Role of protein synthesis in the accumulation of ferritin mRNA during exposure of cells to iron.

The iron-induced biosynthesis of ferritin is regulated at the translational level via multiple mechanism. A prolonged exposure of cells to iron leads to a marked increase in ferritin mRNA levels caused by stabilization of the message. Here we show that this stabilization requires the synthesis de novo of an iron-inducible protein factor.     

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.     

A CYANOPHYTE CAPABLE OF FIXING NITROGEN UNDER HIGH LEVELS OF OXYGEN1

Nostoc cordubensis Prosperi (Cyanophyta, Nostocaceae) is characterized by its mucilaginous colonies. Much of this mucilage is produced by heterocysts. By controlling growth conditions, heterocysts with and without mucilage were obtained. Mucilaginous heterocysts retained nitrogen-fixing capability at high oxygen concentrations, whereas heterocysts lacking mucilage were unable to fix nitrogen at oxygen concentrations higher than 20%. Comparison of results obtained using tetrazolium salts as indicators of highly reduced zones showed similar results.     

DNA stainability with base-specific fluorochromes: dependence on the DNA topology in situ

The influence of DNA topology on stainability with the externally binding fluorochromes Hoechst 33258 (HO) and mithramycin (MI) was investigated in HeLa nuclei in comparison with the intercalating dye propidium iodide (PI). Changes in DNA topology were induced with a mild DNAse I treatment. Stainability properties of untreated and nuclease-treated nuclei were compared with those of the supercoiled-circular and the relaxed-linear forms of the plasmid pBR322. DNAse-treated nuclei stained with HO showed a higher fluorescence intensity than control samples, independently of the dye concentration, in contrast with the findings obtained with PI. Similar behaviour was observed with the relaxed-linear form of pBR322, compared with the supercoiled-circular molecule. With MI, the stainability of HeLa nuclei did not depend on the DNA topology, whereas the stainability of the plasmid was similar to that of HO. In order to assess whether this discrepancy depended on differences in the availability of DNAse-sensitive sites to the fluorochromes, fluorescence resonance energy transfer (FRET) studies were performed in nuclei stained with HO+PI, or with HO+MI dye pairs. After DNAse I digestion, the relative FRET efficiency between donor (HO) and acceptor molecules (PI or MI) was reduced significantly only when MI was the acceptor. This result may be due to greater stainability of DNAse-sensitive sites with HO than with MI. These findings indicate that DNA stainability with base-specific fluorochromes may be affected by the topology of chromatin regions.     

Dose-dependent zinc inhibition of DNA ladder in apoptotic HeLa cells regulates the activity of poly(ADP-ribose) polymerase and does not protect from death induced by VP-16

Zinc ions exert an inhibitory effect on Ca(2+)Mg(2+)-dependent endonuclease which is supposed to be responsible for the fragmentation of DNA during apoptosis. In the experimental system we used, that is HeLa cells treated with VP-16, the protection from internucleosomal DNA degradation is modulated by Zn concentration and appears to be dependent on the time after treatment. This effect does not prevent cell death or occurrence of apoptotic parameters, suggesting that DNA ladder appearance is not a crucial event in apoptosis. The activation of poly(ADP-ribose)polymerase following the administration of VP-16, is not observed in cells in which DNA fragmentation has been abolished by zinc, supporting the hypothesis that this event is regulated by the appearance of small-sized DNA fragments.     

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.