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161.
Effect of exogenous H(2)O(2) and catalase was tested in liquid cultures of the deoxynivalenol and 15-acetyldeoxynivalenol-producing fungus Fusarium graminearum. Accordingly to previous results, H(2)O(2) supplementation of the culture medium leads to increased toxin production. This study indicates that this event seems to be linked to a general up regulation of genes involved in the deoxynivalenol and 15-acetyldeoxynivalenol biosynthesis pathway, commonly named Tri genes. In catalase-treated cultures, toxin accumulation is reduced, and Tri genes expression is significantly down regulated. Furthermore, kinetics of expression of several Tri genes is proposed in relation to toxin accumulation. Biological meanings of these findings are discussed.  相似文献   
162.
Intestinal epithelial cells (IEC) play an immunoregulatory role in the intestine. This role involves cell-cell interactions with intraepithelial lymphocytes that may also play a role in some enteropathies. The discovery of the RGD motif-containing Protein ADAM-15 (a disintegrin and metalloprotease-15) raises the question of its involvement in these cell-cell interactions. Cell adhesion assays were performed using the Jurkat E6.1 T cell line as a model of T lymphocytes and Caco2-BBE monolayers as a model of intestinal epithelia. Our results show that an anti-ADAM-15 ectodomain antibody inhibited the attachment of Jurkat cells on Caco2-BBE monolayers. Overexpression of ADAM-15 in Caco2-BBE cells enhanced Jurkat cell binding, and overexpression of ADAM-15 in Jurkat cells enhanced their aggregation. Mutagenesis experiments showed that both the mutation of ADAM-15 RGD domain or the deletion of its cytoplasmic tail decreased these cell-cell interactions. Moreover, wound-healing experiments showed that epithelial ADAM-15-mediated Jurkat cell adhesion to Caco2-BBE cells enhances the mechanisms of wound repair. We also found that ADAM-15-mediated aggregation of Jurkat cells increases the expression of tumor necrosis factor-alpha mRNA. These results demonstrate the following: 1) ADAM-15 is involved in heterotypic adhesion of intraepithelial lymphocytes to IEC as well as in homotypic aggregation of T cells; 2) both the RGD motif and the cytoplasmic tail of ADAM-15 are involved for these cell-cell interactions; and 3) ADAM-15-mediated cell-cell interactions are involved in mechanisms of epithelial restitution and production of pro-inflammatory mediators. Altogether these findings point to ADAM-15 as a possible therapeutic target for prevention of inappropriate T cell activation involved in some pathologies.  相似文献   
163.
Mitochondria participate in key metabolic reactions of the cell and regulate crucial signaling pathways including apoptosis. Although several approaches are available to study mitochondrial function in situ are available, investigating functional mitochondria that have been isolated from different tissues and from cultured cells offers still more unmatched advantages. This protocol illustrates a step-by-step procedure to obtain functional mitochondria with high yield from cells grown in culture, liver and muscle. The isolation procedures described here require 1-2 hours, depending on the source of the organelles. The polarographic analysis can be completed in 1 hour.  相似文献   
164.
Insulin signalling is a very ancient and well conserved pathway in metazoan cells, dependent on insulin receptors (IR) which are transmembrane proteins with tyrosine kinase activity. A unique IR is usually present in invertebrates whereas two IR members are found with different functions in vertebrates. This work demonstrates the existence of two distinct IR homologs (SmIR-1 and SmIR-2) in the parasite trematode Schistosoma mansoni. These two receptors display differences in several structural motifs essential for signalling and are differentially expressed in parasite tissues, suggesting that they could have distinct functions. The gene organization of SmIR-1 and SmIR-2 is similar to that of the human IR and to that of the IR homolog from Echinococcus multilocularis (EmIR), another parasitic platyhelminth. SmIR-1 and SmIR-2 were shown to interact with human pro-insulin but not with pro-insulin-like growth factor-1 in two-hybrid assays. Phylogenetic results indicated that SmIR-2 and EmIR might be functional orthologs whereas SmIR-1 would have emerged to fulfil specific functions in schistosomes.  相似文献   
165.
The guanine nucleotide-binding protein Ras occurs in solution in two different conformational states, state 1 and state 2 with an equilibrium constant K(12) of 2.0, when the GTP analogue guanosine-5'-(beta,gamma-imido)triphosphate or guanosine-5'-(beta,gamma-methyleno)triphosphate is bound to the active centre. State 2 is assumed to represent a strong binding state for effectors with a conformation similar to that found for Ras complexed to effectors. In the other state (state 1), the switch regions of Ras are most probably dynamically disordered. Ras variants that exist predominantly in state 1 show a drastically reduced affinity to effectors. In contrast, Ras(wt) bound to the GTP analogue guanosine-5'-O-(3-thiotriphosphate) (GTPgammaS) leads to (31)P NMR spectra that indicate the prevalence of only one conformational state with K(12) > 10. Titration with the Ras-binding domain of Raf-kinase (Raf-RBD) shows that this state corresponds to effector binding state 2. In the GTPgammaS complex of the effector loop mutants Ras(T35S) and Ras(T35A) two conformational states different to state 2 are detected, which interconvert over a millisecond time scale. Binding studies with Raf-RBD suggest that both mutants exist mainly in low-affinity states 1a and 1b. From line-shape analysis of the spectra measured at various temperatures an activation energy DeltaH(|) (1a1b) of 61 kJ.mol(-1) and an activation entropy DeltaS(|) (1a1b) of 65 J.K(-1).mol(-1) are derived. Isothermal titration calorimetry on Ras bound to the different GTP-analogues shows that the effective affinity K(A) for the Raf-RBD to Ras(T35S) is reduced by a factor of about 20 compared to the wild-type with the strongest reduction observed for the GTPgammaS complex.  相似文献   
166.
A major gap in our knowledge of the evolution of marsupial mammals concerns the Paleogene of the northern continents, a critical time and place to link the early history of metatherians in Asia and North America with the more recent diversification in South America and Australia. We studied new exceptionally well-preserved partial skeletons of the Early Oligocene fossil Herpetotherium from the White River Formation in Wyoming, which allowed us to test the relationships of this taxon and examine its adaptations. Herpetotheriidae, with a fossil record extending from the Cretaceous to the Miocene, has traditionally been allied with opossums (Didelphidae) based on fragmentary material, mainly dentitions. Analysis of the new material reveals that several aspects of the cranial and postcranial anatomy, some of which suggests a terrestrial lifestyle, distinguish Herpetotherium from opossums. We found that Herpetotherium is the sister group to the crown group Marsupialia and is not a stem didelphid. Combination of the new palaeontological data with molecular divergence estimates, suggests the presence of a long undocumented gap in the fossil record of opossums extending some 45Myr from the Early Miocene to the Cretaceous.  相似文献   
167.
We purified an extracellular pyranose dehydrogenase (PDH) from the basidiomycete fungus Agaricus xanthoderma using ammonium sulfate fractionation and ion-exchange and hydrophobic interaction chromatography. The native enzyme is a monomeric glycoprotein (5% carbohydrate) containing a covalently bound FAD as its prosthetic group. The PDH polypeptide consists of 575 amino acids and has a molecular mass of 65 400 Da as determined by MALDI MS. On the basis of the primary structure of the mature protein, PDH is a member of the glucose-methanol-choline oxidoreductase family. We constructed a homology model of PDH using the 3D structure of glucose oxidase from Aspergillus niger as a template. This model suggests a novel type of bi-covalent flavinylation in PDH, 9-S-cysteinyl, 8-alpha-N3-histidyl FAD. The enzyme exhibits a broad sugar substrate tolerance, oxidizing structurally different aldopyranoses including monosaccharides and oligosaccharides as well as glycosides. Its preferred electron donor substrates are D-glucose, D-galactose, L-arabinose, and D-xylose. As shown by in situ NMR analysis, D-glucose and D-galactose are both oxidized at positions C2 and C3, yielding the corresponding didehydroaldoses (diketoaldoses) as the final reaction products. PDH shows no detectable activity with oxygen, and its reactivity towards electron acceptors is rather limited, reducing various substituted benzoquinones and complexed metal ions. The azino-bis-(3-ethylbenzthiazolin-6-sulfonic acid) cation radical and the ferricenium ion are the best electron acceptors, as judged by the catalytic efficiencies (k(cat)/K(m)). The enzyme may play a role in lignocellulose degradation.  相似文献   
168.
Habitat fragmentation often promotes increased inbreeding depression due to interrupted gene flow between populations. In this study, we demonstrate that Asian elephants most likely also suffer from outbreeding depression due to cryptic speciation. We analysed mitochondrial and nuclear DNA loci from 78 Asian elephants. Haplotype genealogy and analysis of molecular variance revealed two matrilinear clades (α h, β h). Microsatellite analyses of individuals grouped according to their haplotype clade (corresponding group of nuclear genotypes called α nuc and β nuc) revealed significant genotypic differentiation between α nuc and β nuc. Such genealogically differentiated forms in a morphologically uniform species are considered indicative of cryptic speciation. The differentiation was caused by bulls, whereas considering cows only resulted in no differentiation. Such result is best explained by Haldane’s rule whereby hybrid formation between genealogical forms causes lower viability and fertility of heterogametic hybrids. Although the lack of hybrid-specific morphological characteristics renders direct testing of reduced hybrid fitness under natural conditions unfeasible, the effects of Haldane’s rule are demonstrated by reduced male-mediated gene flow between genealogical forms under sympatric conditions, as was indeed suggested by the data found in Asian elephants: male-mediated gene flow between groups α nuc and β nuc was much lower than female-mediated gene flow. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.
Joerns FickelEmail:
  相似文献   
169.
Plasmodium undergoes one round of multiplication in the liver prior to invading erythrocytes and initiating the symptomatic blood phase of the malaria infection. Productive hepatocyte infection by sporozoites leads to the generation of thousands of merozoites capable of erythrocyte invasion. Merozoites are released from infected hepatocytes as merosomes, packets of hundreds of parasites surrounded by host cell membrane. Intravital microscopy of green fluorescent protein-expressing P. yoelii parasites showed that the majority of merosomes exit the liver intact, adapt a relatively uniform size of 12-18 microm, and contain 100-200 merozoites. Merosomes survived the subsequent passage through the right heart undamaged and accumulated in the lungs. Merosomes were absent from blood harvested from the left ventricle and from tail vein blood, indicating that the lungs effectively cleared the blood from all large parasite aggregates. Accordingly, merosomes were not detectable in major organs such as brain, kidney, and spleen. The failure of annexin V to label merosomes collected from hepatic effluent indicates that phosphatidylserine is not exposed on the surface of the merosome membrane suggesting the infected hepatocyte did not undergo apoptosis prior to merosome release. Merosomal merozoites continued to express green fluorescent protein and did not incorporate propidium iodide or YO-PRO-1 indicating parasite viability and an intact merosome membrane. Evidence of merosomal merozoite infectivity was provided by hepatic effluent containing merosomes being significantly more infective than blood with an identical low-level parasitemia. Ex vivo analysis showed that merosomes eventually disintegrate inside pulmonary capillaries, thus liberating merozoites into the bloodstream. We conclude that merosome packaging protects hepatic merozoites from phagocytic attack by sinusoidal Kupffer cells, and that release into the lung microvasculature enhances the chance of successful erythrocyte invasion. We believe this previously unknown part of the plasmodial life cycle ensures an effective transition from the liver to the blood phase of the malaria infection.  相似文献   
170.
Modulated differential scanning calorimetry, wide angle x-ray scattering, and environmental scanning electron microscopy were used to investigate the physical and morphological properties of chicory root inulin spray dried under different conditions. When the feed temperature increased up to 80 °C, the average degree of polymerization of the solubilized fraction increased, leading to a higher glass transition temperature (Tg). Above 80 °C, the samples were completely amorphous, and the Tg did not change. The starting material was semicrystalline, and the melting region was composed of a dual endotherm; the first peak subsided as the feed temperature increased up to a temperature of 70 °C, whereas above 80 °C, no melting peak was observed as the samples were completely amorphous. To a lesser extent, the inlet air temperature of 230 °C allowed a higher amorphous content of the samples than at 120–170 °C but induced a blow-out of the particles.  相似文献   
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