Radiolabeling rhesus monkey CD34+ hematopoietic and mesenchymal stem cells with 64Cu-pyruvaldehyde-bis(N4-methylthiosemicarbazone) for microPET imaging

Noninvasive positron emission tomography (PET) provides a potential method for in vivo tracking of radiolabeled cells. The goal of this study was to assess the potential toxicity of 64Cu-pyruvaldehyde-bis(N4-methylthiosemicarbazone) (PTSM) on rhesus monkey CD34+ hematopoietic and mesenchymal stem cells in vitro in preparation for developing imaging protocols posttransplantation. CD34+ hematopoietic cells were radiolabeled with 0 to 40 microCi/mL 64Cu-PTSM and viability and colony formation were assessed. Rhesus monkey mesenchymal stem cells (rhMSCs) were placed in culture postradiolabeling for assessments of growth and differentiation toward adipogenic, osteogenic, and chondrogenic lineages. The results indicated that CD34+ cells radiolabeled with 20 microCi/mL and rhMSCs radiolabeled with 10 microCi/mL 64Cu-PTSM did not result in adverse effects on growth or differentiation. Nonradioactive copper was also evaluated and showed that the presence of copper was not harmful to the cells. CD34+ cells and rhMSCs radiolabeled with the optimized concentrations of 20 and 10 microCi/mL, respectively, were also assessed using the microPET scanner. Studies showed that a minimum of 2.50x10(4) CD34+ cells (1.1 pCi/cell) and 6.25x10(3) rhMSCs (4.4 pCi/cell) could be detected. These studies indicate that CD34+ hematopoietic cells and rhMSCs can be safely radiolabeled with 64Cu-PTSM without adverse cellular effects.     

Hepatoprotective phytocompounds from Cryptomeria japonica are potent modulators of inflammatory mediators

Cryptomeria japonica is an important plantation conifer tree in Asia. This study aimed to characterize the anti-inflammatory and hepatoprotective activities of the phytocompounds from C. japonica wood on LPS- or TPA-induced activation of proinflammatory mediators and CCl(4)-induced acute liver injury in mice. A CJH7-2 fraction was purified from C. japonica extracts following bioactivity-guided fractionation, and it exhibited significant activities on inhibition of NO production and iNOS expression as well as up-regulating HO-1 expression in LPS-stimulated macrophages. CJH7-2 also potently inhibits COX-2 enzymatic activity (IC(50)=5 microg/mL) and TPA-induced COX-2 protein expression in mouse skin (1mg/200 microL/site). CJH7-2 (10 mg/kg BW) can prevent CCl(4)-induced liver injury and aminotransferases activities in mice. Chemical fingerprinting analysis showed that terpenes are the major bioactive compounds in the CJH7-2 fraction. This is the first study to demonstrate that chemical constituents from the wood extract of C. japonica possess anti-inflammatory activities in vitro and in vivo that may play a role in hepatoprotection.     

Rapid and simultaneous determination of tacrolimus (FK506) and diltiazem in human whole blood by liquid chromatography-tandem mass spectrometry: application to a clinical drug-drug interaction study

Tacrolimus (FK506) is a potent immunosuppressant widely used for organ transplantation patients while diltiazem (DTZ), a calcium-channel inhibitor, is often used in renal transplantation patients to prevent post-transplant hypertension. However, DTZ has a significant pharmacokinetic interaction with FK506. In this study, a rapid and sensitive ammonium-adduct based liquid chromatography-tandem mass spectrometry (LC/MS/MS) method has been developed and validated for the simultaneous determination of FK506 and DTZ in human whole blood using ascomycin as the internal standard (IS). After extraction of the whole blood samples by ethyl acetate, FK506, DTZ and the IS were subjected to LC/MS/MS analysis using electro-spray positive-ion mode ionization (ESI(+)). Chromatographic separation was performed on a Hypersil BDS C18 column (50 mm x 2.1 mm, i.d., 3 microm). The MS/MS detection was conducted by monitoring the fragmentation of 821.7-->768.9 (m/z) for FK506, 415.5-->310.3 (m/z) for DTZ and 809.8-->757.0 (m/z) for IS. The method had a chromatographic running time of approximately 2 min and linear calibration curves over the concentrations of 0.5-200 ng/mL for FK506 and 2-250 ng/mL for DTZ. The recoveries of liquid-liquid extraction method were 58.3-62.6% for FK506 and 50.4-58.8% for DTZ. The lower limit of quantification (LLOQ) of the analytical method was 0.5 ng/mL for FK506 and 2 ng/mL for DTZ. The intra- and inter-day precision was less than 15% for all quality control samples at concentrations of 2, 10, and 50 ng/mL for FK506 and 5, 25, and 100 ng/mL for DTZ. The validated LC/MS/MS method has been successfully used to analyze the concentrations of FK506 and DTZ in whole blood samples from pharmacokinetic studies in renal transplanted patients.     

Identification and Characterization of Proteins Associated with Plant Tolerance to Heat Stress

Heat stress is a major abiotic stress limiting plant growth and productivity in many areas of the world. Understanding mechanisms of plant adaptation to heat stress would facilitate the development of heat-tolerant cultivars for improving productivity in warm climatic regions. Protein metabolism involving protein synthesis and degradation is one of the most sensitive processes to heat stress. Changes in the level and expression pattern of some proteins may play an important role in plant adaptation to heat stress. The identification of stress-responsive proteins and pathways has been facilitated by an increasing number of tools and resources, including two-dimensional electrophoresis and mass spectrometry, and the rapidly expanding nucleotide and amino acid sequence databases. Heat stress may induce or enhance protein expression or cause protein degradation. The induction of heat-responsive proteins, particularly heat shock proteins (HSPs), plays a key role in plant tolerance to heat stress. Protein degradation involving various proteases is also important in regulating plant responses to heat stress. This review provides an overview of recent research on proteomic profiling for the identification of heat-responsive proteins associated with heat tolerance, heat induction and characteristics of HSPs, and protein degradation in relation to plant responses to heat stress.     

Osthole and imperatorin, the active constituents of Cnidium monnieri (L.) Cusson, facilitate glutamate release from rat hippocampal nerve terminals

We examined the effects of osthole and imperatorin, two active compounds of Cnidium monnieri (L.) Cusson, on the release of glutamate from rat hippocampal synaptosomes and investigated the possible mechanism. The results showed that osthole or imperatorin significantly facilitated 4-aminopridine (4-AP)-evoked glutamate release in a concentration-dependent manner. The facilitatory action of osthole or imperatorin was blocked by the vesicular transporter inhibitor bafilomycin A1, not by the glutamate transporter inhibitor l-transpyrrolidine-2,4-dicarboxylic acid (l-trans-PDC), indicating that the release facilitation by osthole or imperatorin results from a enhancement of vesicular exocytosis and not from an increase of Ca²⁺-independent efflux via glutamate transporter. Examination of the effect of osthole and imperatorin on cytosolic [Ca²⁺] revealed that the facilitation of glutamate release could be attributed to an increase in voltage-dependent Ca²⁺ influx. Consistent with this, ω-conotoxin MVIIC, a wide-spectrum blocker of the N- and P/Q-type Ca²⁺ channels, significantly suppressed the osthole or imperatorin-mediated facilitation of glutamate release, but intracellular Ca²⁺ release inhibitor dantrolene had no effect. Osthole or imperatorin did not alter the resting synaptosomal membrane potential or 4-AP-mediated depolarization; thus, the facilitation of 4-AP-evoked Ca²⁺ influx and glutamate release produced by osthole or imperatorin was not due to it decreasing synaptosomal excitability. In addition, osthole or imperatorin-mediated inhibition of 4-AP-evoked release was prevented by protein kinase C (PKC) inhibitors. Furthermore, osthole or imperatorin increased 4-AP-induced phosphorylation of PKC. Together, these results suggest that osthole or imperatorin effects a facilitation of glutamate release from nerve terminals by positively modulating N-and P/Q-type Ca²⁺ channel activation through a signaling cascade involving PKC.     

Simultaneous assessment of the effects of exonic mutations on RNA splicing and protein functions

To simultaneously assess the effects of exonic mutations on RNA splicing and protein functions, we report here an intron-inclusive cDNA (Intinc) expression system. As a test model, twenty-four mutations in exon 9 of the phenylalanine hydroxylase (PAH) gene were examined in an Intinc expression plasmid composed of the PAH cDNA with the exon 9 flanked by its authentic introns. When the PAH enzyme activities from the Intinc plasmid-transfected cells were compared to those of a standard cDNA expression system, five mutations resulted in significant relative differences in PAH activities attributed to altered exon 9-inclusive mRNA levels. Two of the mutations affected exon recognition probably through splice site modifications and the remaining three affected experimentally verified exon splicing enhancer (ESE) motifs. The Intinc expression system allows not only a better link between mutation genotype to disease phenotype but also contributes to further understanding of molecular mechanisms of deleterious effects of mutations.     

颈动脉窦内压的变化速率对反射性降压效应的影响

在成年狗身上制备右侧孤离颈动脉窦,借助灌流装置用饱和氧的任氏液对其进行灌流。灌流压为搏动性的。通过改变每搏泵出量、灌流管道的阻力和弹性来调节灌流压及其变化速率。用多导生理记录仪同步记录股动脉血压、窦内灌流压及其变化速率。本文主要观察窦内压的变化速率对降压反射的影响。在8只狗身上共进行了93次实验。结果表明,在窦内压相同的情况下,灌流压的上升速率愈快,降压效应愈明显,而其下降速率则无显著作用。已有资料证明搏动性窦内压所引起的降压效应较非搏动性压力更为明显。由此可见,狗的颈动脉窦压力感受器不仅对搏动性压力而且对其上升的变化速率也很敏感。     

Overexpression of Smad7 suppressed ROS/MMP9-dependent collagen synthesis through regulation of heme oxygenase-1

We previously reported that AngiotensinII receptor blocker effectively inhibited TGF-β1-mediated epithelial-to-mesenchymal transition progress through regulating Smad7. However, the underlying mechanism by which Smad7 exerted in regulating MMP9 and fibrogenic response has not been fully elucidated. In the current study, we proved that NADPH p47^phox-dependent reactive oxygen species (ROS) production contributed to MMP9 activation and collagen expression, which was suppressed by transfecting pcDNA3–Smad7 in cardiac fibroblasts. The effect of Smad7 overexpression on MMP9 activity and collagen expression was further reversed by adding H₂O₂ (10 μmol/L). In contrast, knockdown of Smad7 caused the enhanced collagen synthesis in cardiac fibroblasts, which was also reversed by treating cells with a ROS inhibitor, YCG063 (2 μmol/L). Further investigation showed that Smad7 regulated NADPH-mediated ROS production through activating Heme oxygenase-1 (HO-1). Meanwhile, the intercellular level of bilirubin (product of hemin) and nitric oxide (NO) in cell supernatant were not significantly increased in cells treated with AngII or transfected with Smad7. Knockdown of HO-1 in Smad7-overexpressed cardiac fibroblasts or cells pretreated with SnPP IX, a competitive inhibitor of HO-1 activity, resulted in increased productions of ROS and NADPH p47^phox, and abolished the inhibitory effects of Smad7 on MMP9 activity and collagen expression. Our results indicated that HO-1 might be critically involved in Smad7-mediated regulation of MMP9 activity and fibrogenic genes expression via antagonizing the enhanced myocardial oxidative stress.     

Nicotine-Induced Neuroprotection Against Ischemic Injury Involves Activation of Endocannabinoid System in Rats

Nicotine has been reported to exert certain protective effect in the Parkinson’s and Alzheimer’s diseases. Whether it has a similar action in focal cerebral ischemia was unclear. In the present study, rats received either an injection of (?)-nicotine hydrogen tartrate salt (1.2 mg/kg, i.p.) or the vehicle 2 h before the 120 min middle cerebral artery occlusion. Neurological deficits and histological injury were assessed at 24 h after reperfusion. The content of endocannabinoids and the expression of cannabinoid receptor CB1 in brain tissues were determined at different time points after nicotine administration. Results showed that nicotine administration ameliorated neurological deficits and reduced infarct volume induced by cerebral ischemia in the rats. The neuroprotective effect was partially reversed by CB1 blockage. The content of the endocannabinoids N-arachidonylethanolamine and 2-arachidonoylglycerol, as well as the expression of cannabinoid receptor CB1 were up-regulated in brain tissues after nicotine delivery. These results suggest that endogenous cannabinoid system is involved in the nicotine-induced neuroprotection against transient focal cerebral ischemia.