A method for the direct evaluation of the fatty acid status in a drop of blood from a fingertip in humans: applicability to nutritional and epidemiological studies

Several studies have shown that the fatty acid composition of circulating lipids reflects dietary fat intake, in turn being related to health status. The fatty acid composition of plasma lipids is therefore an important parameter in studies on dietary interventions. The aim of our study was to develop a rapid and inexpensive method for the analysis of circulating fatty acids applicable to large population groups. Drops of blood collected from fingertips have been directly subjected to transmethylation for gas chromatography analysis. This new method, validated for reproducibility, has been compared with the conventional method, based on withdrawal of blood from the antecubital vein followed by lipid extraction, and identical data have been obtained with the two techniques. Observed and predicted differences between blood and plasma fatty acids are related to the contribution of circulating cell membranes in blood. Finally the application of the methods to samples from 100 healthy subjects and the assessed correlation between dietary habits and blood fatty acid profiles demonstrate the validity of the new method and its applicability to nutritional and epidemiological studies.     

Genomic dynamics of a low-copy-number satellite DNA family in Leptestheria dahalacensis (Crustacea, Branchiopoda, Conchostraca)

The LEP150 satellite DNA (satDNA) family found in Leptestheria dahalacensis (Ruppel, 1837) (Conchostraca) is a low-copy-number satellite with a canonical monomer of 150 bp. Nucleotide variation analyses suggest a 14-bp palindromic region as a possible protein binding site with constraints acting on the whole sequence but a 25-bp variable box. Besides the head-to-tail arrangement of 150 bp monomers, multimers analyses evidenced incomplete monomers, one duplication event, and three inversions. Both observed rearrangements and the higher values of sequence variability scored suggest that rearranged monomers reside in regions with a lower degree of homogenisation efficiency. Sixty-seven percent of the breakpoints occurs at kinkable dinucleotides, thus supporting their role in rearrangements as documented in alphoid satDNA recombination events. Monomers of different lengths may result from crossing over between repeats misaligned through the direct and inverted subrepeats of LEP150 monomers. ANOVA results indicate that the same range of sequence diversity is experienced at the individual and population ranks; therefore, the evolution of the L. dahalacensis satDNA is concerted.     

Toxic cyanobacteria strains isolated from blooms in the Guadiana river (southwestern Spain)

This paper describes the occurrence of toxic cyanobacteria along the Guadiana River over its course between Mérida and Badajoz (Extremadura, Spain). Water sampling for phytoplankton quantification and toxin analysis was carried out regularly between 1999 and 2001 in six different locations, including two shallow, slow-flowing river sites, two streamed river sites and two drinking water reservoirs. The cyanobacterial community differed significantly between these locations, especially during the summer. The predominant genera were Microcystis, Oscillatoria, Aphanizomenon and Anabaena. Using an ELISA assay the total microcystin contents of natural water samples from the most eutrophic locations ranged from 0.10 - 21.86 microg mcyst-LR equivalent x L(-1) in Valdelacalzada and 0.10-11.3 microg mcyst-LR equivalent x L(-1) in Vitonogales, and a seasonal variation of toxin content was observed. The amount of microcystins produced by each strain was determined by ELISA assay and the detection and identification of microcystin variants of three toxic strains of Microcystis aeruginosa was performed by high performance liquid chromatography (HPLC). The analysis of microcystins of the cultured strains revealed that toxin production was variable among different strains of M. aeruginosa isolated either from different blooms or from the same bloom.     

Overexpression of the rice Osmyb4 gene increases chilling and freezing tolerance of Arabidopsis thaliana plants

The expression of the gene Osmyb4, detected at low level in rice (Oryza sativa) coleoptiles grown for 3 days at 29 degrees C, is strongly induced by treatments at 4 degrees C. At sublethal temperatures of 10 and 15 degrees C, its expression in rice seedlings is already evident, but this effect cannot be vicariated by other stresses or ABA treatment. We demonstrate by transient expression that Myb4 transactivates the PAL2, ScD9 SAD and COR15a cold-inducible promoters. The Osmyb4 function in vivo is demonstrated overexpressing its cDNA in Arabidopsis thaliana plants (ecotype Wassilewskija) under the control of the constitutive CaMV 35S promoter. Myb4 overexpressing plants show a significant increased cold and freezing tolerance, measured as membrane or Photosystem II (PSII) stability and as whole plant tolerance. Finally, in Osmyb4 transgenic plants, the expression of genes participating in different cold-induced pathways is affected, suggesting that Myb4 represents a master switch in cold tolerance.     

Malathion detoxification by human hepatic carboxylesterases and its inhibition by isomalathion and other pesticides

The organophosphorothioate (OPT) pesticide malathion (MAL) in mammals is readily hydrolyzed by mammalian carboxylesterases (CE). The reaction competes with the CYP‐catalyzed formation of malaoxon (MOX), the toxic metabolite. Alterations or individual variations in CE activity may result in increased MOX formation, enhancing MAL toxicity. We have characterized the human hepatic CE activity in a panel of 18 human liver microsomes as well as the inhibitory effect of IsoMAL, a major impurity of MAL commercial formulations, parathion (PAR), chlorpyrifos (CPF), and chlorpyrifos‐oxon (CPFO). CE activity showed a low level of variation among individuals (4‐fold). The reaction consists of two different phases, differing in their affinity for MAL (K_m1 = 0.25–0.69 μM; K_m2 = 10.3–26.8 μM). The relatively low K_m1 values confirmed that human CE efficiently detoxify MAL. IsoMAL was shown to be a potent noncompetitive inhibitor of MAL detoxification (K_i = 0.6 μM), with a higher inhibitory potency than CPF and PAR (K_i = 7.5 μM and 50 μM, respectively). These two latter compounds very likely act as mixed inhibitors. CPFO showed the highest inhibitory potency toward CE‐mediated detoxification, being characterized by a K_i = 22 nM. The present results provide useful information for a better understanding of possible interactions between different OPTs and for assessing the potential cumulative risk for exposure to OPT mixtures. © 2005 Wiley Periodicals, Inc. J Biochem Mol Toxicol 19:406–414, 2005; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20106     

Biochemical and Structural Studies of the Mycobacterium tuberculosis O6-Methylguanine Methyltransferase and Mutated Variants

Mycobacterium tuberculosis displays remarkable genetic stability despite continuous exposure to the hostile environment represented by the host''s infected macrophages. Similarly to other organisms, M. tuberculosis possesses multiple systems to counteract the harmful potential of DNA alkylation. In particular, the suicidal enzyme O⁶-methylguanine-DNA methyltransferase (OGT) is responsible for the direct repair of O⁶-alkylguanine in double-stranded DNA and is therefore supposed to play a central role in protecting the mycobacterial genome from the risk of G·C-to-A·T transition mutations. Notably, a number of geographically widely distributed M. tuberculosis strains shows nonsynonymous single-nucleotide polymorphisms in their OGT-encoding gene, leading to amino acid substitutions at position 15 (T15S) or position 37 (R37L) of the N-terminal domain of the corresponding protein. However, the role of these mutations in M. tuberculosis pathogenesis is unknown. We describe here the in vitro characterization of M. tuberculosis OGT (MtOGT) and of two point-mutated versions of the protein mimicking the naturally occurring ones, revealing that both mutated proteins are impaired in their activity as a consequence of their lower affinity for alkylated DNA than the wild-type protein. The analysis of the crystal structures of MtOGT and MtOGT-R37L confirms the high level of structural conservation of members of this protein family and provides clues to an understanding of the molecular bases for the reduced affinity for the natural substrate displayed by mutated MtOGT. Our in vitro results could contribute to validate the inferred participation of mutated OGTs in M. tuberculosis phylogeny and biology.     

Cu(Nor)2·5H2O, a complex of Cu(II) with Norfloxacin: theoretic approach and biological studies. Cytotoxicity and genotoxicity in cell cultures

Norfloxacin is a fluoroquinolone antibiotic used in the treatment of bacterial infections. In this article, we studied the potential antitumoral action of a complex of Norfloxacin with Cu(II), Cu(Nor)₂·5H₂O on osteosarcoma cells (UMR106) and calvaria-derived cells (MC3T3-E1), evaluating its cytotoxicity and genitoxicity. We have also elucidated the more stable conformation of this complex under physiologic conditions by Molecular Dynamic simulations based on the model of the canonical ensemble and PM6 force field. When solvent effect was taken into account, the complex conformation with both carbonyl groups in opposite sides displayed lower energy. Cu(Nor)₂·5H₂O caused an inhibitory effect on the proliferation on both cell lines from 300 μM (P < 0.01). Nevertheless, the decline on cell proliferation of UMR106 cells was more pronounced (45 % vs basal) than in MC3T3-E1 cells (20 % vs basal) at 300 μM (P < 0.01). Cu(Nor)₂·5H₂O altered lysosomal metabolism (Neutral Red assay) in a dose-dependent manner from 300 μM (P < 0.001). Morphological studies showed important transformations that correlated with a decrease in the number of cells in a dose-dependent manner. Moreover, Cu(Nor)₂·5H₂O caused statistically significant genotoxic effects on both osteoblast cell lines in a lower range of concentrations (Micronucleus assay) (P < 0.05 at 10 μM, P < 0.001 from 25 to 50 μM). UMR106 cells displayed a dose-related genotoxic effect between 5 and 25 μM while the MC3T3-E1 cells showed a narrower concentration dependent range. Altogether, these results suggest that Cu(Nor)₂·5H₂O is a good candidate to be further evaluated for alternative therapeutics in cancer treatment.     

p39, the Primary Activator for Cyclin-dependent Kinase 5 (Cdk5) in Oligodendroglia,Is Essential for Oligodendroglia Differentiation and Myelin Repair

Cyclin-dependent kinase 5 (Cdk5) plays key roles in normal brain development and function. Dysregulation of Cdk5 may cause neurodegeneration and cognitive impairment. Besides the well demonstrated role of Cdk5 in neurons, emerging evidence suggests the functional requirement of Cdk5 in oligodendroglia (OL) and CNS myelin development. However, whether neurons and OLs employ similar or distinct mechanisms to regulate Cdk5 activity remains elusive. We report here that in contrast to neurons that harbor high levels of two Cdk5 activators, p35 and p39, OLs express abundant p39 but negligible p35. In addition, p39 is selectively up-regulated in OLs during differentiation along with elevated Cdk5 activity, whereas p35 expression remains unaltered. Specific knockdown of p39 by siRNA significantly attenuates Cdk5 activity and OL differentiation without affecting p35. Finally, expression of p39, but not p35, is increased during myelin repair, and remyelination is impaired in p39^−/− mice. Together, these results reveal that neurons and OLs harbor distinct preference of Cdk5 activators and demonstrate important functions of p39-dependent Cdk5 activation in OL differentiation during de novo myelin development and myelin repair.     

Esperienze Di Estivazione Su Urginea Maritima Bak.

Abstract

Aestivation experiments on URGINEA MARITIMA Bak. — Bulbs of Urginea maritima Bak. have been subjected to artificial aestivation in different months (March, April, May, June) in order to find out the most appropriate period for forced blossoming. The bulbs, once taken out of the soil, have been kept for a whole month at 28° C, with atmospheric humidity not strictly controlled but never falling below 48%.

Aestivation in March and April, when the plant is in its full vegetative activity, caused suppression of the sexual phase. Aestivation in May, when the vegetative activity of the plant is approaching its end, caused total flowering anticipated of about 20 days. Aestivation in June, when the plant is at the beginning of its resting period, had no effect on flowering.

These results seem to show that the leaves create in the course of their activity the necessary conditions for flowering and that this critical stage is reached, in the plants under examination, between April and May.

In the plants treated in May that is after they had reached the critical stage, the flowering was not suppressed; on the contrary it occurred earlier than in the plants left in their natural habitat. The advancement of the time of flowering corresponded more or less to the anticipation of the artificial as compared with the naturally occurring aestivation.