Mechanisms of the acute ischemia-induced arrhythmogenesis--a simulation study

The underlying ionic mechanisms of ischemic-induced arrhythmia were studied by the computer simulation method. To approximate the real situation, ischemic cells were simulated by considering the three major component conditions of acute ischemia (elevated extracellular K(+) concentration, acidosis and anoxia) at the level of ionic currents and ionic concentrations, and a round ischemic zone was introduced into a homogeneous healthy sheet to avoid sharp angle of the ischemic tissue. The constructed models were solved using the operator splitting and adaptive time step methods, and the perturbation finite difference (PFD) scheme was first used to integrate the partial differential equations (PDEs) in the model. The numerical experiments showed that the action potential durations (APDs) of ischemic cells did not exhibited rate adaptation characteristic, resulting in flattening of the APD restitution curve. With reduction of sodium channel availability and long recovery of excitability, refractory period of the ischemic tissue was significantly prolonged, and could no longer be considered as same as APD. Slope of the conduction velocity (CV) restitution curve increased both in normal and ischemic region when pacing cycle length (PCL) was short, and refractory period dispersion increased with shortening of PCL as well. Therefore, dynamic changes of CV and dispersion of refractory period rather than APD were suggested to be the fundamental mechanisms of arrhythmia in regional ischemic myocardium.     

Denaturation and unfolding of human anaphylatoxin C3a: an unusually low covalent stability of its native disulfide bonds

The complement C3a anaphylatoxin is a major molecular mediator of innate immunity. It is a potent activator of mast cells, basophils and eosinophils and causes smooth muscle contraction. Structurally, C3a is a relatively small protein (77 amino acids) comprising a N-terminal domain connected by 3 native disulfide bonds and a helical C-terminal segment. The structural stability of C3a has been investigated here using three different methods: Disulfide scrambling; Differential CD spectroscopy; and Reductive unfolding. Two uncommon features regarding the stability of C3a and the structure of denatured C3a have been observed in this study. (a) There is an unusual disconnection between the conformational stability of C3a and the covalent stability of its three native disulfide bonds that is not seen with other disulfide proteins. As measured by both methods of disulfide scrambling and differential CD spectroscopy, the native C3a exhibits a global conformational stability that is comparable to numerous proteins with similar size and disulfide content, all with mid-point denaturation of [GdmCl]_1/2 at 3.4-5 M. These proteins include hirudin, tick anticoagulant protein and leech carboxypeptidase inhibitor. However, the native disulfide bonds of C3a is 150-1000 fold less stable than those proteins as evaluated by the method of reductive unfolding. The 3 native disulfide bonds of C3a can be collectively and quantitatively reduced with as low as 1 mM of dithiothreitol within 5 min. The fragility of the native disulfide bonds of C3a has not yet been observed with other native disulfide proteins. (b) Using the method of disulfide scrambling, denatured C3a was shown to consist of diverse isomers adopting varied extent of unfolding. Among them, the most extensively unfolded isomer of denatured C3a is found to assume beads-form disulfide pattern, comprising Cys³⁶-Cys⁴⁹ and two disulfide bonds formed by two pair of consecutive cysteines, Cys²²-Cys²³ and Cys⁵⁶-Cys⁵⁷, a unique disulfide structure of polypeptide that has not been documented previously.     

Nicotiana chloroplast genome

Summary Using the existing restriction map and probes from wheat and pea ct-DNA, seven protein genes have been localized in the chloroplast genome of N. tabacum. On the clock-like map, the location of each gene is indicated by its time zone: the 15.2 kD polypeptide of the cytochrome b/f complex at 315, cytochrome f at 430, LS of RuBPCase at 450, both and subunits of ATP synthase at or near 500, proton-translocating subunit of ATP synthase at 820, subunit of ATP synthase at 840 and the 32 kD protein at 930. The genome organization of Nicotiana chloroplast DNA is similar to spinach.     

Combination of bone tissue engineering and BMP-2 gene transfection promotes bone healing in osteoporotic rats

OBJECTIVE: The aim of this study was to develop a feasible approach to promote bone healing in osteoporotic rats using autogenous bone tissue-engineering and gene transfection of human bone morphogenetic protein 2 (hBMP-2). METHODS: Bone marrow stromal cells (BMSCs) from the left tibia of osteoporotic rats were transfected with the hBMP-2 gene in vitro which was confirmed by immunohistochemistry, in situ hybridization and Western blotting. Autogenous transfected or untransfected BMSCs were seeded on macroporous coral hydroxyapatite (CHA) scaffolds. Each cell-scaffold construct was implanted into a defect site which was created in the ramus of the mandible of osteoporotic rats. Four or eight weeks after implantation in situ hybridization was performed in BMSCs transfected with hBMP-2, X-ray examinations, histological and histomorphological analyses were used to evaluate the effect of tissue-engineered bone on osseous defect repair. RESULTS: Newly formed bone was observed at the margin of the defect 4 weeks after implantation with BMSCs transfected with BMP-2. Mature bone was observed 8 weeks after treatment. In the control group there was considerably less new bone and some adipose tissue was observed at the defect margins 8 weeks after implantation. CONCLUSIONS: Autogenous cells transfected with hBMP-2 promote bone formation in osteoporotic rats. BMSC-mediated BMP-2 gene therapy used in conjunction with bone tissue engineering may be used to successfully treat bone defects in osteoporotic rats. This method provides a powerful tool for bone regeneration and other tissue engineering.     

Evodiamine inhibits in vitro angiogenesis: Implication for antitumorgenicity

Evodiamine, the major bioactive compound isolated from Chinese herbal drug named Wu-Chu-Yu, has been reported to exhibit anti-tumor growth and metastasis. However, the effect of evodiamine on angiogenesis remains to be investigated. We used the fresh medium containing evodiamine or human lung adenocarcinoma cell (CL1 cells) derived conditioned media free of evodiamine to test their capability to induce in vitro angiogenesis, i.e., human umbilical vein endothelial cells (HUVECs) tube formation and invasion. We demonstrated that evodiamine could directly inhibit in vitro HUVECs tube formation and invasion. Locally administered evodiamine also inhibited the in vivo angiogenesis in the chick embryo chorioallantoic membrane (CAM) assay. The gene expression of vascular endothelial growth factor (VEGF) and the p44/p42 mitogen-activated protein kinase (MAPK, ERK) that correlated with endothelial cells angiogenesis were inhibited by evodiamine. We found that the evodiamine-treated CL1 cells derived conditioned medium showed decreased VEGF release and reduced ability of inducing in vitro tube formation. After the collection of conditioned media, the VEGF expression of remaining CL1 cells were determined by Western analyses and revealed that evodiamine decreased VEGF expression. Moreover, administration of recombinant human VEGF(165) (rhVEGF(165)) induced tube formation and ERK phosphorylation by HUVECs, and partially attenuated inhibitory effect of evodiamine. From these results, we suggested that evodiamine is a potent inhibitor of angiogenesis. The mechanism might involve at least the inhibition of VEGF expression, probably through repression of ERK phosphorylation.