<Emphasis Type="Italic">Desulfovibrio legallis</Emphasis> sp. nov.: A Moderately Halophilic,Sulfate-Reducing Bacterium Isolated from a Wastewater Digestor in Tunisia

A new moderately halophilic sulfate-reducing bacterium (strain H₁^T) was enriched and isolated from a wastewater digestor in Tunisia. Cells were curved, motile rods (2–3 x 0.5 μm). Strain H₁^T grew at temperatures between 22 and 43°C (optimum 35°C), and at pH between 5.0 and 9.2 (optimum 7.3–7.5). Strain H₁^T required salt for growth (1–45 g of NaCl/l), with an optimum at 20–30 g/l. Sulfate, sulfite, thiosulfate, and elemental sulfur were used as terminal electron acceptors but not nitrate and nitrite. Strain H₁^T utilized lactate, pyruvate, succinate, fumarate, ethanol, and hydrogen (in the presence of acetate and CO₂) as electron donors in the presence of sulfate as electron acceptor. The main end-products from lactate oxidation were acetate with H₂ and CO₂. The G + C content of the genomic DNA was 55%. The predominant fatty acids of strain H₁^T were C_15:0 iso (38.8%), C_16:0 (19%), and C_14:0 iso 3OH (12.2%), and menaquinone MK-6 was the major respiratory quinone. Phylogenetic analysis of the small-subunit (SSU) ribosomal RNA (rRNA) gene sequence indicated that strain H₁^T was affiliated to the genus Desulfovibrio. On the basis of SSU rRNA gene sequence comparisons and physiological characteristics, strain H₁^T is proposed to be assigned to a novel species of sulfate reducers of the genus Desulfovibrio, Desulfovibrio legallis sp. nov. (= DSM 19129^T = CCUG 54389^T).     

Computed Tomography and Optical Imaging of Osteogenesis-angiogenesis Coupling to Assess Integration of Cranial Bone Autografts and Allografts

A major parameter determining the success of a bone-grafting procedure is vascularization of the area surrounding the graft. We hypothesized that implantation of a bone autograft would induce greater bone regeneration by abundant blood vessel formation. To investigate the effect of the graft on neovascularization at the defect site, we developed a micro–computed tomography (µCT) approach to characterize newly forming blood vessels, which involves systemic perfusion of the animal with a polymerizing contrast agent. This method enables detailed vascular analysis of an organ in its entirety. Additionally, blood perfusion was assessed using fluorescence imaging (FLI) of a blood-borne fluorescent agent. Bone formation was quantified by FLI using a hydroxyapatite-targeted probe and µCT analysis. Stem cell recruitment was monitored by bioluminescence imaging (BLI) of transgenic mice that express luciferase under the control of the osteocalcin promoter. Here we describe and demonstrate preparation of the allograft, calvarial defect surgery, µCT scanning protocols for the neovascularization study and bone formation analysis (including the in vivo perfusion of contrast agent), and the protocol for data analysis.The 3D high-resolution analysis of vasculature demonstrated significantly greater angiogenesis in animals with implanted autografts, especially with respect to arteriole formation. Accordingly, blood perfusion was significantly higher in the autograft group by the 7^th day after surgery. We observed superior bone mineralization and measured greater bone formation in animals that received autografts. Autograft implantation induced resident stem cell recruitment to the graft-host bone suture, where the cells differentiated into bone-forming cells between the 7^th and 10^th postoperative day. This finding means that enhanced bone formation may be attributed to the augmented vascular feeding that characterizes autograft implantation. The methods depicted may serve as an optimal tool to study bone regeneration in terms of tightly bounded bone formation and neovascularization.     

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.     

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.     

Validation of the reshaped shared epitope HLA-DRB1 classification in rheumatoid arthritis

Recently, we proposed a classification of HLA-DRB1 alleles that reshapes the shared epitope hypothesis in rheumatoid arthritis (RA); according to this model, RA is associated with the RAA shared epitope sequence (72-74 positions) and the association is modulated by the amino acids at positions 70 and 71, resulting in six genotypes with different RA risks. This was the first model to take into account the association between the HLA-DRB1 gene and RA, and linkage data for that gene. In the present study we tested this classification for validity in an independent sample. A new sample of the same size and population (100 RA French Caucasian families) was genotyped for the HLA-DRB1 gene. The alleles were grouped as proposed in the new classification: S1 alleles for the sequences A-RAA or E-RAA; S2 for Q or D-K-RAA; S3D for D-R-RAA; S3P for Q or R-R-RAA; and X alleles for no RAA sequence. Transmission of the alleles was investigated. Genotype odds ratio (OR) calculations were performed through conditional logistic regression, and we tested the homogeneity of these ORs with those of the 100 first trio families (one case and both parents) previously reported. As previously observed, the S2 and S3P alleles were significantly over-transmitted and the S1, S3D and X alleles were under-transmitted. The latter were grouped as L alleles, resulting in the same three-allele classification. The risk hierarchy of the six derived genotypes was the same: (by decreasing OR and with L/L being the reference genotype) S2/S3P, S2/S2, S3P/S3P, S2/L and S3P/L. The homogeneity test between the ORs of the initial and the replication samples revealed no significant differences. The new classification was therefore considered validated, and both samples were pooled to provide improved estimates of RA risk genotypes from the highest (S2/S3P [OR 22.2, 95% confidence interval 9.9-49.7]) to the lowest (S3P/L [OR 4.4, 95% confidence interval 2.3-8.4]).     

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%).     

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     

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.     

Endothelium-derived GM-CSF influences expression of oncostatin M

During and after transendothelial migration, neutrophils undergo a number of phenotypic changes resulting from encounters with endothelium-derived factors. This report uses an in vitro model with human umbilical vein endothelial cells and isolated human neutrophils to examine the effects of two locally derived cytokines, granulocyte (G)-macrophage (M) colony-stimulating factor (GM-CSF) and G-CSF, on oncostatin M (OSM) expression. Neutrophils contacting activated HUVEC expressed and released increased amounts of oncostatin M (OSM), a proinflammatory cytokine known to induce polymorphonuclear neutrophil adhesion and chemotaxis. Neutrophil transendothelial migration resulted in threefold higher OSM expression and protein levels compared with nontransmigrated cells. Addition of anti-GM-CSF neutralizing antibody reduced OSM expression level but anti-G-CSF was without effect. GM-CSF but not G-CSF protein addition to cultures of isolated neutrophils resulted in a significant increase in OSM protein secretion. However, inhibition of β(2) integrins by neutralizing antibody significantly reduced GM-CSF-induced OSM production indicating this phenomenon is adhesion dependent. Thus cytokine-stimulated endothelial cells can produce sufficient quantities of GM-CSF to influence in an adhesion-dependent manner, the phenotypic characteristics of neutrophils resulting in the latter's transmigration. Both transmigration and adhesion phenomenon lead to increased production of OSM by neutrophils that then play a major role in inflammatory response.