A selective inducible NOS dimerization inhibitor prevents systemic,cardiac, and pulmonary hemodynamic dysfunction in endotoxemic mice

Increased nitric oxide (NO) production by inducible NO synthase (NOS2), an obligate homodimer, is implicated in the cardiovascular sequelae of sepsis. We tested the ability of a highly selective NOS2 dimerization inhibitor (BBS-2) to prevent endotoxin-induced systemic hypotension, myocardial dysfunction, and impaired hypoxic pulmonary vasoconstriction (HPV) in mice. Mice were challenged with Escherichia coli endotoxin before treatment with BBS-2 or vehicle. Systemic blood pressure was measured before and 4 and 7 h after endotoxin challenge, and echocardiographic parameters of myocardial function were measured before and 7 h after endotoxin challenge. The pulmonary vasoconstrictor response to left mainstem bronchus occlusion, which is a measure of HPV, was studied 22 h after endotoxin challenge. BBS-2 treatment alone did not alter baseline hemodynamics. BBS-2 treatment blocked NOS2 dimerization and completely inhibited the endotoxin-induced increase of plasma nitrate and nitrite levels. Treatment with BBS-2 after endotoxin administration prevented systemic hypotension and attenuated myocardial dysfunction. BBS-2 also prevented endotoxin-induced impairment of HPV. In contrast, treatment with NG-nitro-l-arginine methyl ester, which is an inhibitor of all three NOS isoforms, prevented the systemic hypotension but further aggravated the myocardial dysfunction associated with endotoxin challenge. Treatment with BBS-2 prevented endotoxin from causing key features of cardiovascular dysfunction in endotoxemic mice. Selective inhibition of NOS2 dimerization with BBS-2, while sparing the activities of other NOS isoforms, may prove to be a useful treatment strategy in sepsis.     

Ras recruits mitotic exit regulator Lte1 to the bud cortex in budding yeast

ACdc25 family protein Lte1 (low temperature essential) is essential for mitotic exit at a lowered temperature and has been presumed to be a guanine nucleotide exchange factor (GEF) for a small GTPase Tem1, which is a key regulator of mitotic exit. We found that Lte1 physically associates with Ras2-GTP both in vivo and in vitro and that the Cdc25 homology domain (CHD) of Lte1 is essential for the interaction with Ras2. Furthermore, we found that the proper localization of Lte1 to the bud cortex is dependent on active Ras and that the overexpression of a derivative of Lte1 without the CHD suppresses defects in mitotic exit of a Deltalte1 mutant and a Deltaras1 Deltaras2 mutant. These results suggest that Lte1 is a downstream effector protein of Ras in mitotic exit and that the Ras GEF domain of Lte1 is not essential for mitotic exit but required for its localization.     

Distribution of interstitial telomere-like repeats and their adjacent sequences in a dioecious plant, <Emphasis Type="Italic">Silene latifolia</Emphasis>

The dioecious plant Silene latifolia has large, heteromorphic X and Y sex chromosomes that are thought to be derived from rearrangements of autosomes. To reveal the origin of the sex chromosomes in S. latifolia, we isolated and characterized telomere-homologous sequences from intra-chromosomal regions (interstitial telomere-like repeats; ITRs) and ITR-adjacent sequences (IASs). Nine genomic DNA fragments with degenerate 84- to 175-bp ITRs were isolated from a genomic library and total genome of male plants. Comparing the nucleotide sequences, the IASs of the nine ITRs were classified into seven elements (IAS-a, IAS-b, IAS-c, IAS-d, IAS-e, IAS-f, and IAS-g) by sequence similarity. The ITRs were grouped into two classes (class-I and -II ITRs) according to the classification of IASs. The class-I ITRs were sub-grouped into three subclasses (subclasses-IA, -IB, and -IC ITRs) based on the arrangement of IAS elements. By contrast, the class-II ITR was located between two different IASs (IAS-f and IAS-g). Genomic Southern analyses showed that both the male and female genomes contained six (IAS-f) to 153 (IAS-d) copies of each IAS per haploid genome. Fluorescence in situ hybridization analyses showed that one IAS element, IAS-d, was distributed in the interstitial and proximal regions of the sex chromosomes of S. latifolia. The distribution of IAS-d is important evidence for past telomere-mediated chromosome rearrangements during the evolution of the sex chromosomes of S. latifolia.     

Thymidine phosphorylase suppresses Fas-induced apoptotic signal transduction independent of its enzymatic activity

Thymidine phosphorylase (TP) has chemotactic and angiogenic activities resulting from its enzymatic activity in vitro, and it also promotes tumor growth and inhibits apoptosis in vivo. Recently, we have reported that TP plays an important role in Fas-induced apoptosis. Caspase-8 cleavage, subsequent cytochrome c release, and caspase-3 cleavage were prevented in KB cells transfected with a TP cDNA (KB/TP cells). In this study, treatment with thymidine phosphorylase inhibitor (TPI) or thymidine did not affect cell survival of KB/TP cells during Fas-induced apoptosis. Moreover, treatment with thymine or 2-deoxy-D-ribose (degradation products of thymidine generated by TP) also did not affect cell survival of control transfectant (KB/CV) cells during Fas-induced apoptosis. These findings indicate that TP suppresses Fas-induced apoptotic signal transduction independent of its enzymatic activity.     

A role for Ran-GTP and Crm1 in blocking re-replication

All eukaryotic cells have regulatory mechanisms that limit genomic replication to a single round each cell cycle. These systems function by blocking formation of prereplication complexes. The regulatory mechanisms in the yeast S. cerevisiae have been identified, but these do not appear to be conserved in metazoans. Using Xenopus egg extracts, we have identified a metazoan-specific regulatory system that limits replication to a single round. We show that during S phase, soluble MCM helicase, an essential initiation factor, is inactivated when it associates with exportin-1/Crm1. Formation of this complex is dependent on both high Ran-GTP and cdk2 kinase activity. Lowering Ran-GTP within nuclei or nuclear extracts allows MCM to reassociate with chromatin during S phase and induces re-replication. Importantly, prevention of re-replication requires MCM-Crm1 complex formation, but it does not require export of MCM from the nucleus. Therefore, in metazoans, Crm1 functions in both nuclear export and blocking of re-replication.     

Development of genetic differentiation and postzygotic isolation in experimental metapopulations of spider mites

We studied the development of genetic differentiation and postzygotic isolation in experimental metapopulations of the two-spotted spider mite, Tetranychus urticae Koch. A genetically diverse starter population was made by allowing six inbred sublines to interbreed. Then three migration patterns were tested: no migration, or one or three immigrants per subpopulation per generation. Variations in four traits were investigated: allozymes, acaricide resistance, diapause, and hatchability. In the allozymes, acaricide resistance, and diapause, genetic variation among subpopulations became high in metapopulations with no migration, but not in the others, which showed that one immigrant is enough to prevent genetic differentiation. Hatchability, which was decreased by interbreeding among the six sublines, gradually recovered in succeeding generations. In metapopulations with no migration, hatchability was reduced again after in-migration at the 15th generation. Different karyotypes or coadapted gene complexes can survive in different subpopulations by genetic drift, and both Wolbachia-infected and -noninfected subpopulations may be selected, which would lead to postzygotic isolation between isolated subpopulations. Our results indicate that sampling effects such as genetic drift or stochastic loss of Wolbachia produce postzygotic isolation in laboratory populations of spider mite.     

The human megakaryocytic cell line UT‐7/TPO expresses functional platelet agonist signals mediated through GPVI and thromboxane receptor

We have demonstrated that a unique megakaryocytic cell line UT‐7/TPO could respond to one of the primary platelet signals through GP (glycoprotein) VI and a secondary signal of the AA (arachidonic acid) cascade. Unlike other megakaryocytic cell lines, UT‐7/TPO was found to express GPVI and its associate signal molecule of FcRγ (Fc receptor γ chain). When UT‐7/TPO was stimulated with the GPVI agonist convulxin, the [Ca²⁺]i (intracellular Ca²⁺) was elevated in a convulxin concentration‐dependent manner, and [Ca²⁺]i elevation was blocked by pretreatment with the Src family kinase inhibitor PP2 and the phospholipase inhibitor U73122. These results strongly indicate that endogenously expressed GPVI signal molecules are functional in UT‐7/TPO. Concerning the AA cascade, the expression of COX (cyclooxygenase)‐1 and TX (thromboxane) synthase was observed, and this cell line was able to produce TX by exogenous AA, followed by [Ca²⁺]i elevation mediated through the TX receptor. It is worth noting that convulxin stimulation did not cause TX generation, even through the GPVI pathway and the AA cascade are functional in this cell line. As there are many reports that convulxin‐stimulated platelets failed to produce TX, it is suggested that UT‐7/TPO has the same property as the platelets in regards to convulxin stimulation. Thus, UT‐7/TPO is useful for the observation of both the GPVI pathway and AA cascade without requiring either the induction of differentiation or GPVI transfection. Furthermore, this cell line provides a new tool for research on platelet activation signals.