Oxidative damage to ferritin by 5-aminolevulinic acid

5-Aminolevulinic acid (ALA), a heme precursor overproduced in various porphyric disorders, has been implicated in iron-mediated oxidative damage to biomolecules and cell structures. From previous observations of ferritin iron release by ALA, we investigated the ability of ALA to cause oxidative damage to ferritin apoprotein. Incubation of horse spleen ferritin (HoSF) with ALA caused alterations in the ferritin circular dichroism spectrum (loss of a alpha-helix content) and altered electrophoretic behavior. Incubation of human liver, spleen, and heart ferritins with ALA substantially decreased antibody recognition (51, 60, and 28% for liver, spleen, and heart, respectively). Incubation of apoferritin with 1-10mM ALA produced dose-dependent decreases in tryptophan fluorescence (11-35% after 5h), and a partial depletion of protein thiols (18% after 24h) despite substantial removal of catalytic iron. The loss of tryptophan fluorescence was inhibited 35% by 50mM mannitol, suggesting participation of hydroxyl radicals. The damage to apoferritin had no effect on ferroxidase activity, but produced a 61% decrease in iron uptake ability. The results suggest a local autocatalytic interaction among ALA, ferritin, and oxygen, catalyzed by endogenous iron and phosphate, that causes site-specific damage to the ferritin protein and impaired iron sequestration. These data together with previous findings that ALA overload causes iron mobilization in brain and liver of rats may help explain organ-specific toxicities and carcinogenicity of ALA in experimental animals and patients with porphyria.     

Kinetic properties of the glucose 6-phosphatase of the liver from arthritic rats

According to previous reports, adjuvant-induced arthritic rats present reduced activities of the hepatic glucose 6-phosphatase. A kinetic study was done in order to characterize this phenomenon. Microsomes were isolated from livers of arthritic and control rats (Holtzman strain) and the glucose 6-phosphatase was measured at various temperatures (13-37 degrees C) and glucose 6-phosphate concentrations. Irrespective of the temperature, the enzyme from arthritic rats presented a reduction of both V(max) and K(M). Detergent treatment of liver microsomes from control rats increased the activity, but no increase was found when microsomes from arthritic rats were treated in the same way. The mannose 6-phosphatase activity of detergent-treated microsomes from arthritic rats was only 25% of the activity found with detergent-treated microsomes from control rats. Without detergent treatment, the mannose 6-phosphatase activities of both control and arthritic rats were minimal. The activation energy, derived from V(max), was not changed by arthritis. In vivo arthritic rats presented higher hepatic glucose 6-phosphate concentrations, a phenomenon that is consistent with a reduced activity of glucose 6-phosphatase. It was concluded that in arthritic rats, the hydrolase is probably reduced, without a similar change in the translocase activity.     

Change and stabilization of the amyloid-beta(1-40) secondary structure by fluorocompounds

The misfolding of the amyloid peptide, which is the result of a well-known alpha-to-beta transition, causes neurodegenerative disorder. Fluorinated alcohols have been described in the literature as potent solvents which can refold the beta-conformation. The present studies demonstrate the effectiveness of differently fluorinated alcohols for the beta-to-alpha refolding process on fibrillar aggregated amyloid beta(1-40). The regenerated helical structure is shown to be maintained in the absence of the fluoroalcohols, a behaviour which was found to contrast with immunoglobulin. We interpret this difference on the basis of the hydrophilic/hydrophobic domains in the amyloid sequence and present some speculations regarding the free-energy levels of the folded states of both proteins. The effect of the -CF(3) group on the observed conformational changes is interpreted as a result of alterations of the hydration shell of the peptides. Moreover, based on the results achieved with fluoroalcohols, we have used novel fluorinated amphiphiles possessing blood-compatibility properties and studied their effect on amyloid beta(1-40). First results point in the direction of a beta-to-alpha transition. Therefore, the use of fluorine groups in the development of new drugs is considered a new possibility requiring further investigation for the prevention of amyloidosis.     

Movement distances and density estimation of small mammals using the spool-and-line technique

Edge effect is an inherent problem when using trapping grids to estimate density of small mammals, resulting in a sampling area larger than the area of the grid. Distances between captures of individuals are used to estimateA(W), the effective sampling area of a trapping grid, but grid size sets a limit for the largest detectable distance. The spool-and-line technique is proposed here as a new method to estimateA(W). Movement distances based on the spool-and-line technique were compared to similar movement distances based on capture-recapture of three species of marsupials of the Atlantic Forest of Brazil. Distances based on the two methods were uncorrelated, and only ln-transformed distances based on the spool-and-line were normally distributed. The maximum distance moved (MaxD) estimated by the spool-and-line was chosen as the more accurate and practical distance to estimate edge effect. Estimates of the effective sampling area and densities for the common opossumDidelphis aurita (Wied-Neuwied, 1826), were compared using MaxD based on spool-and-line (MaxDs p o o l ) , capture-recapture (MaxD_cap), and also the distance between traps (DT). MaxD_spool reflected more accurately density variation between seasons. Movement distances of small mammals based on the spool-and-line technique permit more accurate estimates of density and its dynamics.     

An in vitro evaluation of the effects of homocysteine thiolactone on key steps of angiogenesis and tumor invasion

Homocysteine thiolactone is a highly reactive homocysteine derivative that can react easily with proteins. Protein homocysteinylation has been suggested as a possible mechanism underlying the pathological consequences of impaired homocysteine metabolism. Homocysteine inhibits key steps of angiogenesis and tumor invasion. It can be hypothesized that homocysteine thiolactone could mimic the described anti-angiogenic and anti-invasive effects of homocysteine. Therefore, we studied the effects of homocysteine thiolactone on different key steps of angiogenesis and tumor invasion, using model endothelial and tumor cell lines. This study demonstrates that homocysteine thiolactone, in high contrast to homocysteine, is not an anti-angiogenic compound. Furthermore, our results suggest that homocysteine thiolactone could behave as a pro-angiogenic compound.     

Cooperative partition model of nystatin interaction with phospholipid vesicles

Nystatin is a membrane-active polyene antibiotic that is thought to kill fungal cells by forming ion-permeable channels. In this report we have investigated nystatin interaction with phosphatidylcholine liposomes of different sizes (large and small unilamellar vesicles) by time-resolved fluorescence measurements. Our data show that the fluorescence emission decay kinetics of the antibiotic interacting with gel-phase 1,2-dipalmitoyl-sn-glycero-3-phosphocholine vesicles is controlled by the mean number of membrane-bound antibiotic molecules per liposome, . The transition from a monomeric to an oligomeric state of the antibiotic, which is associated with a sharp increase in nystatin mean fluorescence lifetime from approximately 7-10 to 35 ns, begins to occur at a critical concentration of 10 nystatin molecules per lipid vesicle. To gain further information about the transverse location (degree of penetration) of the membrane-bound antibiotic molecules, the spin-labeled fatty acids (5- and 16-doxyl stearic acids) were used in depth-dependent fluorescence quenching experiments. The results obtained show that monomeric nystatin is anchored at the phospholipid/water interface and suggest that nystatin oligomerization is accompanied by its insertion into the membrane. Globally, the experimental data was quantitatively described by a cooperative partition model which assumes that monomeric nystatin molecules partition into the lipid bilayer surface and reversibly assemble into aggregates of 6 +/- 2 antibiotic molecules.     

Alterations in the phenotype of CMV-specific and total CD8+ T-cell populations in Wegener's granulomatosis

Wegener's granulomatosis (WG) is an autoimmune disease of as yet unknown etiology. To date it has remained obscure what causes WG or determines disease progression. Case reports suggest that viral infections such as cytomegalovirus (CMV) reactivation may contribute to disease flares. In this study we found a skewing of the phenotype of CMV-specific CD8+tet(ramer)+ T-cells in WG. A marked proportion of these cells displayed a late differentiated "effector memory" T-cell phenotype with decreased expression of CD28 and CD62L, and heterogeneous CD27 expression, features which were also seen in CD8+tet- T-cells in WG, but not in controls. Our results might reflect profound generalized changes in the CD8+ T-cell compartment also affecting virus-specific T-cell responses in WG.     

Actinobacterial chitinase-like enzymes: profiles of rhizosphere versus non-rhizosphere isolates

The objective of this study was to determine if antifungal actinomycetes isolated from rhizosphere and non-rhizosphere soils exhibit different chitinase-like production and (or) induction patterns. Selected isolates from both habitats were compared. Chitinase-like levels and isoform characteristic patterns were evaluated over time in culture fluids of isolates grown on media containing different combinations of colloidal chitin and fungal cell wall (FCW) preparation. Supernatants were also subjected to native and non-native polyacrylamide gel electrophoresis (PAGE), using glycol chitin amended gels. For non-native PAGE, protein samples were denatured by two different approaches. Multiple active bands, ranging from 20 to 53 kDa and present in varying amounts, were detected in gels for most strains. Different substrate preferences were observed among strains, and different chitinase-like enzymes were produced, depending upon the substrate combinations used. The presence of FCW in the medium induced specific chitinase-like enzymes not observed otherwise. Enzymatic activities and profiles of the isolates, however, were strain and substrate specific rather than habitat specific. However, a sagebrush rhizosphere soil had a larger actinomycete community with higher chitinolytic activities than the nearby bulk soil. The use of PAGE to compare chitinase-like proteins induced in media with and without FCW was useful for identifying chitinase-like enzymes potentially involved in antifungal activity.     

Worldwide distribution of transposable element copy number in natural populations of Drosophila simulans

Abstract.— Transposable elements (TEs), which promote various kinds of mutations, constitute a large fraction of the genome. How they invade natural populations and species is therefore of fundamental importance for understanding the dynamics of genetic diversity and genome composition. On the basis of 85 samples of natural populations of Drosophila simulans , we report the distributions of the genome insertion site numbers of nine TEs that were chosen because they have a low average number of sites. Most populations were found to have 0–3 insertion sites, but some of them had a significantly higher number of sites for a given TE. The populations located in regions outside Africa had the highest number of sites for all elements except HMS Beagle and Coral , suggesting a recent increase in the activity of some TEs associated with the colonization patterns of Drosophila simulans . The element Tirant had a very distinctive pattern of distribution: it was identified mainly in populations from East Africa and some islands in the Indian Ocean, and its insertion site number was low in all these populations. The data suggest that the genome of the entire species of Drosophila simulans may be being invaded by TEs from populations in which they are present in high copy number.     

Testing for epistasis between deleterious mutations in a parasitoid wasp

Abstract.— Determining the way in which deleterious mutations interact to effect fitness is crucial to numerous areas in evolutionary biology. For example, if each additional mutation leads to a greater decrease in log fitness than the last, termed synergistic epistasis, then sex and recombination provide an advantage because they enable deleterious mutations to be eliminated more efficiently. However, there is a severe shortage of relevant empirical data, especially of the form that can help test mutational explanations for the widespread occurrence of sex. Here, we test for epistasis in the parasitic wasp Nasonia vitripennis , examining the fitness consequences of chemically induced deleterious mutations. We examine two components of fitness, both of which are thought to be important in natural populations of parasitic wasps: longevity and egg production. Our results show synergistic epistasis for longevity, but not for egg production.