Induction of apoptosis and activation of JNK and p38 MAPK pathways in deoxynivalenol-treated cell lines

Deoxynivalenol (DON) is a mycotoxin produced by what are thought to be the most prevalent toxin-producing fungi of the Fusarium genus. Here, we present the results of apoptosis induction, phosphorylation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinases (MAPKs), and expression of the c-Jun protein after DON treatment, in a pre-B lymphocyte REH cell line. In addition, human pre-T lymphocyte Jurkat, hamster kidney-derived BHK21 and mouse hepatoma MH-22a cells were used in comparative experiments in vitro. We found that the DON effect was cell origin-dependent and dose-dependent, with a significant slow-down of cell proliferation and increase of apoptotic cells in blood cell lines. BHK21 and MH-22a cells were less sensitive to the DON effect. In blood-derived REH and Jurkat cells, DON-induced apoptotic changes were preceded by an increase in JNK and p38 MAPKs phosphorylation, as well as in c-Jun expression. However, the activation of JNK phosphorylation and c-Jun expression were transient, but did not coincide with each other. An inhibitor of JNK1/2, SP600125, had a negligible negative effect on REH cell viability after DON treatment, demonstrating that JNK does not contribute to DON-induced apoptosis. In contrast, studies on the role of p38 MAPK revealed that p38 signalling is required for DON-induced apoptosis in REH cells.     

Posture, gait and the ecological relevance of locomotor costs and energy-saving mechanisms in tetrapods

A reanalysis of locomotor data from functional, energetic, mechanical and ecological perspectives reveals that limb posture has major effects on limb biomechanics, energy-saving mechanisms and the costs of locomotion. Regressions of data coded by posture (crouched vs. erect) reveal nonlinear patterns in metabolic cost, limb muscle mass, effective mechanical advantage, and stride characteristics. In small crouched animals energy savings from spring and pendular mechanisms are inconsequential and thus the metabolic cost of locomotion is driven by muscle activation costs. Stride frequency appears to be the principal functional parameter related to the decreasing cost of locomotion in crouched animals. By contrast, the shift to erect limb postures invoked a series of correlated effects on the metabolic cost of locomotion: effective mechanical advantage increases, relative muscle masses decrease, metapodial limb segments elongate dramatically (as limbs shift from digitigrade to unguligrade designs) and biological springs increase in size and effectiveness. Each of these factors leads to decreases in the metabolic cost of locomotion in erect forms resulting from real and increasing contributions of pendular savings and spring savings. Comparisons of the relative costs and ecological relevance of different gaits reveal that running is cheaper than walking in smaller animals up to the size of dogs but running is more expensive than walking in horses. Animals do not necessarily use their cheapest gaits for their predominant locomotor activity. Therefore, locomotor costs are driven more by ecological relevance than by the need to optimize locomotor economy.     

Seed treatment with lactic acid bacteria against seed-borne pathogens of spring wheat

Antifungal potential of lactic acid bacteria (LAB) such as Lactobacillus sakei KTU05-6, Pediococcus acidilactici KTU05-7 and Pediococcus pentosaceus KTU05-8, KTU05-9 and KTU05-10 was tested on naturally contaminated wheat seeds. LAB influence on fungal growth on kernels, seedling diseases and seed germination was examined by laboratory and field experiments. KTU05-10 was selected and later used for seed treatment as solitary strain and in two- or three-component mixtures with KTU05-7 and KTU05-6. The occurrence of Fusarium spp. on wheat kernels in agar plate assays was decreased by seed treatment with all LAB cultures, and the efficacy of each strain depended on incubation temperature. Inoculation of wheat kernels with strains of solitary KTU05-10 and in mixtures with KTU05-7 and KTU05-6 significantly reduced the incidence of Fusarium spp., Bipolaris sorokiniana and Alternaria spp. LAB influence on seed germination and seedling diseases was observed in laboratory and field experiments, but in most cases, this influence was insignificant.     

Prooxidant cytotoxicity of chromate in mammalian cells: the opposite roles of DT-diaphorase and glutathione reductase

The geno- and cytotoxicity of chromate, an important environmental pollutant, is partly attributed to the flavoenzyme-catalyzed reduction with the concomitant formation of reactive oxygen species. The aim of this work was to characterize the role of NAD(P)H:quinone oxidoreductase (NQO1, DT-diaphorase, EC 1.6.99.2) and glutathione reductase (GR, EC 1.6.4.2) in the mammalian cell cytotoxicity of chromate, which was evidenced controversially so far. The chromate reductase activity of NQO1 was higher than that of GR, but lower than that of lipoamide dehydrogenase (EC 1.6.4.3), ferredoxin:NADP+ reductase (EC 1.18.1.2), and NADPH: cytochrome P-450 reductase (EC 1.6.2.4). The reduction of chromate by NQO1 was accompanied by the formation of reactive oxygen species. The concentration of chromate for 50% survival of bovine leukemia virus-transformed lamb kidney fibroblasts (line FLK) during a 24-h incubation was (22 +/- 4) microM. The cytotoxicity was partly prevented by desferrioxamine, the antioxidant N,N'-diphenyl-p-phenylene diamine and by an inhibitor of NQO1, dicumarol, and potentiated by 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU), which inactivates GR. The NADPH-dependent chromate reduction by digitonin-permeabilized FLK cells was partly inhibited by dicumarol and not affected by BCNU. Taken together, these data indicate that the oxidative stress-type cytotoxicity of chromate in FLK cells may be partly attributed to its reduction by NQO1, but not by GR. The effect of BCNU on the chromate cytotoxicity may indicate that the general antioxidant action of reduced glutathione is more important than its prooxidant activities arising from the reactions with chromate.     

Engrafting fetal liver cells into multiple tissues of healthy adult mice without the use of immunosuppressants

We have shown the fetal liver cell engraftments into multiple tissues of adult healthy mice, achieved without suppressing the animals’ immune systems. Fetal cells from the livers of male C57Bl/6J Black lineage mice at day 13 to 15 of gestation were injected intravenously into female adult CC57W/MY White mice. The grafting was evaluated by Y-chromosome-specific PCR, cytometric analysis of fluorescently stained donor cells, and histological analysis. All the methods consistently showed the presence of multiple engraftments randomly distributed through the various organs of the recipients. After 60 days, the grafts still constituted 0.1 to 2.75% of the tissues. The grafted cells did not change their appearance in any of the organs except the brain, where they became enlarged. Inflammatory reactions were not detected in any of the histological preparations. The frequency of engraftments was higher in the liver, indicating that similarity between the donor and recipient cells facilitates engraftment. The high inherent plasticity of fetal liver cells underlies their ability to integrate into healthy recipient organs, which can be governed by environmental conditions and connections with neighboring cells rather than by the initial cellular developmental programs. The fact that fetal liver cells can be grafted into multiple tissues of healthy animals indicates that they can be used to replace the natural loss of cells in adult organisms.     

Skeletal allometry and interlimb scaling patterns in mustelid carnivorans

To address the effects of an evolutionary increase in body size on long bone skeletal allometry, scaling patterns relating body mass, bone length, limb length, midshaft diameters, and cross-sectional properties of the humerus and femur were analyzed for four species of scansorial mustelids. Humeral and, to a lesser extent, femoral allometry is consistent with expectations of elastic similarity: bone and limb length scale with negative allometry on body mass while bone robusticity (cross-sectional parameters against bone length) scales with strong positive allometry. Differences between fore- and hindlimb scaling patterns, however, are observed, with size-dependent increases in forelimb length and humeral strength and robusticity exceeding those of the hindlimb and femur. It is hypothesized that this greater fore- than hindlimb lengthening results in postural modifications that serve to straighten the hindlimb of larger bodied scansorial mustelids relative to smaller mustelids. Straightening of hindlimb joints would more precisely align the long axis of the femur with peak (vertical) ground reaction forces, thereby accounting for the reduction in relative bending stresses acting on the femur compared to the humerus. J. Morphol. 235:121–134, 1998. © 1998 Wiley-Liss, Inc.     

Enzymatic redox properties of novel nitrotriazole explosives implications for their toxicity

The toxicity of conventional nitroaromatic explosives like 2,4,6-trinitrotoluene (TNT) is caused by their enzymatic free radical formation with the subsequent oxidative stress, the formation of alkylating nitroso and/or hydroxylamino metabolites, and oxyhemoglobin oxidation into methemoglobin. In order to get an insight into the mechanisms of toxicity of the novel explosives NTO (5-nitro-1,2,4-triazol-3-one) and ANTA (5-nitro-1,2,4-triazol-3-amine), we examined their reactions with the single-electron transferring flavoenzymes NADPH: cytochrome P-450 reductase and ferredoxin:NADP+ reductase, two-electron transferring flavoenzymes mammalian NAD(P)H:quinone oxidoreductase (DT-diaphorase), and Enterobacter cloacae NAD(P)H:nitroreductase, and their reactions with oxyhemoglobin. The reactivity of NTO and ANTA in the above reactions was markedly lower than that of TNT. The toxicity of NTO and ANTA in bovine leukemia virus-transformed lamb kidney fibroblasts (line FLK) was partly prevented by desferrioxamine and the antioxidant N,N'-diphenyl-p-phenylene diamine, and potentiated by 1,3-bis-(2-chloroethyl)-1-nitrosourea. This points to the involvement of oxidative stress in their cytotoxicity, presumably to the redox cycling of free radicals. The FLK cell line cytotoxicity and the methemoglobin formation in isolated human erythrocytes of NTO and ANTA were also markedly lower than those of TNT, and similar to those of nitrobenzene. Taken together, our data demonstrate that the low toxicity of nitrotriazole explosives may be attributed to their low electron-accepting properties.     

Enzymatic redox reactions of the explosive 4,6-dinitrobenzofuroxan (DNBF): implications for its toxic action

With an aim to understand the toxicity mechanisms of the explosive 4,6-dinitro- benzofuroxan (DNBF), we studied its single-electron reduction by NADPH:cytochrome P450 reductase and ferredoxin:NADP(+) reductase, and two- electron reduction by DT-diaphorase and Enterobacter cloacae nitroreductase. The enzymatic reactivities of DNBF and another explosive 2,4,6-trinitrotoluene (TNT) were similar, except for the much lower reactivity of DNBF towards nitroreductase. DNBF was less cytotoxic in FLK cells than TNT. However, their action shared the same mechanisms, oxidative stress and activation by DT-diaphorase. The lower cytotoxicity of DNBF may be explained by the negative electrostatic charge of its adduct with water which may impede cellular membrane penetration, and by the formation of its less reactive adducts with intracellular reduced glutathione.     

Abdominal muscle and epipubic bone function during locomotion in Australian possums: Insights to basal mammalian conditions and eutherian‐like tendencies in Trichosurus

Mammals have four hypaxial muscle layers that wrap around the abdomen between the pelvis, ribcage, and spine. However, the marsupials have epipubic bones extending anteriorly into the ventral hypaxial layers with two additional muscles extending to the ventral midline and femur. Comparisons of South American marsupials to basal eutherians have shown that all of the abdominal hypaxials are active bilaterally in resting ventilation. However, during locomotion marsupials employ an asymmetrical pattern of activity as the hypaxial muscles form a crosscouplet linkage that uses the epipubic bone as a lever to provide long‐axis support of the body between diagonal limb couplets during each step. In basal eutherians, this system shifts off the femur and epipubic bones (which are lost) resulting in a shoulder to pelvis linkage associated with shifts in both the positions and activity patterns of the pectineus and rectus abdominis muscles during locomotion. In this study, we present data on hypaxial function in two species (Pseudocheirus peregrinus and Trichosurus vulpecula) representing the two major radiations of possums in Australia: the Pseudocheiridae (within the Petauroidea) and the Phalangeridae. Patterns of gait, motor activity, and morphology in these two Australian species were compared with previous work to examine the generality of 1) the crosscouplet lever system as the basal condition for the Marsupialia and 2) several traits hypothesized to be common to all mammals (hypaxial tonus during resting ventilation, ventilation to step synchrony during locomotion, and bilateral transversus abdominis activity during locomotor expiration). Our results validate the presence of the crosscouplet pattern and basic epipubic bone lever system in Australian possums and confirm the generality of basal mammalian patterns. However, several novelties discovered in Trichosurus, reveal that it exhibits an evolutionary transition to intermediate eutherian‐like morphological and motor patterns paralleling many other unique features of this species. J. Morphol., 2010. © 2009 Wiley‐Liss, Inc.