Partial isolation from intact cells of a cell surface-exposed lysophosphatidylinositol-phospholipase C

A novel cell surface phosphoinositide-cleaving phospholipase C (ecto-PLC) activity was isolated from cultured cells by exploiting its presumed external exposure. Biotinylation of intact cells followed by solubilization of the biotinylated proteins from a membrane fraction and recovery onto immobilized-avidin beads, allowed assay of this cell surface enzyme activity apart from the background of the substantial family of intracellular PLCs. Several cell lines of differing ecto-PLC expression were examined as well as cells stably transfected to overexpress the glycosylphosphatidylinositol (GPI)-anchored protein human placental alkaline phosphatase (PLAP) as a cell surface enzyme marker. The resulting bead preparations from ecto-PLC positive cells possessed calcium-dependent PLC activity with preference for lysophosphatidylinositol (lysoPI) rather than phosphatidylinositol (PI). The function of ecto-PLC of intact cells evidently is not to release GPI-anchored proteins at the cell surface, as no detectable Ca²⁺-dependent release of overexpressed PLAP from ecto-PLC-positive cells was observed. To investigate the cell surface linkage of the ecto-PLC itself, intact cells were treated with bacterial PI-PLC to cleave simple GPI anchors, but no decrease in ecto-PLC activity was observed. High ionic strength washes of biotinylated membranes prior to the generation of bead preparations did not substantially reduce the lysoPI-PLC activity. The results verify that the ecto-PLC is truly cell surface-exposed, and unlike other members of the PLC family that are thought to be peripheral membrane proteins, this novel lysoPI-PLC is most likely a true membrane protein. J. Cell. Biochem. 65:550–564. © 1997 Wiley-Liss Inc.     

钛合金和钴铬合金表面白色念珠菌粘附的研究

目的比较钛合金(Ti-6Al-4V)和钴铬合金(Chromium-Cobaltalloy)表面白色念珠菌粘附能力的大小,研究表面粗糙度与细菌粘附的关系。方法将不同表面粗糙度的钛合金和钴铬合金试件进行白色念珠菌体外粘附试验,采用菌落形成计数法测定试件表面的细菌粘附量。结果各钛合金试件组的细菌粘附量均少于相同表面粗糙度的钴铬合金试件组,两种金属试件表面的细菌粘附量均随表面粗糙度的增大而增加。结论钛合金较钴铬合金更能减少由白色念珠菌引起的义齿性口炎等并发症,同时修复体表面严格的研磨抛光也能有效减少这些并发症。     

Simulating structural and thermodynamic properties of carcinogen-damaged DNA

A pair of stereoisomeric covalent adducts to guanine in double-stranded DNA, derived from the reaction of mutagenic and tumorigenic metabolites of benzo[a]pyrene, have been well characterized structurally and thermodynamically. Both high-resolution NMR solution structures and an array of thermodynamic data are available for these 10S (+)- and 10R (-)-trans-anti -[BP]-N(2)-dG adducts in double-stranded deoxyoligonucleotides. The availability of experimentally well-characterized duplexes containing these two stereoisomeric guanine adducts provides an opportunity for evaluating the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method for computing thermodynamic properties from molecular dynamics ensembles. We have carried out 3-ns molecular dynamics simulations, using NMR solution structures as the starting models for the 10S (+)- and 10R (-)-trans-anti-dG adducts in a DNA duplex 11-mer using AMBER 6.0. We employed the MM-PBSA method to compute the free energies, enthalpies, and entropies of the two adducts. Our complete thermodynamic analysis agrees quite well with the full experimental thermodynamic characterization of these adducts, showing essentially equal stabilities of the two adducts. We also calculated the nuclear Overhauser effect (NOE) distances from the molecular dynamics trajectories, and compared them against the experimental NMR-derived NOE distances. Our results showed that the simulated structures are in good agreement with the NMR experimental NOE data. Furthermore, the molecular dynamics simulations provided new structural and biological insights. Specifically, the puzzling observation that the BP aromatic ring system in the 10S (+)-trans-anti-dG adduct is more exposed to the aqueous solvent than the 10R (-)-trans-anti-dG adduct, is rationalized in terms of the adduct structures. The structural and thermodynamic features of these stereoisomeric adducts are also discussed in relation to their reported low susceptibilities to nucleotide excision repair.     

Interstitial noradrenaline concentration of rat hearts as influenced by cellular catecholamine uptake mechanisms

Marked concentration differences of noradrenaline (NA) between the vascular and the interstitial compartment were detected by sampling interstitial transudate from isolated perfused rat hearts. The ratios of vascular/interstitial concentration amounted to 7.4 to 1.3 depending on the concentration of NA administered (3 × 10^–9 to 10^–6 M). These concentration differences were abolished by inhibitors of uptake₁ desipramine (DMI) I and uptake, (O-methyl-isoprenaline (OMI)). Neuronal uptake, was characterized by a K_m of 0.22 mol/l and a V_max of 370 pmol × min^–1 × gWWT^–1, extraneuronal uptake₂ by a K_UPTAKE of = 0.313 min^–4.The apparent permeability surface area (P×S)-product calculated from uptake rate and transcapillary concentration difference was significantly decreased by administrating 100 mol/l (NA) in presence of DMI. A presumed endothelial uptake mechanism contributing to catecholamine translocation was investigated in endothelial cells in culture. These cells showed a specific noradrenaline uptake with a K_m of 4.35 mol/l and a V_max of about 75 pmol × min^–1 x gWWT^–1. Any inhibiton by inhibitors of both of the two noradrenaline uptakes was lacking. The uptake rate of this mechanism is insufficient to contribute to the diffusive conductivity of the capillary wall (P × S-product). We conclude from our investigations on interstitial concentrations of catecholamines and transcapillary concentration differences, that the capillary wall, owing to its metabolic and diffusional characteristics, influences the exchange of catecholamines to a substantial and physiologically relevant extent.     

过敏性眼表疾病的临床特点及诊治研究

目的:对过敏性眼表疾病的临床特点和相关的诊治方法进行分析探讨.方法:选取100例过敏性眼表疾病的患者,对其相应的临床资料进行回顾性分析、通过对过敏性眼表疾病的病史、症状、体征等临床特点进行统计,进而对诊治方法进行分析探讨.结果:50例患者有过敏性眼病史,90例患者表现为眼痒,89例患者表现有异物感,88例患者表现为结膜充血,76例患者表现有上眼睑结膜乳头,70例患者表现有上眼睑结膜滤泡.45例患者为常年过敏性结膜炎,30例患者为特异性结膜炎,10例患者为巨乳头结膜炎,80例患者为过敏性结膜炎,嗜酸性粒细胞的阳性率为100％.经过治疗后,57例患者得到缓解,7例患者得到明显改善,30例患者病情得到控制,且有9例重症患者在急性期应用了激素治疗,6例患者临床症状无明显改善.结论:过敏性眼表疾病的临床症状表现差异较大,对过敏性眼表疾病患者的临床特点做出准确的判断并给出正确及时的治疗,对过敏性眼表疾病患者的恢复有一定的临床意义.     

棉铃虫发生与北太平洋海温的遥相关 及其长期灾变预警

本文分析了山东郓城26年（1974～1999）、德州22年（1978～1999）和江苏丰县20年（1980～1999）棉铃虫百株累计卵量与北太平洋海温的遥相关关系及其时空动态规律,并选出相关显著程度P<0.05概率水平、空间分布范围较大、持续时间较长而稳定的组合作为关键预测因子组建了郓城、德州棉铃虫三代卵,丰县棉铃虫二代卵的预测模型,并筛选出最优长期灾变预警模型。结果表明：① 北太平洋海温场与棉铃虫种群数量消长存在显著或极显著的遥相关区域,其位置及范围随时间变化,但存在若干呈现出空间稳定性和时间持续性的大面积相关显著区域。② 郓城棉铃虫三代卵量和丰县棉铃虫二代卵量与北太平洋海温场的相关区分布形式很相似,与前两年1月份北太平洋月平均海温场存在大片相关显著的区域（35°～ 55°N,135°E～135°W）,持续时间达4个月之久;而德州棉铃虫三代卵量与前两年7～9月份北太平洋低纬度海温有大范围相关显著区（1°～17°N,165°E～120°W）。 ③ 用前两年1～11月份北太平洋海温场相关显著区内各格点的月平均海温距平的平均值做因子建立了棉铃虫长期灾变预警模型,预测检验结果表明：郓城棉铃虫三代卵6年（1994～1999）中报准5年,丰县棉铃虫二代卵5年（1995～1999）中报准3年,德州棉铃虫三代卵5年（1995～1999）全部符合。据此可提前20～27个月做棉铃虫的长期灾变预警。     

Surface polysaccharides and quorum sensing are involved in the attachment and survival of Xanthomonas albilineans on sugarcane leaves

Xanthomonas albilineans, the causal agent of sugarcane leaf scald, is a bacterial plant pathogen that is mainly spread by infected cuttings and contaminated harvesting tools. However, some strains of this pathogen are known to be spread by aerial means and are able to colonize the phyllosphere of sugarcane before entering the host plant and causing disease. The objective of this study was to identify the molecular factors involved in the survival or growth of X. albilineans on sugarcane leaves. We developed a bioassay to test for the attachment of X. albilineans on sugarcane leaves using tissue‐cultured plantlets grown in vitro. Six mutants of strain XaFL07‐1 affected in surface polysaccharide production completely lost their capacity to survive on the sugarcane leaf surface. These mutants produced more biofilm in vitro and accumulated more cellular poly‐β‐hydroxybutyrate than the wild‐type strain. A mutant affected in the production of small molecules (including potential biosurfactants) synthesized by non‐ribosomal peptide synthetases (NRPSs) attached to the sugarcane leaves as well as the wild‐type strain. Surprisingly, the attachment of bacteria on sugarcane leaves varied among mutants of the rpf gene cluster involved in bacterial quorum sensing. Therefore, quorum sensing may affect polysaccharide production, or both polysaccharides and quorum sensing may be involved in the survival or growth of X. albilineans on sugarcane leaves.     

Adhesiveness and surface patterns of eggs in neotropical freshwater teleosts

Eggs of 23 Characiformes and eight Siluriformes, belonging to nine families with diverse reproductive behaviour, were ultrastructurally analysed. The migratory species exhibited non-adhesive eggs, whereas, most of the sedentary species presented some degree of egg adhesiveness. Among the Characiformes, non-adhesive eggs showed zona radiata with pore-canals or a fibrillar net at the surface; weakly adhesive eggs presented only zona radiata with pore-canals while adhesive eggs exhibited zona radiata with apparatus like globules, filaments, villi or honeycomb-like pores depending on the systematic group. The 'jelly' coat is strongly related to the Siluriformes eggs apparently without relationship with adhesiveness. A micropylar disc was present in adhesive eggs of a few species of both Characiformes and Siluriformes. Some patterns were characteristic of the animal pole, others of the vegetal pole, and others were common to both poles. The radial ridges converging to the micropyle in Astyanax bimaculatus lacustris appear to be related to fertilization. In general, egg surface structures in the Characiformes varied according to the genus, whereas all Siluriformes showed a similar egg surface pattern, regardless of the group analysed. Multivariate analysis allowed the identification of eight clusters among the Characiformes and three among the Siluriformes showing relationships between reproductive style and egg characteristics. It is suggested that egg surface and adhesiveness may be related to reproductive patterns and to phylogenesis.