Antioxidant activity and neuroprotective effects of zolpidem and several synthesis intermediates

Structural relationship between the antioxidant melatonin and the non-benzodiazepine hypnotic zolpidem (ZPD) suggests possible direct antioxidant and neuroprotective properties of this compound. In the present work, these effects were analyzed for zolpidem and four of its synthesis intermediates. In vitro assays include lipid peroxidation and protein oxidation studies in liver and brain homogenates. Intracellular antioxidant effects were analyzed by evaluation of free radical formation prevention in HT-22 hippocampal cells treated with glutamate 10mM and measured by flow cytometer DCF fluorescence. The neuroprotective effect of these compounds was evaluated as neuronal death prevention of HT-22 cells treated with the same concentration of glutamate. Zolpidem was found to prevent induced lipid peroxidation in rat liver and brain homogenates showing figures similar to melatonin, although it failed to prevent protein oxidation. ZPD-I was the most effective out of the several zolpidem intermediates studied as it prevented lipid peroxidation with an efficiency higher than melatonin or zolpidem and with an effectiveness similar to estradiol and trolox. ZPD-I prevents protein oxidation, which trolox is known to be unable to prevent. When cellular experiments were undertaken, ZPD-I prevented totally the increase of intracellular free radicals induced by glutamate 10mM in culture medium for 12h, while zolpidem and ZPD-III partially prevented this increase. Also the three compounds protected hippocampal neurons from glutamate-induced death in the same conditions, being their comparative efficacy, ZPD-III > ZPD-I = ZPD.     

Identification of an equilibrium intermediate in the unfolding process of galectin-1, which retains its carbohydrate-binding specificity

The unfolding process of galectin-1 (Gal-1) in the presence of a denaturing agent was examined using fluorescence and far-UV circular dichroism (CD) spectroscopy determinations, and was found to be completely reversible. The data showed that the transitions of guanidine hydrochloride (GdnHCl)-induced lectin unfolding, in the absence of ligand, were biphasic in nature, clearly showing the existence of at least one stable intermediate. On the other hand, the unfolding in the presence of disaccharide yielded data that could fit very well to a two-state model, indicating a stabilizing effect of the ligand. The folding intermediate was further characterized by size exclusion chromatography, near-UV CD and anilinonaphtalene sulfonate binding, and shown to belong to the molten globule type. Strikingly, this intermediate retained its carbohydrate-binding specificity, as evidenced by the tryptophan fluorescence changes detected upon its interaction with lactose.     

Molecular characterization of inulosucrase from Leuconostoc citreum: a fructosyltransferase within a glucosyltransferase

The gene coding for inulosucrase in Leuconostoc citreum CW28, islA, was cloned, sequenced, and expressed in Escherichia coli. The recombinant enzyme catalyzed inulin synthesis from sucrose like the wild-type enzyme. Inulosucrase presents an unusual structure: its N-terminal region is similar to the variable region of glucosyltransferases, its catalytic domain is similar to fructosyltransferases from various microorganisms, and its C-terminal domain presents similarity to the glucan binding domain from alternansucrase, a glucosyltransferase from Leuconostoc mesenteroides NRRL B-1355. From sequence comparison, it was found that this fructosyltransferase is a natural chimeric enzyme resulting from the substitution of the catalytic domain of alternansucrase by a fructosyltransferase. Two different forms of the islA gene truncated in the C-terminal glucan binding domain were successfully expressed in E. coli and retained their ability to synthesize inulin but lost thermal stability. This is the first report of an inulosucrase bearing structural features of both glucosyltransferases and fructosyltransferases.     

Intranuclear inclusions in fine needle aspirates of bronchial low grade mucoepidermoid carcinoma with clear cell change: a report of two cases

BACKGROUND: Mucoepidermoid carcinoma of the bronchus is a rare neoplasm that can be recognized on histology as well as cytology by the presence of three characteristic cell types: mucus secreting, epidermoid and intermediate. We encountered two cases displaying unusual cytologic features, including clear intranuclear inclusions. CASES: Two females, aged 33 and 39, presented with an intrabronchial tumor and pulmonary parenchymatous mass, respectively. Fine needle aspiration of both tumors showed similar cytologic features, with a dominant population of cells with bland nuclei and wide cytoplasm, and frequent intranuclear inclusions. A minor component of mucus-secreting cells was also recognized. Histologically, both tumors corresponded to the clear cell variant of mucoepidermoid carcinoma. CONCLUSION: The cytologic picture in our cases has not been described previously in fine needle aspirates of mucoepidermoid carcinoma, in neither the bronchus nor salivary gland. The differential diagnosis of a monotonous population of epithelial cells with intranuclear inclusions involves bronchioloalveolar carcinoma, but the absence of the characteristic sheet pattern, as well as the clinical and image findings, excludes this possibility. The lack of atypia and intrabronchial location limits the scope to carcinoid and salivary gland-type tumors of the bronchus. Since we were aware of the possibility of unusual cytologic presentations of mucoepidermoid carcinomas, search for different cellular populations suggested the precise diagnosis.     

Non-occlusal dental microwear variability in a sample of Middle and Late Pleistocene human populations from Europe and the Near East

Non-occlusal, buccal tooth microwear variability has been studied in 68 fossil humans from Europe and the Near East. The microwear patterns observed suggest that a major shift in human dietary habits and food processing techniques might have taken place in the transition from the Middle to the Late Pleistocene populations. Differences in microwear density, average length, and orientation of striations indicate that Middle Pleistocene humans had more abrasive dietary habits than Late Pleistocene populations. Both dietary and cultural factors might be responsible for the differences observed. In addition, the Middle Paleolithic Neanderthal specimens studied show a highly heterogeneous pattern of microwear when compared to the other samples considered, which is inconsistent with a hypothesis of all Neanderthals having a strictly carnivorous diet. The high density of striations observed in the buccal surfaces of several Neanderthal teeth might be indicative of the inclusion of plant foods in their diet. The buccal microwear variability observed in the Neanderthals is compatible with an overall exploitation of both plant and meat foods on the basis of food availability. A preliminary analysis of the relationship between buccal microwear density and climatic conditions prevailing in Europe during the Late Pleistocene has been attempted. Cold climatic conditions, as indicated by oxygen isotope stage data, seem to be responsible for higher densities of microwear features, whereas warmer periods could correspond to a reduced pattern of scratch density. Such a relationship would be indicative of less abrasive dietary habits, perhaps more meat dependent, during warmer periods.     

Transgenic tobacco plants expressing antisense ferredoxin-NADP(H) reductase transcripts display increased susceptibility to photo-oxidative damage

Ferredoxin-NADP(H) reductase (FNR) catalyses the final step of the photosynthetic electron transport in chloroplasts. Using an antisense RNA strategy to reduce expression of this flavoenzyme in transgenic tobacco plants, it has been demonstrated that FNR mediates a rate-limiting step of photosynthesis under both limiting and saturating light conditions. Here, we show that these FNR-deficient plants are abnormally prone to photo-oxidative injury. When grown under autotrophic conditions for 3 weeks, specimens with 20-40% extant reductase undergo leaf bleaching, lipid peroxidation and membrane damage. The magnitude of the effect was proportional to the light intensity and to the extent of FNR depletion, and was accompanied by morphological changes involving accumulation of aberrant plastids with defective thylakoid stacking. Damage was initially confined to chloroplast membranes, whereas Rubisco and other stromal proteins began to decline only after several weeks of autotrophic growth, paralleled by partial recovery of NADPH levels. Exposure of the transgenic plants to moderately high irradiation resulted in rapid loss of photosynthetic capacity and accumulation of singlet oxygen in leaves. The collected results suggest that the extensive photo-oxidative damage sustained by plants impaired in FNR expression was caused by singlet oxygen building up to toxic levels in these tissues, as a direct consequence of the over-reduction of the electron transport chain in FNR-deficient chloroplasts.     

The reduction of CrVI to CrIII by the alpha and beta anomers of D-glucose in dimethyl sulfoxide. A comparative kinetic and mechanistic study

The reduction of CrVI by alpha-D-glucose and beta-D-glucose was studied in dimethyl sulfoxide in the presence of pyridinium p-toluensulfonate, a medium where mutarotation is slower than the redox reaction. The two anomers reduce CrVI by formation of an intermediate CrVI ester precursor of the slow redox step. The equilibrium constant for the formation of the intermediate chromic ester and the rate of the redox steps are different for each anomer. alpha-D-Glucose forms the CrVI-Glc ester with a higher equilibrium constant than beta-D-glucose, but the electron transfer within this complex is slower than for the beta anomer. The difference is attributed to the better chelating ability of the 1,2-cis-diolate moiety of the alpha anomer. The CrV species, generated in the reaction mixture, reacts with the two anomers at a rate comparable with that of CrVI. The EPR spectra show that the alpha anomer forms several linkage isomers of the five-coordinate CrV bis-chelate, while beta-D-glucose affords a mixture of six-coordinate CrV monochelate and five-coordinate CrV bis-chelate. The conversion of the CrV mono- to bis-chelate is discussed in terms of the ability of the 1,2-cis- versus 1,2-trans-diolate moieties of the glucose anomers to bind CrV.