Key role of estrogens and endothelial estrogen receptor α in blood flow-mediated remodeling of resistance arteries.

OBJECTIVE: Flow- (shear stress-)mediated outward remodeling of resistance arteries is involved in collateral growth during postischemic revascularization. As this remodeling is especially important during pregnancy, we hypothesized that estrogens may be involved. A surgical model eliciting a local increase in blood flow in 1 mesenteric resistance artery was used in 3-month-old ovariectomized female rats either treated with 17-ß-estradiol (E2) or left untreated. METHODS AND RESULTS: After 14 days, arterial diameter was greater in high-flow arteries than in normal-flow vessels. An ovariectomy suppressed high-flow remodeling, while E2 restored it. High-flow remodeling was absent in mice lacking the estrogen receptor α but not estrogen receptor ß. The kinetics of inflammatory marker expression, macrophage infiltration, oxidative stress, and metaloproteinases expression were not altered by the absence of E2 after 2 and 4 days, that is, during remodeling. Nevertheless, E2 was required for the increase in endothelial nitric oxide synthase expression and activation at day 4 when diameter expansion occurs. Finally, the impact of E2 on the endothelium appeared crucial for high-flow remodeling, as this E2 action was abrogated in mice lacking endothelial NOS, as well as in Tie2-Cre(+) ERα(f/f) mice. CONCLUSIONS: We demonstrate the essential role of E2 and endothelial estrogen receptor α in flow-mediated remodeling of resistance arteries in vivo.

Identification of the ectomycorrhizal basidiomycete Tylospora fibrillosa Donk by RFLP analysis of the PCR-amplified ITS and IGS regions of ribosomal DNA.

Sitka spruce mycorrhizas, macroscopically identified as being formed by Tylospora fibritiosa Donk, were sampled from a young and on old plantation and the mycobionts were isolated into pure culture. DNA was extracted and fragments of the ribosomal DNA (rDNA) were amplified using the polymerase chain reaction (PCR). The primers were directed to conserved regions of fungal rDXA and hybridize to a wide range of fungi. One amplified region includes the internal spacer (ITS) region which has a low degree of conservation. The JTS amplification products, which were approximately 600 base pairs (bp), were digested with a variety of restriction endonucleases in order to detect restriction fragment length polymorphisms (RFLPs). The RFLPs clearly separated T. fibrillosa from other ectomycorrhizal species but there were only minor differences between the T. fibrillosa isolates. PCR amplification of the ITS region, digestion with the endonudeasc HinfI and examination of the RFLPs produced proved to be a rapid method by which to distinguish T. fibriHosa from a large number of other basidiomyictes. The method was also applied to DNA extracted, from single mycotrhizal root tips. The imergenic spacer region (1GS) of the rDNA is more variable than the ITS region in several fungal species. The 5'end of the 25S and the intergenic region between the 25S and the 5S genes were amplified and analyzed as above. Polymorphisms between T. fibritiosa isolates within this region were limited and RFLPs were not useful m discriminating between isolates, suggesting a low genetic variability in this species.

Automated ribosomal DNA fingerprinting by capillary electrophoresis of PCR products.

Capillary electrophoresis (CE) provides a rapid and automated technique for the analysis of subnanogram amounts of DNA fragments generated by the polymerase chain reaction (PCR). Here we describe the implementation of size-selective CE for DNA profiling and restriction fragment length polymorphism analysis of amplified polymorphic spacers of ribosomal DNA from fungi. Separations of unpurified and isopropanol-precipitated PCR-amplified DNA fragments in the size range of 20-1600 base pairs were achieved in less than 20 min with the use of hydroxypropyl methylcellulose as a sieving medium. The amplified internal transcribed spacer (ITS) and intergenic spacer (IGS) of RNA genes could be sized by coelectrophoresing a standard size ladder mixed with every sample, thereby eliminating errors in size estimation. This, together with the strictly controlled conditions of CE, permit the discrimination of amplified rDNA fragments differing only a few base pairs in length. Inter- and intraspecific variation in the size and number of restriction sites of the amplified rDNA spacers from the ectomycorrhizal basidiomycetes Laccaria laccata and Laccaria bicolor was observed and most strains could thus be reliably genotyped by PCR-CE. Multiple amplified IGS fragments of heterogeneous size were detected in several strains. This polymorphism is due to the occurrence of 5S rDNA subrepeats (i.e., multiple annealing of primer) within IGS. With CE, in contrast to slab gel electrophoresis, run times are short, the capillary can be reused, and full automation is feasible. Data acquisition and analysis are computer-controlled, which facilitates the locus identification and reduces error especially when large numbers of PCR products must be analyzed.(ABSTRACT TRUNCATED AT 250 WORDS)

Rapid identification of genetic variation of ectomycorrhizal fungi by amplification of ribosomal RNA genes.

There is a clear requirement to develop sensitive methods for detecting denned isolates of ectomycorrhizal fungi within the complex microbial communities of natural ecosystems and reforestation sites. We present a method that permits the rapid identification of an ectomycorrhizal isolate using enzymatic amplification (polymerase chain reaction) of DNA extracted either from pure cultures or ectomycorrhizas. A set of oligonucleotide primers capable of amplifying full-length nuclear 17S and 25S ribosomal RNA genes, together with the ribosomal internal transcribed spacer and intergenic spacer, have been designed and could be used for amplifying target sequences from a wide range of ectomycorrhizal genera. Length polymorphism in the amplified rDNA and restriction endonuclease analysis of nearly 6-0 kbp of amplified rDNA provided useful criteria for the rapid typing of isolates from different genera and species. Restriction endonuclease analysis of amplified DNA from 26 isolates representing four species of Laccaria (L. bicolor, L. laccata, L. proxima, L. tortilis) yielded up to 20 scored RFLPs and revealed interspecific and intraspecific polymorphism. Most of the polymorphisms were located within the regions corresponding to the internal transcribed spacer and intergenic spacer. The degree of variation observed was sufficient to discriminate several isolates from the same species. Genetic variation was correlated to some extent with geographical origin of the isolates. However, RFLPs of the rRNA genes cannot unambiguously discriminate all selected isolates within Laccaria species, requiring the development of additional DNA probes. Alone, or in combination with other DNA probes, the amplified rDNA genes may serve in the determination of pure fungal cultures and in the characterization of genetic variation of field ectomycorrhizal populations.