antibodies against the 34-kDa protein precipitate U3 RNA-containing particles. The 34-kDa protein has been isolated from Novikoff hepatoma cell nucleoli by ion exchange and reverse-phase column chromatography. The protein contains 4.1 mol % NG,NG-dimethylarginine (DMA) and 22.8 mol % glycine. It is the most highly arginine-methylated protein thus far detected in higher eukaryotes. This nucleolar 34-kDa protein resembles several nucleoplasmic proteins that are associated with heterogeneous nuclear RNA with respect to isoelectric point, Mr, presence of NG,NG-dimethylarginine, and its high glycine content. The amino-terminal sequence of the first 31 residues of the 34-kDa protein is: Met-Lys-Pro-Gly-Phe-Ser-Pro-DMA-Gly-Gly-Gly-Phe-Gly-Gly-DMA-Gly-Gly- Phe-Gly-Asp-DMA-Gly-Gly-DMA-Gly-Gly-Gly-DMA-Gly-Gly-DMA. In the first 31 residues, there are 16 glycine, 6 DMA, and 3 phenylalanine residues. This is a novel demonstration of clusters of glycine and DMA in a protein.

Amino Acid Sequence↗
PubMed1985

Clustering of glycine and NG,NG-dimethylarginine in nucleolar protein C23.

Protein C23 (Mr 110 000, pI = 5.5), a major phosphoprotein in the nucleolus of mammalian cells, has been shown to contain 1.3 mol% of NG,NG-dimethylarginine (DMA) [Lischwe, M.A., Roberts, K.D., Yeoman, L.C., & Busch, H. (1982) J. Biol. Chem. 257, 14600-14602]. A tryptic peptide from protein C23 that contains DMA has been isolated and sequenced. Its sequence is Gly-Glu-Gly-Gly-Phe-Gly-Gly-DMA-Gly-Gly-Gly-DMA-Gly-Gly-Phe-Gly-Gly-DMA- Gly-Gly- Gly-DMA-Gly-Gly-DMA-Gly-Gly-Phe-Gly-Gly-DMA-Gly-DMA-Gly-Gly-Phe-Gly-Gly- DMA-Gly-Gly-Phe-DMA-Gly-Gly-DMA-Gly-Gly-Gly-Gly-Asp-Phe-Lys. This peptide contains 34 glycine, 10 DMA, and 6 phenylalanine residues and has clusters of glycine and NG,NG-dimethylarginine interspersed with phenylalanine residues. A similar domain has been found at the amino terminus of a nucleolar protein of Mr 34,000, pI = 8.5. This sequence array may represent a conserved domain characteristic of a certain class of nuclear proteins. All of the methylated arginine residues in protein C23, the 34-kilodalton protein, and myelin basic protein [Carnegie, P.R. (1971) Biochem. J. 123, 57-67] have at least one adjacent glycine. Access of certain arginine methylases to arginine residues may be sterically possible because of the lack of a side chain on the adjacent glycine residue(s).

Amino Acid Sequence↗
PubMed1985

Primary and secondary structure of U8 small nuclear RNA.

U8 small nuclear RNA is a new, capped, 140 nucleotides long RNA species found in Novikoff hepatoma cells. Its sequence is: m3GpppAmUmCGUCAGGA GGUUAAUCCU UACCUGUCCC UCCUUUCGGA GGGCAGAUAG AAAAUGAUGA UUGGAGCUUG CAUGAUCUGC UGAUUAUAGC AUUUCCGUGU AAUCAGGACC UGACAACAUC CUGAUUGCUU CUAUCUGAUUOH. This RNA is present in approximately 25,000 copies/cell, and it is enriched in nucleolar preparations. Like U1, U2, U4/U6, and U5 RNAs, U8 RNA was also present as a ribonucleoprotein associated with the Sm antigen. The rat U8 RNA was highly homologous (greater than 90%) to a recently characterized 5.4 S RNA from mouse cells infected with spleen focus-forming virus (Kato, N., and Harada, F. (1984) Biochim. Biophys. Acta, 782, 127-131). In addition to the U8 RNA, three other U small nuclear RNAs were found in anti-Sm antibody immunoprecipitates from labeled rat and HeLa cells. Each of these contained a m3GpppAm cap structure; their apparent chain lengths were 60, 130, and 65 nucleotides. These U small nuclear RNAs are designated U7, U9, and U10 RNAs, respectively.

Animals↗
PubMed1985

U4 small nuclear RNA pseudogenes from rat genome have common truncated 3'-ends.

Four U4 RNA pseudogenes were isolated and characterized from a rat genomic bank. The four pseudogenes contained sequences completely homologous to U4 RNA from nucleotides 1 to 67 and had common truncated 3'-ends. Three of the four pseudogenes were flanked by 14 to 18 nucleotide-long direct repeats. The structural features of these four U4 RNA pseudogenes are consistent with the hypothesis that these pseudogenes arose by RNA self-primed complementary DNA synthesis and integration into the genome (Van Arsdell et al., Cell 26:11-17, 1981).

Animals↗
PubMed1985

Purification of an 86-70 kDa nuclear DNA-associated protein complex.

In the course of studies on nucleolar antigens, monoclonal antibodies were developed, one of which recognized an 86 kDa antigen as shown by analysis of nuclear extracts from HeLa or Namalwa cells. Immunofluorescence studies on HeLa cells showed a nucleoplasmic and phase-dependent nucleolar localization of the monoclonal antibody was decreased after digestion with DNAase I but not with RNAase A. For purification, the antigen was released from nuclei by digestion with DNAase I and then purified by chromatography on DEAE cellulose, phosphocellulose and antibody-Sepharose affinity chromatography. Interestingly, the immunoaffinity purified product contained two polypeptide chains; the immunoreactive polypeptide had an Mr of 86 000 and a pI of 6.0. The complex also contained a 70 kDa, pI 6.5 nonantigenic polypeptide in a 1:1 ratio. The overall purification of the complex was 5700-fold. Both polypeptides contained approx. 15 mol% glutamic acid and the 70 kDa polypeptide contained approx. 15 mol% serine.

Amino Acids↗
PubMed1985

Isolation and characterization of three rat U3 RNA pseudogenes colinear with U3 RNA.

Three different 15-kilobase rat genomic clones that contained sequences colinear with U3 RNA were isolated. These inserts hybridized only to U3 RNA in a mixture of total cellular 4-8 S RNA labeled in vivo which showed that genes or pseudogenes for most other small RNAs were absent in these U3 DNA clones. DNA sequence analysis showed that the three subcloned genes contained full-length U3-coding sequences but each had sequence variations, insertions, and/or deletions when compared to rat U3A or U3B RNA. Two of these pseudogenes contained poly(A) sequences on the 3'-end and were flanked by 6-15-nucleotide long direct repeats. None of the three clones was transcribed when injected into Xenopus oocyte nuclei. One clone was a template for a small RNA slightly larger than U3 RNA, but this transcript was not related to the U3 RNA sequences. The structural features of two of these three U3 DNAs are supportive of the hypothesis that some pseudogenes arose from RNA-mediated DNA synthesis and insertion into the genome at random sites (Van Arsdell, S. W., Denison, R.A., Bernstein, L.B., Weiner, A.M., Manser, T., and Gesteland, R.F. (1981) Cell 26, 11-20). This is the first instance where full-length, colinear, U3 RNA pseudogenes have been isolated and characterized.

Animals↗
PubMed1985

Discrimination of epitopes identified by monoclonal antibodies by competitive binding to nitrocellulose bound antigens.

A new method is described for determining the distribution of epitopes identified by monoclonal antibodies. The method utilizes nitrocellulose membranes as a solid support for antigens which are rapidly adsorbed to nitrocellulose by vacuum-blotting and then used in competitive antibody binding assays. The distribution of epitopes is established by the reciprocal cross-blocking of radiolabeled antibody by increasing concentrations of unlabeled antibody. When unlabeled antibody does not block the binding of labeled antibody to antigen, the 2 antibodies recognize distinct epitopes. When unlabeled antibody blocks the binding of labeled antibody to antigen, the 2 antibodies recognize the same epitope. The method is rapid, sensitive and should be applicable to screening monoclonal antibodies to any epitope.

Antibodies, Monoclonal↗
PubMed1985

Identification and characterization of a polyadenylated small RNA (s-poly A+ RNA) in dinoflagellates.

A 104 nucleotide-long small RNA, referred to as s-poly A+ RNA, containing 30 adenosine residues on its 3' -end was found in dinoflagellates, purified and its nucleotide sequence was determined. The sequence is: (sequence text) The polyadenylation signal AAUAAA was not found in this RNA; this result indicates that the 30 nucleotide-long poly A on the 3' -end is either coded for by this gene, or the poly A chain is added on this small RNA by a mechanism different from that for polyadenylation of messenger RNAs. Two polyadenylated small RNAs identified previously were implicated in differentiation of chicken heart muscle cells (Deshpande, A. K., Jakowlew, S. B., Arnold, H., Crawford, P. A. and Siddiqui, M. A. Q. (1977) J. Biol. Chem. 252, 6521-6527), and in brain specific mRNA transcription (Sutcliffe, J. G., Milner, R. J., Gottesfeld, J. M. and Lerner, R. A. (1984) Nature 309, 237-241). This RNA is the first polyadenylated small RNA to be sequenced.

Animals↗
PubMed1985

A new moderately repetitive rat DNA sequence detected by a cloned 4.5 SI DNA.

4.5 SI RNA is an abundant, noncapped, small nuclear RNA found in rodent cells. The 4.5 SI RNA is 98 or 99 nucleotides long and contains no modified nucleotides; it is synthesized by RNA polymerase III, is partly hydrogen-bonded to poly(A+) hnRNA, and was the first small nuclear RNA to be purified and sequenced (Busch, H., Reddy, R., Ruthblum, L., and Choi, Y. C. (1982) Annu. Rev. Biochem. 51, 617-654). In studies on the structure and organization of genes coding for this abundant RNA, it was found that this RNA is homologous to an apparently novel family of repetitive sequences. Two clones were characterized; one clone showed that its sequence is identical to the RNA in the first 92 residues and differed only in the last six nucleotides. In addition, the 3'-end of the sequence contained an A,T-rich region, and the sequence was flanked by a 15-nucleotide long direct repeat of AAAATATAGACACTG. The second clone characterized contained nucleotide sequences 1-57 corresponding to the RNA and was flanked by a 15-nucleotide long direct repeat. The structural features of these two DNAs are consistent with RNA-mediated DNA synthesis and integration of this DNA into the genome at random sites. It is estimated that there are about 10,000 copies of this family of sequences in the haploid rat genome.

Animals↗
PubMed1985

Topoisomerase I phosphorylation in vitro and in rapidly growing Novikoff hepatoma cells.

Changes in phosphorylation modulate the activity of topoisomerase I in vitro. Specifically, enzymatic activity is stimulated by phosphorylation with a purified protein kinase (casein kinase type II). The purpose of this study was to compare the sites that are phosphorylated in vitro by casein kinase type II with the site(s) phosphorylated in vivo in rapidly growing Novikoff hepatoma cells. Topoisomerase I labeled in vitro was characterized by three major tryptic phosphopeptides (I-III). Separation of these peptides by a C18-reverse phase h.p.l.c. column resulted in their elution at fractions 18 (I), 27 (II) and 44 (III) with 17%, 22.5% and 33% acetonitrile, respectively. In contrast, only one major phosphopeptide was identified by h.p.l.c. in topoisomerase I labeled in vivo. This phosphopeptide eluted at fraction 18 corresponding to the elution properties of phosphopeptide I labeled in vitro. It also co-migrated with tryptic phosphopeptide I when subjected to high-voltage electrophoresis on thin-layer cellulose plates. Preliminary experiments suggest that phosphorylation occurs at a serine residue six amino acids from the N-terminus of the peptide. These data indicate that topoisomerase I is phosphorylated in vivo and in vitro within the same tryptic peptide and suggest that topoisomerase I is phosphorylated in vivo by casein kinase II.

Amino Acid Sequence↗