安宫牛黄丸原方及简方对脑出血大鼠损伤保护作用的研究

探讨安宫牛黄原方及简方对脑出血大鼠的保护作用,寻找更合理的组方。方法:采用胶原酶尾状核注射法造模,观察安宫牛黄丸原方及简方对脑出血损伤大鼠脑组织匀浆中超氧化物歧化酶(SOD)、丙二醛(MDA)含量的影响。结果:安宫牛黄原方及简方能不同程度的降低谷氨酸损伤神经元MDA含量,提高SOD的活性。结论:安宫牛黄丸全方及简方可在一定程度上通过抗氧化损伤来起到脑保护作用。     

Cloning and characterization of PhPI9 involved in floral development from Phalaenopsis Orchid

In the attempt to discover new genes involved in the floral development in monoeotyledonousin species,we have cloned and characterized the homologous PISTALLATA-like (PI-like) gone from Phalaenopsis hybrid cultivar named PhPI9 (Phalaenopsis PI STILLATA # 9).The eDNA of PhPI9 has a fragment of 834 bp and has 60% identity with the PISTILATA from Arabidopsis.The deduced amino acid sequence of PhPI9 had the typical PI-motif.It also formed a subelade with other monoeot PI-type genes in phylogenetie analysis.Southern analysis showed that PhPI9 was present in the Phalaenopsis orchid genome as a single copy.Furthermore,it was expressed only in the lip of the Phalaenopsis flower and no expression was detected in vegetative organs.Thus,as a B-function MADS-box gone,PhP19 specifies floral organ identity in orchids.     

Novel N-(3-carboxyl-9-benzyl-carboline-1-yl)ethylamino acids: synthesis, anti-proliferation activity and two-step-course of intercalation with calf thymus DNA

To explore the intercalating mechanism of -carbolines, four novel N-(3-carboxyl-9-benzyl-carboline-1-yl)ethylamino acids [-phenylalanine (6a), -alanine (6b), -isoleucine (6c) and -glycine (6d)] were prepared here. Their in vitro anticancer activities were examined by their anti-proliferation for 5 human carcinoma cell lines. The average IC50s against 5 human carcinoma cell lines are 53.54 microM, 118.77 microM, 147.34 microM and greater than 291.63 microM for 6a, 6b, 6c and 6d, respectively. The DNA intercalating mechanism of 6a-d was approved by the comparison of the parameters and signals of UV, CD and fluorescence spectra of calf thymus DNA (CT DNA) alone and the CT DNA/6a-d system. Using fluorescence titration based kinetic analysis a two-step-course consisting of stacking and intercalating was described and the stacking was considered as the key step to the CT DNA intercalating mechanism of 6a-d. Using fluorescence titration based thermomechanical analysis, the stacking complexes of 6a-d with CT DNA were described to be formed spontaneously and to be stabilized predominantly by their hydrophobic interactions. The intercalation itself goes very fast and only has limited contribution to their anticancer activities.     

Organotypic culture of adult rabbit retina

Organotypic culture systems of functional neural tissues are important tools in neurobiological research. Ideally, such a system should be compatible with imaging techniques, genetic manipulation, and electrophysiological recording. Here we present a simple interphase tissue culture system for adult rabbit retina that requires no specialized equipment and very little maintenance. We demonstrate the dissection and incubation of rabbit retina and particle-mediated gene transfer of plasmids encoding GFP or a variety of subcellular markers into retinal ganglion cells. Rabbit retinas cultured this way can be kept alive for up to 6 days with very little changes of the overall anatomical structure or the morphology of individual ganglion- and amacrine cells.     

Two new antibacterial sesquiterpenoids from Centipeda minima

Two new guaiane-type sesquiterpene lactones, compounds 1 and 2, along with three known guaianolide- or pseudoguaianolides, were isolated from Centipeda minima (whole plant). Their structures were identified by spectroscopic and mass-spectrometric analyses. The configuration at C5 of the guaiane framework of 1 was rationalized by quantum-mechanical calculations (Table 2). All compounds were found to be active against eight different microbial pathogens (Table 3), with MIC values in the range of 6.25-100 microg/ml.     

A fundamental regulatory role of formate on thuringiensin production by resting cell of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> YBT-032

In this work, a fundamental regulatory role of formate on thuringiensin production by resting cell of Bacillus thuringiensis YBT-032 was investigated. Nicotinamide adenine dinucleotide (NADH) production and formate dehydrogenase activity increased with formate addition from 0.5 to 2.0 g/L, respectively. However, with the formate addition of 1.5 g/L, the activities of pyruvate kinase and glucose 6-phosphate dehydrogenase reached a peak and increased by 316 and 150% relative to those of the control, respectively. In addition, intracellular production of pyruvate, aspartate, citrate and adenine were significantly enhanced by 75, 66, 32 and 78% as well. An improvement (90%) of thuringiensin production was also successfully obtained. Interestingly to point out, thuringiensin yield was closely correlative with adenine production, and the linear relationship was also observed. The results suggest that appropriate formate addition did act as a modulator and facilitate carbon flux in glycolysis and pentose phosphate pathway to synthesize adenine and thuringiensin via intracellular NADH availability.     

Differential recovery of membrane proteins after extraction by aqueous methanol and trifluoroethanol

Cell membrane proteome analysis is limited by inherent membrane hydrophobicity. Conventional membrane protein extraction techniques use detergents, chaotropes and organic acids that require sample clean-up or pH adjustment, and are associated with significant sample loss. We extracted membrane proteins from red blood cells (RBCs) using methanol (MeOH), trifluoroethanol (TFE) and urea, and identified membrane proteins using 2-D LC coupled with MALDI-TOF/TOF-MS. We show that organic solvents MeOH- and TFE-based methods have membrane protein analysis efficiencies comparable to urea, and are complementary for the recovery of both hydrophilic and hydrophobic peptides. The mean grand average of hydropathicity (GRAVY) value of identified peptides from the TFE-based method (-0.107) was significantly higher than that of the MeOH-based method (-0.465) (p<0.001). Sequential and adjunctive use of the organic solvents MeOH and TFE increases the number of proteins identified, and the confidence of their identification. We show that this strategy is effective for shotgun membrane proteome analysis.     

H5N1禽流感病毒HA基因在昆虫细胞中的表达及生物活性鉴定

经RT-PCR扩增了H5N1亚型禽流感病毒血凝素基因(HA)片断,限制性内切酶酶切后将其克隆到pFastBacHTA杆状病毒转座载体,经酶切鉴定及测序,筛选出阳性重组转座载体pFastBac-H5。将pFastBacH5转化含有杆状病毒穿梭载体（bacmid）的DH10Bac感受态细胞,通过蓝白斑筛选和PCR鉴定获得重组杆状病毒穿梭载体rBacmid-H5。rBacmid-H5在脂质体介导下转染sf9昆虫细胞,SDS－PAGE蛋白电泳、Western blot、血凝试验和血凝抑制试验分析表明：分子量约63Kd重组血凝素蛋白（rH5）在sf9昆虫细胞中实现了高效表达。rH5具有血凝活性,而且其血凝活性能够被H5N1禽流感病毒高免血清所抑制;rH5免疫鸡诱导产生针对H5N1禽流感病毒亚型特异的血凝抑制抗体,说明表达的重组蛋白具有与天然蛋白相似的生物活性。     

Proteomics demonstration that normal breast epithelial cells can induce apoptosis of breast cancer cells through insulin-like growth factor-binding protein-3 and maspin

Normal breast epithelial cells are known to exert an apoptotic effect on breast cancer cells, resulting in a potential paracrine inhibition of breast tumor development. In this study we purified and characterized the apoptosis-inducing factors secreted by normal breast epithelial cells. Conditioned medium was concentrated by ultrafiltration and separated on reverse phase Sep-Pak C18 and HPLC. The proapoptotic activity of eluted fractions was tested on MCF-7 breast cancer cells, and nano-LC-nano-ESI-MS/MS allowed the identification of insulin-like growth factor-binding protein-3 (IGFBP-3) and maspin as the proapoptotic factors produced by normal breast epithelial cells. Western blot analysis of conditioned media confirmed the specific secretion of IGFBP-3 and maspin by normal cells but not by breast cancer cells. Immunodepletion of IGFBP-3 and maspin completely abolished the normal cell-induced apoptosis of cancer cells, and recombinant proteins reproduced the effect of normal cell-conditioned medium on apoptosis of breast cancer cells. Together our results indicated that normal breast epithelial cells can induce apoptosis of breast cancer cells through IGFBP-3 and maspin. These findings provide a molecular hypothesis for the long observed inhibitory effect of normal surrounding cells on breast cancer development.     

Intermolecular failure of L-type Ca2+ channel and ryanodine receptor signaling in hypertrophy

Pressure overload–induced hypertrophy is a key step leading to heart failure. The Ca²⁺-induced Ca²⁺ release (CICR) process that governs cardiac contractility is defective in hypertrophy/heart failure, but the molecular mechanisms remain elusive. To examine the intermolecular aspects of CICR during hypertrophy, we utilized loose-patch confocal imaging to visualize the signaling between a single L-type Ca²⁺ channel (LCC) and ryanodine receptors (RyRs) in aortic stenosis rat models of compensated (CHT) and decompensated (DHT) hypertrophy. We found that the LCC-RyR intermolecular coupling showed a 49% prolongation in coupling latency, a 47% decrease in chance of hit, and a 72% increase in chance of miss in DHT, demonstrating a state of “intermolecular failure.” Unexpectedly, these modifications also occurred robustly in CHT due at least partially to decreased expression of junctophilin, indicating that intermolecular failure occurs prior to cellular manifestations. As a result, cell-wide Ca²⁺ release, visualized as “Ca²⁺ spikes,” became desynchronized, which contrasted sharply with unaltered spike integrals and whole-cell Ca²⁺ transients in CHT. These data suggested that, within a certain limit, termed the “stability margin,” mild intermolecular failure does not damage the cellular integrity of excitation-contraction coupling. Only when the modification steps beyond the stability margin does global failure occur. The discovery of “hidden” intermolecular failure in CHT has important clinical implications.