Circulating TNF-alpha and its soluble receptors during experimental acute pancreatitis

Clinical and experimental studies have shown increased concentrations of TNF-alpha and its soluble receptors in serum of patients with acute pancreatitis. In this work, we have investigated the time-course of TNF-alpha and its soluble receptors during taurocholate-induced acute pancreatitis. In addition, since TNF-alpha itself could mediate the shedding of its receptors, we have assessed the effect of inhibiting TNF-alpha production on the release of soluble TNF-alpha receptors in experimental acute pancreatitis. Our results indicate that soluble receptors are released in the early stages of the disease and this increase is concomitant with the release of TNF-alpha, which is mainly bound to specific proteins. The increased concentrations of its receptors strongly suggest that they could be these binding proteins. Inhibition of TNF-alpha generation with pentoxifylline abrogated the shedding of sTNF-alphaR1, but had no effect on sTNF-alphaR2. This finding suggests that the shedding of sTNF-alphaR1 is induced by TNF-alpha itself, but in the case of sTNF-alphaR2, the shedding appears to be induced by another mechanism.     

Endogenous and dietary indoles: a class of antioxidants and radical scavengers in the ABTS assay

Indoles are very common in the body and diet and participate in many biochemical processes. A total of twenty-nine indoles and analogs were examined for their properties as antioxidants and radical scavengers against 2,2'-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) ABTS*+ radical cation. With only a few exceptions, indoles reacted nonspecifically and quenched this radical at physiological pH affording ABTS. Indoleamines like tryptamine, serotonin and methoxytryptamine, neurohormones (melatonin), phytohormones (indoleacetic acid and indolepropionic acid), indoleamino acids like L-tryptophan and derivatives (N-acetyltryptophan, L-abrine, tryptophan ethyl ester), indolealcohols (tryptophol and indole-3-carbinol), short peptides containing tryptophan, and tetrahydro-beta-carboline (pyridoindole) alkaloids like the pineal gland compound pinoline, acted as radical scavengers and antioxidants in an ABTS assay-measuring total antioxidant activity. Their trolox equivalent antioxidant capacity (TEAC) values ranged from 0.66 to 3.9 mM, usually higher than that for Trolox and ascorbic acid (1 mM). The highest antioxidant values were determined for melatonin, 5-hydroxytryptophan, trp-trp and 5-methoxytryptamine. Active indole compounds were consumed during the reaction with ABTS*+ and some tetrahydropyrido indoles (e.g. harmaline and 1-methyl-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid ethyl ester) afforded the corresponding fully aromatic beta-carbolines (pyridoindoles), that did not scavenge ABTS*+. Radical scavenger activity of indoles against ABTS*+ was higher at physiological pH than at low pH. These results point out to structural compounds with an indole moiety as a class of radical scavengers and antioxidants. This activity could be of biological significance given the physiological concentrations and body distribution of some indoles.     

Prevalence of B chromosomes in Orthoptera is associated with shape and number of A chromosomes

We analyze the prevalence of B chromosomes in 1,601 species of orthopteran insects where chromosome number and shape are known. B chromosomes have been reported in 191 of these species. Bs are not uniformly distributed among orthopteran superfamilies, with evident hotspots in the Pyrgomorphoidea (32.3% of species carrying Bs), Grylloidea (14.9%), Acridoidea (14.6%) and Tetrigoidea (14.3%). As expected under the theory of centromeric drive, we found a correlation between B chromosome presence and A chromosome shape-Bs are more frequent in karyotypes with more acrocentric A chromosomes. We also found that Bs are less common in species with high chromosome numbers and appear to be most common at the modal chromosome number (2n = 24). Study effort, measured for each genus, was not associated with B prevalence, A chromosome shape or A chromosome number. Our results thus provide support for centromeric drive as an important and prevalent force in the karyotypic evolution of Orthoptera, just as it appears to be in mammals. We suggest that centromeric drive may provide a mechanistic explanation for White's principle of karyotypic orthoselection.     

Fulfilling the Promise of Personalized Medicine? Systematic Review and Field Synopsis of Pharmacogenetic Studies

Background

Studies of the genetic basis of drug response could help clarify mechanisms of drug action/metabolism, and facilitate development of genotype-based predictive tests of efficacy or toxicity (pharmacogenetics).

Objectives

We conducted a systematic review and field synopsis of pharmacogenetic studies to quantify the scope and quality of available evidence in this field in order to inform future research.

Data Sources

Original research articles were identified in Medline, reference lists from 24 meta-analyses/systematic reviews/review articles and U.S. Food and Drug Administration website of approved pharmacogenetic tests.

Study Eligibility Criteria, Participants, and Intervention Criteria

We included any study in which either intended or adverse response to drug therapy was examined in relation to genetic variation in the germline or cancer cells in humans.

Study Appraisal and Synthesis Methods

Study characteristics and data reported in abstracts were recorded. We further analysed full text from a random 10% subset of articles spanning the different subclasses of study.

Results

From 102,264 Medline hits and 1,641 articles from other sources, we identified 1,668 primary research articles (1987 to 2007, inclusive). A high proportion of remaining articles were reviews/commentaries (ratio of reviews to primary research approximately 25∶1). The majority of studies (81.8%) were set in Europe and North America focussing on cancer, cardiovascular disease and neurology/psychiatry. There was predominantly a candidate gene approach using common alleles, which despite small sample sizes (median 93 [IQR 40–222]) with no trend to an increase over time, generated a high proportion (74.5%) of nominally significant (p<0.05) reported associations suggesting the possibility of significance-chasing bias. Despite 136 examples of gene/drug interventions being the subject of ≥4 studies, only 31 meta-analyses were identified. The majority (69.4%) of end-points were continuous and likely surrogate rather than hard (binary) clinical end-points.

Conclusions and Implications of Key Findings

The high expectation but limited translation of pharmacogenetic research thus far may be explained by the preponderance of reviews over primary research, small sample sizes, a mainly candidate gene approach, surrogate markers, an excess of nominally positive to truly positive associations and paucity of meta-analyses. Recommendations based on these findings should inform future study design to help realise the goal of personalised medicines.

Systematic Review Registration Number

Not Registered     

W::Neo: a novel dual-selection marker for high efficiency gene targeting in Drosophila

We have recently developed a so-called genomic engineering approach that allows for directed, efficient and versatile modifications of Drosophila genome by combining the homologous recombination (HR)-based gene targeting with site-specific DNA integration. In genomic engineering and several similar approaches, a "founder" knock-out line must be generated first through HR-based gene targeting, which can still be a potentially time and resource intensive process. To significantly improve the efficiency and success rate of HR-based gene targeting in Drosophila, we have generated a new dual-selection marker termed W::Neo, which is a direct fusion between proteins of eye color marker White (W) and neomycin resistance (Neo). In HR-based gene targeting experiments, mutants carrying W::Neo as the selection marker can be enriched as much as fifty times by taking advantage of the antibiotic selection in Drosophila larvae. We have successfully carried out three independent gene targeting experiments using the W::Neo to generate genomic engineering founder knock-out lines in Drosophila.     

Pore formation by equinatoxin, a eukaryotic pore-forming toxin, requires a flexible N-terminal region and a stable beta-sandwich

Actinoporins are eukaryotic pore-forming proteins that create 2-nm pores in natural and model lipid membranes by the self-association of four monomers. The regions that undergo conformational change and form part of the transmembrane pore are currently being defined. It was shown recently that the N-terminal region (residues 10-28) of equinatoxin, an actinoporin from Actinia equina, participates in building of the final pore wall. Assuming that the pore is formed solely by a polypeptide chain, other parts of the toxin should constitute the conductive channel and here we searched for these regions by disulfide scanning mutagenesis. Only double cysteine mutants where the N-terminal segment 1-30 was attached to the beta-sandwich exhibited reduced hemolytic activity upon disulfide formation, showing that other parts of equinatoxin, particularly the beta-sandwich and importantly the C-terminal alpha-helix, do not undergo large conformational rearrangements during the pore formation. The role of the beta-sandwich stability was independently assessed via destabilization of a part of its hydrophobic core by mutations of the buried Trp117. These mutants were considerably less stable than the wild-type but exhibited similar or slightly lower permeabilizing activity. Collectively these results show that a flexible N-terminal region and stable beta-sandwich are pre-requisite for proper pore formation by the actinoporin family.     

A proteomic study of Xanthomonas oryzae pv. oryzae in rice xylem sap

Xanthomonas oryzae pv. oryzae (Xoo) is the second most important rice pathogen, causing a disease called bacterial leaf blight. Xoo colonizes and infects the vascular tissue resulting in tissue necrosis and wilting causing significant yield losses worldwide. In this study Xoo infected vascular fluid (xylem sap) was recovered and analyzed for secreted Xoo proteins. Three independent experiments resulted in the identification of 324 different proteins, 64 proteins were found in all three samples which included many of the known virulence-associated factors. In addition, 10 genes encoding for the identified proteins were inactivated and one mutant displayed statistically a significant loss in virulence when compared to the wild type Xoo, suggesting that a new virulence-associated factor has been revealed. The usefulness of this approach in understanding the lifestyle and unraveling the virulence-associated factors of phytopathogenic vascular bacteria is discussed.     

Influence of sucrose concentration and phosphate source on biomass and metabolite accumulation in adventitious roots of Pseudostellaria heterophylla

In this study, we investigated the induction of Pseudostellaria heterophylla adventitious root and the effects of sucrose concentration and phosphate source on biomass increase and metabolites accumulation. These roots were initially cultured in Murashige and Skoog medium for 4 weeks. IBA 3.0 mg L^?1 proved to be the best auxin for inducing adventitious roots and the frequency of adventitious roots induced from roots (100 %) was higher than that from leaves (78 %) and stems (27 %). The medium with 4 % sucrose resulted in the optimum biomass i.e. 1.04 g/flask DW, and the content of saponin and polysaccharides reached the peak i.e. 0.676 and 24.4 %, respectively. With regards to phosphate source, 1.25 mM phosphate concentration was more favorable for biomass of roots (0.87 g/flask of DW), whereas the optimum saponin (0.74 %) and polysaccharides (22.09 %) were achieved with 2.5 mM phosphate. However, the saponin content at 2.5 mM phosphate did not show significant difference from the saponin content at 0.625 mM (0.69 %) or 3.75 mM phosphate (0.69 %).