DNA fragmentation in N-diazoacetylglycine amide-treated cells determined, by the rate of strand separation in alkali with hydroxylapatite chromatography in batch

DNA damage induced in mammalian cells (CHO-K1) by one hour treatment with several concentrations of N-diazoacetylglycine amide (DGA) was evaluated by the method of DNA denaturation in alkali and successive neutralization followed by separation of single from double stranded DNA with the recently described technique of hydroxylapatite chromatography performed in batch. This latter technique does not need complex apparatus and simplifies the simultaneous handling of large number of samples; it also appears as sensitive and reliable as the DNA alkaline elution on filter, to which it can be regarded as both alternative and complementary.     

Upregulation of TPX2 by STAT3: Identification of a Novel STAT3 Binding Site

TPX2, a protein involved in mitosis, is considered a good marker for actively proliferating tissues, highly expressed in a number of cancer cells. We show the presence of high-affinity binding site for STAT3 in the 5′-flanking region of the Tpx2 gene, which is in vivo bound by activated STAT3. A specific STAT3 peptide inhibitor represses the expression of the Tpx2 gene and inhibits the binding of STAT3 to its consensus sequence in human cell lines where STAT3 is activated. These results indicate that activated STAT3 contributes to the over-expression of Tpx2 through the binding to an enhancer site.     

Rapid transformation of nor-Saccharomyces yeasts by electroporation

Summary Various non-Saccharomyces yeasts have been transformed by electroporation of colonies picked from plate. The procedure gives an efficiency of 10³ transformants/g DNA.     

Formation of cation channels in planar lipid bilayers by brefeldin A.

Brefeldin A (BFA) is a novel agent with the unique property of effecting a rapid increase of Golgi cisternae volume and subsequent loss of a recognizable Golgi apparatus in treated cells. Although a receptor-mediated mechanism has been proposed, the molecular basis of BFA action remains unknown (Lippincott-Schwartz, J., Glickman, J., Donaldson, J. G., Robbins, J., Kreis, T. E., Seamon, K. B., Sheetz, M. P., and Klausner, R. D. (1991) J. Cell Biol. 112, 567-577). Since a variety of ionophores distort Golgi architecture by initially causing osmotic swelling of the cisternae (Mollenhauer, H. H., Morre, D. J., and Rowe, L. D. (1990) Biochim. Biophys. Acta 1031, 225-246), Golgi membrane permeabilization by BFA seemed possible. We examined the effects of BFA on the conductance of planar lipid bilayers bathed in several aqueous salt solutions. Addition of BFA (1 microgram/ml) quickly augmented alkali cation conductance (K+ greater than Na+ much greater than Li+) but not anion conductance of the bilayer. Lower concentrations (1 ng/ml) indicated that BFA formed discrete, cation-selective channels in these bilayers. Given that Golgi cisternae volume increases immediately upon treatment with BFA, these findings suggest that alteration of ion gradients or Golgi membrane potential followed by an influx of water may be the mechanism by which BFA initiates disruption of Golgi structural integrity. Subsequent functional perturbations may then ensue either as a consequence of these initial structural changes or by a combination of several distinct mechanisms.     

Electrophysiological changes of Sertoli cells produced by the acute administration of amino acid and FSH.

Electrophysiological studies were carried out using seminiferous tubules of "Sertoli cell enriched testes" of rats irradiated in utero. Sertoli cells were inserted with glass microelectrodes in a superfusion chamber continuously perfused with KRb buffer. The topical application of FSH (4.0 mU/ml) produced a biphasic effect characterized by a rapid hyperpolarization (less than 3 s) followed by depolarization. The depolarizing effect of FSH was prolonged and potentiated in the presence of 5 mmol/l alpha-methylamino-isobutyric acid in the bath medium of the superfusion chamber. Verapamil, at a dose (250 mumol/l) that nullified the stimulatory action of FSH in the amino acid transport, suppressed the depolarizing effect of FSH. It was concluded that in immature rat testes FSH produces electrophysiological changes that mediate the stimulatory action of the amino acid transport.     

The effects of insulin,glucose and anti-insulin upon the activity of uridine diphosphate glucose glycogen α-4-glucosyl-transferase EC2.4.1.11 in the rat placenta

Summary The enzyme uridine diphosphate glucose glycogen -4-glyoosyl-transferase EC2.4.1.11 was found to be active in the rat placenta. The total activity of the enzyme, present from the earliest day investigated, day 12, increased significantly between days 14 and 16 and days 18 and 20. The enzyme was observed to be present in both the active and the inactive forms. In in vivo studies of the effects of insulin, glucose and anti-insulin serum it was observed that insulin and glucose produced an increase in the activity of the enzyme while anti-insulin serum inhibited its activity. Insulin was observed to exert its stimulatory effect also in vitro.The activity of the enzyme was observed to be localized strongly in the decidua basalis, the spongy zone, the labyrinth as well as in the yolk sac. There was a shift in the activity of the enzyme from the decidua basalis and the visceral layer of the yolk sac where it was strongest in the younger placentae (14 or 16 days) to the spongy layer where it was stronger towards the end of gestation. The activity of the enzyme was very weak, at 12 days, in all areas of the placenta.     

PURIFICATION AND CHARACTERIZATION OF CARNOSINE SYNTHETASE FROM MOUSE OLFACTORY BULBS

Carnosine synthetase was purified about 500-fold from mouse olfactory bulb to a specific activity of approx 25 nmol/min/mg. This is an increase of 800-fold over that previously reported for this enzyme from rat brain and 11 times higher than the most highly purified enzyme from chicken pectoral muscle. ATP was essential for activity and could not be replaced by ADP. NAD had no effect on the synthesis of carnosine. Of the β-alanine analogues tested, the purified mouse enzyme incorporated only γ-aminobutyric acid and β-amino-n-butyric acid into peptide linkage with histidine. Synthesis of carnosine by the mouse olfactory bulb enzyme was competitively inhibited by the histidine analogues, 1-methyl histidine and 3-methyl histidine, with K_i values which were at least 40 times the K_m value for histidine (16 μM). Ornithine and lysine were more efficient β-alanine acceptors than 1-methyl histidine for the mouse enzyme. Enzyme from olfactory epithelium and leg skeletal muscle of mice also showed higher K_i values for 1–methyl histidine than the K_m value for histidine. In contrast, carnosine-anserine synthetase from chicken pectoral muscle gave K_m values for histidine, 1-methyl histidine and 3-methyl histidine, which were all in the range of 4–12 μM. The differences in substrate specificity between the enzyme from mouse and chicken implies alternate routes of anserine synthesis in these species and predicts the occurrence of certain novel peptides in mouse brain.