Improved separation at low temperature of glycoproteins by Con A-Sepharose affinity chromatography in the presence of sodium dodecyl sulfate (SDS)

Summary The Con A-Sepharose affinity chromatography of glycoproteins was even more effective at 4°C than that at room temperature (26–28°C) in the presence of sodium dodecyl sulfate (SDS). Application of this methodology to the seperation of several glycoproteins from SDS-solubilized membrane proteins in rat cerebellum, including a glycoprotein characteristics of the Purkinje cells, was sucessful.Acknowledgments. A part of this research was supported by a Grantin-Aid for Scientific Research from the Ministry of Education, Science and Culture (No. 58480150), and also by a grant from National Center of Nervous, Mental and Muscular Disorders (NCNMMD) of the Ministry of Health and Welfare (No.83-11) in Japan.     

Effects of photoperiod, temperature and testosterone-treatment on plasma T3 and T4 levels in the Djungarian hamster, Phodopus sungorus

The effects of photoperiod, temperature and testosterone treatment on plasma T3 and T4 levels were investigated in the Djungarian hamster. Plasma T3 level was affected by temperature (25 degrees C less than 7 degrees C) but not by photoperiod. Plasma T4 level was affected by photoperiod (short day less than long day) at 25 degrees C. Administration of testosterone increased plasma T4 level under short photoperiod at 25 degrees C. Thus, higher plasma T4 level under long photoperiod at 25 degrees C might be induced by testosterone.     

Influences of temperature change on the relaxation and amplitude inhibition by noradrenaline in the rabbit jejunum.

In the rabbit jejunum, the elevation of temperature within the range of 25-37 degrees C diminished the sensitivity to noradrenaline (NA) for both the relaxation and amplitude inhibition. The relaxation by NA was mainly mediated via adrenergic beta-receptors at 25, 30 or 37 degrees C. The amplitude inhibition was mediated via alpha-receptors at 37 degrees C, and both alpha- and beta-receptors at 30 or 25 degrees C.     

Cholecystokinin-octapeptide-induced hyperthermia in guinea-pigs

Intracerebroventricular administration of cholecystokinin-octapeptide (CCK-8) at room temperature (21 degrees C) induced dose-related hyperthermia in guinea-pigs and also produced hyperthermia at low (10 degrees C) and high (30 degrees C) ambient temperatures. CCK-8-induced hyperthermia was direct and not mediated by prostaglandins, norepinephrine, cyclic AMP or naloxone-sensitive receptors.     

Hypothalamic histamine modulates adaptive behavior of rats at high environmental temperature

Histamine content in the rat hypothalamus was lower at 4 degrees C and higher at 31 degrees C compared to that at 21 degrees C. Pretreatment with alpha-fluoromethylhistidine, a 'suicide' inhibitor of histamine decarboxylase, attenuated both the increased level of hypothalamic histamine and rat adaptive behavior at 31 degrees C. Increase of histamine content in the hypothalamus appears to be an important factor contributing to rat adaptive behavior to high environmental temperature.     

Co-localization of galectin-1 with GM1 ganglioside in the course of its clathrin- and raft-dependent endocytosis

Mammalian galectin-1 (Gal-1), a beta-galactoside-binding lectin has a prominent role in regulating cell adhesion, cell growth and immune responses. Downregulation of these biological functions may occur via internalization of Gal-1. In the present study we have investigated the mechanism and possible mediator(s) of Gal-1 endocytosis. We show that internalization occurs at a temperature higher than 22 degrees C in an energy dependent fashion. After one hour incubation Gal-1 localizes in the Golgi system within the cells, and then disappears without accumulation in degradation compartments, such as lysosomes. Based on their strong intracellular co-localization, two glycoconjugates, GM1 ganglioside and CD7 are implicated in the sorting of internalized Gal-1 into Golgi. Other known Gal-1 binding glycoproteins on T cells (CD2, CD3, CD43 and CD45) do not cointernalize with the lectin. Internalization of Gal-1 depends on its lectin activity and follows dual pathways involving clathrin-coated vesicles and raft-dependent endocytosis.     

Temperature dependence of neurotransmitter release in the antarctic fish Pagothenia borchgrevinki

The quantal contents of endplate potentials from extraocular muscles of an antarctic fish Pagothenia borchgrevinki were measured over a range of temperatures. Quantal release was maximal at about 5 degrees C but showed little dependence on temperature between -2 degrees C and 10 degrees C. Above 10 degrees C quantal content declined until release ceased about 18 degrees C. In view of the fact that the ambient temperature at which these fish live is constant at -1.9 degrees C, the results suggest that Pagothenia borchgrevinki is only partially adapted to its environment despite 25 million years acclimatization.     

Haloperidol produces hypothermic effects in rats

Intraperitoneal administration of either haloperidol or chlorpromazine produced hypothermia both in the cold (8 degrees C) and at room temperature (22 degrees C). The hypothermia was brought about both by a decrease in metabolic heat production and an increase in the cutaneous temperature of tail and foot skin. However, at a higher temperature (29 degrees C), there were no changes in rectal temperature and other thermoregulatory responses.     

Increased erythrocyte permeability to Li and Na in the spontaneously hypertensive rat.

Red blood cells incubated in a physiological medium in which Li replaces Na (LiPSS) gain Li in exchange for Na and K. The rate of Li uptake is modestly but significantly increased in the spontaneously hypertensive rat (SHR) at 37 degrees C and at 22 degrees C. The slow rate of Na gain and K loss during cooling at 2 degrees C was about doubled in unmodified whole blood samples from the SHR.     

Lack of participation of cytoplasmic choline in the synthesis of acetylcholine in the striatal synaptosomes of the rat]

Synaptosomes prepared from Rat striatum were loaded with (14C) choline at 0 degrees C in the course of a 60 min. preincubation. After the removal, by washing, of the extracellular molecules, synaptosomes were incubated 5 min. at 37 degrees C in the presence of (3H) choline. The measurement of the amounts of (3H) ACh and of (14C) ACh formed allowed us to conclude that the acetylation of the molecules of choline was directly coupled to their passage through the synaptosomal membrane.     

In vitro radiolabeling of galactosyl and N-acetylgalactosaminyl moieties of glycoproteins with carbon-14.

The primary alcohol group on the carbon 6 of terminal galactosyl and N-acetylgalactosaminyl moieties of glycoproteins can be oxidized to an aldehyde by treatment with galactose oxidase. By reacting these aldehyde groups with 14C-labeled sodium cyanide, 14C-labeled cyanohydrin derivatives were obtained. Similarly, reduction of these aldehyde groups with tritiated sodium borohydride following standard procedures, yields 3H-labeled glycoproteins. 14C- and 3H-labeled derivatives of asialofetuin and asialo ovine submaxillary mucin with high specific radioactivities were prepared using these procedures. Mixtures containing microgram amounts of 14C- and 3H-labeled glycoproteins were subjected to column chromatography and gradient ultracentrifugation and the position of the individual glycoproteins was determined by simultaneous counting for 14C and 3H. These experiments demonstrate the usefulness of this approach for comparative analytical studies using biological specimens available in minute quantities.     

Effect of relative humidity on the free-living stages of Strongyloides papillosus (Rhabdiasoidea: Nematoda)

The eggs and larvae are affected by the changes in the humidity level around them. The eggs do not develop below 87% relative humidity at 30 degrees C and 25 degrees C. At sub-developmental rel. hum. they remain viable for 30 h at 81%, but at 73% rel. hum. level they do not survive beyond 18 h. Survival of larvae in 100% rel. hum. is longer at 30 degrees C than at 25 degrees C and 35 degrees C, and also they exhibit a poor resistance to desiccation.     

Effects of raised and lowered incubation temperatures on the sexual differentiation in the embryos of Emys orbicularis L. (Chelonien)]

Eggs of Emys orbicularis L. were incubated at 18 and 19 degrees 5C during the period of sexual differentiation of the gonads of the embryos; genital glands of testicular structure and retrogressed Müllerian ducts were observed in all cases, like in the embryos issued from eggs incubated at 25 +/- 1 degrees C and 27,5 +/- 0,5 degrees C. On the contrary, the sexual phenotype is female in all the embryos which developed at 35 degrees C, as it is the case between 29 and 30 degrees C.     

Effects of thyrotrophic-releasing hormone (TRH) on thermoregulation in the rat

At ambient temperatures (Ta) of both 8 and 22 degrees C, intraventricular administration of TRH (10-80 microgram) produced a dose-dependent hypothermia in rats. The hypothermia was due to both decreased metabolic heat production and cutaneous vasodilatation. In contrast, at 30 degrees C Ta, TRH increased metabolic heat production (due to behavioral excitation) and led to hyperthermia.     

Acute cold exposure increases the glucagon sensitivity of thermogenic metabolism in the rat

Administration of glucagon to rats at 25 degrees C had no effect upon their VO2, while administration of noradrenaline or noradrenaline plus glucagon raised the VO2. At 5 degrees C, noradrenaline had no effect upon the cold-enhanced VO2, while glucagon caused a rise of 13.7%, implying increased glucagon sensitivity at 5 degrees C. The glucagon-induced enhancement of VO2 was abolished by concurrent administration of noradrenaline.     

Marked elevation of HDL-cholesterol in cold-adapted golden hamsters

Golden hamsters with spontaneous hypercholesterolemia at 22 degrees C developed a further increase in plasma cholesterol when they were maintained at 6 degrees C. This hypercholesterolemia was associated with a redistribution of plasma cholesterol between VLDL and HDL. Plasma cholesterol transported in the VLDL decreased while cholesterol in the HDL increased by 45%. The LDL profile was not significantly modified.     

Response of the ampullae of Lorenzini to static combined electric and thermal stimuli in Scyliorhinus canicula.

The effect of long-lasting electric currents on the Lorenzinian ampullae at constant temperatures between and 25 degrees C was investigated in the dogfish (Scyliorhinus canicula). Steady state neural impulse patterns in single afferent units were analyzed by plotting interval length histograms and computing mean values and standard deviations for currents between -100 and +100 nA. The mean values depended on temperature and on current strength; the relative standard deviations remained almost constant (ca. 20--30%). Negative currents, inserted at the orifice of the ampullary canal led to higher, and positive currents to lower, steady impulse rates in the whole temperature range investigated here. This static component of electrosensitivity again disappeared at higher currents (of 50 nA and more; electric overstimulation). The maximum static response was two orders of magnitude less than the maximum dynamic component of electroreception. The electrosensitivity depended on temperature: the ampullae were most sensitive to electric currents between 13 and 19 degrees C. The maximal neural activity at 19 degrees C was not shifted to higher or lower temperatures by electric stimulation. A constant equivalent of electric and thermal stimulation throughout the tested temperature and current range could not be found.     

Hypothermia induced in mice by enzyme-mediated depletion of serotonin

Injection of a purified serotonin (and tryptophan)-degrading enzyme into mice produced a pronounced hypothermia when the mice were maintained at 22 degrees C, but not at 30 degrees C. Brain levels of serotonin and tryptophan were strikingly depleted, but concentrations of norepinephrine and dopamine remained unchanged.     

In vitro radiolabeling of galactosyl and N-acetylgalactosaminyl moieties of glycoproteins with carbon-14

Summary The primary alcohol group on the carbon 6 of terminal galactosyl and N-acetylgalactosaminyl moieties of glycoproteins can be oxidized to an aldehyde by treatment with galactose oxidase. By reacting these aldehyde groups with¹⁴C-labeled sodium cyanide,¹⁴C-labeled cyanohydrin derivatives were obtained. Similarly, reduction of these aldehyde groups with tritiated sodium borohydride following standard procedures, yields³H-labeled glycoproteins.¹⁴C- and³H-labeled derivatives of asialofetuin and asialo ovine submaxillary mucin with high specific radioactivities were prepared using these procedures. Mixtures containing microgram amounts of¹⁴C- and³H-labeled glycoproteins were subjected to column chromatography and gradient ultracentrifugation and the position of the individual glycoproteins was determined by simultaneous counting for¹⁴C and³H. These experiments demonstrate the usefulness of this approach for comparative analytical studies using biological specimens available in minute quantities.     

Arguments in favor of the extramitochondrial origin of the acetyl radical of acetylcholine synthetized in rat striatum synaptosomes]

Synaptosomes prepared from Rat striatum were loaded with (2-14C) pyruvate at 0 degree C, in the course of a 1 hr. preincubation. When temperature was raised to 37 degrees C, the intra-synaptosomal (2-14C) pyruvate gave rise to 14CO2 but not to (14C) ACh. These results exclude a mitochondrial origin of the acetyl moiety of ACh.