Dexamethasone Up-Regulates a Constitutive Nitric Oxide Synthase in Cerebellar Astrocytes but Not in Granule Cells in Culture

Abstract: Treatment of rat cerebellar astrocyte-enriched primary cultures with dexamethasone enhances the nitric oxide-dependent cyclic GMP formation induced by noradrenaline in a time-(>6 h) and concentration-dependent manner (half-maximal effect at 1 n M ). Stimulation of cyclic GMP formation by the calcium ionophore A23187 is similarly enhanced. In contrast, cyclic GMP accumulation in cells treated with lipopolysaccharide is inhibited by dexamethasone. The potentiating effect of dexamethasone is prevented by the protein synthesis inhibitor cycloheximide and is not due to increased soluble guanylate cyclase activity. Agonist stimulation of [³H]arginine to [³H]citrulline conversion is enhanced by dexamethasone in astrocytes but not in cerebellar granule cells. These results indicate that glucocorticoids may up-regulate astroglial calcium-dependent nitric oxide synthase while preventing expression of inducible nitric oxide synthase and are the first report of a differential long-term regulation of the expression of neuronal and astroglial constitutive nitric oxide synthase activities.     

Peroxynitrite-Induced Cytotoxicity in PC12 Cells: Evidence for an Apoptotic Mechanism Differentially Modulated by Neurotrophic Factors

Abstract: Peroxynitrite is a powerful oxidant formed by the near-diffusion-limited reaction of nitric oxide with superoxide. Large doses of peroxynitrite (>2 m M ) resulted in rapid cell swelling and necrosis of undifferentiated PC12 cells. However, brief exposure to lower concentrations of peroxynitrite (EC₅₀ = 850 µ M ) initially (3–4 h) caused minimal damage to low-density cultures. By 8 h, cytoplasmic shrinkage with nuclear condensation and fragmentation became increasingly evident. After 24 h, 36% of peroxynitrite-treated cells demonstrated these features associated with apoptosis. In addition, 46% of peroxynitrite-treated cells demonstrated DNA fragmentation (by terminal-deoxynucleotide transferase-mediated dUTP-digoxigenin nick end-labeling) after 7 h, which was inhibited by posttreatment with the endonuclease inhibitor aurintricarboxylic acid. Serum starvation also resulted in apoptosis in control cells (23%), the percentage of which was not altered significantly by peroxynitrite treatment. Although peroxynitrite is known to be toxic to cells, the present study provides a first indication that peroxynitrite induces apoptosis. Furthermore, pretreatment of cells with nerve growth factor or insulin, but not epidermal growth factor, was protective against peroxynitrite-induced apoptosis. However, both acidic and basic fibroblast growth factors greatly increased peroxynitrite-initiated apoptosis, to 63 and 70%, respectively. Thus, specific trophic factors demonstrate differential regulation of peroxynitrite-induced apoptosis in vitro.     

Histochemical study of glycoconjugates in the epididymis of the hamster (Mesocricetus auratus)

Summary The glycoconjugates of hamster epididymis were investigated with conventional and lectin histochemistry. A zone of the caput epididymis, with particular histochemical characteristics, has been differentiated. β-Elimination in combination with lectins was used to establish the presence and distribution of N- and O-linked glycoconjugates. The epithelium, spermatozoa and the intertubular matrix were rich in glycoconjugates. The Golgi apparatus and stereocilia of the principal cells were intensely positive with HPA, PNA and SBA lectins. β-limination indicated that these cells contained abundant O-linked glycoconjugates. Apical and clear cells presented a common lectin affinity; their reactivities towards WGA and UEA-I were very positive. These cells probably contain abundant N-glycoconjugates. The spermatozoa were stained by periodic acid-Schiff (PAS) and by all the lectins (especially in the acrosome), except by those with an affinity for α-l-fucosyl residues; the most intense reaction was found with HPA, WGA, PNA and SBA. Changes in the sperm lectin binding along the ductus were observed: sperm flagellum abruptly acquired WGA and PNA labelling from the posterior caput, and HPA reactivity was negative only in the zone between the caput and the corpus.     

Solution conformation and dynamics of a tetrasaccharide related to the LewisX antigen deduced by NMR relaxation measurements

¹H-NMR cross-relaxation rates and nonselectivelongitudinal relaxation times have been obtained at two magnetic fields (7.0and 11.8 T) and at a variety of temperatures for the branchedtetrasaccharide methyl3-O--N-acetyl-galactosaminyl--galactopyranosyl-(14)[3-O--fucosyl]-glucopyranoside (1), an inhibitor of astrocyte growth. Inaddition, ¹³C-NMR relaxation data have also been recorded atboth fields. The ¹H-NMR relaxation data have been interpretedusing different motional models to obtain proton–proton correlationtimes. The results indicate that the GalNAc and Fuc rings display moreextensive local motion than the two inner Glc and Gal moieties, since thosepresent significantly shorter local correlation times. The¹³C-NMR relaxation parameters have been interpreted in termsof the Lipari–Szabo model-free approach. Thus, order parameters andinternal motion correlation times have been deduced. As obtained for the¹H-NMR relaxation data, the two outer residues possess smallerorder parameters than the two inner rings. Internal correlation times are inthe order of 100 ps. The hydroxymethyl groups have also different behaviour,with the exocyclic carbon on the glucopyranoside unit showing the highestS². Molecular dynamics simulations using a solvated systemhave also been performed and internal motion correlation functions have beendeduced from these calculations. Order parameters and interproton distanceshave been compared to those inferred from the NMR measurements. The obtainedresults are in fair agreement with the experimental data.     

Modeling Lanthanide Complexes: Towards the Theoretical Design of Light Conversion Molecular Devices

Theoretical techniques have been developed and/or improved to predict the molecular structure of lanthanide complexes which were used to calculate their electronic properties, in particular, their electronic spectra and energy levels necessary to calculate the rates of energy transfer from the ligands to the metal ion. The molecular structure has been obtained by the SMLC/AM1 (Sparkle Model for the Calculation of Lanthanide Complexes – Austin Model 1) model where the lanthanide ion is simulated by a sparkle implemented into the AM1 Hamiltonian used to perform a HF-SCF (Hartree-Fock Self-Consistent Field) calculation. The previous implementation of the SMLC/AM1 model (sparkle/1) involving only two parameters has been generalized to be consistent with the AM1 Hamiltonian and the new model (sparkle/2) significantly improved the prediction of molecular structures of Eu(III) complexes. For the electronic spectra and energy level calculations of the lanthanide complexes the model replaces the metal ion by a point charge with the ligands held in their positions as determined by the SMLC/AM1 model, and uses a INDO/S-CI (intermediate neglect of differential overlap/spectroscopic-configuration interaction) model. A preliminary study of the solvent effects on the absorption spectra of the free ligand is also presented. For the ligand-lanthanide ion energy transfer Fermi's golden rule is used with the multipolar and exchange mechanisms being implemented and tested for several complexes. These theoretical techniques have been applied to several complexes yielding very good results when compared to experimental data as well as predictions for the molecular and electronic structures and the relative contributions of the mechanisms for the energy transfer rates. This revised version was published online in June 2006 with corrections to the Cover Date.     

Oleic Acid Inhibits Gap Junction Permeability and Increases Glucose Uptake in Cultured Rat Astrocytes

Abstract: The role of oleic acid in the modulation of gap junction permeability was studied in cultured rat astrocytes by the scrape-loading/Lucifer yellow transfer technique. Incubation with oleic acid caused a dose-dependent inhibition of gap junction permeability by 79.5% at 50 µ M , and no further inhibition was observed by increasing the oleic acid concentration to 100 µ M . The oleic acid-mediated inhibition of gap junction permeability was reversible and was prevented by bovine serum albumin. The potency of oleic acid-related compounds in inhibiting gap junction permeability was arachidonic acid > oleic acid > oleyl alcohol > palmitoleic acid > stearic acid > octanol > caprylic acid > palmitic acid > methyloleyl ester. Oleic acid and arachidonic acid, but not methyloleyl ester, increased glucose uptake by astrocytes. Neither oleic acid nor arachidonic acid increased glucose uptake in the poorly coupled glioma C6 cells. These results support that the inhibition of gap junction permeability is associated with the increase in glucose uptake. We suggest that oleic acid may be a physiological mediator of the transduction pathway leading to the inhibition of intercellular communication.     

Identification of somaclonal variants of sugarcane (Saccharum spp.) resistant to sugarcane mosaic virus via RAPD markers

Somaclonal variants resistant to sugarcane mosaic virus (SCMV) were obtained from susceptible sugarcane cv PR62258 through somatic embryogenesis by increasing the number of subcultures of the embryogenic callus tissue in MS medium with 3 mg/L 2,4-dichlorophenoxyacetic acid. Transfers were made at 30-day intervals for 1, 2 or 3 subcultures. Two somaclones, namely AT626 and BT627, were selected by their resistance to SCMV. These subclones have maintained the resistance trait over seven years of testing in the field. In this report we identified the somaclonal SCMV resistant variants from the maternal line and the nonresistant somaclones, using the RAPD technique.     

Complex interaction between different proteinaceous components within the cell-wall structure ofCandida albicans

We have previously described a monoclonal antibody, MAb DC3:H10, which recognized an epitope preferentially expressed on the surface ofCandida albicans germ tubes. In the present study we examined the MAb-reactive material further. Immunoblot analysis of the material purified partially by Sephadex G-200 and DEAE-Sephacel chromatography reacted with antibodies to theC. albicans C3d receptor (CR2). In an ELISA, MAb DC3:H10 captured antigen that was recognized by both anti-CR2 and anti-mp58 fibrinogen binding mannoprotein polyclonal antibodies. The MAb DC3:H10 failed to compete with either of these antisera in an ELISA. Indirect immunofluo-rescence (IIF) analysis showed differences in surface distribution for the MAb DC3:H10, the CR2, and the mp 58 epitopes. Dual labeling IIF experiments showed MAb DC3:H10 binding to be unaffected by binding of fibrinogen or anti-mp58 antibody. However, the binding patterns of MAb DC3:H10 were modified in the presence of anti-CR2 antibody, suggesting a complex interaction between these cell wall components.     

Environmental and developmental regulation of carotenogenesis in the dimorphic fungus Mucor rouxii

Aerobic mycelium of wild-type Mucor rouxii accumulated about ten times higher amounts of the carotenoid pigment -carotene when grown continuously in the presence of light than the corresponding cultures grown in the dark. Carotenoid accumulation was dependent on light intensity, with the threshold located at about 10^-2 W.m^-2. Photocarotenogenesis in complex medium was more efficient with glucose as a carbon source. Carotenoid synthesis by M. rouxii mycelium was unaffected by both retinol acetate and retinal, which are stimulators of carotenogenesis in other zygomycetes. Carotenogenesis was significant in aerobic mycelium but was almost undetectable in anaerobic mycelium as well as in aerobic or anaerobic yeast cells. This suggested an involvement of oxygen in carotenoid synthesis by M. rouxii and the existence of developmental regulation of the expression or operation of the pathway.