Liquid chromatography-mass spectrometry assay for quantification of Gluten Exorphin B5 in cerebrospinal fluid

A sensitive, precise and accurate method for the quantification of the alimentary opioid peptide Gluten Exorphin B5 (GE-B5, Tyr-Gly-Gly-Trp-Leu) in cerebrospinal fluid (CSF) was developed using liquid chromatography-mass spectrometry (LC-MS). Aliquots (10 microL) of sheep CSF were injected into a LC-MS instrument equipped with a reversed-phase C12 column at a flow rate of 250 microL/min. The mobile phase consisted of Eluent A water with 0.01% acetic acid as an ion-pairing reagent, and Eluent B acetonitrile. The LC-MS system was programmed to divert column flow to waste for 3.5 min after injection, after which time flow was directed into the mass spectrometer that operated in positive ion mode. DADLE (Tyr-D-Ala-Gly-Phe-D-Leu) was used as Internal Standard. No significant interfering peaks were detected at the retention times of GE-B5 in CSF blanks. The calibration curves were linear in the range of 0.39-78.00 ng/mL. The lower limit of detection and the lower limit of quantitation values for GE-B5 in CSF were established at 0.30 and 0.78 ng/mL, respectively. The intra-day and inter-day precision values were <12% relative standard deviation. The intra-day and inter-day accuracy were 99.46-100.86% and 98.95-100.02%, respectively. Recovery of GE-B5 in CSF samples was greater than 80%. Stability studies indicate that GE-B5 in CSF undergoes significant degradation (>55% after 600 min), which is reduced by the addition of protease inhibitors. This is the first reported method for the quantification of GE-B5 in CSF.     

A simple and sensitive assay for determining plasma tipranavir concentration in the clinical setting by new HPLC method

A simple method for the quantification of tipranavir, the first non-peptidic HIV protease inhibitor, was developed and validated. Quinoxaline, as internal standard, was added to 50 microl of plasma before a liquid-liquid extraction by 600 microl of protein precipitation solution. The extracts were diluted before being injected in the chromatographic system. Chromatographic separation was made on a C18 column using potassium phosphate buffer (pH 3.2) and acetonitrile with gradient. Detection was performed by an UV detector at 260 nm. Relative error at three control quality concentrations ranged from -1.81 to 1.72%. Intra-day (CV%) and inter-day (CV%) precision ranged from 0.94 to 2.55% and from 3.07 to 4.24%, respectively. LOQ and LOD were 0.090 microg/ml and 0.035 microg/ml, respectively. Mean recovery was 87.1%+/-2.4%. Calibration curve was linear up to 180 microg/ml. Concentration range when optimized (0.703-180 microg/ml) proved to be adequate to measure tipranavir concentration in HIV-1-positive patients, therefore this method could be suitable for therapeutic drug monitoring of this drug.     

Characterisation ofPseudomonas spp. isolated from foods

PutativePseudomonas spp. (102 isolates) from different foods were first characterised by API 20NE and then tested for some enzymatic activities (lipase and lecithinase production, starch hydrolysis and proteolytic activity). However subsequent molecular tests did not always confirm the results obtained, thus highlighting the limits of API 20NE. Instead RFLP ITS1 and the sequencing of 16S rRNA gene grouped the isolates into 6 clusters:Pseudomonas fluorescens (cluster I),Pseudomonas fragi (duster II and V)Pseudomonas migulae (cluster III),Pseudomonas aeruginosa (cluster IV) andPseudomonas chicorii (cluster VI). The pectinolytic activity was typical of species isolated from vegetable products, especiallyPseudomonas fluorescens. InsteadPseudomonas fragi, predominantly isolated from meat was characterised by proteolytic and lipolytic activities.     

Xeno-cannibalism: a survival "escamotage"

Several lines of evidence have demonstrated that self-cannibalism (macroautophagy) is a well regulated process of cell repair as well as of molecule and organelle recycling that allows the cells to survive. However, autophagic activity also represents a cell death pathway characterized by specific features that differentiate autophagy from other cell death processes. We found that cells that are able to exert intense autophagic activity were also able to engulf and digest entire cell siblings. This phenomenon represents a sort of xeno-cannibalism. We wonder whether these two phenomena, self and xeno-cannibalism, could be related the latter being an exacerbation of the first and providing a further survival option to the cells.     

Alkyltransferase-like proteins

Recent in silico analysis has revealed the presence of a group of proteins in pro and lower eukaryotes, but not in Man, that show extensive amino acid sequence similarity to known O(6)-alkylguanine-DNA alkyltransferases, but where the cysteine at the putative active site is replaced by another residue, usually tryptophan. Here we review recent work on these proteins, which we designate as alkyltransferase-like (ATL) proteins, and consider their mechanism of action and role in protecting the host organisms against the biological effects of O(6)-alkylating agents, and their evolution. ATL proteins from Escherichia coli (eAtl, transcribed from the ybaz open reading frame) and Schizosaccharomyces pombe (Atl1) are able to bind to a range of O(6)-alkylguanine residues in DNA and to reversibly inhibit the action of the human alkyltransferase (MGMT) upon these substrates. Isolated proteins were not able to remove the methyl group in O(6)-methylguanine-containing DNA or oligonucleotides, neither did they display glycosylase or endonuclease activity. S. pombe does not contain a functional alkyltransferase and atl1 inactivation sensitises this organism to a variety of alkylating agents, suggesting that Atl1 acts by binding to O(6)-alkylguanine lesions and signalling them for processing by other DNA repair pathways. Currently we cannot exclude the possibility that ATL proteins arose through independent mutation of the alkyltransferase gene in different organisms. However, analyses of the proteins from E. coli and S. pombe, are consistent with a common function.     

Diversity of settlement-stage reef fishes captured by light-trap in a tropical south-west Atlantic Ocean coastal reef system

This study reports the results of 5 years of monitoring reef fish post-larvae using light traps in the Bay of Tamandaré, north-east Brazil. An annotated checklist of pre-settlement fish species, their frequency of occurrence and taxonomic characteristics are provided. In total, 4,422 post-larval fishes belonging to 36 families, 56 genera and 76 species were captured. The most species-rich families were Carangidae (7), Lutjanidae (6) and Pomacentridae (4), while the families Gerreidae (30.47%), Holocentridae (16.54%), Blenniidae (12.01%), Labrisomidae (8.36%), Lutjanidae (8.29%) and Acanthuridae (5.95%) were the most abundant. This is the first study of the taxonomic diversity and assemblage structure of settlement-stage reef fishes in the tropical south-west Atlantic Ocean. Although a few common species were not captured due to selectivity of light traps, the composition and taxonomic diversity of this first collection suggests that light traps are useful for studies of the early life history of a wide range of pre-settlement reef fishes.     

NEOSQUIRREL: a data set of ecological knowledge on Neotropical squirrels

相似文献   

Defaunation and fragmentation erode small mammal diversity dimensions in tropical forests

Forest fragmentation and defaunation are considered the main drivers of biodiversity loss, yet the synergistic effects of landscape changes and biotic interactions on assemblage structure have been poorly investigated. Here, we use an extensive dataset of 283 assemblages and 105 species of small mammals to understand how defaunation of medium and large mammals and forest fragmentation change the community composition and diversity of rodents and marsupials in tropical forests of South America. We used structured equation models to investigate the relationship between small mammal species, functional and phylogenetic diversity with forest size, forest cover and the occurrence of medium and large mammals. The best‐fit model showed that defaunation reduced functional diversity, and that species diversity of small mammals increased with forest patch size. Forest cover did not affect functional and phylogenetic diversity. Our results indicate that occurrence of medium and large sized mammals (probably acting as predators, or competitors of small mammals) and forest patch size help to retain species and functional diversity in small mammal communities. Further, the number of species in a small mammal community was critical to the maintenance of phylogenetic diversity, and may have a pronounced influence on the ecological functions played by small mammals. Identifying how phylogenetic and functional diversity change in function of human pressures allows us to better understand the contribution of extant lineages to ecosystem functioning in tropical forests.     

IGF-I receptor-induced cell-cell adhesion of MCF-7 breast cancer cells requires the expression of junction protein ZO-1

Hyperactivation of the insulin-like growth factor I receptor (IGF-IR) contributes to primary breast cancer development, but the role of the IGF-IR in tumor metastasis is unclear. Here we studied the effects of the IGF-IR on intercellular connections mediated by the major epithelial adhesion protein, E-cadherin (E-cad). We found that IGF-IR overexpression markedly stimulated aggregation in E-cad-positive MCF-7 breast cancer cells, but not in E-cad-negative MDA-MB-231 cells. However, when the IGF-IR and E-cad were co-expressed in MDA-MB-231 cells, cell-cell adhesion was substantially increased. The IGF-IR-dependent cell-cell adhesion of MCF-7 cells was not related to altered expression of E-cad or alpha-, beta-, or gamma-catenins but coincided with the up-regulation of another element of the E-cad complex, zonula occludens-1 (ZO-1). ZO-1 expression (mRNA and protein) was induced by IGF-I and was blocked in MCF-7 cells with a tyrosine kinase-defective IGF-IR mutant. By co-immunoprecipitation, we found that ZO-1 associates with the E-cad complex and the IGF-IR. High levels of ZO-1 coincided with an increased IGF-IR/alpha-catenin/ZO-1-binding and improved ZO-1/actin association, whereas down-regulation of ZO-1 by the expression of an anti-ZO-1 RNA inhibited IGF-IR-dependent cell-cell adhesion. The results suggested that one of the mechanisms by which the activated IGF-IR regulates E-cad-mediated cell-cell adhesion is overexpression of ZO-1 and the resulting stronger connections between the E-cad complex and the actin cytoskeleton. We hypothesize that in E-cad-positive cells, the IGF-IR may produce antimetastatic effects.     

Sequence dependence of translational positioning of core nucleosomes

The basis for the choice of translational position of a histone octamer on DNA is poorly understood. To gain further insights into this question we have studied the translational and rotational settings of core particles assembled on a simple repeating 20 bp positioning sequence. We show that the translational positions of the core particles assembled on this sequence are invariant with respect to the DNA sequence and occur at 20 bp intervals. Certain modifications of the original sequence reduce the spacing of possible dyads to 10 bp. At least one of these alters both the translational and rotational settings. We conclude that the translational position of a core particle is specified by sequence determinants additional to those specifying rotational positioning. The rotational settings on either side of the dyads of core particles assembled on the wild-type and a mutant sequence differ by +2 bp, corresponding to an overall helical periodicity of approximately 10.15 bp. The average helical periodicity of the central two to four turns is 10.5-11 bp whilst that of the flanking DNA is closer to 10 bp. The DNA immediately flanking the dyad is also characterised by a more extensive susceptibility to cleavage by hydroxyl radical.