Optimization of a nonradioactive method for consistent and sensitive determination of activated K-ras protein

Accurate measurement of activity of wild-type K-ras protein is important due to its tumor suppressor action in tissues such as lung. A published method by Taylor and co-workers uses plasmid-containing Escherichia coli cells to produce a glutathione-S-transferase/raf-1 ras binding domain (GST-RBD) fusion protein attached to glutathione beads to isolate activated ras protein. We systematically optimized the method before use on lung tissues. Changing the GST-RBD protein induction temperature from the original 37 to 30 degrees C produced a consistently greater yield of fusion protein. To improve stability of the GST-RBD beads so as to perform large-scale experiments, 0.1% NaN(3) was added. NaN(3)-treated beads retained full affinity for at least 24 days. Sensitivity was improved by using a polyvinylidene difluoride membrane rather than nitrocellulose for immunoblotting. We also compared our GST-RBD beads with two commercial assay kits and found that our beads had both superior sensitivity and reduced variability. In summary, our modification of the GST-RBD affinity method to recover activated K-ras greatly increased the yield of fusion protein, prolonged the useful life of GST-RBD beads to at least 24 days, and enhanced detection sensitivity.     

Microstructural characterization of annulus fibrosus by ultrasonography: a feasibility study with an in vivo and in vitro approach

The main function of the intervertebral disc is biomechanical function, since it must resist repetitive high loadings, while giving the spine its flexibility and protecting the spinal cord from over-straining. It partially owes its mechanical characteristics to the lamellar architecture of its outer layer, the annulus fibrosus. Today, no non-invasive means exist to characterize annulus lamellar structure in vivo. The aim of this work was to test the feasibility of imaging annulus fibrosus microstructure in vivo with ultrasonography. Twenty-nine healthy adolescents were included. Ultrasonographies of L3–L4 disc were acquired with a frontal approach. Annulus fibrosus was segmented in the images to measure the thickness of the lamellae. To validate lamellar appearance in ultrasonographies, multimodality images of two cow tail discs were compared: ultrasonography, magnetic resonance and optical microscopy. In vivo average lamellar thickness was 229.7 ± 91.5 μm, and it correlated with patient body mass index and age. Lamellar appearance in the three imaging modalities in vitro was consistent. Lamellar measurement uncertainty was 7%, with good agreement between two operators. Feasibility of ultrasonography for the analysis of lumbar annulus fibrosus structure was confirmed. Further work should aim at validating measurement reliability, and to assess the relevance of the method to characterize annulus alterations, for instance in disc degeneration or scoliosis.

     

Circadian rhythms in toxic effects of the serotonin antagonist ondansetron in mice

The aim of the study was to learn whether the lethal and the motor incoordination (ataxia) side effect of ondansetron (Zophren) administration is dosing-time dependent. Ondansetron is a serotonin 5-HT3 receptor antagonist used primarily to control nausea and vomiting arising from cytotoxic chemo- and radiotherapy. A total of 210 male Swiss mice 10 to 12 weeks of age were synchronized for 3 weeks by 12 h light (rest span)/12 h dark (activity span). Different doses of ondansetron were injected intraperitoneally (i.p.) at fixed times during the day to determine both the sublethal (TD50) and lethal (LD50) doses, which were, respectively, 3.7 +/- 0.6 mg/kg and 4.6 +/- 0.5 mg/kg. In the chronotoxicologic study a single dose of ondansetron (3.5 mg/kg, i.p.) was administered to different and comparable groups of animals at four different circadian stages [1, 7, 13, and 19 h after light onset (HALO)]. The lethal toxicity was statistically significantly dosing time-dependent (chi2 = 21.51, p < 0.0001). Drug dosing at 1 HALO resulted in 100% survival rate whereas drug dosing at 19 HALO was only one-half that (52%). Similarly, lowest and highest ataxia occurred when ondansetron was injected at 1 and 19 HALO, respectively (chi2 = 22.24, p < 0.0001). Effects on rectal temperature were also dosing-time related (Cosinor analysis, p < 0.0001). The characteristics of the waveform describing the temporal patterns differed between the studied variables, e.g., lethal toxicity and survival rate showing two peaks and rectal temperature showing one peak in the 24 h time series waveform pattern. Cosinor analysis also revealed a statistically significant ultradian (tau = 8 h) rhythmic component in the considered variables. Differences in curve patterns in toxicity elicited by ondansetron on a per end point basis are hypothesized to represent the phase relations between the identified 24 h and 8 h periodicities.     

Buthionine sulfoximine embryotoxicity is associated with prolonged AP-1 activation

BACKGROUND: Many teratogens induce oxidative stress, altering redox status and redox signaling; this has led to the suggestion that developmental toxicants act by disturbing redox status. The goal of these studies was to determine the consequences of altering glutathione homeostasis during organogenesis on embryo development, total DNA methylation, and activator protein-1 (AP-1) DNA binding activity and gene expression. METHODS: Gestational day 10.5 rat embryos were cultured in vitro for up to 44 hour in the presence of L-buthionine-S,R-sulfoximine (BSO), an irreversible inhibitor of gamma-glutamyl-cysteine synthetase, the rate limiting step in glutathione biosynthesis. Effects of BSO on total, oxidized and reduced glutathione, embryo development, DNA methylation, AP-1 DNA binding activity and gene expression were investigated. RESULTS: Significant depletion of glutathione by BSO was first noted at 6 hr in the embryo and at 3 hr in the yolk sac; total glutathione in the conceptus was depleted to the same extent after treatment with either 0.1 or 1.0 mM BSO. Exposure to 0.1 mM BSO did not cause a significant increase in embryotoxicity, although some impairment of growth and development was observed. In contrast, exposure to 1.0 mM BSO severely inhibited growth and development, significantly increasing the incidence of swollen hindbrains and of blebs in the forebrain, limb and maxillary regions. No significant treatment-related differences in total DNA methylation were observed. Interestingly, AP-1 DNA binding activity was similar in control and 0.1 mM BSO-treated conceptuses; however, exposure to 1.0 mM BSO increased AP-1 DNA binding at 6, 24, and 44 hr. The expression of several AP-1 family genes and of gamma-glutamylcysteine synthetase was induced in embryos cultured with 1.0 mM BSO. CONCLUSION: Exposure of embryos in vitro to BSO at a concentration that was embryotoxic induced prolonged AP-1 DNA binding activity and altered gene expression. These data suggest that AP-1 induction may serve as a biomarker of embryo stress.     

Changes in yield ofin-vivo fluorescence of chlorophyll a as a tool for selective herbicide monitoring

Triazines and derivatives of phenylurea, which are often found in outdoor water samples, induce specific changes in the yield of thein-vivo chlorophyll -fluorescence of PSII. These changes are correlated quantitatively with the concentration of the herbicides and can therefore be used to set-up a low-price monitor system. In order to detect selectively the herbicide-sensitive part of the fluorescence emission a pulse amplitude modulated fluorimeter was used. The bioassay system was optimised with respect to test organism, growing and measuring conditions. The relationship between fluorescence yield and herbicide concentrations were experimentally determined for the triazines atrazine and simazine and the phenylurea herbicide DCMU and mathematically fitted (r=0.99). The I₅₀-values were 0.9 µM for DCMU, 2.2 µM for simazine and 3.3 µM for atrazine. The detection limit of about 0.5 µM clearly shows that the sensitivity of this bioassay system is too low to reach the requirements of the drinking water regulation. However, due to its insensitivity against complex water matrices, there is good hope to combine this fluorometric bioassay with a potent herbicide preconcentration method like a solid-phase extraction procedure.Author for correspondence     

Lipid composition of a plasma membrane enriched fraction of maize roots

The lipid composition of a plasma membrane enriched fraction isolated from corn (Zea mays) roots was examined. On a wt basis, the lipid: protein ratio was 1.11. Phospholipids comprised 60% of total lipids with the major phospholipids being phosphatidylcholine (62%) and phosphatidylethanolamine (21%). Free sterol was the major neutral lipid. The sterol:phospholipid molar ratio was 0.31. The fatty acid composition of the membrane was predominantly linoleic (60%) and palmitic (30%).     

Cloning and characterization of AOEB166, a novel mammalian antioxidant enzyme of the peroxiredoxin family.

Using two-dimensional electrophoresis, we have recently identified in human bronchoalveolar lavage fluid a novel protein, termed B166, with a molecular mass of 17 kDa. Here, we report the cloning of human and rat cDNAs encoding B166, which has been renamed AOEB166 for antioxidant enzyme B166. Indeed, the deduced amino acid sequence reveals that AOEB166 represents a new mammalian subfamily of AhpC/TSA peroxiredoxin antioxidant enzymes. Human AOEB166 shares 63% similarity with Escherichia coli AhpC22 alkyl hydroperoxide reductase and 66% similarity with a recently identified Saccharomyces cerevisiae alkyl hydroperoxide reductase/thioredoxin peroxidase. Moreover, recombinant AOEB166 expressed in E. coli exhibits a peroxidase activity, and an antioxidant activity comparable with that of catalase was demonstrated with the glutamine synthetase protection assay against dithiothreitol/Fe3+/O(2) oxidation. The analysis of AOEB166 mRNA distribution in 30 different human tissues and in 10 cell lines shows that the gene is widely expressed in the body. Of interest, the analysis of N- and C-terminal domains of both human and rat AOEB166 reveals amino acid sequences presenting features of mitochondrial and peroxisomal targeting sequences. Furthermore, human AOEB166 expressed as a fusion protein with GFP in HepG2 cell line is sorted to these organelles. Finally, acute inflammation induced in rat lung by lipopolysaccharide is associated with an increase of AOEB166 mRNA levels in lung, suggesting a protective role for AOEB166 in oxidative and inflammatory processes.     

Genetic Diversity and Biological Control Activity of Novel Species of Closely Related Pseudomonads Isolated from Wheat Field Soils in South Australia

Rhizobacteria closely related to two recently described species of pseudomonads, Pseudomonas brassicacearum and Pseudomonas thivervalensis, were isolated from two geographically distinct wheat field soils in South Australia. Isolation was undertaken by either selective plating or immunotrapping utilizing a polyclonal antibody raised against P. brassicacearum. A subset of 42 isolates were characterized by amplified 16S ribosomal DNA restriction analysis (ARDRA), BIOLOG analysis, and gas chromatography-fatty acid methyl ester (GC-FAME) analysis and separated into closely related phenetic groups. More than 75% of isolates tested by ARDRA were found to have >95% similarity to either Pseudomonas corrugata or P. brassicacearum-P. thivervalensis type strains, and all isolates had >90% similarity to either type strain. BIOLOG and GC-FAME clustering showed a >70% match to ARDRA profiles. Strains representing different ARDRA groups were tested in two soil types for biological control activity against the soilborne plant pathogen Gaeumannomyces graminis var. tritici, the causative agent of take-all of wheat and barley. Three isolates out of 11 significantly reduced take-all-induced root lesions on wheat plants grown in a red-brown earth soil. Only one strain, K208, was consistent in reducing disease symptoms in both the acidic red-brown earth and a calcareous sandy loam. Results from this study indicate that P. brassicacearum and P. thivervalensis are present in Australian soils and that a level of genetic diversity exists within these two novel species but that this diversity does not appear to be related to geographic distribution. The result of the glasshouse pot trial suggests that some isolates of these species may have potential as biological control agents for plant disease.     

Microcomputed tomography-based structural analysis of various bone tissue regeneration models

Microcomputed tomography (microCT) analysis is a powerful tool for the evaluation of bone tissue because it provides access to the 3D microarchitecture of the bone. It is invaluable for regenerative medicine as it provides the researcher with the opportunity to explore the skeletal system both in vivo and ex vivo. The quantitative assessment of macrostructural characteristics and microstructural features may improve our ability to estimate the quality of newly formed bone. We have developed a unique procedure for analyzing data from microCT scans to evaluate bone structure and repair. This protocol describes the procedures for microCT analysis of three main types of mouse bone regeneration models (ectopic administration of bone-forming mesenchymal stem cells, and administration of cells after both long bone defects and cranial segmental bone defects) that can be easily adapted for a variety of other models. Precise protocols are crucial because the system is extremely user sensitive and results can be easily biased if standardized methods are not applied. The suggested protocol takes 1.5-3.5 h per sample, depending on bone tissue sample size, the type of equipment used, variables of the scanning protocol and the operator's experience.     

Mechanics and energetics of myosin molecular motors from nonpregnant human myometrium

Mechanical properties of spontaneously contracting isolated nonpregnant human myometrium (NPHM) were investigated throughout the whole continuum of load from zero load up to isometry. This made it possible to assess the three-dimensional tension-velocity-length (T-V-L) relationship characterizing the level of contractility and to determine crossbridge (CB) kinetics of myosin molecular motors. Seventy-seven muscle strips were obtained from hysterectomy in 42 nonpregnant patients. Contraction and relaxation parameters were measured during spontaneous mechanical activity. The isotonic tension-peak velocity (T-V) relationship was hyperbolic in 30 cases and nonhyperbolic in 47 cases. When the T-V relationship was hyperbolic, the Huxley formalism could be used to calculate CB kinetics and CB unitary force. At the whole muscle level and for a given isotonic load level, part of the V-L phase plane showed a common pathway, so that a given instantaneous length corresponded to only one possible instantaneous velocity, independent of time and initial length. At the molecular level, rate constants for CB attachment and detachment were dramatically low, ～100 times lower than those of striated muscles, and ～5 to 10 times lower than those of other smooth muscles. The CB unitary force was ～1.4 ± 0.1 pN. NPHM shared similar basic contractile properties with striated muscles, reflected in the three-dimensional T-V-L relationship characterizing the contractile level. Low CB attachment and detachment rate constants made it possible to generate normal CB unitary force and normal muscle tension in NPHM, even though it contracted extremely slowly compared with other muscles.