Search PubMed⌕ Search

Biomedical subjects

R K Zahn

Publications and source records attributed to R K Zahn.

At least 91 records · Page 5Linked to original sources
PubMed1982

Analysis of polyadenylate . protein complex of polysomal messenger RNA from mouse L cells.

The organization of polysomal poly(A)-ribonucleoprotein complex [poly(A)-RNP] was studied. The poly(A)-associated proteins were liberated directly from the poly(A)-RNP complex. Polysomal mRNA was isolated from L5178y mouse lymphoma cells by oligo (dT)-cellulose chromatography; poly(A)-RNP was prepared by nuclease digestion. The poly(A)-RNP fraction was considered to be pure basing on the size of its poly(A) component which was determined to consist of 155 AMP moieties. By radiolabeling with [3H]dansyl chloride, two poly(A)-associated proteins with molecular masses of 77,000 Da (P77) and 54,000 Da (P54) were identified. In a quantitative approach, it was shown that the polysomal poly(A)-RNP complex is composed of approximately 4 molecules P54 and 2 molecules P77. Digestion experiments with dimers and tetramers containing ribonuclease A indicated that P54 covers 15-20 AMP residues and P77 a sequence of 40-45 ribonucleotides on the poly(A)--155 stretch of polysomal poly(A)-RNP.

Animals↗
PubMed1981

Xenograft rejection in marine sponges. Isolation and purification of an inhibitory aggregation factor from Geodia cydonium.

In sponges there exists a graft rejection mechanism in which an inhibitory aggregation factor is involved. The inhibitory aggregation factor has been isolated from a culture medium containing dissociated cells of the sponge Geodia cydonium. Using ion-exchange and gel fractionation the factor was purified and shown to be electrophoretically pure. The factor has a molecular weight of 27000 and was characterized as a glycoprotein. The activity of the inhibitory aggregation factor was not affected by heat treatment, but treatment with trichloroacetic acid resulted in the irreversible loss of activity. The inhibitory aggregation factor affects the aggregation-factor-mediated reaggregation of dissociated sponge cells by extension of the lag phase preceding the aggregation process; the endpoint of the reaggregation process is not changed. The inhibitory aggregation factor competes with the aggregation receptor for the binding site on the aggregation factor. The inhibitory aggregation factor is not synthesized if homologous aggregation factor is added to the dissociated cells. The results indicate that the inhibitory aggregation factor is synthesized in xenografts of the system Geodia cydonium--Geodia rovinjensis after a grafting period of at least 3 days. Following the synthesis of the factor, the concentration of the extractable aggregation factor decreases.

Animals↗
PubMed1981

DNA-replication complex from cells infected with herpes virus.

Herpes simplex virus (HSV) DNA synthesis is initiated in an intact cell system by a 36-residue ribonucleotide stretch [W.E.G. Müller, R.K. Zahn, J. Arendes, and D. Falke (1979) Virology, 98, 200-210]. In the present study a nucleoplasmic fraction was isolated from rabbit kidney cells infected with HSV (type 1), which catalyzes DNA synthesis. By means of specific assays, containing single-stranded deoxyribopolymers, it was elucidated that the replication complex contains both an RNA-synthesizing and a DNA-synthesizing enzyme. These enzymes were characterized as host cell RNA polymerase II and HSV-induced DNA polymerase. The RNA polymerase II synthesizes an RNA initiator with an average chain length of 25 nucleotides, to which the newly synthesized DNA is covalently attached. The chemical nature of the RNA primer was proven by degradation experiments with endoribonuclease V. In the absence of any initiator in the reaction mixture the HSV-induced DNA polymerase is inactive. Kinetic experiments revealed that DNA synthesis in assays containing poly(dT), dNTPs and NTPs starts after a lag phase of 10 min during which the RNA initiator is synthesized. The HSV DNA NTPs starts after a lag phase of 10 min during which the RNA initiator is synthesized. The HSV DNA replication complex was further fractionated into HSV-induced DNA polymerase and the three cellular RNA polymerases. In the reconstructed system DNA synthesis, catalyzed by virus-induced DNA polymerase starts in a reaction assay, containing single-stranded DNA template, only in the presence of RNA polymerase II. The conclusion that the subnucleoplasmic complex, isolated from HSV-infected cells, is of biological significance for HSV DNA synthesis stems (a) from the the close correlations of the known biochemical events occurring during the initiation of HSV DNA synthesis in intact cells and in the nucleoplasmic system and (b) from control experiments with a subnucleoplasmic complex, isolated from uninfected cells, which is devoid of any potency to initiate DNA synthesis.

Animals↗
PubMed1981

Arabinosyl nucleosides. XXXII. Mechanism of inhibition of L5178y mouse lymphoma cells by 9-beta-D-arabinofuranosylguanine.

The mechanism of the growth-inhibitory action of 9-beta-D-arabinofuranosylguanine (ara-G) on mouse lymphoma cells (L5178y) at the level of nucleic acid synthesis was studied. Ara-G inhibits the cell proliferation at an ED50 concentration of 12.3 microM. The cells die because of unbalanced growth. At cytostatic concentrations ara-G inhibits the incorporation rate of dThd into DNA to a much higher degree compared to those of the precursors into RNA or proteins. Ara-G has no effect on the induction of DNA polymerase alpha occurring at the beginning of the S-phase; this compound exerts its inhibitory activity during the S-phase. Ara-G is intracellularly phosphorylated to ara-GTP. It was demonstrated that ara-G is incorporated into DNA but not into RNA; one molecule of ara-G is incorporated per 7050 molecules of deoxyguanosine. The ara-GMP moieties, incorporated into DNA of L5178y cells are not removed during a culture period of 7.7 doubling steps; compared to the controls no reduction of the viability and growth rate was observed.

Animals↗
PubMed1981

Aggregation of sponge cells: immunological characterization of the species-specific Geodia aggregation factor.

Antibodies were raised against the purified aggregation factor from Geodia cydonium in order to clarify its function during cell aggregation in the homologous and heterologous system. These antibodies inhibited only cell aggregation in the homologous Geodia system and were inactive in the heterologous Tethya lyncurium system. These findings directly indicated that the species-specific reaggregation of sponge cells was initiated by the soluble aggregation factor as already assumed in earlier studies. The amount of neutralizing antibodies was determined by a precipitation reaction with the antigen in capillaries and by microdiffusion. By using the latter technique we got evidence that the Geodia aggregation factor contained a component that was antigenetically related to a galactose-specific lectin present in Geodia cydonium.

Animals↗
PubMed1981

Purification of a nuclease from human serum.

The purification procedure for a nuclease from human serum is described. It includes ammonium sulfate precipitation, chromatography on DEAE-Sephadex and on Sephacryl-S 200, and preparative electrophoresis. The enzyme purified about 2000-fold, is homogeneous in a sodium dodecyl sulfate electrophoretic system, where it has a mol. wt of 78,000. The pH optimum lies around pH 6.5; it is a sugar-nonspecific endonuclease.

Deoxyribonucleases↗
PubMed1981

Assessing consequences of marine pollution by hydrocarbons using sponges as model organisms.

Pollution has been assessed as a mutagenic activity determined by the Ames test, using radiolabelled benzo[a]pyrene (BaP) as a model pollutant. Experimental animals were sponges, mainly Tethya lyncurium from the Northern Adriatic and from the Pacific near Catalina Island, California, U.S.A. Changes in ornithine decarboxylase (ODC) activity (ODC; EC 4.1.1.17) and polyamine concentrations with and without pollution were observed. There is a slow rise in ODC activity during the course of three-weeks exposure and a fast increase of polyamine levels during the course of one day. Mixed function oxygenase (MFO; EC 1.14) activity could not be detected in sponges. There was a significant concentration dependent coupling of radioactive BaP derivatives (BaPD) to the macromolecular fractions; the highest in protein, X 1000 greater than DNA and X 500 greater than RNA. Coupling is light-mediated and drops to zero in the dark. However when activated microsomal fractions from fish, that had been exposed to high level polycyclic aromatic hydrocarbon (PAH) pollution are added, dark incorporation rises to significant levels which can be decreased by the MFO inhibitor 7,8-benzoflavone (BP). The question of possible absence of DNA repair in the sponges and some implications are discussed.

Animals↗
PubMed1981

Lectin, a possible basis for symbiosis between bacteria and sponges.

From the marine sponge Halichondria panicea a lectin was isolated and characterized. The homogeneous lectin (composed of protein to 80.7% and of neutral carbohydrates to 14.1%) had a molecular weight of 78,000 (determined by gel filtration) and consisted of four subunits with a molecular weight of 21,000 each (determined by gel electrophoresis in the presence of sodium dodecyl sulfate). The hemagglutinating activity was only slightly dependent upon ionic strength and incubation temperature and did not require divalent cations, but it was inhibited by reagents for thiol groups. The Halichondria lectin was completely inhibited in hemagglutination competition experiments in the presence of fetuin, D-galacturonic acid, D-glucuronic acid, polygalacturonic acid, or L-fucose. The purified Halichondria lectin did not cause reaggregation of dissociated H. panicea cells. From the same sponge species bacteria were isolated and identified as Pseudomonas insolita. These bacteria were cultivated in marine broth 2216. Under these culture conditions the bacteria grew only in the presence of the homologous lectin; the lectin-caused effect was not abolished by D-glucuronic acid or D-galacturonic acid. However, after addition of a polysaccharide-containing fraction isolated from P. insolita, the lectin-caused, growth-promoting effect was abolished. Other lectins were found to exhibit no growth-promoting effect. On the basis of colony counts, P. insolita was the predominant bacterial species in the sponge extract; 1.9 X 10(6) Pseudomonas colonies were measured in extracts isolated from 1 g of sponge. The assumption of an interrelationship between the sponge and the bacterium is supported by the results indicating that the Halichondria lectin has no effect on the growth of such bacteria isolated from six other marine sponge species. Evidence is presented which indicates that the Halichondria lectin is not utilized during growth of the Pseudomonas species. Lectin activity was detected on the surface of mucoid cells from H. panicea. From the data obtained the possibility is discussed that the Halichondria lectin is a basis for a symbiotic relationship between the sponge and the bacterium.

Animals↗
PubMed1981

Serum DNase activity after experimental, acute hemorrhagic-necrotizing pancreatitis in dogs: detection of a serum DNase isoenzyme.

The activity of a neutral serum DNase has been determined in dogs before and after experimental, acute hemorrhagic-necrotizing pancreatitis. Dog serum DNase corresponds in its conditions of activity and in its isoelectric point with the DNase I. Electrophoretically three DNase isoenzymes can be distinguished. 30 min after induction, an increase of DNase activity was observed. This activity increase is related to only one of the three DNase isoenzymes. After induction a DNase with an isoelectric point of 5.6 occurs. This enzyme is not found in healthy animals.

Acute Disease↗
PubMed1981

Inhibition of DNA synthesis in permeabilized mouse P815 mast cells by arabinofuranosyladenine 5'-triphosphate.

P815 mast cells (mouse leukemic cells) have been successfully permeabilized to 9-beta-arabinofuranosyladenine 5'-triphosphate (araATP) using lysolecithin. The permeabilized cells remained viable according to the following criteria: (a) exclusion of trypan blue after replacing the lysolecithin with culture medium; (b) cell growth rate identical to control cultures, and (c) intact DNA-synthesizing complex in permeable cells. It was shown that permeabilized cells incorporate [3H]thymidine and [3H]deoxyadenosine-triphosphate (dATP) into DNA when the suspension is supplied with the appropriate substrates and cofactors. Permeable cells break down araATP to 9-beta-D-arabinofuranosyladenine 5'-diphosphate only slightly after incubation for 60 min; no further dephosphorylation to 9-beta-D-arabinofuranosyladenine 5'-monophosphate or 9-beta-D-arabinofuranosyladenine occurs. In kinetic experiments it was shown that the DNA-replicating complex of permeabilized cells is competitively inhibited by araATP with respect to dATP; Ki(araATP) was determined to be 0.6 microM and the Km(dATP), 0.9 microM.

Animals↗
PubMed1981

Stimulation of DNA polymerase beta activity in permeabilized mouse P815 mast cells after neocarzinostatin treatment.

Neocarzinostatin (NCS), which causes DNA strand scission both in vitro and in vivo, reversibly inhibits the growth of both unpermeabilized and lysolecithin-permeabilized P815 mast cells (mouse leukemic cells). Kinetic experiments with NCS-pretreated cells revealed that the permeabilized cells are more strongly affected than the unpermeabilized cells, indicating that the membrane protects the cells against the influence of NCS. The two major DNA polymerase activities (form alpha and form beta) were determined in permeabilized cells during the lag phase of growth, after NCS treatment, and an 8.5-fold higher DNA polymerase beta activity was observed in NCS-treated cells than in controls. The activity of the second enzyme, DNA polymerase alpha, was low during the period of cell proliferation.

Animals↗
PubMed1981

Cell aggregation of the marine sponge Geodia cydonium. Identification of lectin-producing cells.

A D-galactose-specific lectin, purified from the marine sponge Geodia cydonium, is present on the cell surface of mucoid cells, free choanocytes and choanocyte clusters, as revealed first, by the adhesion assay which is based on the formation of "rosettes" with erythrocytes, and second, by immunofluorescence studies. Using the same techniques no lectin could be identified on the surface of archaeocytes. Rosette formation was inhibited in the presence of 20 mM D-lactose as well as after preincubation of erythrocytes with purified lectin. Titration experiments in a hemagglutination assay showed that the highest level of extractable lectin (5% of the total protein) is found in mucoid cells, lower concentrations are determined in choanocyte clusters (0.07%), free choanocytes (0.05%) and archaeocytes (0.01%). Only the mucoid cells were found to synthesize lectin which is secreted and subsequently transferred to the cell surface of other cell types. As one consequence of the binding of the lectin to the cell surface of aggregation-deficient choanocytes or archaeocytes, the conversion of these cells to aggregation-susceptible ones is observed. These results support previous evidence that the lectin is involved in the reaggregation process of single cells in the homologous biological system.

Animals↗
PubMed1980

Isolation and characterization of the novel polyadenylate- and polyuridylate-degrading acid endoribonuclease V from calf thymus.

A novel endoribonuclease was detected and purified to homogeneity from calf thymus. The homogeneity was checked by analysis in polyacrylamide gels (both in the presence and in the absence of sodium dodecyl sulfate) as well as by isoelectric focusing. This nuclease activity, which is called Endoribonuclease V, cleaves poly(A) and poly(U); other single- or double-stranded synthetic polyribo- as well as polydeoxyribonucleotides are not degraded. Endoribonuclease V cleaves poly(A) to create first oligoribonucleotides and ultimately 3'-AMP; no P-2':3'-Ado degradation products were detected. The enzyme has a pH optimum of 5.8, an isoelectric point of pH 6.3, a molecular weight of 52,300, and requires neither monovalent nor divalent cations. The enzyme activity is not inhibited by N-ethylmaleimide.

Animals↗