Josef Arendes

Activation of DNA metabolism in T-cells by bestatin.

Abstract Bestatin, [(2S, 3R)-3- amino -2- hydroxy -4- phenylbutanoyl ]- l - leucine , is a microbial product which selectively influences the DNA metabolism of lymphoid tissues in vivo . The present studies, using CBA/J mice, revealed that bestatin increases the incorporation rate of dThd into DNA in spleen, thymus and bone marrow, but not in liver, kidney, spinal cord and lung. The stimulation was found to be dose-and timedependent, and it occurs only in T-cells, not in B-cells, from spleen and thymus. In addition, it is shown that bestatin causes a several-fold induction of DNA polymerase α only in T-cells from spleen and thymus, while the level in B-cells remains constant. Bone marrow cells respond to bestatin treatment with an increase of the DNA polymerase α activity and with an 8-fold induction of terminal deoxynucleotidyl transferase.

Aggregation of sponge cells. XX. Self-aggregation of the circular proteid particle.

In the extracellular space of the tissue of the sponge Geodia cydonium, circular proteid particles are found which carry as subunits the aggregation factor and a series of glycosyltransferases. Using the technique of velocity sucrose gradient centrifugation, the sedimentation coefficient (S020,w) of the particle-monosomes was determined to be 90. By means of the Svedberg equation a molecular weight of 1.3 . 10(8) daltons could be estimated. The monosomes aggregate in the presence of Ca2+ to higher complexes via disomes, trisomes, and pentasomes. The complexes can be redissociated by dodecyl sulfate but not by EDTA. During the Ca2+-mediated self-aggregation, the particles lose their biological activity with respect to their aggregation promoting function.

Age-Dependent Enzymatic Poly(A) Metabolism in Quail Oviduct

The poly(A) metabolism in oviducts from adult and senescent quails has been studied. The incorporation studies by double-labelling of mRNA with [3H]adenosine and [3H]uridine revealed, that after inhibition of transcription by actinomycin D, the incorporation ratio adenosine/uridine increases drastically in adult animals compared with the ratio determined for mRNA from senescent animals. This finding is a hint that in senescent animals the poly(A) stretch of mRNA is shorter than in adult animals. This assumption is supported by the finding that the activity of the extractable poly(A) exoribonuclease is higher in senescent than in adult animals. The activity of poly(A) exoribonuclease is even enhanced after stimulation of senescent animals with progesterone. The activity of the other catabolic enzyme studied, the poly (A) endoribonuclease, as well as the activity of anabolic poly(A) enzyme, the poly(A) polymerase, are almost identical in oviducts from adult and senescent animals. The properties of the catabolic poly(A) enzymes and of the anabolic poly(A) polymerase seem to be identical.

Phosphorylation of Arabinofuranosylthymine in Non-infected and Herpesvirus (TK+ and TK-)-Infected Cells

The phosphorylation of arabinofuranosylthymine (araThd) has been studied both in non-infected cells and in those infected with herpes simplex virus (HSV-1, Lennette; HSV-1, IES and HSV-2, D-316). In these experiments, HSV strains were used which either contain (Lennette, TK+ and D-316 TK+) or lack (IES, TK-) the capacity to induce pyrimidine deoxyribonucleoside kinase. It was found that extracellularly administered araThd is phosphorylated to ara TTP via araTMP and araTDP in both non-infected and in HSV-infected cells. The phosphorylating capacity is more than tenfold lower in non-infected cells than in infected cells. Interestingly, cells infected with the TK- strain have a tenfold higher phosphorylating capacity than normal, uninfected cells, a fact which might indicate that host cell deoxythymidine kinase is induced during HSV infection. AraTMP is incorporated into cellular DNA but not into HSV DNA. This finding is in contrast to observations with arabinofuranosyladenine, which is incorporated into both cellular and HSV DNA. In vitro experiments with HSV-induced DNA polymerase show that araTTP strongly inhibits the enzyme activity. Therefore we conclude that the inhibition of HSV DNA polymerase by araTTP (formed intracellularly from araThd) is the explanation for the observed antiviral activity of araThd.

Size determination of poly(A) after in vitro methylation with radioactive dimethyl sulfate

A simple method for the size determination of poly(A) using in vitro labeling by methylation with [3H]dimethyl sulfate is described. After methylation, modified poly (A) has the same mobility, using polyacrylamide gel electrophoresis, as has the unmodified polymer, thus showing that the methylation does not cause degradation. Therefore the method is a sensitive assay to size the poly(A) segments from in vivo unlabeled tissue. The method was applied to determine the size of poly(A) sequences on mRNA from mouse L5178Y cells and from rat liver.

Arabinosyl nucleosides. XII. Influence of arabinofuranosylthymine on growth of L5178y cells

The antibiotic 1-beta-D-arabinofuranosylthymine (araThd) is a potent inhibitor of the growth of mouse lymphoma cells (L5178y). The ED50 concentration was found to be 9.8 muM. The cells die as a consequence of an unbalanced growth. The cytostatic activity of araThd can be abolished by coincubation with dThd and dUrd but not with Urd. At cytostatic concentrations araThd selectively blocks DNA synthesis; RNA- and protein synthesis are unaffected. Intracellularly araThd is rapidly phosphorylated to araTTP. This enzymic phosphorylation does not influence the synthesis of the naturally occuring, related triphosphate dTTP. AraTMP is incorporated into DNA during DNA synthesis; 1 mol of ara-TMP is incorporated/19,500 molecules of dTMP.

Filter paper disk techniques for assay of nucleotidase

A DE filter disk technique for assaying the activity of nucleotidase is described. This method is based on the observation that nucleotides bind to the filters at 5 mM Tris-HCl (pH 7.8) while nucleosides do not. As parameter for the nucleotidase activity the decrease of bound nucleotides is determined. In parallel experiments the amount of the product (nucleoside) formed can be measured by DEAE Sephadex column chromatography.The filter disk technique can be applied for the determination of vmax and Km of a nucleotidase by using different ribonucleosidase monophosphate substrates.

Differential mode of inhibition of terminal deoxynucleotidyl transferase by 3′-dATP, ATP, βaraATP and αaraATP

Terminal deoxynucleotidyl transferase (TdT) catalyzes the primer-dependent but template-independent DNA polymerization of deoxynucleoside 5’-triphosphates; the enzyme was discovered and purified to homogeneity by Bollum [ 1,2]. The TdT is only present in thymus, in bone marrow [3] and in blood lymphoblasts of patients with acute lymphoblastic leukemia [4]. TdT has also been reported to be present in nonthymic cells [5], as well as in germinating wheat embryo [6]; for a critical reexamination of these observations an optimized TdT assay [2,7] and specific inhibitors are necessary. To establish these prerequisites proper experiments with purified TdT preparations must be performed. In the present study it is shown that the activity of a 700-fold enriched TdT preparation from thymus is competitively inhibited by ATP, 3’-deoxyadenosine triphosphate (3’-dATP; cordycepin triphosphate) and 9.O-D-arabinofuranosyladenine 5’-triphosphate @araATP) with respect to both the natural substrate 2’-dATP (dATP) and the initiator oligo [d(pA)a]. 9-cwD-Arabinofuranosyladenine 5’-triphosphate (oaraATP) was found to exert no inhibitory effect toward TdT.

Poly(Adenosine Diphosphate Ribose) Synthesis during Herpes Simplex Virus Infection

Immediately after infection of baby hamster kidney cells with herpes simplex virus (HSV), cellular DNA synthesis was blocked, while extensive HSV DNA synthesis began. These dramatic alterations of the control mechanisms for these two DNA synthesizing systems were not accompanied by a change in the poly(adenosine diphosphate ribose) polymerase activity.

Differential and selective inhibition of cellular and herpes simplex virus DNA synthesis by arabinofuranosyladenine

The influence of 9-beta-D-arabinofuranosyladenine (beta araAdo) and of its anomer 9-alpha-D-arabinofuranosyladenine (alpha araAdo) was studied in non-infected cells and cells infected with herpes simplex virus type 1 (HSV-1) and HSV type 2 (HSV-2). alpha AraAdo is a strong inhibitor of proliferation of non-infected cells. Multiplication of HSV-1 and HSV-2 is not affected at all by alpha araAdo, while their growth is strongly inhibited by beta araAdo. alpha AraAdo exerts no effect on the incorporation of dThd into HSV DNA, but blocks the incorporation into host cell DNA. Its anomer, beta araAdo, affects the incorporation rate of both the viral DNA system and the host cell DNA system (the latter one to a lesser extent). alpha AraAMP is incorporated into newly synthesized cellular DNA but not into HSV DNA. Enzymic studies relevant that alpha araATP has no effect on the HSV DNA polymerase system but a high inhibitory potency in the host cell DNA polymerase alpha system. The anomeric form, beta araATP, is a sensitive inhibitor of HSV DNA polymerase while the cellular DNA polymerases alpha and beta are more refractory.