Autoradiographic Evidence for Reutilization of DNA Catabolites by Granulocytopoiesis in the Rat

The proliferating granulocyte precursor pool of rat bone marrow was labelled during DNA synthesis by continuous infusion and by single injection of ³H-thymidine (³H-TdR), as well as by single injection of ¹²⁵I-iododeoxyuridine (¹²⁵I-UdR). The appearance of neutrophilic granulocytes in the blood stream after these various labelling procedures was studied by autoradiography. Labelling patterns of blood neutrophils were identical during continuous infusion and after single injection of ³H-TdR, and 100 % labelling of the blood compartment was achieved. This result indicated reutilization of DNA catabolites to occur in granulocytopoiesis leading to continuous availability of ³H-labelled DNA precursors even after a single injection of ³H-TdR. Attempts to suppress reutilization of label by infusion of cold thymidine 1 h after injection of ³H-TdR were unsuccessful. However, a change in the labelling pattern of blood neutrophils was seen after single injection of ¹²⁵I-UdR, a DNA precursor poorly reutilized in comparison to ³H-TdR. This result provided further evidence for reutilization of DNA catabolites by the cell system investigated. A comprehensive discussion of the results indicates that thymidinemonophosphate is the biochemical level of reutilization in granulocytopoiesis.     

Electron Microscope and High Resolution Autoradiographic Studies of the Erythroblasts in Haemoglobin H Disease

Electron microscope studies of bone marrow from a patient with haemoglobin H (HbH) disease have revealed the presence of highly condensed branching intracytoplasmic inclusions within a proportion of the early and late polychromatic erythroblasts and reticulocytes. Intraerythroblastic inclusions of this type have not yet been described in any other haematological disorder and may well be a specific feature of HbH disease. Electron microscope autoradiographic studies of the marrow cells from the patient with HbH disease revealed that (1) active protein synthesis continues in inclusion-containing erythroblasts and (2) there is a rapid precipitation of a considerable proportion of the newly synthesized free beta chains onto the surfaces of preformed inclusions, presumably after conversion to HbH. These electron microscope and electron microscope autoradiographic findings are quite different from the findings previously reported in homozygous beta thalassaemia using the same techniques.     

Evidence for the Presence of a Low Mr GTP-binding Protein, ram p25, in Human Platelet Membranes

The ram gene was isolated from a rat megakaryocyte cDNA library and encodes a GTP-binding protein with a Mr of 25 000. In order to clarify the presence of the ram protein (ram p25) in human platelets, we tried to purify ram p25 from the sodium cholate extract of human platelet membranes by a combination of DEAE-Sephacel, Sephacryl S300HR, hydroxyapatite HCA-100S and DEAE-Toyopearl 650(S) column chromatographies. In the course of the purification, a specific antibody raised against a synthetic COOH-terminal peptide of ram p25 was used. In conclusion, ram p25 was partially purified and was observed to be present in membrane fractions of human platelets.     

Evidence for the Presence of a Low Mr GTP-binding Protein,ram p25, in Human Platelet Membranes

The ram gene was isolated from a rat megakaryocyte cDNA library and encodes a GTP-binding protein with a Mr of 25 000. In order to clarify the presence of the ram protein (ram p25) in human platelets, we tried to purify ram p25 from the sodium cholate extract of human platelet membranes by a combination of DEAE-Sephacel, Sephacryl S300HR, hydroxyapatite HCA-100S and DEAE-Toyopearl 650(S) column chromatographies. In the course of the purification, a specific antibody raised against a synthetic COOH-terminal peptide of ram p25 was used. In conclusion, ram p25 was partially purified and was observed to be present in membrane fractions of human platelets.     

Effects of Ethanol on DNA, RNA, and Protein Synthesis in Rat Astrocyte Cultures

Brain growth retardation is a major feature of the fetal alcohol syndrome (FAS). Insulin and insulin-like growth factors (IGF-I and IGF-II) exert significant growth-promoting effects on the central nervous system (CNS). The present study examined the effects of ethanol and its interactions with growth factors on the incorporation of labeled precursors into DNA, RNA, and protein in primary astrocyte cultures prepared from term fetal rats. Cultures were exposed to ethanol for 18hr in serum-free medium before measuring nucleoside or amino acid incorporation into acid-precipitable cell constituents. Under basal conditions, ethanol induced dose-dependent changes in the rates of incorporation of tritiated thymidine, uridine, and valine. The fraction of the total thymidine uptake that was incorporated into DNA was reduced in the presence of 100 and 200 mM ethanol. Effects on uridine and valine incorporation paralleled cell uptake. Insulin (10(-6) M) and IGF-I (10(-9) M) increased (p less than 0.01) incorporation of radiolabeled thymidine, uridine, and valine. Analysis of variance indicated highly significant interactions between ethanol and the effects of growth factors on incorporation of both nucleosides and valine. Interference with the action of neurotrophic factors may be a significant factor in fetal brain growth retardation associated with maternal ethanol ingestion.     

Evidence of Particle-Associated RNA-Directed DNA Polymerase and High Molecular Weight RNA in Human Gastrointestinal and Lung Malignancies

Previous communications have demonstrated that neoplastic cells of human breast cancers, leukemias, lymphomas, sarcomas, and brain tumors contain particles with similar diagnostic attributes as those found in RNA oncornaviruses. The present paper concerns malignancies of the gastrointestinal and pulmonary systems for which, like brain tumors, no suitable animal model or corresponding virus exists. By means of the simultaneous detection assay, these tumors have been found to contain 70S RNA and RNA-directed DNA polymerase encapsulated in particulate components possessing densities of 1.16-1.17 g/ml. Twelve out of 17 (70%) colon carcinomas, three out of five (60%) gastric carcinomas, all of three rectal carcinomas, and seven out of ten (70%) lung carcinomas contained detectable levels of these virus-like entities. None of the corresponding normal tissues was positive.     

Evidence for the Presence of the Toxin-Binding Protein on the Plasma Membrane of Sugarcane Cells

Helminthosporoside is a host-specific toxin produced by Helminthosporium sacchari, both in culture and in infected sugarcane tissue. The susceptibility of any given clone of sugarcane to disease is invariably associated with the presence of a toxin-binding protein. This report presents evidence for the presence of the toxin-binding protein on the plasma membrane of the plant cell. This evidence includes, among others: (1) The protection of susceptible plant tissues by prior treatment with antiserum to the binding protein, (2) the successful pyridoxylation and reduction of the binding protein in vivo followed by its isolation, (3) the reaction of sugarcane cells and free protoplasts with [(3)H]antiserum prepared against the binding protein, and (4) the agglutination of sugarcane protoplast preparations with antiserum to the binding protein.     

Sequential Studies on Serum-Levels of Lysozyme,Lactoferrin and Eosinophil Cationic Protein in Alcoholics after Alcohol Withdrawal

12 male alcoholics were followed for 16 d after alcohol withdrawal with respect to the number of the different circulating blood cells and to serum levels of leucocyte markers such as β₂-microglobulin, eosinophil cationic protein (ECP), lactoferrin and lysozyme. The results suggest a direct toxic effect of ethanol on the neutrophil granulocytes as indicated by high S-lactoferrin levels. Relatively low levels of S-lysozyme as compared to S-lactoferrin could suggest a reduced activity of the monocytes/macrophages. The eosinophils and lymphocytes seemed quite unaffected by ethanol. Increased haematopoietic activity after withdrawal was indicated by increasing cell numbers in the peripheral blood and by increasing serum levels of lactoferrin and lysozyme. The increasing monocyte/macrophage activity after withdrawal as suggested by S-lysozyme levels was closely related (P < 0.001) to the increased thrombopoietic activity as evaluated by peripheral thrombocyte counts. This latter finding could point to a direct relationship between monocyte/macrophage activity and thrombocyte production.     

Evidence for and Localization of Vegetative Viral DNA Replication by Autoradiographic Detection of RNA·DNA Hybrids in Sections of Tumors Induced by Shope Papilloma Virus

The occurrence and localization of vegetative viral DNA replication was studied in sections of tumors induced by the rabbit Shope papilloma virus, in cottontail and domestic rabbit papillomas, in primary domestic rabbit carcinoma, and in transplantable VX2 carcinoma, by in situ hybridization of radioactive RNA complementary to viral DNA. Vegetative viral DNA replication and viral protein synthesis were compared by means of cytological hybridization and immunofluorescence techniques on adjacent frozen sections.     

Effects of Anticonvulsant Drugs on the Synthesis of DNA and Protein by Human Bone Marrow Cells in Vitro

Suspensions of human bone marrow cells were incubated with various concentrations of phenobarbitone or phenytoin sodium for 2 h, and the effects of this incubation on the subsequent incorporation of ³H-thymidine and ³H-leucine into DNA and protein, respectively, were studied. Both drugs caused a depression of ³H-thymidine incorporation and this phenomenon was not prevented by the addition of 100 μg of pteroylglutamic acid, folinic acid or 5-methyltetrahydrofolate per ml of marrow culture. The lowest concentration of drug which caused a statistically significant depression of ³H-thymidine incorporation was 200 μg per ml for phenobarbitone and 50 μg per ml for phenytoin sodium. Both phenobarbitone and phenytoin sodium also caused an increase in the incorporation of ³H-leucine at concentrations of 50 and 20 μg per ml, respectively, suggesting the possibility that a stimulation of protein synthesis within erythropoietic cells may play an important role in the development of anticonvulsant-induced macrocytosis.     

Biochemical Studies on Adenovirus Multiplication, XIX. Resolution of Late Viral RNA Species in the Nucleus and Cytoplasm

Late after infection of cultured human cells (KB) with adenovirus type 2, the nucleus contains heterogeneous viral RNA species ranging in size from 10 to 43 S. Four viral RNA species found in the nucleus (36, 38, 40, and 43 S) are synthesized predominantly during a 15-min labeling period with [(3)H]uridine, while smaller RNA species accumulate when labeling is continued for longer periods. In contrast, 6-8 viral RNA species, of sedimentation coefficient from 10 to 29 S, are found in the cytoplasm after a 30-min pulse label and a 2-hr chase. DNA-RNA hybridization-competition experiments demonstrate that viral RNA sequences present in nuclear 36-43S RNA are also present in cytoplasmic and polyribosomal RNA, suggesting that at least some of the cytoplasmic viral-specific RNA molecules are derived by cleavage of high molecular weight precursors from the nucleus.     

Eosinophil Cationic Protein and Phospholipase A2 Activity in Human Gastric Juice: With Emphasis on Helicobacter pylori Status and Effects of Antacids

To elucidate possible new effects of antacids, gastric juice from 15 volunteers with known Helicobacter pylori status were analysed for eosinophil cationic protein (ECP), phospholipase A₂ (PLA₂) activity, phosphatidylcholine (PC), and bile acids (BA) before and after administration of one tablet of antacid or placebo in a double blind cross-over design. Geomtric mean ECP concentrations were more than 13 times higher in gastric juice from H. pylori-positive (12.9 μg/l) than from H. pylori-negative (0.97 μg/l) subjects (p = 0.0032). Geometric mean PLA₂ activity was 1.31 U/l for the negative subjects and 4.02 U/l for the positive subjects (p = 0.13). There were no differences between positive and negative subjects with regard to either PC or BA concentration. Regardless of H. pylori status, mean PC concentration increased significantly after antacids as compared with placebo (p = 0.024). The effect of antacids did not differ significantly from placebo for ECP, PLA₂ activity, or BA concentration. Hence, antacids may not act by binding ‘toxic’ H. pylori-associated gastric juice components like ECP or PLA₂. Increased concentration of PC may indicate an increased protective capacity induced by antacids.     

The Importance of Gene Rearrangement in Evolution: Evidence from Studies on Rates of Chromosomal, Protein, and Anatomical Evolution

We have compared the relative rates of protein evolution and chromosomal evolution in frogs and mammals. The average rate of change in chromosome number has been about 20 times faster in mammals than in frogs. Whereas it takes only 3.5 million years, on the average, for a pair of mammal species to develop a difference in chromosome number, the corresponding period for frogs is 70 million years. In contrast, the rate of protein evolution in mammals has been roughly equal to that in frogs. The rapid rate of gene rearrangement in mammals parallels both their rapid anatomical evolution and their rapid evolutionary loss of the potential for interspecific hybridization. Thus, gene rearrangements may be more important than point mutations as sources for evolutionary changes in anatomy and way of life.     

Studies on nucleic acid reassociation kinetics: rate of hybridization of excess RNA with DNA, compared to the rate of DNA renaturation.

The rate of reaction of double-stranded replicative form (RF) [3H]DNA of bacteriophage phiX174 with excess (+)strand DNA and (+)strand RNA was measured by standard methods of hydroxyapatite chromatography. The reactions follow pseudo-first-order kinetics and the observed rate constant for the RNA-DNA reaction differs less than 25% from that of the DNA-DNA reaction. The pseudo-first-order rate constants are close to the value predicted on the basis of the second-order rate constant measured in the renaturation of the double-stranded phiX RF [3H]DNA.     

Rapid and sensitive colorimetric method for visualizing biotin-labeled DNA probes hybridized to DNA or RNA immobilized on nitrocellulose: Bio-blots.

Biotin-labelled DNA probes, prepared by nick-translation in the presence of biotinylated analogs of TTP, are hybridized to DNA or RNA immobilized on nitrocellulose filters. After removal of residual probe, the filters are incubated for 2--5 min with a preformed complex made with avidin-DH (or streptavidin) and biotinylated polymers of intestinal alkaline phosphatase. The filters are then incubated with a mixture of 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium, which results in the deposition of a purple precipitate at the sites of hybridization. This procedure will detect target sequences in the 1- to 10-pg range after enzyme incubation periods of 1 hr or less. The incubation period can be extended up to 24 hr, if required, to increase the color intensity of the hybridization signal. Furthermore, at high probe concentrations (250--7560 ng/ml), biotin-labeled DNA exhibits lower nonspecific binding to nitrocellulose than does radiolabeled DNA, so hybridization times required for the analysis of unique mammalian gene sequences can be decreased to 1--2 hr. This nonradiographic method of probe detection should be of general utility for genetic studies using Southern, RNA, or dot-blot hybridization protocols.     

Isolation of Escherichia coli RNA Polymerase Binding Sites on T5 and T7 DNA: Further Evidence for Sigma-Dependent Recognition of A-T-Rich DNA Sequences

Previous isolation and analysis of E. coli RNA polymerase (EC 2.7.7.6) binding sites on λ DNA had demonstrated the existence of a sigma-dependent process of recognition of A-T-rich DNA sequences. We have now extended this finding to T5 and T7 DNA and häve provided evidence for the double-strandedness of the isolated binding sites. The possible equation of these sites to the genetically defined promoters is discussed.