Preparative isolation of di-, tri- and tetrapyrimidine nucleotides from depurinated herring sperm DNA (author's transl)

The pyrimidine nucleotides p(dT)3, p(dT)2p, pdCp, p(dC)2p and p(dC)3 and the mixtures of sequence isomers p(dC, dT), p(dC, dT)p, p(dT2, dU), (dT2, dU)p, p(dC, dT2), p(dC, dT2)p, p(dC, dT3), p(dC, dT), p(dC2, dT)p, and p(dC2, dT2) are isolated on a preparative scale from depurinated hydrolysates of herring sperm DNA by the following procedure. The DNA hydrolysate is first separated into a low and a high molecular weight mixture of pyrimidine nucleotides by column chromatography on DEAE-cellulose. The nucleoside bisphosphates pdCp and pdTp, the dinucleotides p(dC)2 and p(dT)2 and the sequence isomers p(dC, dT) are largely separated out of the mixture of the low molecular weight pyrimidine nucleotides. The remaining mixture is rechromatographed at pH 3.5 on QAE-Sephadex. This separates the pyrimidine nucleotides containing a majority of cytidylic acid units from those containing more thymidylic acid units, which are then fractionated at pH 7.5 according to the number of bases in the chain. The pyrimidine nucleotides and mixtures of sequence isomers separated to 83-99% purity by column chromatography are further separated by paper chromatography and are obtained in chromatographically pure form after this step. The structures of the isolated DNA fragments are determined from chromatographic data, absorption and enzymatic degradation.     

Arrangement of glycan chains in the sacculus of Escherichia coli.

A novel of Escherichia coli endopeptidase was used for a selective partial hydrolysis of the peptide bridges which interlink the glycan chains in E. coli sacculi. The loosening of the murein network revealed, in the electron microscope, a preferential orientation of the glycan chains, more or less perpendicular to the length axis of the cell. Control incubations with E. coli transglycosylase or egg-white lysozyme did not leave ordered structures behind.     

On the physico-chemical basis of voltage-dependent molecular gating mechanisms in biological membranes

Summary The possible nature and theoretical treatment of electric field-induced molecular processes in a membrane are examined. Special attention is given to fairly fast switching phenomena as reflected by asymmetry currents as well as ionic gating in squid axon and similar systems. The apparent charge displacement associated with the underlying mechanisms is argued to be brought about by conformational transitions of integral macromolecular structures. Under these circumstances, voltage changes can actually control the functional state of membranes by direct interference with conformational equilibria. A basic model is quantitatively discussed and shown to account for certain observed asymmetry currents. Effects due to temperature, pressure, or chemical interactions can be readily described. It is indicated how more complicated voltage-dependent membrane processes may be approached along these lines.     

Effects of Δ9-tetrahydrocannabinol administration on marker proteins of rat testicular cells

The effects of chronic administration of 2 mg Δ⁹-tetrahydrocannabinol per kg body weight upon rat testicular cell function was examined by use of selected testicular cell marker proteins. γ-Glutamyl transpeptidase was used as a marker of Sertoli cell plasma membranes; sorbitol dehydrogenase was used as a marker of pachytene spermatocytes. The interstitial cells were marked by cytochrome P-450, a microsomal component, and β-glucuronidase, a lysosomal component. The results of this study show a rapid reduction in microsomal P-450 content following 2 days of tetrahydrocannabinol administration. In addition, γ-glutamyl transpeptidase was significantly reduced at 2 days and continued to decline to day 9. β-Glucuronidase and sorbitol dehydrogenase exhibited no significant change over the course of the experiment. It is suggested that the reduction of testosterone synthesis in testes of tetrahydrocannabinol treated rats may be the result of a reduction in P-450 content.     

A peptide action in a lobster neuromuscular preparation

The neuropeptide proctolin causes a sustained contraction of the opener muscle of the dactyl of the lobster walking leg. This substance acts directly on the muscle at concentrations as low as 10^?10M. The contraction is dependent on extracellular calcium. Neither a significant depolarization nor a detectable change in the input resistance accompanies the response. No presynaptic action of proctolin is indicated; excitatory and inhibitory junctional potential sizes and the frequency of spontaneous miniature excitatory junctional potentials are unaffected.     

Local anesthetics. Effect of pH on use-dependent block of sodium channels in frog muscle.

Sodium currents were studied under voltage clamp in the presence of neutral, amine, and quaternary local anesthetic compounds. Use-dependent block was observed as a cumulative depression of INa seen with repetitive depolarizing test pulses applied at frequencies of 2-10s-1. With quaternary QX-314, the time constant of use dependence was long, and with neutral benzocaine, very short. With lidocaine and procaine, increasing external pH (pHo) changed the time constant from long to short, but alterations of internal pH have no effect. Inactivation in Na channels was measured by the influence of prepulses on peak INa during test pulses. Single-stimulus inactivation curves were shifted more with lidocaine at high pHo than at low pHo, but inactivation curves measured during pulse trains with any of the drugs and at any pHo were strongly shifted. All measurements show that the drug-receptor reaction was slow for amine drugs at low pHo, as for quaternary drugs at any pHo, and fast for amine drugs at high pHo, as for neutral drugs at any pHo. The major effect of low pHo on amine drugs was to reduce the concentration of drugs in the fiber and to protonate drug molecules on the receptor, thus trapping them in the blocking position for a longer time. Direct effects of pH on the receptor seemed minimal.     

Linear cooperative binding of large ligands involving mutual exclusion of different binding modes

A rigorous treatment is given for mutually exclusive multiple mode cooperative binding on a linear structure of equivalent binding "contacts". This will be of special interest with regard to larger ligands implicating the possibility that there are different kinds of binding interactions with more than one monomeric sub unit of a linear biopolymer. Quantitative evaluation of binding properties is shown to be essentially based on calculating the largest root of an algebraic equation. The whole procedure can be practically executed by means of a fairly simple computer program. Various typical examples comprising only two modes are discussed in more detail. For some nucleotide-polylysine systems definite binding parameters have been determined from pertinent experimental data.     

Requirements for maintaining the embryonic state of avian tendon cells in culture

Summary Primary avian tendon cells (PAT) maintain their embryonic state when cultured in medium F-12 with very low serum (0.2%) and ascorbate (50 μg per ml); that is, they retain the potential for devoting 20–30% of their total protein synthesis to collagen. However, if the cells are left at a confluent cell density or are derived from confluent cultures, this potential is irreversibly decreased. This effect, along with poor medium formulations, probably accounts for the “dedifferentiation” process that occurs when fibroblasts are cultured. In contrast, PAT cells kept at subconfluent cell densities retain the ability to synthesize high levels of collagen. The one limitation in obtaining long-term cultures of high collagen-producing tendon cells in the inability of serum at low concentrations to remain a potent mitogen after a few subcultures. The quantitative loss of function has long been considered to be a cell culture artifact; however, we propose that this drop in collagen synthesis is a reflection of the developmental programing of these cells. In separate series of experiments using organ cultures, we show that tendon tissue from the embryo makes over 30% collagen, whereas, “young” tendons make 18% and “older” tendons from the adult make less than 1%. Therefore, a quantitative drop in collagen synthesis would be expected if normal development were to occur in culture. Our data are consistent with the idea that cultures of embryonic tendon cells are triggered to mature by a mechanism that correlates with high cell density. This investigation was supported in part by National Science Foundation Grant PCM 77-14982; in part by the Division of Biomedical and Environmental Research of the Department of Energy under contract W-7405-ENG-48; and by a National Institutes of Health Fellowship IF32 CA 05807-01, from the National Cancer Institute to R. I. S.