L1 (LINE-1) retrotransposable elements provide a "fossil" record of the phylogenetic history of murid rodents

The single most difficult problem in phylogenetic analysis is deciding whether a shared taxonomic character is due to common ancestry or one that appeared independently due to convergence, parallelism, or reversion to an ancestral state. Mammalian L1 retrotransposons undergo periodic amplifications in which multiple copies of the elements are interspersed in the genome. Because these elements apparently are transmitted only by inheritance and are retained in the genome, a shared L1 amplification event can only be an inherited ancestral character. We propose that L1 amplification events can be an excellent tool for analyzing mammalian evolution and demonstrate here how we addressed several refractory problems in rodent systematics using L1 DNA as a taxonomic character.      

Plant virus infection development as affected by heavy metal stress

The work was focused on the investigation of possible dependencies between the development of viral infection in plants and the presence of high heavy metal concentrations in soil. Field experiments have been conducted in order to study the development of systemic tobacco mosaic virus (TMV) infection in Lycopersicon esculentum L. cv. Miliana plants under effect of separate salts of heavy metals Cu, Zn and Pb deposited in soil. As it is shown, simultaneous effect of viral infection and heavy metals in tenfold maximum permissible concentration leads to decrease of total chlorophyll content in experiment plants mainly due to the degradation of chlorophyll a. The reduction of chlorophyll concentration under the combined influence of both stress factors was more serious comparing to the separate effect of every single factor. Plants' treatment with toxic concentrations of lead and zinc leaded to slight delay in the development of systemic TMV infection together with more than twofold increase of virus content in plants that may be an evidence of synergism between these heavy metal's and virus' effects. Contrary, copper although decreased total chlorophyll content but showed protective properties and significantly reduced amount of virus in plants.     

DNA methylation-associated colonic mucosal immune and defense responses in treatment-na?ve pediatric ulcerative colitis

Inflammatory bowel diseases (IBD) are emerging globally, indicating that environmental factors may be important in their pathogenesis. Colonic mucosal epigenetic changes, such as DNA methylation, can occur in response to the environment and have been implicated in IBD pathology. However, mucosal DNA methylation has not been examined in treatment-naïve patients. We studied DNA methylation in untreated, left sided colonic biopsy specimens using the Infinium HumanMethylation450 BeadChip array. We analyzed 22 control (C) patients, 15 untreated Crohn’s disease (CD) patients, and 9 untreated ulcerative colitis (UC) patients from two cohorts. Samples obtained at the time of clinical remission from two of the treatment-naïve UC patients were also included into the analysis. UC-specific gene expression was interrogated in a subset of adjacent samples (5 C and 5 UC) using the Affymetrix GeneChip PrimeView Human Gene Expression Arrays. Only treatment-naïve UC separated from control. One-hundred-and-twenty genes with significant expression change in UC (> 2-fold, P < 0.05) were associated with differentially methylated regions (DMRs). Epigenetically associated gene expression changes (including gene expression changes in the IFITM1, ITGB2, S100A9, SLPI, SAA1, and STAT3 genes) were linked to colonic mucosal immune and defense responses. These findings underscore the relationship between epigenetic changes and inflammation in pediatric treatment-naïve UC and may have potential etiologic, diagnostic, and therapeutic relevance for IBD.     

Heparin inhibits the inositol 1,4,5-trisphosphate-dependent, but not the independent, calcium release induced by guanine nucleotide in vascular smooth muscle

The effects of heparin on the release of intracellular Ca2+, assessed by tension development in saponin-permeabilized rabbit main pulmonary artery, were determined. Heparin inhibited (IC50 = 5 micrograms/ml) inositol 1,4,5-trisphosphate (InsP3)-induced, but not caffeine-induced, Ca2+ release. The initial (InsP3-dependent) component of GTP gamma S-induced Ca2+-release was also inhibited by heparin, but the InsP3-independent component was resistant to both heparin and procaine. These results support the existence of a G protein activated mechanism of Ca2+ release that is not mediated by InsP3 or by Ca2+-induced Ca2+ release.     

Deletion of the protein kinase A/protein kinase G target SMTNL1 promotes an exercise-adapted phenotype in vascular smooth muscle

In vivo protein kinases A and G (PKA and PKG) coordinately phosphorylate a broad range of substrates to mediate their various physiological effects. The functions of many of these substrates have yet to be defined genetically. Herein we show a role for smoothelin-like protein 1 (SMTNL1), a novel in vivo target of PKG/PKA, in mediating vascular adaptations to exercise. Aortas from smtnl1(-/-) mice exhibited strikingly enhanced vasorelaxation before exercise, similar in extent to that achieved after endurance training of wild-type littermates. Additionally, contractile responses to alpha-adrenergic agonists were greatly attenuated. Immunological studies showed SMTNL1 is expressed in smooth muscle and type 2a striated muscle fibers. Consistent with a role in adaptations to exercise, smtnl1(-/-) mice also exhibited increased type 2a fibers before training and better performance after forced endurance training compared smtnl1(+/+) mice. Furthermore, exercise was found to reduce expression of SMTNL1, particularly in female mice. In both muscle types, SMTNL1 is phosphorylated at Ser-301 in response to adrenergic signals. In vitro SMTNL1 suppresses myosin phosphatase activity through a substrate-directed effect, which is relieved by Ser-301 phosphorylation. Our findings suggest roles for SMTNL1 in cGMP/cAMP-mediated adaptations to exercise through mechanisms involving direct modulation of contractile activity.     

Cryo-atomic force microscopy of unphosphorylated and thiophosphorylated single smooth muscle myosin molecules

The purpose of this study was to determine whether steric blockage of one head by the second head of native two-headed myosin was responsible for the inactivity of nonphosphorylated two-headed myosin compared with the high activity of single-headed myosin, as suggested on the basis of electron microscopy of two-dimensional crystals of heavy meromyosin (Wendt, T., Taylor, D., Messier, T., Trybus, K. M., and Taylor, K. A. (1999) J. Cell Biol. 147, 1385-1390; and Wendt, T., Taylor, D., Trybus, K. M., and Taylor, K. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 4361-4366). Our earlier cryo-atomic force microscopy (cryo-AFM) (Zhang, Y., Shao, Z., Somlyo, A. P., and Somlyo, A. V. (1997) Biophys. J. 72, 1308-1318) indicates that thiophosphorylation of the regulatory light chain increases the separation of the two heads of a single myosin molecule, but the thermodynamic probability of steric hindrance by strong binding between the two heads was not determined. We now report this probability determined by cryo-AFM of single whole myosin molecules shown to have normal low ATPase activity (0.007 s-1). We found that the thermodynamic probability of the relative head positions of nonphosphorylated myosin was approximately equal between separated heads as compared with closely apposed heads (energy difference of 0.24 kT (where k is a Boltzman constant and T is the absolute temperature)), and thiophosphorylation increased the number of molecules having separated heads (energy advantage of -1.2 kT (where k is a Boltzman constant and I is the absolute temperature)). Our results do not support the suggestion that strong binding of one head to the other stabilizes the blocked conformation against thermal fluctuations resulting in steric blockage that can account for the low activity of nonphosphorylated two-headed myosin.     

Human RhoA/RhoGDI complex expressed in yeast: GTP exchange is sufficient for translocation of RhoA to liposomes

The human small GTPase, RhoA, expressed in Saccharomyces cerevisiae is post-translationally processed and, when co-expressed with its cytosolic inhibitory protein, RhoGDI, spontaneously forms a heterodimer in vivo. The RhoA/RhoGDI complex, purified to greater than 98% at high yield from the yeast cytosolic fraction, could be stoichiometrically ADP-ribosylated by Clostridium botulinum C3 exoenzyme, contained stoichiometric GDP, and could be nucleotide exchanged fully with [3H]GDP or partially with GTP in the presence of submicromolar Mg2+. The GTP-RhoA/RhoGDI complex hydrolyzed GTP with a rate constant of 4.5 X 10(-5) s(-1), considerably slower than free RhoA. Hydrolysis followed pseudo-first-order kinetics indicating that the RhoA hydrolyzing GTP was RhoGDI associated. The constitutively active G14V-RhoA mutant expressed as a complex with RhoGDI and purified without added nucleotide also bound stoichiometric guanine nucleotide: 95% contained GDP and 5% GTP. Microinjection of the GTP-bound G14V-RhoA/RhoGDI complex (but not the GDP form) into serum-starved Swiss 3T3 cells elicited formation of stress fibers and focal adhesions. In vitro, GTP-bound-RhoA spontaneously translocated from its complex with RhoGDI to liposomes, whereas GDP-RhoA did not. These results show that GTP-triggered translocation of RhoA from RhoGDI to a membrane, where it carries out its signaling function, is an intrinsic property of the RhoA/RhoGDI complex that does not require other protein factors or membrane receptors.     

Modulation of Ca2+ sensitivity by cyclic nucleotides in smooth muscle from protein kinase G-deficient mice

The cGMP-dependent protein kinase (PKG) is the main mediator of nitric oxide-induced relaxation of smooth muscle. Although this pathway is well established, the cellular action of PKG, nitric oxide, and cGMP is complex and not fully understood. A cross-talk between the cGMP-PKG and other pathways (e.g. cAMP-protein kinase A) seems to exist. We have explored cGMP- and cAMP-dependent relaxation of smooth muscle using PKG-deficient mice (cGKI-/-). In intact ileum strips of wild type mice (cGKI+/+), 8-Br-cGMP inhibited the sustained phase of carbachol contractions by approximately 80%. The initial peak was less inhibited (approximately 30%). This relaxation was associated with a reduction in intracellular [Ca2+] and decreased Ca2+ sensitivity. Contractions of cGKI-/- ileum were not influenced by 8-Br-cGMP. EC50 for 8-Br-cGMP for PKG was estimated to be 10 nm. PKG-independent relaxation by 8-Br-cGMP had an EC50 of 10 microm. Relaxation by cAMP (approximately 50% at 100 microm), Ca2+ sensitivity of force, and force potentiation by GTPgammaS were similar in cGKI+/+ and cGKI-/- tissues. The results show that PKG is the main target for cGMP-induced relaxation in intestinal smooth muscle. cGMP desensitize the contractile system to Ca2+ via PKG. PKG-independent pathways are activated at 1000-fold higher cGMP concentrations. Relaxation by cAMP can occur independently of PKG. Long term deficiency of PKG does not lead to an apparent up-regulation of the cAMP-dependent pathways or changes in Ca2+ sensitivity.