Intracellular pH electrode : Experiments on the giant squid axon

A new, easy method to produce and calibrate a 1-μm tip intracellular pH electrode is described. This antimony electrode and a micro-calomel electrode were inserted into the giant axon of Loligo pealii. The potential obtained when the axon was bathed in seawater corresponded to a pH of 7.0 ± 0.2. It was found that acidification of the external perfusate induced a drop in axoplasmatic pH leading to changes in the membrane electrical properties. Changes of resting or action potentials did not influence intracellular pH.     

Coincidence of down-regulation and desensitization in pituitary gonadotrophs stimulated by gonadotropin releasing hormone

The dynamics of gonadotropin releasing hormone (GnRH) induced luteinizing hormone (LH) release was studied $\text{in}$$\text{vitro}$ by superfusion of cultured pituitary cells. Continuous exposure of the cells to GnRH resulted in desensitization of the gonadotroph responsiveness to further stimulation by the hormone. The refractory state was achieved within 4 hr of hormone introduction (10^?7 M) and was accompanied by down-regulation of GnRH receptors (50%) assayed by equilibration with [¹²⁵I]iodo-[D-Ala⁶]des-Gly¹⁰-GnRH N-ethylamide. The data indicate that GnRH can regulate the number of its own receptors, and that desensitization is accompanied by down-regulation.     

Cyanoketone competition with estradiol for binding to the cytosolic estrogen receptor

Cyanoketone, an inhibitor of many steroidogenic processes, has been found to inhibit binding of estradiol to its receptor in a competitive manner. The Ki observed was 1.2 X 10(-6)M. This action may explain some of cyanoketone's effects in vivo.     

Stabilization by glutaraldehyde of high-rate electron transport in isolated chloroplasts

Treatment of isolated chloroplasts with glutaraldehyde affects their ability to photoreduce artificial electron acceptors. The remaining rate of O₂ evolution approaches zero with methyl viologen, is low with ferricyanide, but nearly normal with lipophilic Photosystem II acceptors, like oxidized p-phenylenediamine and oxidized diaminodurene. Since Photosystem I donor reactions are also affected, a specific site of inhibition of electron transport to Photosystem I is indicated. At the same time, glutaraldehyde prolongs the longevity of the chloroplasts stored in dark. In control samples the half-life of Photosystem II activity varied between 5 days at 4 °C and 1 day at 25 °C. Glutaraldehyde treatment increased these half times approx. 3-fold. The glutaraldehyde doses required to induce inhibition and stabilization were very similar.     

Biosynthesis and stability of globin mRNA in cultured erythroleukemic friend cells

Biosynthesis and stability of the mRNA population in DMSO-induced Friend erythroleukemic cells were studied after labeling the RNA with ³H-uridine and then chasing it with nonlabeled uridine. Globin RNA metabolism was studied by hybridization to excess complementary DNA covalently coupled to oligo(dT)-cellulose. After a labeling period of 120 min, 2–4% of the poly(A)-containing labeled RNA was in globin RNA; it decayed with a half-life of 16–17 hr. The rest of the poly(A)-containing RNA was composed of two kinetic populations: 85–90% decayed with a half-life of about 3 hr, while 10% decayed with a half-life of about 37 hr. The portion of globin RNA in labeled poly(A)-containing RNA behaved in an unexpected fashion during the chase period. During the initial chase period, the percentage of globin RNA increased rapidly, reaching a maximum of about 15% at 20 hr, but if subsequently declined gradually.Based on these findings, a model was built that describes the changes in the proportion of globin mRNA in poly(A)-containing RNA during continuous synthesis and after chase of the labeled RNA. It appears that if the parameters described remain constant during the maturation of erythroblasts, then this model would not account for the almost exclusive presence of globin RNA in the reticulocyte. By far the most effective way to achieve this high level of globin RNA is the destabilization of the mRNA population which is more stable than globin RNA, and not the stabilization of globin RNA itself.     

Nitrite and nitrate synthesis from pyruvic-oxime by anAlcaligenes sp.

AnAlcaligenes sp. isolated from soil was characterized as to its ability to oxidize and grow on pyruvic-oxime. Abundant nitrification of pyruvic-oxime was demonstrated with maximal nitrite and nitrate production of 1867 mg NO₂ ^–-N per liter and 42 mg NO₃ ^–-N per liter. TheAlcaligenes sp. oxidized hydroxylamine and this metabolism was stimulated when either acetate or pyruvate was present. This organism was also capable of limited pyruvic-oxime oxidation when cultured in an acidic medium. The metabolism of pyruvic-oxime and nitrification by theAlcaligenes sp. in the environment are discussed.     

Effects of phospholipid and detergent on the substrate specificity of adrenal cytochrome P-450scc. Substrate binding and kinetics of cholesterol side chain oxidation

Cytochrome P-450scc was isolated from mitochondria of bovine adrenal cortex by hydrophobic chromatography on octyl Sepharose followed by affinity chromatography on cholesterol-7-(thiomethyl)carboxy-3 beta-acetate-Sepharose. The partially purified eluate from the octyl Sepharose resin was free of adrenodoxin and adrenodoxin reductase and displayed biphasic binding characteristics for cholesterol, cholesterol sulfate, and cholesterol acetate (CA). Chromatography of the octyl Sepharose eluate on CA-Sepharose removed extraneous proteins and resolved the cytochrome P-450scc into two fractions, each of which displayed monophasic binding with all three substrates. These fractions behaved identically with respect to their ability to bind substrates, their kinetic properties, and their rate of migration during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The dissociation constants of the cytochrome P-450scc.substrate complexes are 1.1, 2.6, and 1.3 microM for cholesterol, cholesterol sulfate, and cholesterol acetate, respectively. Addition of phospholipids isolated from adrenal cortex mitochondria or adrenodoxin had no effect on the equilibrium binding constants. Addition of Emulgen 913, however, decreased the binding affinities 10-20-fold. Emulgen 913 also inhibited the interaction of adrenodoxin with the cytochrome. An active side chain cleavage system was reconstituted with purified P-450 by addition of saturating amounts of adrenodoxin, adrenodoxin reductase, and NADPH-generating system. The apparent Km values for this reconstituted system of cholesterol, cholesterol sulfate, and cholesterol acetate are 1.8, 1.9, and 0.6 microM, respectively. Since the Km values of substrate oxidation are similar to the Kd values of the cytochrome P-450.substrate complexes, it seems likely that the binding of substrates, particularly when the side chain cleavage system is free of mitochondrial membranes, is not rate-limiting. Based on these results and electrophoretic data, it appears that one cytochrome P-450 present in adrenal mitochondria can oxidize cholesterol, its sulfate, and its acetate. This enzyme represented about 60% of the cytochrome P-450 present in the octyl Sepharose eluate. The factors responsible for the biphasic kinetics of oxidation by intact mitochondria and biphasic binding of sterol substrates by partially purified preparations of cytochrome P-450scc are still unknown.     

Enhanced proliferation,survival, and yield of cultured myelin basic protein (MBP)-reactive Lewis rat lymph node cells following dual activation with concanavalin A and MBP

Lymph node cells (LNC) from Lewis rats sensitized 7 days previously to myelin basic protein (MBP) exhibit greatly increased survival and undergo much greater proliferation when cultured in the presence of both concanavalin A (Con A) and MBP. Dual activation, in contrast to culture with either the mitogen or the neural antigen, results in a substantially greater yield of lymphoblasts with the capacity to transfer experimental allergic encephalomyelitis (EAE) to normal syngeneic recipients. Dual activation provides a practical advantage over other methods reported to date for securing larger numbers of functional MBP-reactive lymphoid cells for immunologic studies.     

Tyrosine phosphorylation of measles virus nucleocapsid protein in persistently infected neuroblastoma cells.

Subacute sclerosing panencephalitis is a slowly progressing fatal human disease of the central nervous system which is a delayed sequel of measles virus (MV) infection. A typical pathological feature of this disease is the presence of viral ribonucleocapsid structures in the form of inclusion bodies and the absence of infectious virus or budding viral particles. The mechanisms governing the establishment and maintenance of a persistent MV infection in brain cells are still largely unknown. To understand the mechanisms underlying MV persistence in neuronal cells, a tissue culture model was studied. Clone NS20Y/MS of the murine neuroblastoma C1300 persistently infected with the wild-type Edmonston strain of MV secretes relatively high levels of alpha/beta interferon (IFN). As shown previously, treatment of the persistently infected cultures with anti-IFN serum converted the persistent state into a productive infection indicated by the appearance of multinucleated giant cells. In this study, we have investigated whether alpha/beta IFN produced by NS20Y/MS cells activates cellular protein tyrosine kinases which will induce tyrosine phosphorylating activity specific to virus-infected cells. We present data to show augmented protein tyrosine kinase activity in the persistently infected cells. We demonstrate that the MV N protein is phosphorylated on tyrosine in addition to serine and threonine in the persistent state but not in NS20Y cells acutely infected with MV.     

Comparative thermoregulatory adaptations of field mice of the genus Apodemus to habitat challenges

Thermoregulatory abilities, which may play a role in physiological adaptations, were compared between two field mouse species (Apodemus mystacinus and A. hermonensis) from Mount Hermon. While A. hermonensis is common at altitudes above 2100 m, A. mystacinus is common at 1650 m. The following variables were compared in mice acclimated to an ambient temperature of 24°C with a photoperiod of 12L:12D, body temperature during exposure to 4°C for 6 h, O₂ consumption and body temperature at various ambient temperature, non-shivering thermogenesis measured as a response to a noradrenaline injection, and the daily rhythm of body temperature. Both species could regulate their body temperature at ambient temperatures between 6 and 34°C. The thermoneutral zone for A. mystacinus lies between 28 and 32°C, while for A. hermonensis a thermoneutral point is noted at 28°C. Both species increased O₂ consumption and body temperature as a response to noradrenalin. However, maximal VO ₂ consumption as an response to noradrenaline and non-shivering thermogenesis capacity were higher in A. mystacinus, even though A. hermonensis is half the size of A. mystacinus. The body temperature rhythm in A. hermonensis has a clear daily pattern, while A. mystacinus can be considered arhythmic. The results suggest that A. hermonensis is adapted to its environment by an increase in resting metabolic rate but also depends on behavioural thermoregulation. A. mystacinus depends more on an increased non-shivering thermogenesis capacity.Abbreviations C thermal conductance - NA noradrenaline - NST non-shivering thermogenesis - OTC overall thermal conductance - RMR resting metabolic rate - STPD standard temperature and pressure dry - T _a ambient temperature - T _b body temperature - I _b Min minimal T _b , measured before NA iniection - T _b NA maximal - T _b as a response to NA injection - T _lc lower critical point - TNP thermoneutral point - TNZ thermoneutral zone - VO₂ O₂ consumption - VO₂ Min minimal VO₂ measured before NA injection - VO₂NA maximal VO₂, as a response to NA injection