The potential of mass spectrometry imaging in plant metabolomics: a review

Phytochemistry Reviews - Imaging mass spectrometry (MSI) has contributed important information to several scientific fields and more recently to the biological sciences. The use of MSI as an...     

Estimation of the affinity of naloxone at supraspinal and spinal opioid receptors in vivo: studies with receptor selective agonists

The apparent affinity of naloxone at cerebral and spinal sites was estimated using selective mu [D-Ala2, Gly-o15]-enkephalin (DAGO) and delta [D-Pen2, D-Pen5]enkephalin] (DPDPE) opioid agonists in the mouse warm water tail-withdrawal test in vivo; the mu agonist morphine was employed as a reference compound. The approach was to determine the naloxone pA2 using a time-dependent method with both agonist and antagonist given intracerebroventricularly (i.c.v.) or intrathecally (i.th.); naloxone was always given 5 min before the agonist. Complete time-response curves were determined for each agonist at each site in the absence, and in the presence, of a single, fixed i.c.v. or i.th. dose of naloxone. From these i.c.v. or i.th. pairs of time-response curves, pairs of dose-response lines were constructed at various times; these lines showed decreasing displacement with time, indicative of the disappearance of naloxone. The graph of log (dose ratio-1) vs. time was linear with negative slope, in agreement with the time-dependent form of the equation for competitive antagonism. From this plot, the apparent pA2 and naloxone half-life was calculated at each site and against each agonist. The affinity of naloxone was not significantly different when compared between agonists after i.c.v. administration. A small difference was seen between the affinity of i.th. naloxone against DPDPE and DAGO; the i.th. naloxone pA2 against morphine, however, was not different than that for DPDPE and DAGO. The naloxone half-life varied between 6.6 and 16.9 min, values close to those previously reported for this compound. These results suggest that the agonists studied may produce their i.c.v. analgesic effects at the same receptor type or that alternatively, the naloxone pA2 may be fortuitously similar for mu and delta receptors in vivo. Additionally, while the affinity of naloxone appears different for the receptors activated by i.th. DAGO and DPDPE, further work may be necessary before firm conclusions regarding the nature of the spinal analgesic receptor(s) can be drawn.     

Ultra-high-throughput picoliter-droplet microfluidics screening of the industrial cellulase-producing filamentous fungus Trichoderma reesei

Journal of Industrial Microbiology & Biotechnology - The selection of improved producers among the huge number of variants in mutant libraries is a key issue in filamentous fungi of industrial...     

Treatment with a sphingosine analog after the inception of house dust mite-induced airway inflammation alleviates key features of experimental asthma

Background

In vivo phosphorylation of sphingosine analogs with their ensuing binding and activation of their cell-surface sphingosine-1-phosphate receptors is regarded as the main immunomodulatory mechanism of this new class of drugs. Prophylactic treatment with sphingosine analogs interferes with experimental asthma by impeding the migration of dendritic cells to draining lymph nodes. However, whether these drugs can also alleviate allergic airway inflammation after its onset remains to be determined. Herein, we investigated to which extent and by which mechanisms the sphingosine analog AAL-R interferes with key features of asthma in a murine model during ongoing allergic inflammation induced by Dermatophagoides pteronyssinus.

Methods

BALB/c mice were exposed to either D. pteronyssinus or saline, intranasally, once-daily for 10 consecutive days. Mice were treated intratracheally with either AAL-R, its pre-phosphorylated form AFD-R, or the vehicle before every allergen challenge over the last four days, i.e. after the onset of allergic airway inflammation. On day 11, airway responsiveness to methacholine was measured; inflammatory cells and cytokines were quantified in the airways; and the numbers and/or viability of T cells, B cells and dendritic cells were assessed in the lungs and draining lymph nodes.

Results

AAL-R decreased airway hyperresponsiveness induced by D. pteronyssinus by nearly 70%. This was associated with a strong reduction of IL-5 and IL-13 levels in the airways and with a decreased eosinophilic response. Notably, the lung CD4⁺ T cells were almost entirely eliminated by AAL-R, which concurred with enhanced apoptosis/necrosis in that cell population. This inhibition occurred in the absence of dendritic cell number modulation in draining lymph nodes. On the other hand, the pre-phosphorylated form AFD-R, which preferentially acts on cell-surface sphingosine-1-phosphate receptors, was relatively impotent at enhancing cell death, which led to a less efficient control of T cell and eosinophil responses in the lungs.

Conclusion

Airway delivery of the non-phosphorylated sphingosine analog, but not its pre-phosphorylated counterpart, is highly efficient at controlling the local T cell response after the onset of allergic airway inflammation. The mechanism appears to involve local induction of lymphocyte apoptosis/necrosis, while mildly affecting dendritic cell and T cell accumulation in draining lymph nodes.     

Selected anthropometric variables and aerobic fitness as predictors of cardiovascular disease risk in children

The aim of this study was to assess the suitability of body mass index, waist circumference, waist-to-height ratio and aerobic fitness as predictors of cardiovascular risk factor clustering in children. A cross-sectional study was conducted with 290 school boys and girls from 6 to 10 years old, randomly selected. Blood was collected after a 12-hour fasting period. Blood pressure, waist circumference (WC), height and weight were evaluated according to international standards. Aerobic fitness (AF) was assessed by the 20-metre shuttle-run test. Clustering was considered when three of these factors were present: high systolic or diastolic blood pressure, high low-density lipoprotein (LDL) cholesterol, high triglycerides, high plasma glucose, high insulin concentrations and low high-density lipoprotein (HDL) cholesterol. A ROC curve identified the cut-off points of body mass index (BMI), WC, waist-to-height ratio (WHtR) and AF as predictors of risk factor clustering. BMI, WC and WHR resulted in significant areas under the ROC curves, which was not observed for AF. The anthropometric variables were good predictors of cardiovascular risk factor clustering in both sexes, whereas aerobic fitness should not be used to identify cardiovascular risk factor clustering in these children.     

Isoaccepting species of serine tRNA coded by bacteriophage T5sto.

By aminoacyl-tRNA-DNA hybridization and chromatographic analysis, evidence was provided that the bacteriophage T5stO codes for two tRNAser species. Trinucleotide- or polynucleotide-stimulated binding experiments assigned the codons UCC or UCU to these two tRNAser species. They also suggested that the synthesis of these two tRNAser species does not modify the reading capacity for codons less used in Escherichia coli F and corresponds to a different situation compared with the T4-coded tRNA's.     

Improved enantioselective conversion of styrene epoxides and meso-epoxides through epoxide hydrolases with a mutated nucleophile-flanking residue

In epoxide hydrolase from Agrobacterium radiobacter (EchA), phenylalanine 108 flanks the nucleophilic aspartate and forms part of the substrate-binding pocket. The influence of mutations at this position on the activity and enantioselectivity of the enzyme was investigated. Screening for improved enantioselectivity towards para-nitrophenyl glycidyl ether (pNPGE) using spectrophotometric progress curve analysis yielded five different mutants with 3- to 7-fold improved enantioselectivity. The increase in enantioselectivity was in most cases the result of an enhanced catalytic efficiency toward the preferred enantiomer. Several mutations at position F108 resulted in a higher activity toward cis-disubstituted meso-epoxides, which were converted to a single product enantiomer. Mutant F108C converted cis-2,3-epoxybutane to (2R,3R)-2,3-butanediol of >99% ee with a 7-fold improved activity, and mutant F108A hydrolyzed cyclohexene oxide to (1R,2R)-1,2-cyclohexanediol of >99% ee with a more than 150-fold higher activity than wild-type enzyme. It is concluded that single amino acid substitutions in the active site of epoxide hydrolase can result in enzyme variants with catalytic properties that are suitable for preparative scale production of (S)-epoxides and chiral vicinal diols in high yield and with excellent ee.     

Sensitive fluorescence in situ hybridization signal detection in maize using directly labeled probes produced by high concentration DNA polymerase nick translation

The signal produced by fluorescence in situ hybridization (FISH) often is inconsistent among cells and sensitivity is low. Small DNA targets on the chromatin are difficult to detect. We report here an improved nick translation procedure for Texas red and Alexa Fluor 488 direct labeling of FISH probes. Brighter probes can be obtained by adding excess DNA polymerase I. Using such probes, a 30 kb yeast transgene, and the rp1, rp3 and zein multigene clusters were clearly detected.