Histamine Stimulation of Cyclic AMP Accumulation in Astrocyte-Enriched and Neuronal Primary Cultures from Rat Brain

Histamine stimulates cyclic AMP accumulation in astrocyte-enriched and neuronal primary cultures from rat brain in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine. The response in the astrocyte cultures (Emax = 304 +/- 44% over basal, EC50 = 43 +/- 5 microM) was much higher than in neuronal cultures (Emax = 24 +/- 2%, EC50 = 14 +/- 7 microM). The histamine effect in astrocytes was competitively inhibited by the H2 antagonists cimetidine (Ki = 1.1 +/- 0.2 microM) and ranitidine (Ki = 46 +/- 10 nM) but was insensitive to the H1 antagonist mepyramine (1 microM). The two selective H2 agonists impromidine and dimaprit behaved as partial agonists and showed relative potencies (139 and 0.5, respectively) consistent with an interaction with H2 receptors. The more selective H1 agonist 2-thiazolylethylamine (0.01-1 mM) did not potentiate the response to impromidine (10 microM). Thus, in contrast to what is generally observed in intact cell preparations from brain, the histamine-induced cyclic AMP accumulation in astroglial cells is mediated solely by H2 receptors. The small effect shown in neuronal cultures also appears to be mediated by H2 receptors.     

Impaired local natural killer cell activity in human colorectal carcinomas

Summary The present study was undertaken to study natural killer (NK) cell activity in patients with colorectal cancer at peripheral and local levels. Mononuclear cells were isolated from uninvolved colorectal mucosa, tumor tissue and peripheral blood, and tested against the colon carcinoma cell line CaCo-2 and the erythroleukemia cell line K-562. Peripheral blood NK cell activity from the patients showed similar levels compared with healthy controls, whereas, mononuclear cells of tumor tissue were found to have a significantly decreased NK cell activity compared to the normal intestinal mucosa (P<0.01). No relation was found between the NK cell activity and the advancement of the disease according to the Duke's stage. Interferon- (IFN-) stimulated the NK cell activity of the mononuclear cells from blood, mucosa and tumor. However, the increase of NK cell activity after IFN- stimulation was lower in the tumor compared to the mucosa (P<0.02). The lectin, phytohaemagglutinin, increased the cytotoxicity of mononuclear cells from blood, mucosa and tumor to a similar level. These results suggest that patients with colorectal tumors exhibit a normal NK cell activity in peripheral blood and intestinal mucosa; however, a diminished NK cell activity exists at the tumor level. Although mononuclear cells isolated from the tumor have a normal response to lectin stimulation they show hyporesponsiveness to IFN- stimulation with regard to their NK cell activity.     

Assembly and disassembly of brain tubulin is affected by high ammonia levels

Rats were fed a diet containing ammonium for up to 6 months. High ammonia levels were attained in brain. The amount of polymerized tubulin in microtubules increased, while the amount of free tubulin remained unchanged. Polymerization of tubulin from brain of ammonium fed rats (30 min, 37°C) was approximately 60% of control. Depolymerization of the microtubules was also affected and took approximately 3 times longer than in controls. These results indicate that both assembly and disassembly of tubulin in brain are impaired by high ammonia levels. Interestingly, the amount of microtubule-associated proteins was not affected.     

Proteolytic stimulation and solubilization of membrane-bound acetylcholinesterase from muscle sarcotubular system

Incubation of membranes derived from sarcotubular system of rabbit skeletal muscle with increasing concentrations of Triton X-100 produced both stimulation of the AChE activity and solubilization of this enzyme. Mild proteolytic treatment of microsomal membranes produced a several fold activation of the still membrane-bound acetylcholinesterase (AChE) activity. Attempts were made to solubilize AChE from microsomal membranes by proteolytic treatment. About 30–40% of the total enzyme activity could be solubilized by means of trypsin or papain. Short trypsin treatment of the microsomal membranes produced first an activation of the membrane-bound enzyme followed by solubilization. Incubation of muscle microsomes for a short time with papain yielded a significant portion of soluble enzyme. Membrane-bound enzyme activation was measured after a prolonged incubation period. These results are compared with those of solubilization obtained by treatment of membranes with progressive concentrations of Triton X-100. The occurrence of molecular forms in protease-solubilized AChE was investigated by means of centrifugation analysis and slab gel electrophoresis. Centrifugation on sucrose gradients revealed two main components of 4.4S and 10–11S in either trypsin or papain-solubilized AChE. These components behaved as hydrophilic species whereas the Triton solubilized AChE showed an amphipatic character. Application of slab gel electrophoresis showed the occurrence of forms with molecular weights of 350,000; 175,000; 165,000; 85,000 and 76,000. The stimulation of membrane-bound AChE by detergents or proteases would indicate that most of the enzyme molecules or their active sites are sequestered into the lipid bilayer through lipid-protein or protein-protein interactions and these are broken by proteolytic digestion of the muscle microsomes.     

A protein-free diet changes synaptosomal membrane fluidity and tyrosine and glutamate transport

Synaptosomes were isolated from cerebrums of rats fed standard (20% protein) or protein-free diets for 30 days. Arrhenius plots of their (Na⁺/K⁺)ATPase activities revealed a transition temperature of 25.5°C for control rats and 23.4°C for rats on protein-free diet, indicating that the latter increases synaptosomal membrane fluidity. The only change observed in the composition of the synaptosomal membranes was a 26% decrease of sialic acid. In synaptosomes from rats on protein-free diet the uptake of tyrosine was slightly reduced while that of glutamate was not affected. However, the exit of glutamate was reduced.     

Biological role of endoreplication in the process of spermatogenesis inChara vulgaris L.

Summary During cell division in antheridial filaments ofChara vulgaris an increase in DNA content occurs in both shield cells and manubria within an antheridium, reaching 16C–64C and 8C–32C levels, respectively. Endoreplication ceases prior to the formation of spermatids and initiation of spermiogenesis, probably as a result of symplasmic isolation of the antheridium from the thallus. As the DNA content of the nuclei increases, the shield cells³H-leucine incorporation increases, and they grow intensively in the tangential plane. Translation decreases considerably after termination of shield cell growth. DNA content of mature manubria is half of that in shield cells, although their size is 10 times that of manubria. Translational activity of manubria also increases as DNA content rises and cells grow. However, during spermiogenesis, this activity remains at its maximum, which is associated with the secretory function of the manubria. Spermiogenesis is also accompanied by far-reaching ultrastructural changes within the manubrial cytoplasm.The level of endopolyploidy in both shield cells and manubria of antheridia formed in the spring is higher by one replication cycle, than in autumnal antheridia. AMO-1618, at a concentration of 10^–5M reduces the DNA content in the autumnal manubria. The higher the manubrial level of endopolyploidy in spermiogenesis, the greater their size, and the higher the translational activity and number of joined spermatids. The number of spermatozoids in the antheridium is also positively correlated with the internal volume of an antheridium, which is itself dependent on the endopolyploidy level of shield cells.The results obtained confirm the assumption that endoreplication favours the higher growth dynamics and potential translational activity, which occurs in the dynamic growth phase only in shield cells, while in manubria, i.e. cells producing substances necessary to spermatozoids development, it remains high until the end of spermiogenesis.     

Inverse Diauxy in the Yeast Hansenula anomala: Mutants Derepressed for Malic Acid Utilization in the Presence of Glucose

Utilization of l-malic acid by yeast strain Hansenula anomala IGC 4380 is subject to glucose repression. Derepressed mutants were obtained with UV light by use of the nonmetabolizable glucose analog 2-deoxyglucose as a selective agent. Three mutant strains degraded l-malic acid in the presence of up to 30% (wt/vol) glucose and are of potential interest for the biological deacidification of grape must. The mutant strains, as compared with the parent strain, displayed inverse diauxy in glucose-malate medium, glucose being metabolized only after malate consumption had been completed.     

Unfolding of trp repressor studied using fluorescence spectroscopic techniques.

The unfolding properties of the trp repressor of Escherichia coli have been studied using a number of different time-resolved and steady-state fluorescence approaches. Denaturation by urea was monitored by the average fluorescence emission energy of the intrinsic tryptophan residues of the repressor. These data were consistent with a two-state transition from dimer to unfolded monomer with a free energy of unfolding of 19.2 kcal/mol. The frequency response profiles of the fluorescence emission brought to light subtle urea-induced modifications of the intrinsic tryptophan decay parameters both preceding and following the main unfolding transition. The increase of lifetime induced by urea required higher concentrations of urea than the increase in the total intensity described by Gittelman and Matthews [(1990) Biochemistry 29, 7011]. This indicates that the intensity increase has both dynamic and static origins. To assess the effect of tryptophan binding upon repressor stability, and to determine whether repressor oligomerization would be detectable in an unfolding experiment, we examined denaturation profiles of repressor labeled with the long-lived fluorescence probe 5-(dimethylamino)naphthalene-1-sulfonyl (DNS), by monitoring the average rotational correlation time of the probe. These experiments revealed a protein concentration dependent transition at low urea concentrations. This transition was promoted by tryptophan binding. We ascribe this transition to urea-induced dissociation of repressor tetramers. The main unfolding transition of the dimer to unfolded monomer was also observable using this technique, and the free energies associated with this transition were 18.3 kcal/mol in the absence of tryptophan and 24.1 kcal/mol in its presence, demonstrating that co-repressor binding stabilizes the repressor dimer against denaturation.(ABSTRACT TRUNCATED AT 250 WORDS)     

TWO DISTINCT COLONIAL MORPHOTYPES OF AMPHORA COFFEAEFORMIS (BACILLARIOPHYCEAE) CULTURED ON SOLID MEDIA1

When cultured on different types of solid media, the marine-fouling diatom Amphora coffeaeformis (Ag.) Kütz. consistently formed two distinct colonial morphotypes named tight and fuzzy. Tight colonies were comprised mainly of small, morphologically distorted, nonmotile cells, whereas morphologically normal and highly motile cells formed the fuzzy colonies. Cells from tight colonies were less adherent to glass, grew more slowly in liquid media, and had a slightly decreased viability on plates with copper than cells from fuzzy colonies. Whereas the protein profiles of the two types of cells were nearly identical in polyacrylamide gels stained with Coomassie blue, cells from tight colonies produced a significantly lower amount of a protease-resistant, low M_r polysaccharide or glycoconjugate as detected in silver-stained gels. The frequency of appearance of the fuzzy and tight morphotypes was not influenced by the mode of nutrition or the type of substratum to which the algal cells adhered. However, certain formulations of solid medium and the presence of growth-inhibitory concentrations of copper in agar plates favored the formation of tight colonies. Due to their frequencies and patterns of appearance, it was clear that the two naturally formed morphotypes were not the consequence of spontaneous mutations, genetic rearrangement, or selection of stable natural variants, and we have hypothesized that they were linked to a normal physiological behavior. The tight colonial morphotype was used as a valuable marker to screen for true motility/adhesion mutants within an ultraviolet-mutagenized population of A. coffeaeformis. Seven mutants were isolated that were non-motile on agar plates, poorly adherent to glass, and distinguished from naturally formed cells from tight colonies by their inability to form fuzzy colonies upon subculture on solid media.