Immunological approach to investigating membrane cell damages induced by lipoperoxidative stress

Oxygen-reactive species are being described as agents responsible for cell degeneration mechanisms resulting from membrane, enzyme, and nuclear alterations. Lipid peroxidation on its own is considered to be one of the consequences of the free radicals attack, and among the different reactive aldehydes that can be formed from the decomposition of lipid peroxides, the most extensively assayed have been malondialdehyde (MDA). However, the different techniques currently used for MDA assay (HPLC, GLC) are barely sensitive enough to follow its production at the cellular level. In order to develop an immunofluorescent technique able to detect cellular damages provoked by lipoperoxidation, polyclonal antibodies against lysozyme modified by MDA treatment have been raised in rabbits. We show that this immunserum recognizes specifically all the MDA-treated proteins tested, but not the intact proteins or the proteins treated by other aldehydes. Moreover, we demonstrate using an ELISA technique that the amount of immunoreactive proteins in MDA-treated membrane erythrocytes is proportional to the concentration of MDA applied, suggesting that this assay may represent a quantitative method of determination of lipoperoxidative alterations. In addition, when coupled to an indirect fluorophore antibody (FITC), the immunserum allows a precise location of these modified proteins within the membranes of erythrocytes in which lipid peroxidation was initiated by far UV irradiation. In summary, the interest of this work is to provide an immunological probe that can precociously detect membrane damages induced by MDA, regardless of the cell type and pro-oxidant (physiological or pathological) conditions.     

Isolation and Characterization of a Cytosolic Phospholipase A2 from Bovine Adrenal Medulla

Abstract: We have recently demonstrated that bovine adrenal medulla contains a soluble phospholipase A₂ (PLA₂), which is localized in the cytosol. In the present study, this PLA₂ was purified 1,097-fold using sequential concanavalin A, hydrophobic interaction, anion exchange, gel filtration, and an additional anion exchange chromatography. The enzyme is activated over the range of 20–1,000 µ M Ca²⁺ and has a pH optimum near 8.0. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the protein has a molecular mass of 26 kDa and an isoelectric point of 4.6 as revealed by isoelectric focusing. The cytosolic PLA₂ is not inhibited by NaCl, and the enzymatic activity is stimulated at low concentrations of Triton X-100 (0.01%) and deoxycholate (1 m M ) but inhibited at higher concentrations (0.1% and 3 m M , respectively) of these detergents. Furthermore, heat treatment (57°C, 5 min) reduced the enzymatic activity by 80%, whereas glycerol (30%) increased the activity. p -Bromophenacylbromide, a frequently used irreversible inhibitor of type II PLA₂, has little effect until 100 µ M , and 2–10 m M dithiothreitol totally inactivated the enzyme. The purified PLA₂ displays a preference for phosphatidylcholine as a substrate but hydrolyzes phospholipid substrates with arachidonic acid or linoleic acid esterified at the sn -2 position to the same extent. It is concluded that the chromaffin cell cytosolic PLA₂, which was isolated and characterized in this study, represents a type of PLA₂ that has not been formerly reported in chromaffin cells. Additional research on the chromaffin cell cytosolic PLA₂ will help to reveal whether the enzyme is important for exocytosis.     

Phylogeographic Structure of White Oaks Throughout the European Continent

Patterns of chloroplast DNA (cpDNA) variation were studied in eight white oak species by sampling 345 populations throughout Europe. The detection of polymorphisms by restriction analysis of PCR-amplified cpDNA fragments allowed the identification of 23 haplotypes that were phylogenetically ordered. A systematic hybridization and introgression between the eight species studied is evident. The levels of subdivision for unordered (G(ST)) and ordered (N(ST)) alleles are very high and close (0.83 and 0.85). A new statistical approach to the quantitative study of phylogeography is presented, which relies on the coefficients of differentiation G(ST) and N(ST) and the Mantel's test. Based on pairwise comparisons between populations, the significance of the difference between both coefficients is evaluated at a global and a local scale. The mapped distribution of the haplotypes indicates the probable routes of postglacial recolonization followed by oak populations that had persisted in southern refugia, especially in the Iberian peninsula, Italy and the Balkans. Most cpDNA polymorphisms appear to be anterior to the beginning of the last recolonization. A subset of the preexisting haplotypes have merely expanded north, while others were left behind in the south.     

Clostridium perfringens epsilon-toxin acts on MDCK cells by forming a large membrane complex.

Epsilon-toxin is produced by Clostridium perfringens types B and D and is responsible for a rapidly fatal enterotoxemia in animals, which is characterized by edema in several organs due to an increase in blood vessel permeability. The Madin-Darby canine kidney (MDCK) cell line has been found to be susceptible to epsilon-toxin (D. W. Payne, E. D. Williamson, H. Havard, N. Modi, and J. Brown, FEMS Microbiol. Lett. 116:161-168, 1994). Here we present evidence that epsilon-toxin cytotoxic activity is correlated with the formation of a large membrane complex (about 155 kDa) and efflux of intracellular K+ without entry of the toxin into the cytosol. Epsilon-toxin induced swelling, blebbing, and lysis of MDCK cells. Iodolabeled epsilon-toxin bound specifically to MDCK cell membranes at 4 and 37 labeled C and was associated with a large complex (about 155 kDa). The binding of epsilon-toxin to the cell surface was corroborated by immunofluorescence staining. The complex formed at 37 degrees C was more stable than that formed at 4 degrees C, since it was not dissociated by 5% sodium dodecyl sulfate and boiling.     

pAMβ1 resolvase has an atypical recombination site and requires a histone-like protein HU

The broad-host-range plasmid pAMβ1 from Gram-positive bacteria encodes a resolvase, designated Resβ, which shares homology with the proteins of the resolvase—invertase family. Here we report the purification and in vitro characterization of Resβ. This resolvase is particular in two aspects: it has an atypical binding site and requires a cofactor to promote resolution in vitro . Resβ binds to two regions within its resolution site res . One contains two inverted repeats (R1 and R2), the other contains only one repeat (R3). The cofactor required for resolution in vitro is present in crude extracts of both Bacillus subtilis and Escherichia coli and can be substituted by the E. coli histone-like protein HU. The possible mode of action of HU in the resolution process is discussed.     

First evidence of human meconium glycoasparagines

During a systematic study of carbohydrate material present inhuman meconium, in addition to the previously described mucins,glycolipids and free oligosaccharides, we have now characterizeda significant quantity of free glycoasparagines. These glycoasparagineshave been isolated from human meconium by a combination of ion-exchange,concanavalin A (ConA)-affinity and high-performance liquid (HPLC)chromatographies. Their structures have been established by400 MHz ¹H-NMR spectroscopy. These compounds are related toN-acetyllactosaminic type structures and are based on the commoncore These glycoasparagines are probably derived from both proteaseand partial exoglycosidase hydrolysis of fetal gastrointestinalN-glycosyl proteins. Their structures are discussed in the contextof the known catabolic pathways of N-glycans glycoasparagine N-glycosyl protein catabolism meconium NMR     

Inactivation pattern of the fragile X in heterozygous carriers

Summary Chromosome analysis with conventional staining, G-banding, and R-banding with 5-bromodeoxyuridine (BrdU) incorporation were performed on the lymphocytes of ten females, who were heterozygous for the fragile X-chromosome. Mental development of these females varied greatly: moderate to severe mental retardation was found in one and moderate mental retardation in four females. Normal to borderline intelligence was found in three and normal intelligence was noted in two further females. The discrepancy in percentage of active fragile X-chromosomes in the five females with moderate mental retardation was found to be 60–100% (mean value: 80%). The three women with normal to borderline intelligence showed a corresponding discrepancy from 57 to 86% (mean value: 77%) of active fragile X-chromosomes. Finally, two female heterozygotes for fragile X with normal intelligence showed 70 and 76% (mean value 73%) of active fragile X-chromosomes. The phenotypic features also did not seem to correspond with the X-chromosome inactivation pattern. Based on the data obtained, we suggest that there is no evident correlation between the frequency of the active fragile X chromosomes and the mental status of these females.     

Adjuvant treatment with polyadenylic-polyuridylic acid in operable breast cancer: updated results of a randomised trial.

The results of a randomised trial of polyadenylic-polyuridylic acid given as adjuvant treatment for operable breast cancer were reviewed after a mean follow up period of 87 months. Of the 300 patients included in the original trial, 145 had been allocated to conventional treatment alone and served as controls. At the time of review the overall survival of the group given polyadenylic-polyuridylic acid was significantly improved (p less than 0.05) as compared with that of the controls given conventional treatment alone. Significant benefit (p less than 0.02) was also observed among patients with evidence of disease in lymph nodes, the best results occurring in those with up to three invaded nodes, who showed a significant increase in both overall and relapse free survival. No evidence of toxicity was recorded. These findings confirm the value of polyadenylic-polyuridylic acid as adjuvant treatment for operable breast cancer. Results in an experimental model and in patients receiving the adjuvant suggested a possible role of interferon and natural killer (NK) cells in the mechanism of action.     

In vivo measurement of cadmium (115mCd) transport and accumulation in the stems of intact tomato plants (Lycopersicon esculentum,Mill.)

The internal distribution processes of cadmium (^115mCd) in the stems of tomato plants have been investigated using in vivo -spectrometric measurements with semiconductor detectors. The initial penetration into the xylem after root uptake and the subsequent lateral redistribution in the surrounding tissues were monitored continuously in each individual plant. The kinetics of the lateral migration toward the epidermis can be described by a diffusion process, characterized by an apparent diffusion coefficient of about 10^-8 cm² s^-1. After two days of cadmium supply this heavy metal is accumulated in relatively large amounts in the epidermal and cortical layers, from which it can be released only very slowly. On the contrary, the exchange processes with other divalent cations cause in the xylem a quick release, its rate being dependent on the duration of the preceding cadmium treatment.