Prostaglandin E1 binding and adenylate cyclase activation in normal and transformed fibroblasts

The binding of [3H]prostaglandin E1 to membranes of clones of normal rat kidney fibroblasts (NRK cells) has been measured. Cell lines that responded to prostaglandin E1, such as NRK and NRK transformed with Schmitt-Ruppin strain of Rous sarcoma virus (SR-NRK cells), have a high affinity prostaglandin E1 binding site. Murine-sarcoma-virus-transformed lines of NRK cells are unresponsive to prostaglandin E1 and have reduced prostaglandin E1 binding Exposure of cells to prostaglandin E1 results both in decreased prostaglandin E1 responsiveness and reduced prostaglandin E1 binding. Activation of adenylate cyclase is correlated to binding of prostaglandin E1 to receptors in both NRK and SR-NRK cell membranes. Mathematical models suggest that GTP decreases the affinity of hormone for its receptor while increasing the catalytic efficiency of adenylate cyclase, and that aggregates of occupied receptors may play an important role in the activation of adenylate cyclase.     

Antagonism of benzodiazepine binding in rat and human brain by Antilirium.

Physostigmine (Antilirium^R) has been reported to reverse benzodiazepine- induced sleep or coma in man and prevent death in animals. Accordingly, we investigated the effect of Antilirium upon benzodiazepine binding to both rat and human brain. We report that Antilirium inhibits ³H-diazepam and ³H-flunitrazepam binding in a dose-dependent manner. The degree of inhibition of binding by Antilirium correlates with the affinity of benzodiazepine for its “receptor” such that diazepam is more affected than flunitrazepam. The inhibition is rapid but the kinetics are complex with only doses of Antilirium showing competitive inhibition when studied at equilibrium. These results may explain, at least in part, the effectiveness of Antilirium at reversing benzodiazepine-induced hypnosis without necessarily implicating a cholinergic mechanism.     

Light-induced changes of cyclic GMP content in frog retinal rod outer segments measured with rapid freezing and microdissection

Cyclic GMP concentration was measured in the rod outer segments (ROS) of the isolated frog retinas. Retinas were quickly frozen in 0.5 s after the short light flash producing 90%-saturated late receptor potential (2,000 rhodopsins bleached per rod). ROS were obtained by microdissection, and cGMP levels were determined by radioimmunoassay method. No detectable changes in cGMP concentration was found in this stimulus condition. Dark-adapted ROS contained 46.3 ± 1.5 pmole cGMP per mg dry weight, flash-illuminated ones –45 ± 2 pmole/mg. 3-s bright illumination (ca. 10⁷ rhodopsins bleached per rod per second) led to approximately 30% drop in cGMP content. It is supposed that the main part of cGMP within the ROS is in the bound state and therefore fast light-induced changes in its minor free fraction may escape the detection.     

Pathways of d-fructose catabolism in species of Pseudomonas

Cell-free extracts of d-fructose grown cells of Pseudomonas putida, P. fluorescens, P. aeruginosa, P. stutzeri, P. mendocina, P. acidovorans and P. maltophila catalyzed a P-enolpyruvate-dependent phosphorylation of d-fructose and contained 1-P-fructokinase activity suggesting that in these species fructuse-1-P and fructose-1,6-P₂ were intermediates of d-fructose catabolism. Neither the 1-P-fructokinase nor the activity catalyzing a P-enolpyruvate-dependent phosphorylation of d-fructose was present in significant amounts in succinate-grown cells indicating that both activities were inducible. Cell-free extracts also contained activities of fructose-1,6-P₂ aldolase, fructose-1,6-P₂ phosphatase, and P-hexose isomerase which could convert fructose-1,6-P₂ to intermediates of either the Embden-Meyerhof pathway or Entner-Doudoroff pathway. Radiolabeling experiments with 1-¹⁴C-d-fructose suggested that in P. putida, P. aeruginosa, P. stutzeri, and P. acidovorans most of the alanine was made via the Entner-Doudoroff pathway with a minor portion being made via the Embden-meyerhof pathway. An edd ^- mutant of P. putida which lacked a functional Entner-Doudoroff pathway but was able to grow on d-fructose appeared to make alanine solely via the Embden-Meyerhof pathway.Non-Standard Abbreviations cpm counts per min - edd ^- mutant lacking Entner-Doudoroff dehydrase (6-PGA dehydrase) - EDP Entner-Doudoroff pathway - EMP Embden-Meyerhof pathway - FDP fructose-1,6-P₂ - FDPase FDP phosphatase - F-1-P fructose-1-P - F-6-P fructose-6-P - FPTs PEP: d-fructose phosphotransferase system - G-6-P glucose-6-P - KDPG 2-keto-3-deoxy-6-P-gluconate - PEP P-enolpyruvate - 1-PFK 1-P-fructokinase - 6-PFK 6-P-fructokinase - 6-PGA 6-P-gluconate     

Metabolsim of apoB and apoC lipoproteins in man: kinetic studies in normal and hyperlipoproteininemic subjects.

The kinetics of apolipoproteins B and C were studied in 14 normal and hyperlipoproteinemic subjects after injection of exogenously (125)I-labeled very low density lipoprotein (VLDL) particles. Plasma radioactivities of apoB and apoC were determined over a period of 4 days in VLDL (d < 1.006) and total radioactivity in intermediate (IDL) (1.006 < d < 1.019), low (LDL) (1.019 < d < 1.063), and high (HDL) (1.063 < d < 1.21) density lipoproteins. The data were analyzed by the use of a model, developed mostly from these data, with the following results. The VLDL particle undergoes a series of incremental density changes, most likely due to a number of delipidation steps, during which apoB stays with the particle until the density reaches the IDL range. There is, however, a loss of apoC associated with these delipidation steps. In our normal subjects, all IDL apoB eventually becomes LDL. In our hyperlipemic subjects some of the apoB on IDL is also degraded directly. The apoC lost by VLDL and IDL recycles to HDL, and most of it is picked up again by newly synthesized VLDL. There is a slowdown of the stepwise delipidation process in all hyperlipemic individuals studied. Three additional features became apparent in the type III subjects. First, there is a significant increase (a factor of 2 compared to normal) in the apoB synthesis rate by way of VLDL; second, there is an induced direct apoB synthesis pathway by way of IDL (and/or LDL); third, a bypass of the regular stepwise VLDL delipidation pathway is induced by which VLDL particles lose apoC but none of their apoB, thereby forming a new particle with metabolic properties similar to LDL, but with a density still in the VLDL density range. Two type III patients treated with nicotinic acid and clofibrate showed a sharp decrease in their VLDL apoB synthesis rates. This was somewhat compensated by an increased IDL apoB synthesis rate. A type I patient on a medium chain triglyceride diet also showed a number of metabolic changes, including reduced VLDL apoB synthesis and the induction of considerable IDL and/or LDL apoB synthesis.     

Substrate-associated macromolecules promote cytodifferentiation of BC3H1 myogenic cells.

Differentiated mouse BC3H1 myogenic cells secrete substrate-associated macro-molecules (SAM) which restrict the proliferation of undifferentiated cells and promote both cell shape changes and expression of predominantly the vascular smooth muscle (VSM)-specific isoform of the contractile protein alpha-actin. While we previously reported that high cell density was required for stimulating maximal expression of VSM alpha-actin in BC3H1 cells (Strauch and Reeser: Journal of Biological Chemistry 264:8345-8355, 1989), the permissive effect of SAM on myoblast cytodifferentiation was not at all dependent on the formation of cell to cell contacts. This observation suggests that biogenesis of an extracellular matrix rather than the formation of physical contacts between cells may be the rate-limiting step for induction of VSM alpha-actin expression at high cell density. The biologically active moieties in SAM that promote cytodifferentiation also are expressed by mouse embryonic fibroblast cell lines and are distinctly different from a class of adheron-like macromolecules released by differentiated BC3H1 myocytes directly into the culture medium. While SAM was cell growth restrictive, reconstituted particulate material (RPM) prepared from myocyte-conditioned medium promoted the adhesion and proliferation of growth-arrested myoblasts. SAM and RPM are composed of different polypeptide subunits which collectively may establish microenvironmental conditions that are permissive for BC3H1 myogenic cell differentiation.     

Behavioural thermoregulation and homing by spring chinook salmon, Oncorhynchus tshawytscha (Walbaum), in the Yakima River

Temperature-sensitive radio transmitters were employed to study the patterns of behavioural thermoregulation, habitat preference and movement of 19 adult spring chinook salmon, Oncurhynchus tshawytscha (Walbaum), in the Yakima River. During the 4 months prior to spawning, fish maintained an average internal temperature 2.5°C below ambient river temperature. This represented a 12 to 20% decrease in basal metabolic demand or a saving of 17.3 to 29.9 calkg⁻¹ h⁻¹. Fish were most commonly associated with islands, pools, and rock out-croppings along stream banks. Homing behaviour appeared to be modified to optimize temperature regimes and energy conservation. As the time of spawning approached, fish left thermal refuges and migrated to spawning grounds upstream and downstream of refuge areas. Although spring chinook salmon residing within cool-water refuges may be capable of mitigating sub-lethal temperature effects, cool-water areas need to be abundant and available to the fish. The availability of suitable thermal refuges and appropriate holding habitat within mainstem rivers may affect long-term population survival.     

Nano- and picoplankton growth and production in the Bay of Villefranche sur Mer (N.W. Mediterranean)

Plankton production in the Bay of Villefranche was relatively constant during March and April 1986 but the particle size at which the production occurred was more variable. At the beginning of the study, production was dominated by the larger (ca. 6 m) flagellates but towards the end it was more or less equally divided between the nano- and picoplankton. There were considerable differences in the estimates of population growth rates, depending on the methods used, but on average the population doubling times were close to 12 hours for autotrophs and 24 hours for heterotrophs. As autotrophs do not grow during the night, each population was therefore doubling once per day. It seemed that each of the nanoor picoplankton populations could adversely affect the growth of the others. This could be either by simple predation or by some form of inhibition. Although nutrient levels in the bay were uniformly low, the addition of nutrients did not always stimulate algal growth. The plankton populations seemed to be both in a state of equilibrium and intense ecological competition.     

A unique binding cavity for divalent cations in the DNA-metal-chromomycin A3 complex

Binding of chromomycin A3 (CRA) to calf thymus DNA was investigated in the presence of divalent cations using visible absorption and 1H-nmr spectroscopies. An apparent equilibrium binding constant (approximately 10(11) M-1) was obtained from metal competition experiments using EDTA to remove the metal cation from the DNA-M-CRA (M: metal) complex. The large binding constant of the drug to DNA enabled us to obtain essentially complete complexation of CRA to the short homogeneous d(ATGCAT)2 duplex using stoichiometric amounts of the metal cation. Large induced chemical shifts were observed in the 1H-nmr spectrum of the above complex using the paramagnetic Co2+ cation, indicating that the metal occupies a unique binding site. Since no induced 1H-nmr chemical shifts were observed for the drug-Co2+ mixture, it was concluded that no metal-drug complex is formed. In addition, it was found that bound CRA is negatively charged at physiological pH and binding to the DNA could be affected only by using metal cations whose ionic radius size (less than 0.85 A) and charge (2+) were simultaneously satisfied. Stringent metal cation selectivity for the DNA-M-CRA complex may be intimately connected with the antitumor selectivity of CRA, since different types of cells generally possess widely differing molar concentrations of metal cations.