The abortive synthesis of new DNA chains in Ehrlich ascites tumour cells treated with nitrogen mustard (HN2)

Nitrogen mustard (HN2)-sensitive Ehrlich ascites tumour cells, exposed to HN2 in vivo, showed an inhibition of DNA synthesis which increased with dosage. The effects of alkylation involved at least three distinct components: (1) interference with new 9S DNA chain formation, (2) slowing of the rate of chain growth and (3) loss of newly formed short chains. The dominant effect seemed to be abortive synthesis of new 9S chains; this effect could account for most of the inhibition of DNA synthesis if an initial rapid synthesis of 9S DNA were accompanied by an initial rapid rate of destruction of these chains. By relating the known frequencies of guaninyl alkylations to the postulated ‘replicon’ size observed in control experiments, it appears that only difunctional alkylation frequencies can be directly correlated with the inhibition of discontinuous DNA synthesis by HN2, Mechanistically, this could reflect interference of di-guaninyl alkylations with the integration of 9S chains into 30S, 44S and higher molecular weight species of DNA by ligases. The resulting obstructed short chains, ≦ 9S, might be exposed and so be vulnerable to destruction by the increased nuclease activities expected after alkylation.     

Glycosaminoglycan composition of human amniotic fluid

The glycosaminoglycan composition of human amniotic fluid between 12–21 weeks gestation has been studied by Dowex column chromatography coupled with enzymatic analyses of the specific glycosaminoglycan in each column fraction. The total uronic acid recovered from the columns consisted of “glycopeptides” (7%), hyaluronic acid (34%), nonsulfated chondroitin (14%), chondroitin-4-sulfate (13%), chondroitin-6-sulfate (20%), dermatan sulfate (5%), and heparan sulfate (6%). Based on these studies a simple screening procedure was devised to detect increased quantities of heparan sulfate and dermatan sulfate in 5–10-ml samples of amniotic fluid and tested in the antenatal diagnosis of Hurler and Hunter's syndrome. A false negative result was recorded in a Hunter fluid obtained early gestation and a false positive result recorded in a normal fluid obtained at weeks. These data suggest that the time in gestation when amniotic fluid is sampled for chemical analysis is an important variable affecting glycosaminoglycan composition in both normal and pathological pregnancies.     

Chemically modified nylons as supports for enzyme immobilization. Polyisonitrile-nylon

Four-component condensations between amine, carboxyl, isocyanide and aldehyde lead to the formation of N-substituted amides (Ugi, 1962). The present paper describes the use of such condensations for the introduction of chemically reactive groups on to the polyamide backbone of nylon. Polyisonitrile-nylon was synthesized by partial hydrolysis of nylon-6 powder, followed by resealing of the newly formed -CO(2)... NH(2) (-) pairs via a four-component condensation, by using acetaldehyde and 1,6-di-isocyanohexane. Polyisonitrile-nylon could also be converted into a diazotizable arylamino derivative, polyaminoaryl-nylon, by a four-component condensation by using a bifunctional amine, pp'-diaminodiphenylmethane, in the presence of an aldehyde and a carboxylate compound. The versatility of four-component condensations involving the isocyanide functional group of polyisonitrile-nylon allowed coupling of proteins, in an aqueous medium at neutral pH, through either their amino or carboxyl groups. Trypsin and papain were bound to polyisonitrile-nylon through their amino groups by a four-component condensation by using acetaldehyde and acetate; conversely, succinyl-(3-carboxypropionyl-)trypsin, pepsin and papain were coupled through their carboxyl groups in the presence of acetaldehyde and an amine (Tris). Diazotized polyaminoaryl-nylon could be utilized for the immobilization of papain, via the tyrosine residues of the enzyme.     

Theory of melting of the triple helix poly (A + 2U) for a 1 : 2 mixture of Poly A to Poly U

The Zimm-Bragg theory is extended to treat the melting of the triple helix poly (A + 2U) for a solution with a 1 : 2 mole ratio of poly A to poly U. Only the case for long chains is considered. For a given set of parameters the theory predicts the fraction of segments in the triple helix, double helix, and random coil states as a function of temperature. Four nucleation parameters are introduced to describe the two order–disorder transitions (poly (A + 2U) ? poly A + 2 poly U and poly (A + U) ? poly A + poly U) and the single order–order transition (poly (A + 2U) ? poly (A + U) + poly U). A relation between the nucleation parameters is obtained which reduces the number of independent parameters to three. A method for determining these parameters from experiment is presented. From the previously published data of Blake, Massoulié and Fresco⁸ for [Na⁺] = 0.04, we find σ_T = 6.0 × 10^?4, σ_D = 1.0 × 10^?3, and σσ* = 1.5 × 10^?3. σ_T and σ_D are the nucleation parameters for nucleating a triple helix and double helix, respectively, from a random coil region. σσ* is the nucleation parameter for nucleating a triple helix from a double helix and a single strand. Melting curves are generated from the theory and compared with the experimental melting curves.     

STUDIES ON THE ORIGIN OF RIBOSOMES IN AMOEBA PROTEUS

The origin of cytoplasmic RNA and ribosomes was studied in Amoeba proteus by transplantation of a radioactive nucleus into an unlabeled cell followed by examination of the cytoplasm of the recipient for the presence of label. When a RNA-labeled nucleus was used, label appeared in the ribosomes, ribosomal RNA, and soluble RNA. Since the kinetics of appearance of labeled RNA indicates that the nucleus was not injured during the transfer, and since the transferred nuclear pool of labeled acid-soluble RNA precursors is inadequate to account for the amount of cytoplasmic RNA label, it is concluded that cytoplasmic ribosomal RNA is derived from acid-insoluble nuclear RNA and is probably transported as an intact molecule. Likewise, cytoplasmic soluble RNA probably originated in the nucleus, although labeling by terminal exchange in the cytoplasm is also possible. The results were completely different when a protein-labeled nucleus was grafted into an unlabeled host. In this case, label was found only in soluble proteins in the host cell cytoplasm, and there were no (or very few) radioactive ribosomes. This suggests that the nuclear pool of ribosomal protein and ribosomal protein precursors is relatively small and perhaps nonexistent (and, furthermore, shows that there was no cytoplasmic ribosomal contamination of the transferred nucleus).     

STUDIES OF AMINES IN THE STRIATUM IN MONKEYS WITH NIGRAL LESIONS

The effects of ventromedial tegmental lesions on the biosynthesis and disposition of biogenic amines in the striatum of monkeys were investigated. The concentrations of endogenous dopamine and of the intraventricularly injected [³H]dopamine were distinctly lower in the striatum on the lesion side than on the intact side. The storage of [³H]dopamine in the caudate nucleus was impaired to a much greater extent than the storage of the newly synthesized [³H]norepinephrine. The concentrations of endogenous serotonin and of the intraventricularly injected [¹⁴C]serotonin were lower in the striatum on the lesion side than on the intact side. However following MAO inhibition, the concentration of [¹⁴C]serotonin did not differ significantly on the two sides of the caudate nucleus. The in vivo biosynthesis of dopamine from tyrosine was significantly reduced in the striatum on the lesion side. Tyrosine hydroxylase and DOPA decarboxylase activities were decreased on the lesion side of the striatum as compared with the intact side. Thus, the ventromedial tegmental lesions affect the storage and the synthesis of dopamine and serotonin in the ipsilateral striatum.