The effects of the Rho-kinase inhibitor, Y-27632 [1] on Ca P+ -sensitization of force induced by arachidonic acid (AA), phorbol 12,13-dibutyrate (PDBu), GTPQ QS, and by the stable thromboxane analog, 9,11-dideoxy-9K K,11K K-methanoepoxy-PGF PK (U-46619), were determined in K K-toxin-permeabilized smooth muscles. Y-27632 relaxed (up to 99%) Ca P+ -sensitization by GTPQ QS (10 W WM) and U-46619 (1 W WM), but not by PDBu (20 W WM), and reduced GTPQ QS-induced myosin light chain (MLC PH ) phosphorylation from 28% to 17% (P = 0.002). GTPQ QS-induced force sensitization was inhibited by Y-27632 more potently when the inhibitor was added during the plateau of force than prior to stimulation. In K K-toxin-permeabilized smooth muscle, Y-27632 inhibited AA (50 W WM)-induced Ca P+ -sensitization of force (by 66 þ 1.3%) and reduced MLC PH phosphorylation. In contrast, Y-27632 did not relax force Ca P+ -sensitized by AA in smooth muscle permeabilized with Triton X-100. We conclude that (i) AA induces Ca P+ -sensitization through dual mechanisms, one mediated by Rho-kinase (or a related kinase), and (ii) Rho-kinase is not required for phorbol ester-induced Ca P+ -sensitization.z 1998 Federation of European Biochemical Societies.

Telomeres are protein–DNA structures found at the ends of linear chromosomes and are crucial for genome integrity. Telomeric DNA length is primarily maintained by the enzyme telomerase. Cells lacking telomerase will undergo senescence when telomeres become critically short. In Saccharomyces cerevisiae, a very small percentage of cells lacking telomerase can remain viable by lengthening telomeres via two distinct homologous recombination pathways. These “survivor” cells are classified as either Type I or Type II, with each class of survivor possessing distinct telomeric DNA structures and genetic requirements. To elucidate the regulatory pathways contributing to survivor generation, we knocked out the telomerase RNA gene TLC1 in 280 telomere-length-maintenance (TLM) gene mutants and examined telomere structures in post-senescent survivors. We uncovered new functional roles for 10 genes that affect the emerging ratio of Type I versus Type II survivors and 22 genes that are required for Type II survivor generation. We further verified that Pif1 helicase was required for Type I recombination and that the INO80 chromatin remodeling complex greatly affected the emerging frequency of Type I survivors. Finally, we found the Rad6-mediated ubiquitination pathway and the KEOPS complex were required for Type II recombination. Our data provide an independent line of evidence supporting the idea that these genes play important roles in telomere dynamics.

Telomeres are eukaryotic protein-DNA complexes found at the ends of linear chromosomes that are essential for maintaining genome integrity and are implicated in cellular aging and cancer. The guanine (G)-rich strand of telomeric DNA, usually elongated by the telomerase reverse transcriptase, can form a higher-order structure known as a G-quadruplex in vitro and in vivo. Several factors that promote or resolve G-quadruplexes have been identified, but the functional importance of these structures for telomere maintenance is not well understood. Here we show that the yeast telomerase subunit Est1p, known to be involved in telomerase recruitment to telomeres, can convert single-stranded telomeric G-rich DNA into a G-quadruplex structure in vitro in a Mg(2+)-dependent manner. Cells carrying Est1p mutants deficient in G-quadruplex formation in vitro showed gradual telomere shortening and cellular senescence, indicating a positive regulatory role for G-quadruplex in the maintenance of telomere length.

The unicellular eukaryotic organisms represent the popular model systems to understand aging in eukaryotes. Candida albicans, a polymorphic fungus, appears to be another distinctive unicellular aging model in addition to the budding yeast Saccharomyces cerevisiae and fission yeast Schizosaccharomyces pombe. The two types of Candida cells, yeast (blastospore) form and hyphal (filamentous) form, have similar replicative lifespan. Taking the advantage of morphologic changes, we are able to obtain cells of different ages. Old Candida cells tend to accumulate glycogen and oxidatively damaged proteins. Deletion of the SIR2 gene causes a decrease of lifespan, while insertion of an extra copy of SIR2 extends lifespan, indicating that like in S. cerevisiae, Sir2 regulates cellular aging in C. albicans. Interestingly, Sir2 deletion does not result in the accumulation of extra-chromosomal rDNA molecules, but influences the retention of oxidized proteins in mother cells, suggesting that the extra-chromosomal rDNA molecules may not be associated with cellular aging in C. albicans. This novel aging model, which allows efficient large-scale isolation of old cells, may facilitate biochemical characterizations and genomics/proteomics studies of cellular aging, and help to verify the aging pathways observed in other organisms including S. cerevisiae.

Recent studies have established that the highly condensed and transcriptionally silent heterochromatic domains in budding yeast are virtually dynamic structures. The underlying mechanisms for heterochromatin dynamics, however, remain obscure. In this study, we show that histones are dynamically acetylated on H4K12 at telomeric heterochromatin, and this acetylation regulates several of the dynamic telomere properties. Using a de novo heterochromatin formation assay, we surprisingly found that acetylated H4K12 survived the formation of telomeric heterochromatin. Consistently, the histone acetyltransferase complex NuA4 bound to silenced telomeric regions and acetylated H4K12. H4K12 acetylation prevented the over-accumulation of Sir proteins at telomeric heterochromatin and elimination of this acetylation caused defects in multiple telomere-related processes, including transcription, telomere replication, and recombination. Together, these data shed light on a potential histone acetylation mark within telomeric heterochromatin that contributes to telomere plasticity.

In Saccharomyces cerevisiae, the essential gene CDC13 encodes a telomeric single-stranded DNA-binding protein that interacts with Stn1p and Ten1p genetically and physically, and is required for telomere end protection and telomere length control. The molecular mechanism by which Ten1 participates in telomere length regulation and chromosome end protection remains elusive. In this work, we observed a weak interaction of Cdc13p and Ten1p in a gelfiltration analysis using purified recombinant Cdc13p and Ten1p. Ten1p itself exhibits a weak DNA-binding activity, but enhances the telomeric TG 1-3 DNA-binding ability of Cdc13p. Cdc13p is co-immunoprecipitated with Ten1p. In the mutant ten1-55 or ten1-66 cells, the impaired interaction between Ten1p and Cdc13p results in much longer telomeres, as well as a decreased association of Cdc13p with telomeric DNA. Consistently, the Ten1-55 and Ten1-66 mutant proteins fail to stimulate the telomeric DNA-binding activity of Cdc13p in vitro. These results suggest that Ten1p enhances the telomeric DNA-binding activity of Cdc13p to negatively regulate telomere length.

The Notch signaling pathway plays a key role in angiogenesis and endothelial cell formation, but it remains unclear whether it is involved in vascular repair by endothelial progenitor cells after traumatic brain injury. Therefore, in the present study, we controlled the Notch signaling pathway using overexpression and knockdown constructs. Activation of the Notch signaling pathway by Notch1 or Jagged1 overexpression enhanced the migration, invasiveness and angiogenic ability of endothelial progenitor cells. Suppression of the Notch signaling pathway with Notch1 or Jagged1 siRNAs reduced the migratory capacity, invasiveness and angiogenic ability of endothelial progenitor cells. Activation of the Notch signaling pathway in vivo in a rat model of mild traumatic brain injury promoted neurovascular repair. These findings suggest that the activation of the Notch signaling pathway promotes blood vessel formation and tissue repair after brain trauma.

In telomerase negative yeast cells, Rad52-dependent recombination is activated to maintain telomeres. This recombination-mediated telomere elongation usually involves two independent pathways, type I and type II, and leads to generation of type I and type II survivors. It remains elusive whether the recombination-mediated telomere elongation prefers to take place on shorter or longer telomeres. In this study, we exploited the de novo telomere addition system to examine the telomere recombination event in telomerase negative cells. We show that recombination preferentially occurs on shorter rather than longer telomeres in both pre-survivors and established type II survivors. In type II survivors, the short VII–L telomeres could invade either terminal TG1–3 sequence or short tracts of TG1–3 sequence in subtelomeric Y′-X and Y′-Y′ junction to initiate recombination. Unexpectedly, short VII–L telomere recombination still takes place in type II survivors lacking either Rad50 or Rad59, which are required for type II survivor generation in senescing telomerase-null cells. Our results support the notion that Rad50 and Rad59 are not essential for the maintenance of type II survivors once established.