Salicylate hydroxylation as an early marker of in vivo oxidative stress in diabetic patients.

In vivo metabolism of salicylic acid produces two main hydroxylated derivatives (2,5- and 2,3-dihydroxybenzoic acid). The former can be produced by enzymatic pathways through the cytochrome P-450 system, while the latter is reported to be solely formed by direct hydroxyl radical attack. Therefore, measurement of 2,3-dihydroxybenzoate, following oral administration of salicylate in its acetylated form (aspirin), has been proposed for assessment of oxidative stress. In this article we report plasma levels of 2,3- and 2,5-dihydroxybenzoates following the administration of 1 g aspirin and plasma levels of thiobarbituric acid-reactive material (TBARM) in well-controlled diabetic patients and in healthy subjects. 2,3-Dihydroxybenzoate levels were significantly higher (23%) in diabetic patients than in controls (63.4 +/- 20.1 versus 49.0 +/- 6.8 nM; p < .05). On the other hand, TBARM values were not significantly different between groups. These results suggest that the method is useful to reveal in vivo oxidative stress independently from the peroxidation of lipids, and they support the hypothesis that oxygen radicals are involved in the pathogenesis of chronic complications of diabetes.     

Isolation of chitin synthetase from Saccharomyces cerevisiae. Purification of an enzyme by entrapment in the reaction product

Chitin synthetase, in the zymogen form, was extracted with digitonin from a particulate fraction from Saccharomyces cerevisiae and converted into active form by treatment with immobilized trypsin. When the activated enzyme was incubated with UDP-GlcNAc and other components of an assay mixture, a chitin precipitate formed, trapping a large portion of the synthetase. The enzyme was easily extracted frm the chitin gel with a recovery of approximately 50% and an enrichment of approximately 100-fold. Further purification was obtained by repeating the chitin step. After polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the purified synthetase showed a major band corresponding to Mr 63,000, a weaker band at Mr 74,000, and some other minor bands. Under nondenaturing conditions, an Mr of 570,000 was calculated for the enzyme from Stokes radius and sedimentation coefficient determinations. After electrophoresis in a nondenaturing gel and incubation with the components of the standard assay, chitin was formed and precipitated in the gel, yielding an opaque band. Soluble oligosaccharides were not precursors for insoluble chitin, suggesting that synthesis of chitin chains takes place by a processive mechanism. N-Acetylglucosamine stimulated the purified synthetase only slightly and did not participate as a primer in the reaction. The same chain length, somewhat more than 100 units of GlcNAc, was determined in samples of chitin that had been synthesized either in vivo, or with a membrane preparation or with purified synthetase. These results suggest that chitin synthetase itself is capable both of initiating chitin chains without a primer and of determining their length.     

Refinement of the structure of bovine seminal ribonuclease

We report here the refinement at 2.5-Å resolution of the x-ray crystal structure of bovine seminal ribonuclease, a dimeric covalent enzyme. The protein, which crystallizes with one molecule in the asymmetric unit, consists of two subunits of identical chemical sequences, related by an almost exact binary axis. The tertiary structure of the subunits is similar to that of the pancreatic enzyme, which shows similar catalytic properties. The refinement was carried out using the restrained least-squares procedure both in the reciprocal and real spaces. The assemblage of the subunits in the dimer is described and discussed.     

Immunoadjuvant effects of Candida albicans and its cell wall fractions in a mouse lymphoma model

Summary Chemical, ultrastructural, and immunoadjuvant properties of Candida albicans (CA) and of a number of its fractions have been characterized through the analysis of the antitumor activity of soluble and insoluble cell wall components.CD2F₁ mice were inoculated IP with Moloney virus-induced lymphoma LSTRA and treated with bis-1-chloroethyl-nitrosurea (BCNU) on day +5 after tumor challenge. A significant increase of the antitumor efficacy of BCNU treatment was found in mice inoculated with CA as immunoadjuvant on days –14 and +1 (–14/+1 schedule) with respect to tumor challenge.However, no significant difference in survival time was found between mice treated with BCNU alone and those treated with BCNU plus either soluble mannan or glucan-protein fractions extracted from CA and administered according to –14/+1 or –7/+1 schedules. On the other hand, mice treated with BCNU plus the insoluble glucan fraction (wall ghosts) given on days –14/+1 or even on day –7 only (i.e., without boosting after tumor challenge) survived longer than animals treated with BCNU alone.The immunoadjuvant effect of CA and of other classic immunoadjuvants, such as BCG and Corynebacterium parvum, was completely abolished by total-body irradiation (400 R) given 5 h before the first administration of the agent on day –7 prior to tumor challenge.These results indicate that: (a) the minimal structure required for the expression of the immunoadjuvant effect of CA is the insoluble, -glucan component of the cell wall; (b) the soluble components of CA cell wall (i.e., mannan and glucan-protein) per se do not show any detectable immunoadjuvant effect in the present animal-tumor system; they may, however, modulate this effect, as shown by the fact that whole CA, but not the insoluble -glucan, needs a boosting injection for the expression of its immunoadjuvant properties; (c) the immunoadjuvanticity of CA is radiosensitive.     

Binding of matrix attachment regions to lamin B1.

Eukaryotic chromatin is organized into topologically constrained loops that are attached to the nuclear matrix. The regions of DNA that interact with the matrix are called matrix attachment regions (MARs). We studied the spatial distribution of MAR-binding sites in the nuclear matrix from rat liver cells, following a combined biochemical and ultrastructural approach. We found that MAR-binding sites are distributed equally over the internal fibrogranular network and the peripheral nuclear lamina. Internal and peripheral binding sites have similar binding characteristics: both sets of binding sites show specific and saturable binding of MARs from different organisms. By means of a DNA-binding protein blot assay and in vitro binding studies, we identified lamin B1 as a MAR-binding protein, which provides evidence for a specific interaction of DNA with the nuclear lamina.     

Cytochemical and molecular characteristics of the process of cornification during feather morphogenesis

Feathers are the most complex epidermal derivatives among vertebrates. The present review deals with the origin of feathers from archosaurian reptiles, the cellular and molecular aspects of feather morphogenesis, and focus on the synthesis of keratins and associated proteins. Feathers consist of different proteins among which exists a specialized group of small proteins called beta-keratins. Genes encoding these proteins in the chick genome are distributed in different chromosomes, and most genes encode for feather keratins. The latter are here recognized as proteins associated with the keratins of intermediate filaments, and functionally correspond to keratin-associated proteins of hairs, nails and horns in mammals. These small proteins possess unique properties, including resistance and scarce elasticity, and were inherited and modified in feathers from ancestral proteins present in the scales of archosaurian progenitors of birds. The proteins share a common structural motif, the core box, which was present in the proteins of the reptilian ancestors of birds. The core box allows the formation of filaments with a different molecular mechanism of polymerization from that of alpha-keratins. Feathers evolved after the establishment of a special morphogenetic mechanism gave rise to barb ridges. During development, the epidermal layers of feathers fold to produce barb ridges that produce the ramified structure of feathers. Among barb ridge cells, those of barb and barbules initially accumulate small amounts of alpha-keratins that are rapidly replaced by a small protein indicated as “feather keratin”. This 10 kDa protein becomes the predominant form of corneous material of feathers. The main characteristics of feather keratins, their gene organization and biosynthesis are similar to those of their reptilian ancestors. Feather keratins allow elongation of feather cells among supportive cells that later degenerate and leave the ramified microstructure of barbs. In downfeathers, barbs are initially independent and form plumulaceous feathers that rest inside a follicle. Stem cells remain in the follicle and are responsible for the regeneration of pennaceous feathers. New barb ridges are produced and they merge to produce a rachis and a flat vane. The modulation of the growth pattern of barb ridges and their fusion into a rachis give rise to a broad variety of feather types, including asymmetric feathers for flight. Feather morphogenesis suggests possible stages for feather evolution and diversification from hair-like outgrowths of the skin found in fossils of pro-avian archosaurians.     

Assessing influence in biofuel production and ecosystem services when environmental changes affect plant–pest relationships

Climate change is currently affecting both biodiversity and human activities; land use change and greenhouse gas emissions are the main drivers. Many agricultural services are affected by the change, which in turn reflects on the basic provisioning services, which supply food, fibre and biofuels. Biofuels are getting increasing interest because of their sustainability potential. Jatropha curcas gained popularity as a biodiesel crop, due to its ease of cultivation even in harsh environmental conditions. Notwithstanding its high economic importance, few studies are available about its co‐occurrence with pests of the genus Aphthona in sub‐Saharan Africa, where these insects feed on J. curcas, leading to relevant economic losses. Using ecological niche modelling and GIS post‐modelling analyses, we infer the current and future suitable territories for both these taxa, delineating areas where J. curcas cultivations may occur without suffering the presence of Aphthona, in the context of future climate and land use changing. We introduce an area‐normalized index, the ‘Potential‐Actual Cultivation Index’, to better depict the ratio between the suitable areas shared both by the crop and its pest, and the number of actual cultivations, in a target country. Moreover, we find high economic losses (~?50%) both in terms of carbon sequestration and in biodiesel production when J. curcas co‐occur with the Aphthona cookei species group.