Determination of plasma, urine, and bovine serum albumin low-molecular-weight carbonyl levels by capillary gas chromatography with electron-capture and mass-selective detection

Peroxidation of lipids produces low-molecular-weight carbonyl compounds, which are reactive with biological nucleophiles. The analysis of these compounds is often difficult. A multicomponent method for the determination of 11 of them in biological samples is reported. The samples are subjected to a pretreatment-derivatization procedure followed by gas chromatographic analysis with either electron-capture detection (ECD) or mass-selective detection (MSD) in the selected-ion monitoring mode. The procedure involves derivatization of the analyte with 2,4,6-trichlorophenylhydrazine, extraction with n-hexane, and separation of the derivatization products on a nonpolar gas chromatographic column. The concentration of the derivatization reagent, pH, reaction time, temperature, and presence of extraneous ions were investigated to determine the optimal derivatization conditions. Under these conditions, the method allows for the selective detection of low-molecular-weight carbonyl compounds at femtomole levels in several biological materials such as plasma, urine, and bovine serum albumin without interferences. The limits of detection were in the ranges 0.01-0.2 microM for ECD and 0.15-1.5 microM for MSD. The mean procedural recoveries obtained during the method validation were within the range 85-95% and the intra- and interassay standard deviations do not exceed 4.6 and 6.1%, respectively.     

Inference of a radiation in Mastus (Gastropoda, Pulmonata, Enidae) on the island of Crete

The Mediterranean land snail genus Mastus (Beck, 1837) is highly divergent. Thirty-two Mastus species have been recorded throughout the genus range, and 23 of them are endemic to the islands of the Aegean Sea and mainland Greece. Of these, all 16 Mastus species reported from Crete are endemic to this island. A robust molecular phylogenetic framework based on mitochondrial and nuclear genes (1623 bp) allowed us to explore the temporal diversification pattern of lineages, using molecular clock approaches. Our results showed an initial radiation in the evolutionary history of the Cretan lineage, followed by a subsequent slowdown of lineage splitting rate. Using a dated major vicariant event of the Aegean area, we estimated the absolute time of the radiation event and proposed a biogeographic scenario accounting for the observed pattern. Additionally, we tried to infer the processes that led to the divergence of the Cretan Mastus species, by applying comparative methods in phylogenetically informated context. Overall, our results favoured a nonecological radiation scenario in the Cretan Mastus species due to an allopatric divergence of secondary sexual characters.     

Monomeric and oligomeric flavanols are agonists of membrane androgen receptors

The present work reports a new mode of action of the naturally occurring flavanols catechin and epicatechin and their dimers B2 and B5, in the breast cancer T47D cell line, namely, their interaction with membrane androgen receptors. We show that monomeric and dimeric flavanols are complete (B2) or partial displacers of radiolabeled testosterone bound on T47D membranes, with affinities ranging from 1.7 (B5) to 82.2 nM (B2). In addition, they trigger the phosphorylation of the same signaling molecules (FAK, PI3K) as testosterone-BSA, minutes after binding to membrane receptors, leading to actin cytoskeleton polymerization and redistribution, with formation of filopodia and lamellipodia. The PI3K inhibitor wortmannin reverts the effect of polyphenols and testosterone-BSA, providing additional evidence about activation of a similar signaling cascade. Incubation of T47D cells for more than 2 h with polyphenols or testosterone-BSA induces apoptosis, which follows the same time-dependent pattern. We conclude that flavanols (monomers or dimers) are agonists of membrane androgen receptors and could be used as testosterone-protein conjugates for the management of tumors, in which, application of testosterone-BSA induces regression, providing additional data about the mechanism of their antiproliferative action.     

Human immunodeficiency virus type 1 genomic RNA sequences in the female genital tract and blood: compartmentalization and intrapatient recombination

Investigation of human immunodeficiency virus type 1 (HIV-1) in the genital tract of women is crucial to the development of vaccines and therapies. Previous analyses of HIV-1 in various anatomic sites have documented compartmentalization, with viral sequences from each location that were distinct yet phylogenetically related. Full-length RNA genomes derived from different compartments in the same individual, however, have not yet been studied. Furthermore, although there is evidence that intrapatient recombination may occur frequently, recombinants comprising viruses from different sites within one individual have rarely been documented. We compared full-length HIV-1 RNA sequences in the plasma and female genital tract, focusing on a woman with high HIV-1 RNA loads in each compartment who had been infected heterosexually and then transmitted HIV-1 by the same route. We cloned and sequenced 10 full-length HIV-1 RNA genomes from her genital tract and 10 from her plasma. We also compared viral genomes from the genital tract and plasma of four additional heterosexually infected women, sequencing 164 env and gag clones obtained from the two sites. Four of five women, including the one whose complete viral sequences were determined, displayed compartmentalized HIV-1 genomes. Analyses of full-length, compartmentalized sequences made it possible to document complex intrapatient HIV-1 recombinants that were composed of alternating viral sequences characteristic of each site. These findings demonstrate that the genital tract and blood harbor genetically distinct populations of replicating HIV-1 and provide evidence that recombination between strains from the two compartments contributes to rapid evolution of viral sequence variation in infected individuals.     

Factors affecting male Prays oleae (Lepidoptera: Yponomeutidae) captures in pheromone-baited traps in olive orchards

Effects of trap design, height and side of trap placement on olive trees, pheromone doses in dispensers, aging of dispensers in the field, and secondary pheromone components were evaluated for the development of an effective pheromone monitoring system for Prays oleae (Bernard) Lesne (Lepidoptera: Yponomeutidae) in olive orchards. Field trials showed that trap design, pheromone dose, and trapping side affected male captures, whereas dispenser age, trap height, and secondary components had no influence. Pherocon 1C and Delta traps baited with 1 mg of (Z)-7-tetradecenal captured more male moths than Pherocon II or Funnel traps. Placement of traps at different cardinal directions significantly affected captures, but this trend was not consistent and varied with flight period and trap position internal or external to the tree canopy. Moth phenology as determined by pheromone traps from early April to mid-October was consistent with published field data. Results indicate that Pherocon 1C or Delta traps baited with 1 mg of (Z)-7-tetradecenal provide an effective tool for monitoring the flight activity of P. oleae and the time of application of control measures.     

Are splicing mutations the most frequent cause of hereditary disease?

Disease-causing point mutations are assumed to act predominantly through subsequent individual changes in the amino acid sequence that impair the normal function of proteins. However, point mutations can have a more dramatic effect by altering the splicing pattern of the gene. Here, we describe an approach to estimate the overall importance of splicing mutations. This approach takes into account the complete set of genes known to be involved in disease and suggest that, contrary to current assumptions, many mutations causing disease may actually be affecting the splicing pattern of the genes.     

Human spastin has multiple microtubule-related functions

Hereditary spastic paraplegias (HSPs) are neurodegenerative diseases caused by mutations in more than 20 genes, which lead to progressive spasticity and weakness of the lower limbs. The most frequently mutated gene causing autosomal dominant HSP is SPG4, which encodes spastin, a protein that belongs to the family of ATPases associated with various cellular activities (AAAs). A number of studies have suggested that spastin regulates microtubule dynamics. We have studied the ATPase activity of recombinant human spastin and examined the effect of taxol-stabilized microtubules on this activity. We used spastin translated from the second ATG and provide evidence that this is the physiologically relevant form. We showed that microtubules enhance the ATPase activity of the protein, a property also described for katanin, an AAA of the same spastin subgroup. Furthermore, we demonstrated that human spastin has a microtubule-destabilizing activity and can bundle microtubules in vitro, providing new insights into the molecular pathogenesis of HSP.     

A multi-clustering fusion scheme for data partitioning

A multi-clustering fusion method is presented based on combining several runs of a clustering algorithm resulting in a common partition. More specifically, the results of several independent runs of the same clustering algorithm are appropriately combined to obtain a distinct partition of the data which is not affected by initialization and overcomes the instabilities of clustering methods. Subsequently, a fusion procedure is applied to the clusters generated during the previous phase to determine the optimal number of clusters in the data set according to some predefined criteria.     

Potential of radial basis function neural networks in discriminating benign from malignant lesions of the lower urinary tract

OBJECTIVE: To investigate the potential value of morphometry and neural network tools for discriminating benign from malignant nuclei and lesions of the lower urinary tract. STUDY DESIGN: The study group consisted of 33 cases of lithiasis, 41 cases of inflammation, 66 cases of benign hyperplasia of the prostate, 4 cases of carcinoma in situ, 48 cases of grade 1 transitional cell carcinoma of the bladder (TCCB) and 123 cases of grade 2 and 3 TCCB. Images of routinely processed voided urine smears stained by the Giemsa technique were analyzed by a custom image analysis system. Analysis of the images gave a data set of features from 31,158 nuclei. A radial basis function (RBF)-type neural network was employed to discriminate benign from malignant nuclei, based on the extracted morphometric and textural features. Subsequently a second RBF classifier was employed to discriminate benign from malignant cases. The nuclei from 156 randomly selected cases (50% of total cases) was used as a training set, and the nuclei from the remaining 159 cases made up the test set. Similarly, in an attempt to discriminate at the patient level, the same 156 cases were used to train an RBF classifier; the remaining 159 cases were used for the test set. The cases used for training and testing the 2 classifiers (nuclear and patient level) were the same for the 2 kinds of classifiers. RESULTS: Application of the RBF classifier permitted the correct classification of 93.64% of benign nuclei and 85.61% of malignant, giving an overall accuracy of 84.45%. At the patient level the RBF classifier permitted an overall accuracy of 94.97%. These results were on the test sets. CONCLUSION: The role of nuclear morphologic features in the cytologic diagnosis of lower urinary tract alterations was confirmed by the results of this study. The observed overlap in feature space indicates that the nuclear characteristics do not form strictly separate clusters; that fact explains the difficulty morphologists have with reproducible identification of nuclei from the lower urinary tract. Application of RBF offers good classification at the nuclear and patient level and promises to become a powerful tool for everyday practice in the cytologic laboratory.