一氧化氮对家兔房室结细胞自发活动的电生理效应

应用经典玻璃微电极技术,观察一氧化氮(NO)对家兔房室结细胞自发活动的电生理效应及其作用机制。结果显示：(1)NO供体硝普(SNP,1-1000μmol／L)及SIN-1(100,1000μmol／L)剂量依赖性地抑制房室结细胞的动作电位幅值(APA)、零相最大人士升速度(Vmax)、4期自动除极速度(VDD)及自发放电频率(RSF);(2)应用L型钙通道开放剂Bay K8644(0．25μmol／L),可拮抗SNP对房室结细胞的电生理效应;(3)提高灌流液中钙离子浓度(5mmol／L)也可逆转SNP对起搏细胞的抑制效应;(4)用无钙K-H液灌流房室结,可完全阻断SNP对房事结细胞的抑制效应。(5)应用鸟苷酸环化酶阻断剂甲基美蓝(50μmol／L)对SNP的上述电生理效应无影响。以上结果提示,NO可能是通过cGMP非依赖性途径减弱钙离子内流,进而抑制了家兔房室结细胞的自发电活动。     

Inhibition of endogenous trypsin- and chymotrypsin-like activities in transgenic lettuce expressing heterogeneous proteinase inhibitor SaPIN2a

SaPIN2a, a proteinase inhibitor II from American black nightshade (Solanum americanum Mill.) is highly expressed in the phloem and could be involved in regulating proteolysis in the sieve elements. To further investigate the physiological role of SaPIN2a, we have produced transgenic lettuce (Lactuca sativa L.) expressing SaPIN2a from the CaMV35S promoter by Agrobacterium-mediated transformation. Stable integration of the SaPIN2a cDNA and its inheritance in transgenic lines were confirmed by Southern blot analysis and segregation analysis of the R₁ progeny. SaPIN2a mRNA was detected in both the R₀ and R₁ transformants on northern blot analysis but the SaPIN2a protein was not detected on western blot analysis using anti-peptide antibodies against SaPIN2a. Despite an absence of significant inhibitory activity against bovine trypsin and chymotrypsin in extracts of transgenic lettuce, the endogenous trypsin-like activity in each transgenic line was almost completely inhibited, and the endogenous chymotrypsin-like activity moderately inhibited. Our finding that heterogeneously expressed SaPIN2a in transgenic lettuce inhibits plant endogenous protease activity further indicates that SaPIN2a regulates proteolysis, and could be potentially exploited for the protection of foreign protein production in transgenic plants.Abbreviations CaMV cauliflower mosaic virus - cDNA complementary DNA - NOS nopaline synthase - PAGE polyacrylamide gel electrophoresis - PI proteinase inhibitor - SaPIN2a Solanum americanum proteinase inhibitor IIa - SDS sodium dodecyl sulphate - T-DNA transferred DNA     

Human mesenchymal stem cells tissue development in 3D PET matrices

Human mesenchymal stem cells (hMSCs) are attractive cell sources for engineered tissue constructs with broad therapeutic potential. Three-dimensional (3D) hMSC tissue development in nonwoven poly(ethylene terephthalate) (PET) fibrous matrices was investigated. HMSCs were seeded onto 3D PET scaffolds and were cultured for over 1 month. Their proliferation rates were affected by seeding density but remained much lower than those of 2D controls. Compared to 2D surfaces, hMSCs grown in 3D scaffolds secreted and embedded themselves in an extensive ECM network composed of collagen I, collagen IV, fibronectin, and laminin. HMSCs were influenced by the orientation of adjacent PET fibers to organize the ECM proteins into highly aligned fibrils. We observed the increased expressions of alpha(2)beta(1) integrin but a slight decrease in the expression of alpha(5)beta(1) integrin in 3D compared to 2D culture and found that alpha(V)beta(3) was expressed only in 2D. Paxillin expression was down-regulated in 3D culture with a concomitant change in its localization patterns. We demonstrated the multi-lineage potentials of the 3D tissue constructs by differentiating the cells grown in the scaffolds into osteoblasts and adipocytes. Taken together, these results showed that hMSCs grown in 3D scaffolds display tissue development patterns distinct from their 2D counterparts and provide important clues for designing 3D scaffolds for developing tissue engineered constructs.     

Stem cells: cross-talk and developmental programs

The thesis advanced in this essay is that stem cells-particularly those in the nervous system-are components in a series of inborn 'programs' that not only ensure normal development, but persist throughout life so as to maintain homeostasis in the face of perturbations-both small and great. These programs encode what has come to be called 'plasticity'. The stem cell is one of the repositories of this plasticity. This review examines the evidence that interaction between the neural stem cell (as a prototypical somatic stem cell) and the developing or injured brain is a dynamic, complex, ongoing reciprocal set of interactions where both entities are constantly in flux. We suggest that this interaction can be viewed almost from a 'systems biology' vantage point. We further advance the notion that clones of exogenous stem cells in transplantation paradigms may not only be viewed for their therapeutic potential, but also as biological tools for 'interrogating' the normal or abnormal central nervous system environment, indicating what salient cues (among the many present) are actually guiding the expression of these 'programs'; in other words, using the stem cell as a 'reporter cell'. Based on this type of analysis, we suggest some of the relevant molecular pathways responsible for this 'cross-talk' which, in turn, lead to proliferation, migration, cell genesis, trophic support, protection, guidance, detoxification, rescue, etc. This type of developmental insight, we propose, is required for the development of therapeutic strategies for neurodegenerative disease and other nervous system afflictions in humans. Understanding the relevant molecular pathways of stem cell repair phenotype should be a priority, in our view, for the entire stem cell field.     

Cytokine mRNA expression in unilateral ischemic-reperfused rat lung with salt solution supplemented with low-endotoxin or standard bovine serum albumin

Our aim was to determine whether cytokine mRNA expression is induced by experimental manipulation including artificial perfusate or ischemia-reperfusion (I/R) in an isolated, perfused rat lung model. Constant pulmonary flow [Krebs-Henseleit solution supplemented with low-endotoxin (LE) or standard (ST) bovine serum albumin 4%, 0.04 ml/g body wt] and ventilation were maintained throughout. Right and left pulmonary arteries were isolated, and the left pulmonary artery was occluded for 60 min and then reperfused for 30 min. Analysis of tumor necrosis factor-alpha, IL-1 beta, IL-6, IL-10, and IFN-gamma mRNA expression by RT-PCR and evaluation of vascular permeability by bronchoalveolar lavage (BAL) fluid albumin content were conducted separately in right and left lung. Both LE and ST groups (each 12 rats) showed increases in vascular permeability by I/R (BAL fluid albumin content: 5.53 +/- 1.55 vs. 15.63 +/- 8.87 and 4.76 +/- 2.71 vs. 16.72 +/- 4.85 mg.ml BAL fluid-1.g lung dry wt-1, mean +/- SD; right vs. left lung in LE and ST groups, P < 0.05 between right and left). Cytokine mRNA expression was significantly higher in the I/R lung than in the control lung in the LE group, whereas it was higher in the control lung in the ST group (P < 0.05). mRNAs of not only proinflammatory but also anti-inflammatory cytokines were expressed in I/R lung, which are expected to aggravate I/R injury. The reversed pattern of cytokine mRNA expression in the ST group was possibly due to the longer perfusion of control lung with perfusate containing endotoxin, which caused no lung damage without I/R.     

T cells infiltrate the brain in murine and human transmissible spongiform encephalopathies

CD4 and CD8 T lymphocytes infiltrate the parenchyma of mouse brains several weeks after intracerebral, intraperitoneal, or oral inoculation with the Chandler strain of mouse scrapie, a pattern not seen with inoculation of prion protein knockout (PrP(-/-)) mice. Associated with this cellular infiltration are expression of MHC class I and II molecules and elevation in levels of the T-cell chemokines, especially macrophage inflammatory protein 1beta, IFN-gamma-inducible protein 10, and RANTES. T cells were also found in the central nervous system (CNS) in five of six patients with Creutzfeldt-Jakob disease. T cells harvested from brains and spleens of scrapie-infected mice were analyzed using a newly identified mouse PrP (mPrP) peptide bearing the canonical binding motifs to major histocompatibility complex (MHC) class I H-2(b) or H-2(d) molecules, appropriate MHC class I tetramers made to include these peptides, and CD4 and CD8 T cells stimulated with 15-mer overlapping peptides covering the whole mPrP. Minimal to modest K(b) tetramer binding of mPrP amino acids (aa) 2 to 9, aa 152 to 160, and aa 232 to 241 was observed, but such tetramer-binding lymphocytes as well as CD4 and CD8 lymphocytes incubated with the full repertoire of mPrP peptides failed to synthesize intracellular gamma interferon (IFN-gamma) or tumor necrosis factor alpha (TNF-alpha) cytokines and were unable to lyse PrP(-/-) embryo fibroblasts or macrophages coated with (51)Cr-labeled mPrP peptide. These results suggest that the expression of PrP(sc) in the CNS is associated with release of chemokines and, as shown previously, cytokines that attract and retain PrP-activated T cells and, quite likely, bystander activated T cells that have migrated from the periphery into the CNS. However, these CD4 and CD8 T cells are defective in such an effector function(s) as IFN-gamma and TNF-alpha expression or release or lytic activity.     

Detailed analysis of the CD8+ T-cell response following adenovirus vaccination

We examined CD8(+) T-cell expansion and function following intramuscular immunization with a recombinant adenovirus. This study has identified a number of properties which may explain the strong immunogenicity of adenovirus vectors: (i) the ability to deliver large amounts of antigen into the lymphoid tissues, (ii) the ability to induce rapid expansion and migration of CD8(+) T cells throughout the lymphatics, and (iii) the ability to produce a sustained, high-level CD8(+) T-cell response.     

Detection of open and closed conformations of tryptophan synthase by 15N-heteronuclear single-quantum coherence nuclear magnetic resonance of bound 1-15N-L-tryptophan

1-15N-L-Tryptophan (1-15N-L-Trp) was synthesized from 15N-aniline by a Sandmeyer reaction, followed by cyclization to isatin, reduction to indole with LiAlH4, and condensation of the 15N-indole with L-serine, catalyzed by tryptophan synthase. 1-15N-L-Trp was complexed with wild-type tryptophan synthase and beta-subunit mutants, betaK87T, betaD305A, and betaE109D, in the absence or presence of the allosteric ligands sodium chloride and disodium alpha-glycerophosphate. The enzyme complexes were observed by 15N-heteronuclear single-quantum coherence nuclear magnetic resonance (15N-HSQC NMR) spectroscopy for the presence of 1-15N-L-Trp bound to the beta-active site. No 15N-HSQC signal was detected for 1-15N-L-Trp in 10 mm triethanolamine hydrochloride buffer at pH 8. 1-15N-L-Trp in the presence of wild-type tryptophan synthase in the absence or presence of 50 mm sodium chloride showed a cross peak at 10.25 ppm on the 1H axis and 129 ppm on the 15N axis as a result of reduced solvent exchange for the bound 1-15N-L-Trp, consistent with formation of a closed conformation of the active site. The addition of disodium alpha-glycerophosphate produced a signal twice as intense, suggesting that the equilibrium favors the closed conformation. 15N-HSQC NMR spectra of betaK87T and betaE109D mutant Trp synthase with 1-15N-L-Trp showed a similar cross peak either in the presence or absence of disodium alpha-glycerophosphate, indicating the preference for a closed conformation for these mutant proteins. In contrast, the betaD305A Trp synthase mutant only showed a 15N-HSQC signal in the presence of disodium alpha-glycerophosphate. Thus, this mutant Trp synthase favored an open conformation in the absence of disodium alpha-glycerophosphate but was able to form a closed conformation in the presence of disodium alpha-glycerophosphate. Our results demonstrate that the 15N-HSQC NMR spectra of 1-15N-L-Trp bound to Trp synthase can be used to determine the conformational state of mutant forms in solution rapidly. In contrast, UV-visible spectra of wild-type and mutant Trp synthase in the presence of L-Trp with NaCl and/or disodium alpha-glycerophosphate are more difficult to interpret in terms of altered conformational equilibria.