Proteins C23 and B23 are the major nucleolar silver staining proteins.
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Biomedical subjects
Publications and source records attributed to H Busch.
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Novikoff hepatoma nucleolar nonhistone proteins, C23 and B23, contain highly acidic phosphorylated regions (Mamrack, M. D., et al. (1977) Biochem. Biophys. Res. Commun. 76, 150--157). Tryptic peptides from protein C23 containing these regions were purified by DEAE-Sephadex columns and paper electrophoresis at pH 1.8. One of these, peptide C23-Ca, was sequenced by combined automated and conventional methods. The proposed amino acid sequence is shown in eq 1. This peptide was found in three 32P-labeled forms with phosphoryl groups at positions 8 and 25, and probably 28. The highly acidic sequences adjacent to the phosphorylation sites represent a unique class of phosphorylation sites different from those in histones or substrates for cytoplasmic cAMP-dependent kinases. Ala-Ala-Pro-Ala-A5la-Pro-Ala-Ser-Glu-A10sp-Glu-Asp-Glu-Glu-A15sp-Asp-Asp-Asp-Glu-A20sp-Asp-Asp-Asp-Asp-S25er-Gln-Glu-Ser-Glu-G30lu-Glu-Asp-Glu-Glu-V35al-Met-Glu-Ile-Thr-P40ro-Ala-Lys (1).
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The perichromatin granules were studied in hepatocytes of experimental rats injected with cycloheximide because the increased number of these nuclear components after such treatment facilitated their cytochemical investigation. Most perichromatin granules were sensitive to the digestion with pepsin and ribonuclease. In contrast, small population of perichromatin granules was resistent to such digestion under conditions which remove known RNA containing components such as ribosomes, nucleolar RNP components and interchromatin granules. The size of these resistent perichromatin granules was reduced and they consisted of filaments the width of which was similar to that of filaments in the chromatin. Moreover, a small population of perichromatin granules was sensitive to the digestion with pepsin and deoxyribonuclease. The size of these granules was only slightly reduced. All these observations indicate that most perichromatin granules contain the RNA and some the DNA. A possibility also exists that the perichromatin granules might contain both RNA and DNA but in various proportions. In addition, partial digestion with pepsin followed by a complete digestion with ribonuclease and deoxyribonuclease removed perichromatin granules as well as other nucleoprotein structures. On the other hand, such digestion facilitated the visualization of the nuclear and cytoplasmic skeleton (matrix) in situ.
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Rabbit antibodies to nucleoli isolated from HeLa cells produced bright nucleolar fluorescence in HeLa cells by the indirect immunofluorescence technique. After absorption with fetal bovine serum and placental nuclei, the IgG still produced bright nucleolar fluorescence in 12 human tumor cells including HeLa, HEp-2, cultures of prostate and mammary carcinomas, the Goldenberg GW-39 colon tumor, and biopsy specimens of prostatic, adrenal cortical, thyroid, and squamous cell carcinomas, a hairy cell leukemia of the spleen, a hepatic metastasis of an adenocarcinoma of the colon, and an osteogenic sarcoma. Bright nucleolar fluorescence was not produced in nine nontumor human cells including biopsy specimens of bone marrow, kidney, placenta, thyroid, liver, and prostate, peripheral blood buffy coat, and cultures of normal skin fibroblasts. Nucleolar fluorescence with the absorbed IgG was prevented in HeLa cells by pretreatment of the cells with acid, base, and proteases but not by pretreatment with nucleases; absorption of this IgG with extracts of HeLa nucleoli prevented the nucleolor fluorescence in HeLa and other human tumor cells.
Nonhistone protein BA has been shown to decrease in amount in the chromatin of growth- stimulated normal rat liver (Yeoman et al. 1975. Cancer Res. 35:1249-1255) and in mitogen-stimulated normal human lymphocytes (Yeoman et al. 1976. Exp. Cell Res. 100:47- 55.). Subsequently, protein BA was purified and was shown to prefer to bind to double- stranded A-T-rich DNAs (Catino et al. 1978. Biochemistry. 17:983-987.). Immunization of rabbits with highly purified protein BA has resulted in the production of a specific antibody. A specific immunoreactivity for chromosomal protein BA has been demonstrated by immunoelectrophoresis and double antibody immunoprecipitation analysis with rabbit anti-BA immunoglobulin and IgG fractions. Light microscope examination of normal rat liver crysections by the indirect immunofluorescence procedure has demonstrated a cytoplasmic as well as a nuclear localization for protein BA with a pronounced perinucleolar fluorescence. Immunoelectron microscopy employing the peroxidase antiperoxidase method of antigen localization has confirmed the immunofluorescence data and has show a heterochromatin localization for protein BA. The relationship of the localization of protein BA to gene control in quiescent cells or to configurations of heterochromatin as well as the marked reduction in the amounts of protein BA which occur in stimulated growth states remains to be defined.
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Different brands of bovine albumins were tested as reagent in the second step of the so called 3-stage-screening test for irregular blood group antibodies. In 3 of 9 brands of bovine albumin there was anticomplementary activity which was the cause of diminished or negative reactions in the third stage of the screening test (antiglobulin stage). The disadvantageous brands of bovine albumin contained Na2-EDTA which revealed to be responsible for the anti-complementary activity.
With rabbit antibodies to nuclear 0.01 M Tris-HCl, pH 8, extract or "nucleolar preparations" of human HeLa S3 cells and fluorescein-labeled goat anti-rabbit antibodies, bright nucleolar immunofluorescence was observed in 61 or 63 human adenocarcinomas, squamous cell carcinomas, sarcomas, hematological neoplasms, and other malignant tumors. With these antibodies, nucleolar immunofluorescence was not found in 23 normal tissue specimens, 10 benign adenomas and hyperplastic tissues, and 8 specimens of inflammatory diseases. In the nontumorous tissues examined, positive nucelolar fluorescence was found in a few sections of a gastric ulcer and chronic ulcerative colitis which have been known propensities for malignant change; these areas may have been undergoing focal malignant changes.
The portion of warm and fresh blood transfusions had increased obviously. This is accompanied by a decrease of the restrictive period of the former 72 and the presently 48 hours for blood units to be transfused. Therefore, the danger of a symphilis transmitted by transfusion is impending again. This is realized insufficiently and the syphilis may occur, if blood is drawn in the seronegative phase of the primary stage of syphilis and transfused within the restrictive period. Model trials on the infectivity of Treponema pallidum revealed that the syphilis cannot be transmitted by blood and its derivates drawn in the seronegative phase after storage at 4 degrees C for 24 hours. Therefore, the reduction of the restrictive period to 48 hours is justified.
The observations suggest that the cells of the human malignomas contain such protein or proteins which keep their specific antigenic characteristics even under isolation techniques. These characteristics have been directly due to the nucleus of the malignant cell, and had remained also under the cell-culture process. The nuclear antigen and specific antibody technique described in this paper seem to be suitable as an immunofluorescent method in the immunodiagnostics of the human tumors, furthermore provide valuable data in the clarification of the problem of human tumors.
Male Sprague-Dawley rats were treated with carminomycin i.v. in doses ranging from 1 to 40 mg/kg. Within 1 hr after the administration of carminomycin, 20 mg/kg, nucleoli of cardiac and skeletal muscle cells were segregated, while nucleoli of liver parenchyma cells were unaffected. Three and one-half hr after drug administration, cardiac muscle nucleoli reverted to normal ultrastructure. However, some skeletal muscle cell nucleoli were still segregated. Following treatment with carminomycin, 10 mg/kg, no significant ultrastructural changes were observed. These results demonstrate that at sufficiently high doses carminomycin induces ultrastructural lesions in nucleoli of both cardiac and muscle cells. The dose of carminomycin required to produce nucleolar segregation in cardiac and skeletal muscle is 6 times greater than the dose of Adriamycin (3.5 mg/kg) required to induce equivalent alterations.
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