Incorporation of adenosine into nucleotides of chromaffin cells maintained in primary cultures

Extracellular adenosine was incorporated into nucleotides of bovine chromaffin cells maintained in primary culture. In intact chromaffin tissue, a very low incorporation was found (0.8 pmol/10⁶ cells/h at an adenosine concentration of 11.45 μM), which increased 282 times in freshly isolated chromaffin cells. When maintained in primary culture, this value decreased to a value similar to that of chromaffin tissue, but later on, and in the presence of nerve growth factor (NGF), a time dependent increase of adenosine incorporation was observed which, in 84-h old cells reached up to 54 times more than that found in intact tissue. This incorporation might reflect changes in the adenosine transport at the cell membrane level, furthered by NGF effect. Incorporation, which was time-dependent, was weakly modified by stimulation of cells with 10^?4 M acetylcholine. However, acetylcholine-induced release of labelled nucleotides from chromaffin granules was observed, probably in relation to granule maturation.     

Circadian changes in carrageenin-induced edema: the anti-inflammatory effect and bioavailability of phenylbutazone in rats

The circadian variations in paw-edema produced by carrageenin and the anti-inflammatory effect of phenylbutazone were studied, in rats kept under a 12 light-12 dark regimen, in comparison with the variations of plasma phenylbutazone and oxyphenbutazone levels. When the experiment was performed during the light span (08.00 and 14.00 h), the rats were highly sensitive to the phlogistic effect of carrageenin, the plasma levels of phenylbutazone and oxyphenbutazone were lower, and the anti-inflammatory effect of phenylbutazone, weaker. Opposite results were obtained when the experiment was performed during the dark span (02.00 and 20.00 h). The results indicate that the chronoeffectiveness of phenylbutazone is influenced by both its chronokinetics and the chronesthesy of the biosystem involved.     

Mutagenesis in multinucleate cells: the effects of N-methyl-N'-nitro-N-nitrosoguanidine on Phycomyces spores

Multinucleate cells, such as the spores of the fungus Phycomyces, are unsuitable for the isolation of recessive mutants. Nuclear killing by N-methyl-N'-nitro-N-nitrosoguanidine (henceforth nitrosoguanidine) eliminates all but one of the nuclei in some of the cells and allows the expression of recessive mutations. Even in the best conditions, only about 35% of the survivors have a single functional nucleus. Functionally uninucleate cells can be positively selected. This involves the exposure to nitrosoguanidine of the spores of a heterokaryon and selection for a recessive marker present in a small fraction of its nuclei. The optimal conditions for nitrosoguanidine mutagenesis in Phycomyces differ from those for bacteria and yeast. Buffer composition and pH are less important than in other organisms. Survival is an exponential function and mutation induction a linear function of the dose of the mutagen (concentration X time). Spore germination leads to an immediate increase in the number of gene copies per cell, thus further hindering the expression of recessive mutations; dominant mutations are then nearly always isolated in heterokaryotic form.     

Energy migration in the poly(ra-rU)-ethidium complex. determination of the unwinding angle of the polyribonucleic helix

The polarized fluorescence of the ethidium bromide (EB)-poly(rA-rU) complex has been studied by pulse fluorometry. As expected for a polynucleotide snowing one single kind of intercalation site, the decay of the whole emission is a single exponential (time constant 27 ns). The anisotropy decay is analysed as follows: (1) A brownian contribution having two correlation times, one of which characterizes local motions and the other a macromolecular motion. (2) A contribution due to transfers between EB molecules fixed to the same polynucleotide molecule, is analysed by a method analogous to the method used in previous work on EB-DNA complexes. That method consists in choosing a molecular model of the complex depending on geometrical parameters, and in simulating the energy migration on that model with a Monte Carlo calculation. Poly(rA-rU) is assumed here to adopt the structure A of RNA. Intercalated EB molecules modify the anale between two consecutive base pairs by δ. The angular position of the EB transition moment is defined by an angle φ. One finds that the angle φ is situated between 0° and 30°, which corresponds to a whole intercalation of the chroniophore as opposed to the semi-intercalation which has been proposed for certain dyes. The angle δ is negative; therefore there is an unwinding of the polyribonucleotide helix. Its absolute value is about 38°, sensibly greater than The value previously found for EB-DNA complexes.     

Prey capture by the cuttlefish (Sepia officinalis L): An experimental study of two strategies

This study shows that the size of the prey ($\text{Carcinus maenas}$) relative to the predator ($\text{Sepia officinalis}$) is of importance in the choice between two types of attack: either capture by ejection of the two extensible tentacles, or capture by jumping on the prey. Small crabs are preferentially captured by the first method and large crabs by the second. Other factors which may explain the observed variations, include previous experience of the predator and the behaviour of the prey.     

Evidence of essential arginyl residues in chicken liver mevalonate-5-pyrophosphate decarboxylase

Chicken liver mevalonate-5-pyrophosphate decarboxylase (ATP:5-diphosphomevalonate carboxy-lyase (dehydrating), EC 4.1.1.33.) is inactivated by phenylglyoxal in triethanolamine buffer at pH 8.15. The reaction follows pseudo-first-order kinetics with a second-order rate constant of 108 M-1 min-1. Appropriate treatment of the kinetic data for the inactivation reaction indicates that the reaction of a single phenylglyoxal molecule per active unit of the enzyme is enough to completely inactivate the protein. The partially inactivated enzyme shows unaltered Km but decreased V as compared to native mevalonate-5-pyrophosphate decarboxylase. The dissociation constants for the enzyme-substrate complexes were estimated from inactivation reactions at different concentrations of substrates. From the data it is concluded that the modified amino acid is important for the binding of both substrates.     

Formation of copper-bleomycin complexes: evidence of a three-step process

The formation of Cu(II)-bleomycin complexes as a function of pH has been studied using circular dichroism, absorption, electron paramagnetic resonance spectroscopy, and potentiometric titration. Our data support the following points: the formation of Cu(II)-bleomycin complexes occurs in a three-step process: a first complex (I) is formed at pH 1.2, which most probably involves the pyrimidine nitrogen, the secondary amine nitrogen, and two water molecules as the four in-plane ligands of copper. A second complex (II) is formed at pH 2.5, through the further coordination of the peptide nitrogen of histidine residue, and histidine imidazole nitrogen giving rise to the release of two protons. The fixation, in apical position, of the alpha-amino nitrogen of beta-aminoalanine occurs in a last step through the release of one additional proton. A value of 2.7 has been obtained for the pK of formation of this third complex, which is the species present at physiological pH. In the Cu(II)-depbleomycin system only one complex (II') has been detected.     

Transferrin binding to K562 cell line : Effect of heme and sodium butyrate induction

Experiments demonstrating the existence of receptors for iron-saturated transferrin on K562 cells are described. Binding of ¹²⁵I-labelled transferrin is rapid, saturable and reversible, and can be specifically inhibited by unlabelled transferrin, but not by other proteins. The number of receptors determined by Scatchard analysis significantly decreased when K562 cells moved from the exponential to the quiescent phase of growth. Induction by hemin or sodium butyrate resulted in a marked reduction of transferrin binding. This phenomenon was due entirely to reduction in the number of receptors and was without effect on the affinity of interaction. The effect of butyrate and hemin on the number of transferrin receptors in other hematopoietic cell lines was investigated. Butyrate on the various cell lines was variable in its effect, whereas hemin constantly elicited a significant reduction in the number of transferrin receptors.     

Reversible inactivation of Saccharomyces cerevisiae glutathione reductase under reducing conditions

Glutathione reductase from Saccharomyces cerevisiae was rapidly inactivated following aerobic incubation with NADPH, NADH, and several other reductants, in a time- and temperature-dependent process. The inactivation had already reached 50% when the NADPH concentration reached that of the glutathione reductase subunit. The inactivation was very marked at pH values below 5.5 and over 7, while only a slight activity decrease was noticed at pH values between these two values. After elimination of excess NADPH the enzyme remained inactive for at least 4 h. The enzyme was protected against redox inactivation by low concentrations of GSSG, ferricyanide, GSH, or dithiothreitol, and high concentrations of NAD(P)+; oxidized glutathione effectively protected the enzyme at concentrations even lower than GSH. The inactive enzyme was efficiently reactivated after incubation with GSSG, ferricyanide, GSH, or dithiothreitol, whether NADPH was present or not. The reactivation with GSH was rapid even at 0 degree C, whereas the optimum temperature for reactivation with GSSG was 30 degrees C. A tentative model for the redox interconversion, involving an erroneous intramolecular disulfide bridge, is put forward.     

The Expression of Connexins and SOX2 Reflects the Plasticity of Glioma Stem-Like Cells

Glioblastoma (GBM) is the most malignant primary brain tumor, with an average survival rate of 15 months. GBM is highly refractory to therapy, and such unresponsiveness is due, primarily, but not exclusively, to the glioma stem-like cells (GSCs). This subpopulation express stem-like cell markers and is responsible for the heterogeneity of GBM, generating multiple differentiated cell phenotypes. However, how GBMs maintain the balance between stem and non-stem populations is still poorly understood. We investigated the GBM ability to interconvert between stem and non-stem states through the evaluation of the expression of specific stem cell markers as well as cell communication proteins. We evaluated the molecular and phenotypic characteristics of GSCs derived from differentiated GBM cell lines by comparing their stem-like cell properties and expression of connexins. We showed that non-GSCs as well as GSCs can undergo successive cycles of gain and loss of stem properties, demonstrating a bidirectional cellular plasticity model that is accompanied by changes on connexins expression. Our findings indicate that the interconversion between non-GSCs and GSCs can be modulated by extracellular factors culminating on differential expression of stem-like cell markers and cell-cell communication proteins. Ultimately, we observed that stem markers are mostly expressed on GBMs rather than on low-grade astrocytomas, suggesting that the presence of GSCs is a feature of high-grade gliomas. Together, our data demonstrate the utmost importance of the understanding of stem cell plasticity properties in a way to a step closer to new strategic approaches to potentially eliminate GSCs and, hopefully, prevent tumor recurrence.     

The effect of diabetes, nutritional factors, and sex on rat liver and kidney mevalonate kinase, mevalonate-5-phosphate kinase, and mevalonate-5-pyrophosphate decarboxylase

Isopycnic sucrose gradient separation of rat liver organelles revealed the presence of two distinct branched-chain α-keto acid decarboxylase activities; a mitochondrial activity, which decarboxylates the three branched-chain α-keto acids and requires CoA and NAD⁺ and a cytosolic activity, which decarboxylates α-ketoisocaproate, but not α-ketoisovalerate, or α-keto-β-methylvalerate. The latter enzyme does not require added CoA or NAD⁺. Assay conditions for the cytosolic α-ketoisocaproate decarboxylase activity were optimized and this activity was partially characterized. In rat liver cytosol preparations this activity has a pH optimum of 6.5 and is activated by 1.5 m ammonium sulfate. The decarboxylase activity has an apparent K_m of 0.03 mm for α-ketoisocaproate when optimized assay conditions are employed. Phenylpyruvate is a very potent inhibitor. α-Ketoisovalerate, α-keto-β-methylvalerate, α-ketobutyrate, and α-ketononanoate also inhibit the α-ketoisocaproate decarboxylase activity. The data indicate that the soluble α-ketoisocaproate decarboxylase is an oxidase. Rat liver cytosol preparations consumed oxygen when either α-ketoisocaproate or α-keto-γ-methiolbutyrate were added. None of the other α-keto acids tested stimulated oxygen consumption. 1-¹⁴C-Labeled α-keto-γ-methiolbutyrate is also decarboxylated by cytosol preparations. The α-ketoisocaproate oxidase was purified 20-fold from a 70,000g supernatant fraction of a rat liver homogenate. In these preparations the activity was increased 4-fold by the addition of dithiothreitol, ferrous iron, and ascorbate. The major product of this enzyme activity is β-hydroxyisovalerate. Isovalerate is not a free intermediate in the reaction. The data indicate an alternative pathway for metabolism of α-ketoisocaproate which produces β-hydroxyisovalerate.     

Visualization of chromatin distribution in living PTO cells by Hoechst 33342 fluorescent staining

Chromatin distribution was visualized in living cells with the selective DNA fluorochrome Hoechst 33342. This dye was shown to be non-toxic on the rat kangaroo PTO cell line by measuring the labelled cell growth rate. The aim of this work was firstly to visualize chromatin distribution without fixation or dehydration and secondly to demonstrate that quantitative determination of DNA content was possible under these non-toxic labelling conditions. During interphase, condensed, decondensed and thin network chromatin configurations were visualized. In nucleolar regions the fluorochrome revealed well-defined chromocentres. During mitosis, fluorescent chromosome banding was observed in vital conditions and chromocentres on fixed chromosomes. Chromatin segregation was visualized after micronucleation, which induced chromosomal set distribution in individual micronuclei. By this means, we demonstrated that the chromocentres observed in interphase nuclei were part of nuclear organizer region (NOR)-bearing chromosomes. This vital staining of chromatin was shown to be compatible with the quantitative determination of DNA content, both in living PTO cells and in isolated nuclei.     

Quantitative determination of tryptophanyl and tyrosyl residues of proteins by second-derivative fluorescence spectroscopy

Second-derivative spectroscopy has been applied to the study of the fluorescence of aromatic amino acids. The spectral features of the second derivative emission spectra of free aromatic amino acids and proteins are described, the emission of each aromatic fluorophore being characterized by a particular minimum-maximum pair. An easy, accurate, and rapid method is proposed for the quantitative determination of tyrosine and tryptophan, based on the addition of small amounts of a standard solution to the samples followed by the measurement of the increase in the distance between a selected minimum and an adjacent maximum, in the second-derivative spectrum. For tyrosine determination, excitation wavelength was 275 nm, and the selected minimum-maximum (m,M) pair was (300; 330 nm), while an excitation of 300 nm and a minimum-maximum pair (357; 377 nm) were employed for the tryptophan determination. This method enables the tryptophan content of proteins to be determined directly, without the need for correction for the presence of tyrosine. The tyrosine content of proteins can also be determined at neutral pH, in the presence of both tryptophan and phenylalanine. The proposed method has also been applied to trypsin activation of frog epidermis tyrosinase.     

Self-exchange rates and electron transfer kinetics of horse heart ferricytochrome C with amino acid-pentacyanoferrate(II) complexes

The electron transfer reactions of horse heart cytochrome c with a series of amino acid-pentacyanoferrate(II) complexes have been studied by the stopped-flow technique, at 25°C, μ = 0.100, pH 7 (phosphate buffer). A second-order behavior was observed in the case of the Fe(CN)₅ (histidine)^3? complex, with k = 2.8 x 10⁵ M^?1 sec^?1. For the Fe(CN)₅ (alanine)^4? and Fe(CN)₅(L-glutamate)^5? complexes, only a minor deviation of the second-order behavior, close to the experimental error (k = 3.2 × 10⁵ and 1.6 x 10⁵ M^?1 sec^?1, respectively) was noted at high concentrations of the reactants (e.g., 6 × 10^?4 M). The results are in accord with recent work on the Fe(CN)₆^4?/cytochrome c system demonstrating weak association of the reactants. The calculated self-exchange rate constants including electrostatic interactions for the imidazole,L -histidine, 4-aminopyridine, glycinate, β-alaninate, andL-glutamate pentacyanoferrate(II) complexes were 3.3 × 105, 3.3 × 10⁵, 2.8 × 10⁶,4.1 × 10²,5.5 × 10², and 6.0 M^?1 sec^?1, respectively. Marcus theory calculations for the cytochrome c reactions were interpreted in terms of two nonequivalent binding sites for the complexes, with the metalloprotein self-exchange rate constants varying from 10⁴ M^?1 sec^?1 (histidine, imidazole, and 4-aminopyridine complexes) to 10⁶ M^?1 sec ^?1 (glycinate, β-alaninate, and L-glutamate complexes).