IL-10 inhibits vascular smooth muscle cell activation in vitro and in vivo

The anti-inflammatory cytokine IL-10 inhibits intimal hyperplasia after stent implantation via a powerful inactivation of monocytes. We tested the hypothesis that IL-10 may also inhibit vascular smooth muscle cell (SMC) activation via the inhibition of the NF-kappaB/I-kappaB system. The IL-10 receptor was detected in rat SMCs in vitro and in vivo. In LPS-stimulated rat SMCs, 1 ng/ml recombinant murine IL-10 (mIL-10) reduced I-kappaBalpha and I-kappaBbeta degradation, NF-kappaB activation, as well as the expression of the NF-kappaB-dependent gene IL-6 by 32%, 31%, 75%, and 19%, respectively (P < 0.05 for all). Similar results were obtained in vivo 6 h and 4 days after balloon abrasion of the rat aorta, a model in which intimal hyperplasia results essentially from SMC activation. Moreover, mIL-10 reduced SMC proliferation and migration in vitro (by 60% for both, P < 0.0001), resulting in reduced SMC proliferation and intimal growth 14 days after balloon abrasion of the rat aorta (by 76% and 75%, respectively; P < 0.005). In conclusion, mIL-10 has a direct inhibitory effect on SMCs in vitro and in vivo. This effect is mediated in part by NF-kappaB inactivation and may participate in the overall protective effect of IL-10 on postangioplasty restenosis.     

Cadmium-responsive thiols in the ectomycorrhizal fungus Paxillus involutus

Molecular and cellular mechanisms underlying the sustained metal tolerance of ectomycorrhizal fungi are largely unknown. Some of the main mechanisms involved in metal detoxification appear to involve the chelation of metal ions in the cytosol with thiol-containing compounds, such as glutathione, phytochelatins, or metallothioneins. We used an improved high-performance liquid chromatography method for the simultaneous measurement of thiol-containing compounds from cysteine and its derivatives (gamma-glutamylcysteine, glutathione) to higher-molecular-mass compounds (phytochelatins). We found that glutathione and gamma-glutamylcysteine contents increased when the ectomycorrhizal fungus Paxillus involutus was exposed to cadmium. An additional compound with a 3-kDa molecular mass, most probably related to a metallothionein, increased drastically in mycelia exposed to cadmium. The relative lack of phytochelatins and the presence of a putative metallothionein suggest that ectomycorrhizal fungi may use a different means to tolerate heavy metals, such as Cd, than do their plant hosts.     

A new dominant selection marker for transformation of Pichia pastoris to soraphen A resistance

Despite the considerable progress molecular genetics of filamentous fungi has made during the past decade, there is still an urgent need for efficiently working selectable markers for fungal transformation. Using Pichia pastoris as a host, we describe the development of a new dominant selectable marker of prokaryotic origin. This system, termed sor(R), is based upon the resistance of the bacterial enzyme acetyl-CoA carboxylase (ACCase) to the macrocyclic polyketide soraphen A, a potent inhibitor of fungal ACCase produced by the myxobacterium Sorangium cellulosum. In this study, we firstly demonstrate that the integration of a single sor(R) cassette into the P. pastoris genome confers resistance to elevated concentrations of soraphen A. Furthermore, it has been shown that the versatility of this marker can be considerably increased by splitting the sor(R) cassette, especially when successive transformations are performed on the same strain. As pronounced sensitivity to soraphen A is the rule among filamentous fungi, we expect the sor(R) marker to be a widely applicable tool for fungal transformation.     

Cooperative strand invasion of supercoiled plasmid DNA by mixed linear PNA and PNA-peptide chimeras

Peptide nucleic acid (PNA) is a DNA analog with broad biotechnical applications, and possibly also treatment applications. Its suggested uses include that of a specific anchor sequence for biologically active peptides to plasmids in a sequence-specific manner. Such complexes, referred to as Bioplex, have already been used to enhance non-viral gene transfer in vitro. To investigate how hybridization of PNAs to supercoiled plasmids would be affected by the binding of multiple PNA-peptides to the same strand of DNA, we have developed a method of quantifying the specific binding of PNA using a PNA labeled with a derivative of the fluorophore thiazole orange (TO). Cooperative effects were found at a distance of up to three bases. With a peptide present at the end of one of the PNAs, steric hindrance occurred, reducing the increase in binding rate when the distance between the two sites was less than two bases. In addition, we found increased binding kinetics when two PNAs binding to overlapping sites on opposite DNA strands were used, without the use of chemically modified bases in the PNAs.     

Correspondence between anomalous m- and DeltaCp-values in protein folding

Proteins folding according to a classical two-state system characteristically show V-shaped chevron plots. We have previously interpreted the symmetrically curved chevron plot of the protein U1A as denaturant-dependent movements in the position of the transition state ensemble (TSE). S6, a structural analog of U1A, shows a classical V-shaped chevron plot indicative of straightforward two-state kinetics, but the mutant LA30 has a curved unfolding limb, which is most consistent with TSE mobility. The kinetic m-values (derivatives of the rate constants with respect to denaturant concentration) in themselves depend on denaturant concentration. To obtain complementary information about putative mobile TSEs, we have carried out a thermodynamic analysis of the three proteins, based on data for refolding and unfolding over the range 10 degrees C to 70 degrees C. The data at all temperatures can be fitted to two-state model systems. Importantly, for all three proteins the activation heat capacities are, within error, identical to the heat capacities measured in independent experiments under equilibrium conditions. Although the equilibrium heat capacities are essentially invariant with regard to denaturant concentration, the activation heat capacities, similar to the structurally equivalent kinetic m-values, show marked denaturant dependence. Furthermore, the values of beta++ at different denaturant concentrations measured by m-values and by heat capacity values are very similar. These observations are consistent with significant transition state movements within the framework of two-state folding. The basis for TSE movement appears to be enthalpic rather than entropic, suggesting that the binding energy of denaturant-protein interactions is a major determinant of the response of energy landscape contours to changing environments.     

Historical climate change and speciation: neotropical seasonally dry forest plants show patterns of both tertiary and quaternary diversification

Historical climate changes have had a major effect on the distribution and evolution of plant species in the neotropics. What is more controversial is whether relatively recent Pleistocene climatic changes have driven speciation, or whether neotropical species diversity is more ancient. This question is addressed using evolutionary rate analysis of sequence data of nuclear ribosomal internal transcribed spacers in diverse taxa occupying neotropical seasonally dry forests, including Ruprechtia (Polygonaceae), robinioid legumes (Fabaceae), Chaetocalyx and Nissolia (Fabaceae), and Loxopterygium (Anacardiaceae). Species diversifications in these taxa occurred both during and before the Pleistocene in Central America, but were primarily pre-Pleistocene in South America. This indicates plausibility both for models that predict tropical species diversity to be recent and that invoke a role for Pleistocene climatic change, and those that consider it ancient and implicate geological factors such as the Andean orogeny and the closure of the Panama Isthmus. Cladistic vicariance analysis was attempted to identify common factors underlying evolution in these groups. In spite of the similar Mid-Miocene to Pliocene ages of the study taxa, and their high degree of endemism in the different fragments of South American dry forests, the analysis yielded equivocal, non-robust patterns of area relationships.     

Differential mesowear in occluding upper and lower molars: opening mesowear analysis for lower molars and premolars in hypsodont horses

A new approach of reconstructing ungulate diet, the mesowear method, was recently introduced by Fortelius and Solounias ([2000] Am Mus Novitat 3301:1-36). Mesowear is based on facet development on the occlusal surfaces of the teeth. Restricting mesowear investigation to maxillary cheek teeth would allow mesowear investigation only in assemblages of large numbers of individuals and therefore would generally restrict this method to relatively few assemblages of recent and fossil ungulates. Most of the fossil, subfossil, and recent ungulate osteological assemblages that may be assigned to a single taxon have smaller numbers of individuals. This results in a demand to extend the mesowear method to further tooth positions in order to obtain stable dietary classifications of fossil taxa. The focus of this article is to test if a consistent mesowear classification is obtainable for mandibular as well as for maxillary teeth. For statistical testing, large assemblages of isolated cheek teeth of the Vallesian hipparionine horse Hippotherium primigenium and of the recent zebra Equus burchelli were employed as models. The upper tooth positions P4, M1, M2, and M3 as suggested by Kaiser and Solounias (2003) as the model for the "extended" mesowear method and the lower tooth positions P4-M3 were tested for their consistency in classification of the mesowear variables. We found a considerable shift of the mesowear signature towards the grazing edge of the mesowear continuum in lower cheek teeth. In order to adjust the signal of lower teeth to the signal of the upper teeth, a calibration factor was introduced which allowed incorporation of lower cheek teeth into the same model of mesowear investigation together with upper cheek teeth. We propose that this model is particularly suited for the reconstruction of paleodiets in hypsodont hipparionine and equine equids. We further investigated the functional relation between the mesowear profiles and the distribution of dental tissues along the course of the occlusal contact. We therefore correlated mesowear profiles with enamel distribution profiles and found the mesowear profile to be strongly controlled by the attritional environment encountered by a given apex area. The differential signal observed in cusp apex morphology between upper and lower cheek teeth was found to be more closely related to attrition by the antagonistic tooth than to the distribution of dental tissues in the tooth under consideration. The results suggest a general extension of the mesowear method of paleodiet reconstruction and a basic scenario for the evolution of anisodont dentitions.     

Bird orientation: compensation for wind drift in migrating raptors is age dependent

Despite the potentially strong effect of wind on bird orientation, our understanding of how wind drift affects migrating birds is still very limited. Using data from satellite-based radio telemetry, we analysed the effect of changing winds on the variation of the track direction of individual birds. We studied adults and juveniles of two raptor species, osprey Pandion haliaetus and honey buzzard Pernis apivorus, on autumn migration between North Europe and Africa, and demonstrate an important difference between the age categories of both species in the extent of wind drift. For juveniles, side- and following-wind components affected the rates of movement perpendicular to and along the mean direction, respectively, to a similar degree, suggesting full wind drift. By contrast, for adults the rate of crosswind displacement was significantly smaller than the effect of wind on forward movement, showing much reduced wind drift (29%). This indicates that adults have acquired a more sophisticated orientation system, permitting detection of and compensation for wind drift, than juveniles. These drift effects are likely to reduce the ability of juveniles to locate species-specific wintering areas in case of rapid climatic wind change.     

A phospholipase D-dependent process forms lipid droplets containing caveolin,adipocyte differentiation-related protein,and vimentin in a cell-free system

We developed a microsome-based, cell-free system that assembles newly formed triglyceride (TG) into spherical lipid droplets. These droplets were recovered in the d 相似文献   

The inhibitor thiomandelic acid binds to both metal ions in metallo-beta-lactamase and induces positive cooperativity in metal binding

Thiomandelic acid is a simple, broad spectrum, and reasonably potent inhibitor of metallo-beta-lactamases, enzymes that mediate resistance to beta-lactam antibiotics. We report studies by NMR and perturbed angular correlation (PAC) spectroscopy of the mode of binding of the R and S enantiomers of thiomandelic acid, focusing on their interaction with the two metal ions in cadmium-substituted Bacillus cereus metallo-beta-lactamase. The 113Cd resonances are specifically assigned to the metals in the two individual sites on the protein by using 113Cd-edited 1H NMR spectra. Each enantiomer of thiomandelate produces large downfield shifts of both 113Cd resonances and changes in the PAC spectra, which indicate that they bind such that the thiol of the inhibitor bridges between the two metals. For R-thiomandelate, this is unambiguously confirmed by the observation of scalar coupling between Halpha of the inhibitor and both cadmium ions. The NMR and PAC spectra reveal that the two chiral forms of the inhibitor differ in the details of their coordination geometry. The complex with R-thiomandelate, but not that with the S-enantiomer, shows evidence in the PAC spectra of a dynamic process in the nanosecond time regime, the possible nature of which is discussed. The thiomandelate complex of the mononuclear enzyme can be detected only at low metal to enzyme stoichiometry; the relative populations of mononuclear and binuclear enzyme as a function of cadmium concentration provide clear evidence for positive cooperativity in metal ion binding in the presence of the inhibitor, in contrast to the negative cooperativity observed in the free enzyme.