Patency of the preterm fetal ductus arteriosus is regulated by endothelial nitric oxide synthase and is independent of vasa vasorum in the mouse

Patency of the fetal ductus arteriosus (DA) is maintained in an environment of low relative oxygen tension and a preponderance of vasodilating forces. In addition to prostaglandins, nitric oxide (NO), a potent vasodilator in the pulmonary and systemic vasculatures, has been implicated in regulation of the fetal DA. To further define the contribution of NO to DA patency, the expression and function of NO synthase (NOS) isoforms were examined in the mouse DA on days 17-19 of pregnancy and after birth. Our results show that endothelial NOS (eNOS) is the predominant isoform expressed in the mouse DA and is localized in the DA endothelium by in situ hybridization. Despite rapid constriction of the DA after birth, eNOS expression levels were unchanged throughout the fetal and postnatal period. Pharmacological inhibition of prostaglandin vs. NO synthesis in vivo showed that the preterm fetal DA on day 16 is more sensitive to NOS inhibition than the mature fetal DA on day 19, whereas prostaglandin inhibition results in marked DA constriction on day 19 but minimal effects on the day 16 DA. Combined prostaglandin and NO inhibition caused additional DA constriction on day 16. The contribution of vasa vasorum to DA regulation was also examined. Immunoreactive platelet endothelial cell adhesion molecule and lacZ tagged FLK1 localized to DA endothelial cells but revealed the absence of vasa vasorum within the DA wall. Similarly, there was no evidence of vasa vasorum by vascular casting. These studies indicate that eNOS is the primary source of NO in the mouse DA and that vasomotor tone of the preterm fetal mouse DA is regulated by eNOS-derived NO and is potentiated by prostaglandins. In contrast to other species, mechanisms for DA patency and closure appear to be independent of any contribution of the vasa vasorum.     

Indirect intracoronary delivery of adenovirus encoding adenylyl cyclase increases left ventricular contractile function in mice

We performed indirect intracoronary delivery of adenovirus vectors in mice and explored techniques including hypothermia and pharmacological means to increase cardiac gene transfer. Mice were maintained in a normothermic state or cooled to 25 degrees C. The aorta or both the pulmonary artery and aorta were clamped while a needle was advanced into the left ventricular cavity to deliver adenovirus vectors encoding enhanced green fluorescent protein (EGFP) or murine adenylyl cyclase type VI (AC(VI)) with saline, sodium nitroprusside, acetylcholine, or serotonin. Clamping was maintained for 30 s (normothermia) or 2 min (25 degrees C) after adenovirus administration. Mice were killed 7 or 21 days later, and hearts were examined for EGFP expression. Compared with clamping the aorta alone and with no cooling, gene transfer was increased as follows: 1) 1.3-fold with hypothermia to extend dwell time; 2) 4.5-fold by clamping the aorta and the pulmonary artery; 3) 11.4-fold with nitroprusside administration; 4) 11.8-fold with serotonin addition, and 5) 14.3-fold with acetylcholine delivery. Gene expression remained substantial at 21 days, and no significant inflammatory response was seen. Efficacy of the method was tested by performing gene transfer of adenovirus encoding AC(VI). Fourteen days after gene transfer, hearts isolated from mice that received adenovirus encoding AC(VI) showed increased contractile function. Indirect intracoronary delivery of adenovirus vectors in mice is associated with efficient cardiac gene transfer and increased left ventricular function after AC(VI) gene transfer.     

G1 cell cycle progression and the expression of G1 cyclins are regulated by PI3K/AKT/mTOR/p70S6K1 signaling in human ovarian cancer cells

Ovarian cancer is one of the most common cancers among women. Recent studies demonstrated that the gene encoding the p110alpha catalytic subunit of phosphatidylinositol 3-kinase (PI3K) is frequently amplified in ovarian cancer cells. PI3K is involved in multiple cellular functions, including proliferation, differentiation, antiapoptosis, tumorigenesis, and angiogenesis. In this study, we demonstrate that the inhibition of PI3K activity by LY-294002 inhibited ovarian cancer cell proliferation and induced G(1) cell cycle arrest. This effect was accompanied by the decreased expression of G(1)-associated proteins, including cyclin D1, cyclin-dependent kinase (CDK) 4, CDC25A, and retinoblastoma phosphorylation at Ser(780), Ser(795), and Ser(807/811). Expression of CDK6 and beta-actin was not affected by LY-294002. Expression of the cyclin kinase inhibitor p16(INK4a) was induced by the PI3K inhibitor, whereas steady-state levels of p21(CIP1/WAF1) were decreased in the same experiment. The inhibition of PI3K activity also inhibited the phosphorylation of AKT and p70S6K1, but not extracellular regulated kinase 1/2. The G(1) cell cycle arrest induced by LY-294002 was restored by the expression of active forms of AKT and p70S6K1 in the cells. Our study shows that PI3K transmits a mitogenic signal through AKT and mammalian target of rapamycin (mTOR) to p70S6K1. The mTOR inhibitor rapamycin had similar inhibitory effects on G(1) cell cycle progression and on the expression of cyclin D1, CDK4, CDC25A, and retinoblastoma phosphorylation. These results indicate that PI3K mediates G(1) progression and cyclin expression through activation of an AKT/mTOR/p70S6K1 signaling pathway in the ovarian cancer cells.     

NF-kappaB activation is required for the development of cardiac hypertrophy in vivo

In the present study, we examined whether NF-kappaB activation is required for cardiac hypertrophy in vivo. Cardiac hypertrophy in rats was induced by aortic banding for 1, 3, and 5 days and 1-6 wk, and age-matched sham-operated rats served as controls. In a separate group of rats, an IkappaB-alpha dominant negative mutant (IkappaB-alphaM), a superrepressor of NF-kappaB activation, or pyrrolidinedithiocarbamate (PDTC), an antioxidant that can inhibit NF-kappaB activation, was administered to aortic-banded rats for 3 wk. The heart weight-to-body weight ratio was significantly increased at 5 days after aortic banding, peaked at 4 wk, and remained elevated at 6 wk compared with age-matched sham controls. Atrial natriuretic peptide and brain natriuretic peptide mRNA expressions were significantly increased after 1 wk of aortic banding, reached a maximum between 2 and 3 wk, and remained increased at 6 wk compared with age-matched sham controls. NF-kappaB activity was significantly increased at 1 day, reached a peak at 3 wk, and remained elevated at 6 wk, and IKK-beta activity was significantly increased at 1 day, peaked at 5 days, and then decreased but remained elevated at 6 wk after aortic banding compared with age-matched sham controls. Inhibiting NF-kappaB activation in vivo by cardiac transfection of IkappaB-alphaM or by PDTC treatment significantly attenuated the development of cardiac hypertrophy in vivo with a concomitant decrease in NF-kappaB activity. Our results suggest that NF-kappaB activation is required for the development of cardiac hypertrophy in vivo and that NF-kappaB could be an important target for inhibiting the development of cardiac hypertrophy in vivo.     

An LQT mutant minK alters KvLQT1 trafficking

Cardiac I_Ks, the slowly activated delayed-rectifier K⁺ current, is produced by the protein complex composed of - and -subunits: KvLQT1 and minK. Mutations of genes encoding KvLQT1 and minK are responsible for the hereditary long QT syndrome (loci LQT1 and LQT5, respectively). MinK-L51H fails to traffic to the cell surface, thereby failing to produce effective I_Ks. We examined the effects that minK-L51H and an endoplasmic reticulum (ER)-targeted minK (minK-ER) exerted over the electrophysiology and biosynthesis of coexpressed KvLQT1. Both minK-L51H and minK-ER were sequestered primarily in the ER as confirmed by lack of plasma membrane expression. Glycosylation and immunofluorescence patterns of minK-L51H were qualitatively different for minK-ER, suggesting differences in trafficking. Cotransfection with the minK mutants resulted in reduced surface expression of KvLQT1 as assayed by whole cell voltage clamp and immunofluorescence. MinK-L51H reduced current amplitude by 91% compared with wild-type (WT) minK/KvLQT1, and the residual current was identical to KvLQT1 without minK. The phenotype of minK-L51H on I_Ks was not dominant because coexpressed WT minK rescued the current and surface expression. Collectively, our data suggest that ER quality control prevents minK-L51H/KvLQT1 complexes from trafficking to the plasma membrane, resulting in decreased I_Ks. This is the first demonstration that a minK LQT mutation is capable of conferring trafficking defects onto its associated -subunit. potassium channel; hereditary arrhythmia; electrophysiology; protein interaction     

大鼠局灶性脑缺血再灌注损伤后纹状体和海马区瞬时受体电位通道蛋白4的表达增加

实验在大鼠大脑中动脉阻塞性脑缺血(middle cerebral arterv occlusion,MCAO)模型上采用Western Blot方法检测脑缺血再灌注不同时程(6h、12h、1d、3d)脑组织中瞬时受体电位通道蛋白4(transient receptor potential channel4,TRPC4)的表达情况,并与正常对照组相比,结果显示,12 h、1 d、3 d组纹状体、海马区域TRPC4含量明显高于正常组(P<0.05)。采用免疫组织化学定位检测,显示TRPC4主要表达在神经元细胞膜上;免疫组化阳性细胞统计分析显示,在不同时程缺血组中纹状体、海马区域TRPC4的表达与正常组相比有所增加,其中纹状体、海马区缺血再灌注1 d、3 d组缺血同侧1RPC4阳性细胞升高显著(P<0.05)。脑缺血再灌注损伤后TRPC4相对含量增加,提示TRPC4可能参与脑缺血引起的急性和迟发性神经元损伤。     

Fine mapping of a malting-quality QTL complex near the chromosome 4H S telomere in barley

Malting quality has long been an active objective in barley (Hordeum vulgare L.) breeding programs. However, it is difficult for breeders to manipulate malting-quality traits because of inheritance complexity and difficulty in evaluation of these quantitative traits. Quantitative trait locus (QTL) mapping provides breeders a promising basis with which to manipulate quantitative trait genes. A malting-quality QTL complex, QTL2, was mapped previously to a 30-cM interval in the short-arm telomere region of barley chromosome 4H in a Steptoe/Morex doubled haploid population by the North American Barley Genome Project, using an interval mapping method with a relatively low-resolution genetic map. The QTL2 complex has moderate effects on several malting-quality traits, including malt extract percentage (ME), -amylase activity (AA), diastatic power (DP), malt -glucan content (BG), and seed dormancy, which makes it a promising candidate gene source in malting barley-cultivar development. Fine mapping QTL2 is desirable for precisely studying barley malting-quality trait inheritance and for efficiently manipulating QTL2 in breeding. A reciprocal-substitution mapping method was employed to fine map QTL2. Molecular marker-assisted backcrossing was used to facilitate the generation of isolines. Fourteen different types of Steptoe isolines, including regenerated Steptoe and 13 different types of Morex isolines, including regenerated Morex, were made within a 41.5-cM interval between MWG634 and BCD265B on chromosome 4H. Duplicates were identified for 12 Steptoe and 12 Morex isoline types. The isolines together with Steptoe and Morex were grown variously at three locations in 2 years for a total of five field environments. Four malting-quality traits were measured: ME, DP, AA, and BG. Few significant differences were found between duplicate isolines for these traits. A total of 15 putative QTLs were mapped; three for ME, four for DP, six for AA, and two for BG. Background genotype seemed to make a difference in expression/detection of QTLs. Of the 15 QTLs identified, ten were from the Morex and only five from the Steptoe background. By combining the results from different years, field environments, and genetic backgrounds and taking into account overlapping QTL segments, six QTLs can be conservatively estimated: two each for ME and AA and one each for DP and BG with chromosome segments ranging from 0.7 cM to 27.9 cM. A segment of 15.8 cM from the telomere (MWG634–CDO669) includes all or a portion of all QTLs identified. Further study and marker-assisted breeding should focus on this 15.8-cM chromosome region.     

The calmodulin binding region of the skeletal ryanodine receptor acts as a self-modulatory domain

A synthetic peptide (CaMBP) matching amino acids 3614-3643 of the skeletal ryanodine receptor (RyR1) binds to both Ca2+-free calmodulin (CaM) and Ca2+-bound CaM with nanomolar affinity [J. Biol. Chem. 276 (2001) 2069]. We report here that CaMBP increases [3H]ryanodine binding to RyR1 in a dose- and Ca2+-dependent manner; it also induces Ca2+ release from SR vesicles, and increases open probability (P(o)) of single RyR channels reconstituted in planar lipid bilayers. Further, CaMBP removes CaM associated with SR vesicles and increases [3H]ryanodine binding to purified RyR1, suggesting that its mechanism of action is two-fold: it removes endogenous inhibitors and also interacts directly with complementary regions in RyR1. Remarkably, the N-terminus of CaMBP activates RyRs while the C-terminus of CaMBP inhibits RyR activity, suggesting the presence of two discrete functional subdomains within this region. A ryr1 mutant lacking this region, RyR1-Delta3614-3643, was constructed and expressed in dyspedic myoblasts (RyR1-knockout). The depolarization-, caffeine- and 4-chloro-m-cresol (4-CmC)-induced Ca2+ transients in these cells were dramatically reduced compared with cells expressing wild type RyR1. Deletion of the 3614-3643 region also resulted in profound changes in unitary conductance and channel gating. We thus propose that the RyR1 3614-3643 region acts not only as the CaM binding site, but also as an important modulatory domain for RyR1 function.     

Luminescent pH sensing and DNA binding properties of a novel ruthenium(II) complex

A new Ru(II) complex, [Ru(bpy)(2)(dhipH3)](ClO4)(2) (in which bpy=2,2'-bipyridine, dhipH(3)=3,4-dihydroxy-imidado[4,5-f][1,10]-phenanthroline), was synthesized and characterized, and the pH effect on the emission spectra of the complex was studied. The interaction of the complex with calf thymus DNA was investigated by UV-visible and emission spectroscopy, and viscosity measurements. The results suggest that the complex acted as a sensitive luminescent pH sensor and a strong ct-DNA intercalator with an intrinsic binding constant of (4.0+/-0.7) x 10(5) M(-1) in buffered 50 mM NaCl.