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Biomedical subjects

R K Zahn

Publications and source records attributed to R K Zahn.

At least 55 records · Page 3Linked to original sources

Attachment of sponge cells to collagen substrata: effect of a collagen assembly factor.

Collagen, isolated from the sponge Geodia cydonium in the absence of denaturing agents, had the typical amino acid composition and was associated with the carbohydrates galactose and glucose. The resulting individual fibrils with a diameter of 23 nm, displayed a 19.5 nm periodicity with one intraperiod band. A collagen assembly factor (CAF) was identified in and partially purified from the extracellular space. The CAF reacted with antibodies against intact Geodia cells but not with antibodies against Geodia lectin and Geodia aggregation factor. In the presence of the CAF, the collagen fibrils reconstituted collagen bundles in an ordered sequence of events, which were followed by electron-microscopical and biochemical methods. Bundle formation was not dependent on the presence of the homologous lectin, glycoconjugates or aggregation factor. Homologous cells (Geodia archaeocytes) were determined to attach only to those Geodia collagen substrates that contained CAF. The attachment of these cells did not require fibronectin or Geodia lectin. Homologous glycoconjugates or NaOH-treated collagen inhibited cell attachment. Collagen from the sponge Chondrosia reniformis, even in the presence of Geodia CAF, was no appropriate substrate for Geodia cell attachment. Whether collagen is a component of cell-matrix interactions in sponge systems also in vivo is discussed.

Animals↗

Potent antileukemic activity of the novel cytostatic agent avarone and its analogues in vitro and in vivo.

Avarone and avarol are novel cytostatic agents which have potent antileukemic activity both in vitro and in vivo (mice). Cell culture experiments revealed that the cytostatic activity of these two compounds on L5178Y mouse lymphoma cells was 13- to 14-fold higher than that determined for HeLa cells and 40- to 43-fold higher than that for human melanoma cells. Nontumor cells (human fibroblasts and human gingival cells) were highly resistant against the two compounds. The inhibitory potency of avarone on L5178Y cells (50% inhibitory concentration, 0.62 microM) was significantly higher than the avarol activity (50% inhibitory concentration, 0.93 microM). Modification of the molecule at the quinone ring or the double bond in the terpenoid skeleton resulted in a significant loss of activity. In vivo studies with L5178Y cells in the ascites of mice confirmed the strong antileukemic effect determined in vitro. At doses of 10 mg/kg given i.p. once daily for 5 days to mice bearing approximately 10(8) leukemia cells, avarone was found to be curative in about 70% of the mice (20% for avarol). The optimal daily i.p. dose of avarone increased life span over controls by 146% when treatment was begun 1 day after tumor implantation and by 87% when treatment was delayed until day 8. Avarol, although active, was less effective. Based on the determined log10 kill values, avarone can be classified as a highly active and avarol as a markedly active cytostatic agent. The efficacy of the two compounds is also emphasized by the therapeutic index of 11.7 for avarone and of 4.5 for avarol. The two agents were determined not to be either direct mutagens or premutagens in the Ames test.

Animals↗

Control of L5178y cell growth by the galactose-specific lectin from Geodia cydonium.

The galactose-specific lectin from the sponge Geodia cydonium was determined to cause an increase of the growth rate of L5178y mouse lymphoma cells. The lectin interacts with cell surface components which were solubilized and enriched by affinity chromatography; their Mr's were 170,000, 140,000 and 88,000. Results from Ouchterlony diffusion studies suggest that the cell surface ligand is monovalent. Given to cells in suspension, the lectin causes cell agglutination. This process could be abolished by coincubation with the soluble cell surface ligand. Plating the cells onto substrate-attached lectin resulted in a stimulation of cell spreading. Scatchard analyses revealed that L5178y cells contained 6.3 X 10(7) lectin binding sites with an affinity (Ka) of 1.7 X 10(7) M-1. The binding of the Geodia lectin to the cell surface can be prevented by addition of horse serum. The blocking serum components were isolated by affinity chromatography and determined to consist of six protein species.

Animals↗

Inhibition of mitosis by avarol, a natural product isolated from the sponge Dysidea avara.

Avarol, a sesquiterpenoid hydroquinone, is a cytostatic agent, isolated from the sponge Dysidea avara. Autoradiographic studies show that in vivo (L5178y mouse lymphoma cells) avarol changes the labelling index in favour of the fraction of unlabelled cells (from 1.24 to 1.04). At concentrations below the 50% inhibition dose, the mitotic index increases from 6.5 +/- 0.5 to 10.4 +/- 0.8; at higher concentrations the formation of mitotic figures is almost completely suppressed. In vitro studies applying the methods of viscosimetry and electron microscopy demonstrate that avarol inhibits assembly of brain microtubule protein at an at least stoichiometric concentration ratio. Moreover, evidence is presented that the new antimitotic agent avarol inhibits protofilament elongation rather than lateral association of tubulin during protofilament formation. The results suggest that avarol interferes with polymerization of tubulin both in interphase and during mitosis.

Animals↗

The role of protein phosphokinase and protein phosphatase during the nuclear envelope nucleoside triphosphatase reaction.

The activities of nuclear envelope-associated protein phosphokinase and protein phosphatase were determined in nuclear ghosts from liver and oviduct of quails. The protein kinase was found to be inhibited by poly(A) by 75%. During the kinase reaction proteins with molecular weights of 106 000 and 64 000 were phosphorylated. The phosphoprotein phosphatase from liver was stimulated to 190% by poly(A), whereas only a slight enhancing effect by this polymer was determined with the oviduct enzyme (to 125%). Comparative determinations of the nuclear ghost-associated enzyme activities revealed the following values (in nmol Pi/min per 10(8) ghosts); oviduct: phosphokinase, 0.015; phosphatase, 0.004 and nucleoside triphosphatase, 39.4; and liver: phosphokinase, 0.044; phosphatase, 0.012 and nucleoside triphosphatase, 11.7. These data indicate that phosphorylation/dephosphorylation proceeds independently of the nucleoside triphosphatase cycle. This assumption is supported by analytical results revealing that no marked dephosphorylation occurs after poly(A) binding to the nuclear envelope. Moreover, stoichiometrical data showed a nearly 1:1 molar ratio between ATP-binding and phosphorylation of nuclear envelope protein. From these findings a new model for the nucleoside triphosphatase-mediated poly(A)(+)mRNA efflux from nuclei is deducted, proposing phosphokinase and phosphatase only to modulate the affinity of the 'carrier structure' for poly(A) (+)mRNA, but not to constitute the nucleoside triphosphatase.

Animals↗

Age-dependent changes of nuclear envelope protein phosphokinase and protein phosphatase activities. Significance for altered nucleo-cytoplasmic mRNA translocation during development.

Nuclear envelopes are associated with a protein phosphokinase and a phosphoprotein phosphatase, whose activities are modulated by poly(A) in an opposite manner. The activities of these enzymes were determined in nuclear ghosts from liver and oviduct of quails of different age and of different hormone status. Under optimal conditions, kinase activity was found to increase in immature animals 8-fold in response to diethylstilbestrol; co-administration of progesterone had no marked effect on enzyme activity. After the initial burst, the activity of the enzyme increased only slightly during ageing. Two proteins present in nuclear ghosts of Mr 64 000 and of Mr 106 000 are phosphorylated during the kinase reaction; both the relative and the absolute extents of phosphate incorporation into these proteins alter drastically during ageing or hormone treatment of immature animals. Like the kinase activity, the activity of protein phosphatase increased in immature animals markedly in response to hormone treatment. Thereafter the activity remained constant in liver while in oviducts the phosphatase activity dropped to 30% in both mature and old animals.

Aging↗

Binding of polyribonucleotides and polydeoxyribonucleotides to bovine brain microtubule protein: age-dependent modulation via phosphorylation of high-molecular-weight microtubule-associated proteins and tau proteins.

Binding of both synthetic poly(A) and naturally occurring poly(A) (+)mRNA as well as DNA to microtubule protein is mediated by microtubule-associated proteins; tubulin itself is not capable of binding these polymers. Bovine brain microtubule protein from immature animals was found to have a significantly lower capacity to bind poly(A) than microtubule protein from old animals. On the other hand, "old" microtubule protein binds DNA more efficiently than "immature" microtubule protein. Microtubule-associated protein 2 [preferred binding site for DNA] and tau proteins [preferred binding site for poly (A)] are specifically phosphorylated by a microtubule-associated, cAMP-dependent protein kinase. It was found that the affinity of microtubule protein for poly(A) is markedly decreased by autophosphorylation of the protein; in the case of DNA, the decrease in affinity was less. Autophosphorylation of "immature" microtubule proteins diminished the binding capacity for poly(A) to a greater extent than do "old" proteins. Scatchard plot analysis revealed that microtubule-protein possesses two different binding sites for poly(A). The corresponding dissociation constants were found to be increased in the phosphorylated system, but phosphorylation does not appear to alter the total number of binding sites. Compared to immature animals, microtubule protein from "old" bovine brains was found to have a reduced number of binding sites for poly(A), whereas the values of the dissociation constants remain unchanged. In contrast to total microtubule protein and homogeneous microtubule-associated protein 2, only one kind of binding site for poly(A) could be detected in homogeneous tau protein. No influence of different RNA or DNA species on microtubule protein-associated cAMP-dependent protein kinase, adenosine triphosphatase and guanosine triphosphatase activities could be detected.

Aging↗

Induction of DNA polymerase alpha and terminal deoxynucleotidyl transferase in the human lymphoblastoid cell line Molt-4 by the immunomodulator bestatin.

The influence of the immunomodulator bestatin on the expression of terminal deoxynucleotidyl transferase and of DNA polymerase alpha and beta in Molt-4 cells has been studied. Bestatin was found to stimulate cell growth within the range of 0.3-33 microM, while concentrations higher than 300 microM were inhibitory during an incubation period of 48 h. The cell surface bound microsomal leucine aminopeptidase (bestatin receptor) activity decreased gradually during incubation at concentrations of bestatin above 3 microM. This effect was also observed after incubation with amastatin, but not with leupeptin or tunicamycin. Determinations of the activities of DNA synthesizing enzymes from bestatin-treated Molt-4 cells revealed a direct correlation between the decrease of the surface bound microsomal leucine aminopeptidase activity and the increase of the terminal deoxynucleotidyl transferase and DNA polymerase alpha activity; the DNA polymerase beta activity remained unchanged. From these experiments it is hypothesized that bestatin might cause a promoting effect on the differentiation processes of precursor T cells in vivo.

Adjuvants, Immunologic↗

Intraspecific recognition system in scleractinian corals: morphological and cytochemical description of the autolysis mechanism.

Intraspecific aggressive interactions have been observed in the scleractinian coral Stylophora pistillata. Extensive field observations suggest that intraspecific interactions result in the formation of necrosis in one of the two competing colonies. The formation of necroses is primarily restricted to those layers that are interspersed with polyps. In a second phase the non-living, central corallum is destroyed by foreign microorganisms. In some cases the necroses are resealed again, either by a living septal layer or by a calcareous plug. Evidence is presented indicating that the enzyme carbonic anhydrase has a role in these activities. First, it is involved in the apical growth of the colony. Using a semiquantitative assay procedure, the highest enzyme level was determined in polyps at the tips of the branches; lower levels were measured in polyps of the foot/trunk region. Secondly, the enzyme was found to be induced in those areas that are in close contact with non-isogeneic tissue. From these data we conclude that carbonic anhydrase is involved in the process of autolytic digestion in response to intraspecific contact of two non-clonemates.

Autolysis↗

Synergistic effect of peplomycin in combination with bleomycin on L5178y mouse lymphoma cells in vivo.

Studying the treatment of NMRI mice with ip injections of bleomycin (BLM) for 5 days we found an approximate LD50 of 35 mg/kg; the toxicity of peplomycin (PEP) was slightly higher (LD50: approximately 25 mg/kg). The effect of the two drugs on growth of L5178y mouse lymphoma cells in NMRI mice was examined. BLM alone caused at a concentration of 2.5 mg/kg an almost complete inhibition of tumor cell growth; the same effect was determined with 1 mg PEP/kg. At these concentrations the drugs caused an increase of the survival time of 110% (BLM) or 104% (PEP). Given in combination, one-sixth of the optimal doses yielded an 100% increase of the median survival time. These results indicate a significant synergistic activity of the PEP-BLM combination on L5178y cell growth in vivo (FIC index: 0.34).

Animals↗

Inhibition of nuclear envelope nucleoside triphosphatase-regulated nucleocytoplasmic messenger RNA translocation by 9-beta-D-arabinofuranosyladenine 5'-triphosphate in rodent cells.

Nucleocytoplasmic translocation of polyadenylated messenger RNA is an energy-dependent process which is regulated by a nuclear envelope nucleoside triphosphatase; this enzyme was found to be stimulated by the 3'-terminal polyadenylic acid [poly(A)] tail of messenger RNA (Bernd, A., Schröder, H. C., Zahn, R. K., and Müller, W. E. G. Eur. J. Biochem., 129: 43-49, 1982). RNA efflux from isolated mouse lymphoma (L5178Y) cell nuclei is strongly reduced if 9-beta-D-arabinofuranosyladenine 5'-triphosphate (ara-ATP) is present in the transport medium. Half-maximal inhibition of RNA efflux occurs with 120 microM ara-ATP. Most likely, the inhibitory effect of ara-ATP is caused by inhibition of nuclear envelope nucleoside triphosphatase; this enzyme was found to be highly sensitive to inhibition by this antibiotic. The inhibition type of the nucleoside triphosphatase of rat liver nuclear ghosts is competitive with respect to ATP; the Ki:Km ratio was determined to be 0.27. Besides nucleoside triphosphatase, nuclear envelopes contain a protein phosphokinase modulating the affinity of pore complex laminae to poly(A). This enzyme was also found to be strongly inhibited by ara-ATP in a competitive way with respect to ATP (Ki:Km, 0.056) and could therefore also contribute to the overall inhibition of RNA transport. The polyadenylation of endogenous RNA by poly(A) polymerase(s) in intact rat liver nuclei as well as in nuclear matrices isolated from the same source was found to be markedly suppressed in the presence of ara-ATP. The inhibitions of both poly(A) polymerase activities (contained in whole nuclei or nuclear matrix bound) are of the competitive type with respect to ATP. In in vitro assays, nuclear envelope nucleoside triphosphatase is inhibited by microtubule protein. Of the 2 ATP-dependent enzyme activities associated with microtubule protein (cyclic adenosine 3':5'-monophosphate-dependent protein kinase and adenosine triphosphatase), only the kinase was slightly affected by ara-ATP. Cellular uptake of adenosine 5'-monophosphate and perhaps 9-beta-D-arabinofuranosyladenine 5'-monophosphate (ara-AMP) is facilitated by a cellular membrane-bound 5'-nucleotidase. Our studies revealed that neither cleavage of ara-AMP nor inhibition of the enzyme activity by ara-AMP occurs. 9-beta-D-Arabinofuranosyladenine and ara-AMP represent neither direct mutagens nor premutagens as determined by the Salmonella-mammalian microsome mutagenicity test.

Animals↗

Fast ephemeral DNA damage upon BaP injection.

Injection of high concentrations of benzo[a]pyrene (10-125 micrograms BaP/g body weight) into young frogs (Xenopus laevis), fish (Gambusia affinis) or tissue culture cells (L 5178 Y) in a state of logarithmic growth causes alterations in DNA. In frogs these alterations reach a maximum at 60-90 min after application, then they decrease and become undetectable after 3 h. Within 8-12 days after the single BaP dose, a new wave of DNA alterations can again be detected. Parallel measurements of mixed function oxygenases (MFO) showed no short term activity changes within the first day. There was, however, an increase of activity starting on the 8th day following injection in frogs, and lasting until day 14. DNA alterations have been assessed by two methods. One is a modification of the alkaline filter elution method of Kohn et al. (1976) and is believed to determine the number of events leading to single strand breaks at alkaline pH. The other method determines the number of S1 nuclease sensitive sites in highly purified native DNA. Both results were found to be highly correlated. The effect does not seem to be caused by an impurity, unless an unknown, very minor, and extraordinarily active component is present. The effect in frogs is linked to nutritional status. Frogs fed a carbohydrate-rich, protein-poor diet do not give the fast response. The fast effect cannot be blocked by application of Actinomycin, Cordycepin, and Chloramphenicol injected either 30 min before or simultaneously with the BaP. Benzo(e)pyrene, a non-tumorigenic isomer of BaP, does not cause any effects at similar doses.

Animals↗

Association of a polyuridylate-specific endoribonuclease with small nuclear ribonucleo-proteins which had been isolated by affinity chromatography using antibodies from a patient with systemic lupus erythematosus.

Immunoglobulins, containing antibodies against U1-snRNP, have been prepared from a patient with systemic lupus erythematosus. After coupling these antibodies to a Sepharose matrix, U-snRNPs have been isolated and purified from rat liver nuclei by use of immunoaffinity chromatography. The resulting RNPs had the typical protein pattern of U-sn RNPs and a sedimentation coefficient of 12 S. The U-snRNP preparation was associated with an endoribonuclease which required Mg2+ for optimal activity. The enzyme, with an pH optimum of 6.2, degraded only poly(U). Other single-stranded polyribo- and polydeoxyribonucleotides, tRNA, as well as double-stranded RNA and DNA were not digested. The products of a terminal digestion are (U)6-12 with 3'-OH and 5'-P termini. The possible involvement of this endoribonuclease in the splicing of hnRNA is discussed.

Animals↗

Characterization of the trimeric, self-recognizing Geodia cydonium lectin I.

A D-galactose-specific lectin I was extracted from the sponge Geodia cydonium and purified by affinity chromatography. The molecular weight of lectin I as determined by high-pressure liquid gel chromatography, was found to be 36500 +/- 1300. Disc gel electrophoresis in the presence and in the absence of sodium dodecyl sulfate showed that lectin I is a trimer composed of three different subunits (Mr: 13800, 13000 and 12200); two of the three subunits are linked by one disulfide bond. Isoelectric focusing gave a pI of 5.6 for the native molecule and a pI of 4.4 and of 7.4 for the subunits. The three subunits carry carbohydrate side chains, composed of D-galactose (94%) and of arabinose (5%). Based on experiments with lectins, the terminal D-galactose residues are bound by beta 1 leads to 6 and/or beta 1 leads to 4 glycosidic linkages. The Geodia lectin I contains, besides two carbohydrate recognition sites, at least one receptor site for a second lectin I molecule.

Amino Acids↗

Purification and properties of a novel pyrimidine-specific endoribonuclease termed endoribonuclease VII from calf thymus that is modulated by polyadenylate.

Endoribonuclease VII, a novel endoribonuclease from calf thymus, was identified and purified by us. The purified enzyme has Mr = 74,000; its homogeneity was checked by analysis in polyacrylamide gels (both in the presence and in the absence of sodium dodecyl sulfate). The nuclease cleaves poly(U) and poly(C) while other single-stranded homopolyribo- as well as polydeoxyribonucleotides are not degraded; poly(A,C) is hydrolyzed to a smaller extent, while poly(U) X poly(A) is not degraded at all. Poly(A) modulates the poly(U)-degrading activity; at a molar ratio of approximately 1 [poly(A)]:10 [poly(U)], a more than 100% stimulation of the enzyme activity was achieved, while at lower ratios an almost complete inhibition of the enzyme activity resulted. Binding studies revealed that endoribonuclease VII has a marked affinity for poly(A) and poly(U). During hydrolysis, oligo(U)12 fragments with 3'-OH and 5'-P termini are formed. The basic enzyme (pI = 8.5) has its activity optimum at pH 7.2, requiring neither monovalent nor divalent cations; the enzyme is not inhibited by thiol group reagents. Several lines of evidence suggesting a role of endoribonuclease VII in mRNA processing are presented.

Animals↗

Identification and properties of the cell membrane bound leucine aminopeptidase interacting with the potential immunostimulant and chemotherapeutic agent bestatin.

Bestatin was found to be a competitive inhibitor (with respect to the Leu-NA substrate) not only of the isolated microsomal and cytosolic leucine aminopeptidases (Leu-APm and Leu-APc) but also of the aminopeptidases (APs) present in membrane preparations (from mouse liver) and on the cell surface of L5178Y cells. Kinetic parameters indicate that cellular AP is identical to Leu-APm. To rule out the possibility that AP-B is involved in the inhibition reactions, comparable studies with amastatin were performed. Electrophoretical studies revealed the solubilized cell membrane bound AP to co-migrate with Leu-APm in polyacrylamide gels. The activity of the separated membrane AP was inhibited by bestatin in situ. The cell membrane bound AP activity was found to be lowest in lymphocytes, higher in tumor cells and highest in bone marrow cells and macrophages. Using synchronized L5178Y cells, the AP activity changes during the cell division cycle; the lowest activity was determined during the G1-phase and 35% higher values were measured during the S/G2-phase. The fluctuation of the cell surface associated AP activity parallels with changes in the number of binding sites for bestatin.

Animals↗

Microdisc-electrophoretic study of deoxyribonucleases in cow snout epidermis.

Acid and neutral deoxyribonucleases (DNases) of the cow snout epidermis were investigated by the microdisc-electrophoresis of polyacrylamide gels containing highly polymerized DNA and by isoelectric focusing techniques. The nucleases were characterized with respect to their pH optimum. An acid DNase at pH 5.0 was detected as a single distinct band after the electrophoretic separation. After isoelectric focussing also, only one acid DNase activity with an isoelectric point (IP) of 6.2 was detectable. Neutral DNases at pH 7.4 were demonstrated as major and minor bands by their different electrophoretic mobilities. In the isoelectric focusing system also, two neutral DNases, a major one (IP, 4.6) and a minor one (IP, 6.4), were found. Characterization with respect to their histologic location showed acid and neutral DNases across the epidermal layers with the highest activities in the upper layers, where DNA concentration had been shown to be lowest. In correlation with their subcellular distribution, the highest activities of both acid and neutral DNase were found in the 105,00 X g supernatant of the subcellular fractions.

Animals↗

Age-dependent alterations of microtubule-associated enzyme activities from bovine brain (protein kinase, adenosine triphosphatase, guanosine triphosphatase).

Microtubules have been isolated from immature (3-4 weeks' old) and old (11-13 years' old) bovine brains. Quantitative studies revealed that the concentration of extractable microtubule protein per gram of wet brain decreased from 0.47 mg (immature animals) to 0.34 mg (old animals). The major components of microtubule protein (tubulin and high-molecular-weight microtubule-associated proteins) do not undergo an age-correlated change. Determination of the endogenous protein kinase activity revealed that the activity associated with "immature" calf brain microtubules was six times higher than the activity present in "old" preparations. In contrast, the stimulatory effect of cyclic AMP on protein phosphorylation in microtubules from old bovine brains exceeds nine-fold the value obtained from immature animals. After addition of casein (exogenous acceptor), the basal activities increased in both preparations without altering the age-correlated difference in the specific activity. By comparing the radioactivity pattern of sodium dodecyl sulfate polyacrylamide gels after autophosphorylation of microtubule protein with [gamma-32P]ATP, 1.5 moles of phosphate per mole of high-molecular-weight microtubule-associated protein were estimated to be incorporated in preparations from immature animals and 0.9 mole of phosphate per mole of associated protein in the experiments with "old" microtubule protein. Adenosine triphosphatase activity, associated with the high-molecular-weight microtubule-associated protein 1, was determined to be 15% reduced in preparations from old animals, compared to the activity in "young" preparations. In contrast, the guanosine triphosphatase activity increased five-fold during ageing; the higher activity of this enzyme was observed both during the initial and the steady-state phases of microtubule formation.

Adenosine Triphosphatases↗