Basement membrane collagen IV deficiency promotes abdominal aortic aneurysm formation.

Abdominal aortic aneurysm (AAA) is a complex disease which is incompletely accounted for. Basement membrane (BM) Collagen IV (COL4A1/A2) is abundant in the artery wall, and several lines of evidence indicate a protective role of baseline COL4A1/A2 in AAA development. Using Col4a1/a2 hemizygous knockout mice (Col4a1/a2+/-, 129Svj background) we show that partial Col4a1/a2 deficiency augmented AAA formation. Although unchallenged aortas were morphometrically and biomechanically unaffected by genotype, explorative proteomic analyses of aortas revealed a clear reduction in BM components and contractile vascular smooth muscle cell (VSMC) proteins, suggesting a central effect of the BM in maintaining VSMCs in the contractile phenotype. These findings were translated to human arteries by showing that COL4A1/A2 correlated to BM proteins and VSMC markers in non-lesioned internal mammary arteries obtained from coronary artery bypass procedures. Moreover, in human AAA tissue, MYH11 (VSMC marker) was depleted in areas of reduced COL4 as assessed by immunohistochemistry. Finally, circulating COL4A1 degradation fragments correlated with AAA progression in the largest Danish AAA cohort, suggesting COL4A1/A2 proteolysis to be an important feature of AAA formation. In sum, we identify COL4A1/A2 as a critical regulator of VSMC phenotype and a protective factor in AAA formation.

Taste receptor cells express pH-sensitive leak K+ channels.

Two-pore domain K+ channels encoded by genes KCNK1-17 (K2p1-17) play important roles in regulating cell excitability. We report here that rat taste receptor cells (TRCs) highly express TASK-2 (KCNK5; K2p5.1), and to a much lesser extent TALK-1 (KCNK16; K2p16.1) and TASK-1 (KCNK3; K2p3.1), and suggest potentially important roles for these channels in setting resting membrane potentials and in sour taste transduction. Whole cell recordings of isolated TRCs show that a leak K+ (Kleak) current in a subset of TRCs exhibited high sensitivity to acidic extracellular pH similar to reported properties of TASK-2 and TALK-1 channels. A drop in bath pH from 7.4 to 6 suppressed 90% of the current, resulting in membrane depolarization. K+ channel blockers, BaCl2, but not tetraethylammonium (TEA), inhibited the current. Interestingly, resting potentials of these TRCs averaged -70 mV, which closely correlated with the amplitude of the pH-sensitive Kleak, suggesting a dominant role of this conductance in setting resting potentials. RT-PCR assays followed by sequencing of PCR products showed that TASK-1, TASK-2, and a functionally similar channel, TALK-1, were expressed in all three types of lingual taste buds. To verify expression of TASK channels, we labeled taste tissue with antibodies against TASK-1, TASK-2, and TASK-3. Strong labeling was seen in some TRCs with antibody against TASK-2 but not TASK-1 and TASK-3. Consistent with the immunocytochemical staining, quantitative real-time PCR assays showed that the message for TASK-2 was expressed at significantly higher levels (10-100 times greater) than was TASK-1, TALK-1, or TASK-3. Thus several K2P channels, and in particular TASK-2, are expressed in rat TRCs, where they may contribute to the establishment of resting potentials and sour reception.

A Study on the Phylogeny of the Dyer's Woad Rust Fungus and Other Species of Puccinia from Crucifers.

ABSTRACT The identity of a Puccinia species occurring on the introduced weed dyer's woad (Isatis tinctoria) was studied using sequences from the internal transcribed spacer of the nuclear ribosomal DNA. The relationship of this fungus to other Puccinia species occurring on the family Brassicaceae in Europe and North America was examined, and we tested the hypothesis that P. thlaspeos and P. monoica are correlated species. The data suggest that the Puccinia species from dyer's woad is closely related to the North American species P. consimilis and may be derived from an indigenous strain of P. consimilis that switched hosts. Thus, the Puccinia species from dyer's woad is probably native to North America and is unlikely to cause disease epidemics on indigenous plants if used as a biological control agent against dyer's woad. P. thlaspeos appears to be polyphyletic and, therefore, P. thlaspeos and P. monoica do not appear to be correlated species. Additional DNA sequence data will be needed to clarify further the phylogeny of Puccinia species on the family Brassicaceae.

Membrane-associated replication of an unencapsidated double-strand RNA of the fungus, Cryphonectria parasitica.

Fungal vesicles isolated from a hypovirulent strain (EP113) of the chestnut blight fungus, Cryphonectria parasitica, contained double-stranded RNA and possessed an RNA-dependent RNA polymerase activity which was absent in comparable preparations from dsRNA-free vesicles of a virulent strain (EP 155). RNA polymerase activity remained associated with hypovirulent vesicles when these were sedimented through a 10 to 40% sucrose gradient and the polymerase activity coincided with the peak of dsRNA content. Incorporation of [32P]-UTP into RNA was proportional to the amount of vesicles present in the reaction mixture. Enzyme activity was dependent upon the presence of dsRNA-containing vesicles, Mg2+ and the four ribonucleotide triphosphates, and was insensitive to inhibitors of DNA-dependent RNA polymerases. The optimum temperature for polymerase activity was 30 degrees, and temperatures higher than 35 degrees inactivated the enzyme. Treatment of vesicles with low concentrations of detergent led to a two- to threefold increase in the rate of RNA synthesis. The RNA polymerase products, synthesized in vitro, hybridized specifically with C. parasitica genomic dsRNAs. Hybridization to single-stranded cDNA clones containing inserts of the terminal domains of the homopolymer and heteropolymer ends of the dsRNA showed that the reaction products were full-length copies of both strands of the dsRNA. Single-stranded RNA synthesis was asymmetrical, with greater than 80% of the polymerase products being of positive polarity. It can be estimated that in the fungal vesicles isolated from hypovirulent C. parasitica, transcription of the dsRNA into mRNA for translation is in the order of two- to eightfold more active than replication. On the basis of our results and of the evidence accumulated so far, we suggest that the replication strategy employed by the hypovirulence-associated dsRNA is following that of positive-strand RNA viruses.