A hemagglutinin specific for sialic acids in a rat brain synaptic vesicle-enriched fraction

A hemagglutinating activity was detected in a synaptic vesicle-enriched fraction prepared from adult rat brain, using trypsinized glutaraldehyde-fixed rabbit erythrocytes. The specific activity of the fraction, in two series of experiments, was 7.5 and 16-fold higher than in the other subcellular fractions. The activity was absent from the synaptosome cytosol. In a study using twenty-five different carbohydrates and glycoproteins, best inhibitors were N-acetylneuraminic acid and N-glycolylneuraminic acid, together with bovine submaxillary mucin and a glycopeptide fraction prepared from rabbit erythrocyte membranes. The activity was thermolabile and very sensitive to proteolytic enzymes (but insensitive to neuraminidase) indicating that a protein (agglutinin) is responsible for the activity. Experiments using detergents and high ionic strength showed that the agglutinin is tightly bound to membranes, inactivated by the so-called non denaturing detergents, and stable in diluted sodium dodecyl sulphate. Hypotheses are discussed on the possible function of the agglutinin.     

In vivo inhibition of nitrogenase by hydroxylamine in Rhodospirillaceae Role of nitric oxide

Rhodobacter capsulatus strains E1F1 and B10 and Rhodobacter sphaeroides DSM 158 did not use hydroxylamine as nitrogen source for growth but metabolized it mainly through the glutamine synthetase reaction. Hydroxylamine had a high toxicity for cells growing either under phototrophic or dark-aerobic conditions. l-methionine-d,l-sulfoximine partially inhibited hydroxylamine uptake and increased the inhibition time of nitrogenase activity by this nitrogen compound. Nitric oxide was also a powerful inhibitor of nitrogenase in intact cells of R. capsulatus. Since low amounts of NO were produced from hydroxylamine, short-term inhibition of nitrogenase in the presence of this compound could be mediated in vivo by nitric oxide.Abbreviations GS glutamine synthetase - MSX l-methionine-d,l-sulfoximine - MTA mixed alkyltrimethylammonium bromide     

Phorbol 12-myristate 13-acetate induces resistance of human melanoma cells to natural-killer-and lymphokine-activated-killer-mediated cytotoxicity

Summary Human melanoma cells are sensitive to the lytic activity of natural killer (NK) and lymphokine-activated killer (LAK) cells in vitro. The events resulting in tumour cell killing by lymphocytic effectors have not been completely clarified, and the same target cell determinants regulating responsiveness to immune cytolysis have not yet been identified. Indeed, changes in the differentiative status of leukemia cells as well as in the expression of major histocompatibility complex (MHC) antigens have been described to modulate sensitivity to cytotoxic effectors; moreover surface expression of adhesion factors or extracellular matrix proteins by the cancer cells can promote the activation of the cytolytic effectors and has been described to correlate with tumour cell sensitivity to cytolytic cells. We reasoned that treatment with differentiation inducers could modulate melanoma cell sensitivity to NK and LAK cells. The present study demonstrates that human melanoma GLL-19 cells, when treated with the phorbol diester phorbol 12-myristate 13-acetate (PMA) in vitro, undergo growth inhibition and neuron-like differentiation. Moreover PMA treatment induces an evident inhibition of GLL-19 cell sensitivity to NK- and LAK-mediated cytotoxicity. GLL-19 cells express constitutively MHC class I antigens. PMA treatment, however, does not modify the expression of MHC class I and class II DR antigens in human melanoma GLL-19 cells. We have finally evaluated the effects of PMA on the expression at the cell surface of adhesion factors such as ICAM-1, and extracellular matrix proteins such as collagen IV, laminin and fibronectin; we have also studied the expression of the integrin vitronectin receptor, a membrane receptor for adhesive proteins. While adhesion factors and extracellular matrix proteins appear to play an important role in the interaction between immune effector and tumour target, it can be supposed that the modulation of such membrane-associated proteins or glycoproteins induces NK and LAK resistance in cancer cells. We indeed found that PMA treatment induced in GLL-19 a marked reduction of membrane expression of collagen IV and ICAM-1; moreover PMA reduced the cell membrane expression of the integrin vitronectin receptor. On the other hand, membrane expression of fibronectin and laminin was not affected by PMA. These data indicate that the acquisition of a NK- and LAK-resistant phenotype by GLL-19 cells occurs together with cell differentiation, down-regulation of membrane expression of collagen IV, ICAM-1 and vitronectin receptor, but in the absence of changes in MHC antigens.This work has been supported by the Italian Association for Cancer Research (A. I. R. C.) and by Istituto Superiore di Sanità, Italy-USA joint program on New Therapies on Neoplasia.     

The distribution of A1 adenosine receptor and 5′-nucleotidase in pig brain cortex subcellular fractions

Pig brain cerebral cortex was subfractionated by isopycnic centrifugation in sucrose gradients. In each subfraction the content of the agonist [³H]R-PIA binding, the activity of adenosine metabolizing enzymes (5-nucleotidase and adenosine deaminase) and the activity of membrane marker enzymes were determined. The fractions were also examined by electron microscope. In general, the results suggest a widespread distribution of A₁ adenosine receptors in membranes from different origins. Marker enzyme profile characterization indicated an enrichment of A₁ adenosine receptor in pre-synaptic membranes isolated from the crude synaptosomal fraction (P2B subfraction) as well as in membranes of glial origin such as myelin. The receptor is also present in the endoplasmic reticulum and in membranes isolated from the microsomal fraction that seem to have a post-synaptic origin (P3B). In subfractions having a high content of adenosine receptor the equilibrium binding paramters were obtained as well as the proportion of high- to low-affinity sites. From the values of the equilibrium constants it was not possible to find differences between the receptor in the different subfractions. Analysis of the affinity state distribution showed a diminished percentage of high-affinity sites in fraction P3A, which can be accounted by the existence of myelin membranes; in contrast the percentage of high-affinity states was higher in P2 and P3B, indicating that in these fractions the receptor is present in synaptosomal membranes. The close correlation shown between the enzyme 5-nucleotidase specific activity and the specific ligand binding distributions led us to postulate an important role for the enzyme in the regulation of adenosine action in pig brain cortex.     

Computational study of radiation chemical processing in comet nuclei

Cometary nuclei have been exposed to high levels of ionizing radiation since their formation. We present here some results of a computer model calculation of the effect of ionizing radiation on cometary material. The external (cosmic rays) and internal (embedded radionuclides) contributions in the processing of cometary nuclei are considered. As a first approximation we have used the available kinetic data of the liquid water system to model the radiation effects in a frozen cometary environment. Out data suggest that massive radiation chemical processing due to cosmic rays may have taken place only in the outer layers of comets. The internal contribution of radionuclides to the radiation processing of comet cores seems to be modest. Therefore, comets could be carriers of intact homochiral biomolecules.Part of this work was carried out during a leave at the Laboratory of Chemical Evolution.     

Experimental and computational study of the radiation-induced decomposition of formaldehyde. Implications to cometary nuclei

The radiation-induced decomposition of aqueous solutions of formaldehyde was studied at 298 and 77 K in order to obtain an insight into the possible role of ionizing radiation on cometary chemistry. Aqueous solutions of 1.0 mol dm^–3 formaldehyde were exposed to -radiation in the dose range from 0.01 kGy to 1.2 MGy. The radiation chemical yield of decomposition of formaldehyde was determined to be: G(–CH₂(OH)₂)=26.3±1.2 at 298 K and G0.48 at 77 K.Based on previous estimates of the total dose of ionizing radiation that comets have accumulated over 4.6 billion years, we predict a radiation-induced depletion of formaldehyde as a function of depth in comet nuclei: 100% destruction in the outer layers (0–20 m) and 10% destruction in the interior layers.A Portion of this work was presented at the XXVIII COSPAR Plenary Meeting (MF. 7) held at the Hague, The Netherlands on June 25–26, 1990.     

Effect of dissolved oxygen concentration on repeated production of gluconic acid by immobilised mycelia of Aspergillus niger

Summary Gluconic acid production from corn starch hydrolysates by immobilised mycelia of Aspergillus niger was studied in a laboratory-scale stirred fermentor at different concentrations of glucose (S ₀) and dissolved oxygen (DO) in the culture broth. Its evolution was simulated quite well by applying the same unstructured model set up in previous experiments using stirred and airlift fermentors. In particular, increasing S ₀ in the range 70–160 g/l, although uninfluential upon the yield coefficient, resulted in an exponential decrease in the gluconic acid formation rate constant. Nevertheless, the greater the oxygen transfer rate used in the fermentor, the smaller the inhibitor effect of the higher concentrations of glucose on gluconate productivity became. This was achieved by enriching the inlet air with pure oxygen so as to maintain the DO level above 75% saturation throughout the fermentation. Offprint requests to: M. Moresi     

Putrescine uptake regulation in response to alpha-difluoromethylornithine treatment in Leishmania infantum promastigotes

Summary The putrescine uptake/efflux regulation and their regulatory role on intracellular polyamine pools have been studied in the parasitic protozoa Leishmania infantum. Putrescine uptake was age-dependent with maximal values in logarithmic phase promastigotes and minimal in stationary phase. Moreover, putrescine uptake was activated in response to depletion of intracellular polyamines by alpha-difluoromethylornithine (DFMO) — a well known irreversible enzyme-activated inhibitor of ornithine decarboxylase. Kinetic studies of putrescine uptake induction showed a notable rise in Vmax without Km changes, suggesting a de novo synthesis of putrescine carriers. Putrescine uptake was able to replenish polyamine content and also to recover the proliferative rate in cells treated during 24 hours with DFMO.     

An electron microscopy study of wall expansion during Candida albicans yeast and mycelial growth using concanavalin A-ferritin labelling of mannoproteins

Depending upon growth temperature, Candida albicans can exhibit two different morphologies, a budding yeast or a mycelium. By studying the distribution of concanavalin A-ferritin particles on the cell wall surface during bud and germ tube formation, we have elucidated the way cell wall extension occurs. Both processes initially require the localized lysis of the wall in order to allow the incorporation of the newly synthesized material. Later on, the cell wall behaves as an elastic structure, allowing extension by an intosusception process and, as a consequence, cell growth.Abbreviation Con A concanavalin A