Microvascular remodeling and accelerated hyperemia blood flow restoration in arterially occluded skeletal muscle exposed to ultrasonic microbubble destruction

We showed previously that microbubble destruction with pulsed 1-MHz ultrasound creates a bioeffect that stimulates arteriogenesis and a chronic increase in hyperemia blood flow in normal rat muscle. Here we tested whether ultrasonic microbubble destruction can be used to create a microvascular remodeling response that restores hyperemia blood flow to rat skeletal muscle affected by arterial occlusion. Pulsed ultrasound (1 MHz) was applied to gracilis muscles in which the lateral feed artery was occluded but the medial feed artery was left intact. Control muscles were similarly occluded but did not receive ultrasound, microbubbles, or both. Hyperemia blood flow and number of smooth muscle (SM) alpha-actin-positive vessels, >30-mum arterioles, and capillaries per fiber were determined 7, 14, and 28 days after treatment. In ultrasound-microbubble-treated muscles, lateral region hyperemia blood flow was increased at all time points and restored to normal at day 28. The number of SM alpha-actin vessels per fiber was increased over control in this region at days 7 and 14 but decreased by day 28, when larger-diameter arterioles became more prevalent in the medial region. The number of capillaries per fiber was increased over control only at day 7 in the lateral region and only at days 7 and 14 in the medial region, indicating that the angiogenesis response was transient and likely did not contribute significantly to flow restoration at day 28. We conclude that ultrasonic microbubble destruction can be tailored to stimulate an arteriogenesis response that restores hyperemia blood flow to skeletal muscle in a rat model of arterial occlusion.     

蓝斑核调制电刺激包钦格复合体引起的吸气抑制效应

实验选用成年健康家兔,用乌拉坦麻醉,以膈神经放电为指标,观察了电刺激和化学刺激脑桥蓝斑核对延髓包钦格复合体吸气抑制效应的影响。结果观察到:(1)长串电刺激蓝斑核后,在一定时间之内电刺激包钦格复合体所导致的膈神经放电抑制效应明显减弱,与对照组(仅刺激包钦格复合体)相比,抑制程度减弱(28.78 ±19.49)%。(2)蓝斑核内微量注射谷氨酸钠后,电刺激包钦格复合体导致的膈神经放电抑制效应明显减弱,与对照组相比,抑制程度减弱(19.18 ±8.06)%,与长串电刺激蓝斑核的效应一致。这些结果提示,蓝斑核对包钦格复合体吸气抑制效应具有调制作用。     

慢性间歇性低氧降低急性缺氧对大鼠肺动脉平滑肌细胞膜上电压门控性钾通道的抑制

为了从离子通道水平上探讨机体低氧适应的离子机制,本实验将雄性 SD 大鼠随机分为常氧对照组和慢性间歇性低氧组[氧浓度(10 ± 0.5) %, 间断缺氧每天 8 h]。用酶解法急性分离单个大鼠肺内动脉平滑肌细胞(pulmonary artery smoothmuscle cells, PASMCs),以全细胞膜片钳技术记录 PASMCs 膜上的电压门控性钾通道 (voltage-gated potassium channel, KV) 电流,观察急性缺氧对慢性间歇性低氧大鼠 PASMCs 的 KV 的影响, 为机体适应低氧能力提供实验依据。结果显示:⑴常氧对照组在电流钳下,急性缺氧可使膜电位明显去极化(由-47.2 ±2.6 mV 去极到 -26.7 ±1.2 mV ); 在电压钳下, 急性缺氧可显著抑制 KV电流( 60 mV 时, KV电流密度从 153.4 ± 9.5 pA/pF降到 70.1 ± 10.6 pA/pF), 峰电流的抑制率为(57.6 ± 3.3) %, 电流-电压关系曲线向右下移。⑵慢性间歇性低氧组KV电流密度随低氧时间延长而逐渐减少(慢性低氧10 d后就有显著性意义),电流- 电压关系曲线逐渐右下移。⑶急性缺氧对慢性间歇性低氧大鼠PASMCs KV电流的抑制作用随慢性间歇性低氧时间延长而逐渐减弱。上述观察结果提示慢性间歇性低氧减弱急性缺氧对 KV 的抑制, 这可能是机体低氧适应的一种重要机制。     

Expression and characteristics of the gene encoding azoreductase from Rhodobacter sphaeroides AS1.1737

A gene that encodes a protein with azoreductase activity was obtained by PCR amplification from Rhodobacter sphaeroides AS1.1737. The enzyme, with a molecular weight of 18.7 kD, was heterologously expressed in Escherichia coli and its azoreductase activity was characterized. Furthermore, the reduction mechanism of azo dyes catalyzed by the azoreductase was studied in detail. The presence of a hydrazo-intermediate was identified, which provided a convincing evidence for the assumption that azo dyes were degraded via an incomplete reduction stage.     

Effects of endocytosis inhibitory drugs on rubella virus entry into VeroE6 cells

It has been suggested that infectious entry of rubella virus (RV) is conducted by receptor mediated endocytosis. To explore the cellular entry mechanism of RV, inhibitory effects of drugs affecting various endocytic pathways on RV entry into VeroE6 cells were analyzed. Results showed that RV infectious entry into VeroE6 cells is mediated by clathrin-dependent endocytosis and not by caveolae-mediated endocytosis. Moreover, chemical inhibition of macropinocytosis such as treatments of amiloride, actin and microtubule-disrupting drug significantly reduced RV infection. Considering that macropinocytosis is inducible endocytosis by cellular stimulations, clathrin-mediated endocytosis is likely to be a major route of RV infectious entry.     

Mechanisms of murine cranial suture patency mediated by a dominant negative transforming growth factor-beta receptor adenovirus

Using a physiologic model of mouse cranial suture fusion, the authors' laboratory has previously demonstrated that transforming growth factor (TGF)-betas appear to be more abundantly expressed in the suture complex of the fusing posterior frontal compared with the patent sagittal suture. Furthermore, the authors have shown that by blocking TGF-beta signaling with a replication-deficient adenovirus encoding a defective, dominant negative type II TGF-beta receptor (AdDN-TbetaRII), posterior frontal suture fusion was inhibited. In this study, the authors attempt to further elucidate the role of TGF-beta in cranial suture fusion by investigating possible mechanisms of AdDN-TbetaRII-mediated cranial suture patency using both an established organ culture model and a novel in vitro co-culture system that recapitulates the in vivo anatomic dura mater/cranial suture relationship. In this article, the authors demonstrate that blocking TGF-beta signaling with the AdDN-TbetaRII construct led to inhibition of cellular proliferation in the suture mesenchyme and subjacent dura mater during the early period of predicted posterior frontal suture fusion. Interestingly, co-culture experiments revealed that transfecting osteoblasts with AdDN-TbetaRII led to alterations in the gene expression levels of two important bone-related molecules (Msx2 and osteopontin). Inhibiting TGF-beta signaling prevented time-dependent suppression of Msx2 and prevented induction of osteopontin, thereby retarding osteoblast differentiation. Furthermore, the authors demonstrated that the AdDN-TbetaRII construct was capable of blocking TGF-beta -mediated up-regulation of collagen IalphaI, an extracellular matrix molecule important for bone formation. Collectively, these data strongly suggest that AdDN-TbetaRII maintains posterior frontal patency, in part by altering early events in de novo bone formation, including cellular proliferation and early extracellular matrix production.     

The role of adapter protein Shc in estrogen non-genomic action

Breast cancer is one of the most common malignancies in the United States. Seventy percent of breast cancers are hormone-responsive due to the presence of estrogen receptors ERalpha and ERbeta, which are important diagnostic and therapeutic targets in cancer treatment. Estrogen acts through its receptors, which reside on the cell membrane as demonstrated recently and in the nucleus, leading to cancer cell proliferation and protection from cell death. The membrane ERalpha has been reported in MCF-7 human breast cancer cells and is believed to mediate estrogen effects to activate mitogen-activated protein (MAP) kinase and phosphoinositide 3-kinase (PI3-kinase). Activation of many growth factor receptors require adapter proteins to delivery the upstream signals to downstream kinases, such as MAP kinase. Both Shc and the p85alpha subunit of PI3-kinase are adapter proteins. In addition to their roles in transducing signals from membrane growth factor receptors, they have been demonstrated to interact with ERalpha in an estrogen dependent manner. In this review, the role of Shc in mediating estrogen effects on MAP Kinase regulation, cell growth and anti-apoptosis will be discussed. The possible role of PI3-kinase in estrogen rapid action is also reviewed in brief.     

Fus3-regulated Tec1 degradation through SCFCdc4 determines MAPK signaling specificity during mating in yeast

Signaling specificity is fundamental for parallel mitogen-activated protein kinase (MAPK) cascades that control growth and differentiation in response to different stimuli. In Saccharomyces cerevisiae, components of the pheromone-responsive MAPK cascade activate Fus3 and Kss1 MAPKs to induce mating and Kss1 to promote filamentation. Active Fus3 is required to prevent the activation of the filamentation program during pheromone response. How Fus3 prevents the crossactivation is not clear. Here we show that Tec1, a cofactor of Ste12 for the expression of filamentation genes, is rapidly degraded during pheromone response. Fus3 but not Kss1 induces Tec1 ubiquination and degradation through the SCFCdc4 ubiquitin ligase. T273 in a predicted high-affinity Cdc4 binding motif is phosphorylated by Fus3 both in vitro and in vivo. Tec1T273V blocks Tec1 ubiquitination and degradation and allows the induction of filamentation genes in response to pheromone. Thus, Fus3 inhibits filamentous growth during mating by degrading Tec1.     

Real-time PCR quantitation of clostridia in feces of autistic children

Based on the hypothesis that intestinal clostridia play a role in late-onset autism, we have been characterizing clostridia from stools of autistic and control children. We applied the TaqMan real-time PCR procedure to detect and quantitate three Clostridium clusters and one Clostridium species, C. bolteae, in stool specimens. Group- and species-specific primers targeting the 16S rRNA genes were designed, and specificity of the primers was confirmed with DNA from related bacterial strains. In this procedure, a linear relationship exists between the threshold cycle (CT) fluorescence value and the number of bacterial cells (CFU). The assay showed high sensitivity: as few as 2 cells of members of cluster I, 6 cells of cluster XI, 4 cells of cluster XIVab, and 0.6 cell of C. bolteae could be detected per PCR. Analysis of the real-time PCR data indicated that the cell count differences between autistic and control children for C. bolteae and the following Clostridium groups were statistically significant: mean counts of C. bolteae and clusters I and XI in autistic children were 46-fold (P = 0.01), 9.0-fold (P = 0.014), and 3.5-fold (P = 0.004) greater than those in control children, respectively, but not for cluster XIVab (2.6 x 10(8) CFU/g in autistic children and 4.8 x 10(8) CFU/g in controls; respectively). More subjects need to be studied. The assay is a rapid and reliable method, and it should have great potential for quantitation of other bacteria in the intestinal tract.