Interaction of extracellular vesicles of Bacteroides gingivalis W50 with human polymorphonuclear leucocytes

The effects of B. gingivalis W50 extracellular vesicles (ECV) on neutrophil chemotaxis and viability were assessed and compared with those of whole cells and the extracellular non-dialysable soluble protein (EP) fraction. None of the fractions tested, including soluble fractions derived from cells and ECV by sonication, induced neutrophil chemotaxis. Only ECV and cells inhibited f-MLP-stimulated chemotaxis. ECV and cells were cytotoxic towards neutrophils. The cytotoxic response was time dependent. The soluble EP fraction did not influence cell viability.     

Haemin-restriction influences haemin-binding haemagglutination and protease activity of cells and extracellular membrane vesicles of Porphyromonas gingivalis W50

Porphyromonas gingivalis strain W50 was grown in a chemostat either under haemin limitation or haemin excess at pH 7.3. Cells and the extracellular vesicle (ECV) and extracellular protein (EP) fractions were separated, quantified, and assayed for haemagglutination, protease activity and haemin binding. Under haemin-limitation, despite a reduction in cell yield, there was a 2.5-fold increase in the gravimetric yield of extracellular vesicles. Cells and vesicles from haemin-limited cultures, haemagglutinated sheep red blood cells to higher titres than their haemin-excess counterparts. Growth in haemin-excess conditions resulted in increased haemin-binding capacities of ECV, cells and EDTA-extracted outer membrane. Cells grown under haemin-excess showed a 2-fold elevation in specific activity towards the substrate N-alpha-benzoyl-L-arginine-p-nitroanilide (L-BAPNA) compared to haemin-limited cells. The specific activities against L-BAPNA for haemin-limited ECV were 3-fold greater than their haemin-excess counterparts. These vesicle activities represented 25% and 3% of the total culture protease activity under haemin limited and haemin excess conditions respectively.     

Glycylprolyl dipeptidase activity of Bacteroides gingivalis W50 and the avirulent variant W50/BEI

Glycylprolyl dipeptidase activity was measured in cells, extracellular vesicles (ECV) and the soluble extracellular protein fraction (EP) of batch cultures of strains W50 and W50/BEI. Total culture enzyme activity of W50 dropped with age whilst that of W50/BEI remained constant. Activity was highest in the cellular fraction, greater for W50/BEI than W50 and rose with culture age. Both strains showed similar ECV activities but these declined with culture age. The EP glycylprolyl dipeptidase activity of W50/BEI in older cultures rose to a level 13-fold greater than W50. The majority of extracellular activity was represented by the ECV for strain W50 but by EP for W50/BEI. Variable but incomplete attenuation of activity was achieved by dithiothreitol. ECV and EP activities were associated with a high molecular mass fraction, but a smaller fraction (molecular mass 30,000) was detected in W50/BEI EP.     

The role of TG2 in ECV304-related vasculogenic mimicry

Tumour vasculogenesis can occur by a process referred to as vasculogenic mimicry, whereby the vascular structures are derived from the tumour itself. These tumours are highly aggressive and do not respond well to anti-angiogenic therapy. Here, we use the well characterised ECV304 cell line, now known as the bladder cancer epithelial cell line T24/83 which shows both epithelial and endothelial characteristics, as a model of in vitro vasculogenic mimicry. Using optimised ratios of co-cultures of ECV304 and C378 human fibroblasts, tubular structures were identifiable after 8 days. The tubular structures showed high levels of TG2 antigen and TG in situ activity. Tubular structures and in situ activity could be blocked either by site-directed irreversible inhibitors of TG2 or by silencing the ECV304 TG2 by antisense transfection. In situ activity for TG2 showed co-localisation with both fibronectin and collagen IV. Deposition of these proteins into the extracellular matrix could be reduced by inclusion of non-cell penetrating TG inhibitors when analysed by Western blotting suggesting that the contribution of TG2 to tube formation is extracellular. Incubation of ECV304 cells with these same irreversible inhibitors reduced cell migration which paralleled a loss in focal adhesion assembly, actin cytoskeleton formation and fibronectin deposition. TG2 appears essential for ECV304 tube formation, thus representing a potential novel therapeutic target in the inhibition of vasculogenic mimicry.     

亚热带森林树种多样性对凋落叶分解胞外酶活性的影响

森林生物多样性与生态系统功能关系是当前群落生态学的热点研究领域。然而, 以往研究更多聚焦在森林植物多样性丧失对群落生产力的影响, 而对森林凋落物分解的相关研究稍显不足。森林凋落叶分解的快慢直接受控于凋落物分解者分泌的胞外酶的活性, 后者更是指示森林生态系统养分循环的重要指标之一。本研究依托我国江西亚热带森林生物多样性与生态系统功能控制实验, 通过对不同植物多样性梯度样方内目标树种凋落叶胞外酶活性、理化性质以及腐生真菌的分析, 探索树种多样性丧失对胞外酶活性的影响及其调控机制, 以探讨森林树种多样性对地表、地下生态过程和功能的影响。结果表明, 样方水平树种多样性丧失显著影响胞外酶的活性, 除单种样方外, 随着样方水平树种丰富度的增加, 胞外酶活性呈现出增长趋势; 与碳周转相关的α-葡萄糖苷酶(AG)、β-葡萄糖苷酶(BG)、纤维二糖水解酶(CB)在树种多样性最大时活性达到最高; 而木糖苷酶(XS)以及与氮、磷和顽拗有机养分分解相关的N-乙酰-β-氨基葡萄糖苷酶(NAG)、酸性磷酸酶(AP)和多酚氧化酶(PPO)在树种多样性较低时活性较高。针对目标树种周围的邻居树种多样性进一步分析发现, 各胞外酶活性随着邻居树种多样性的变化呈“单峰”响应趋势, 酶活性大多在邻居树种丰富度为6时呈现峰值。研究发现真菌分解者在胞外酶活性对植物多样性的响应上可能存在重要的调控作用, 可以推测树种多样性通过改变腐生真菌分解者的群落结构和多度, 从而影响胞外酶活性。     

Overexpressed alpha3beta1 and constitutively activated extracellular signal-regulated kinase modulate the angiogenic properties of ECV304 cells.

ECV304, a spontaneously transformed cell line derived from the human umbilical vein endothelial cell (HUVEC) (Takahashi et al., 1990), has been developed as an in vitro angiogenesis model. In the present study, we further characterized the angiogenic properties of this cell line. Compared to HUVEC, ECV304 cells showed distinct features including a higher activity of cellular adhesion, slower but reproducible progression of angiogenesis on Matrigel, and resistance to apoptosis. Thus, the expression of integrin and activation of extracellular-signal regulated kinase 1/2 (Erk1/2), a downstream effector of the integrin pathway, were examined. Flow cytometry revealed that alpha3beta1 integrin was markedly upregulated in ECV304 cells, while alpha(v)beta1 and alpha5beta1 integrins were slightly downregulated. Consistent with this, the binding activity to collagen type IV and laminin, major extracellular matrices of Matrigel, was increased 1.4- and 1.9-fold in ECV304 cells, respectively. This tight binding may retard the initial stage of sprouting and migration in the angiogenesis of ECV304 cells. It has been further demonstrated that Erk1/2 is constitutively active in ECV304 cells, rendering them resistent to the inhibitory effect of PD98059 on proliferation. However, migration of both HUVEC and ECV304 cells was inhibited to a similar extent by PD98059 in a dose-dependent manner. Up to 50 microM of PD98059, no significant changes in cell binding and tubulogenesis on Matrigel was observed in ECV304 cells. In contrast, the tubulogenesis of HUVEC was severely impaired by PD98059. Elevated Erk1/2 activity in ECV304 cells was suppressed by dominant negative H-Ras, but not by cytochalasin D. These results suggest that the overexpression of alpha3beta1 integrin and the constitutive activation of Erk1/2 play a key role in the alteration of the angiogenic properties of ECV304 cells.     

光照和pH对忍冬纤孔菌液体培养的影响

以液体培养为基础,研究不同光照和培养液初始pH条件下,忍冬纤孔菌培养过程中生物量、胞内外多酚含量,以及胞内外提取物抗氧化活性的变化。结果显示,24h光照轻微抑制菌丝体的生长,但能促进胞内外多酚的形成和提取物对DPPH自由基的抑制;在初始培养液pH5.3条件下,菌丝体生物量最高,胞内外多酚含量和提取物活性最高。     

Platelet activating factor activity in the phospholipids of bovine spermatozoa

Platelet activating factor (PAF) has been detected in sperm from several mammalian species and can affect sperm motility and fertilization. Because bovine sperm contain a high percentage of ether-linked phospholipid precursors required for PAF synthesis, a study was undertaken to determine the PAF activity of bovine sperm phospholipids. Total lipids of washed, ejaculated bull sperm were extracted, and phospholipids were fractionated by thin-layer chromatography. Individual phospholipid fractions were assayed for PAF activity on the basis of [3H]serotonin release from equine platelets. PAF activity was detected in the PAF fraction (1.84 pmol/mumol total phospholipid) and in serine/inositol (PS/PI), choline (CP), and ethanolamine phosphoglyceride (EP) and cardiolipin (CA) fractions. Activity was highest in the CP fraction (8.05 pmol/mumol total phospholipid). Incomplete resolution of PAF and neutral lipids may have contributed to the activity in the PS/PI and CA fractions, respectively. Phospholipids from nonsperm sources did not stimulate serotonin release. Platelet activation by purified PAF and by sperm phospholipid fractions was inhibited by the receptor antagonist SRI 63-675. These results indicate that bovine sperm contain PAF and that other sperm phospholipids, especially CP and EP, which are high in glycerylether components, are capable of receptor-mediated platelet activation.     

Features of Fibrobacter intestinalis DR7 mutant which is impaired with its ability to adhere to cellulose

A spontaneous adhesion-defective mutant (DR7-M) of Fibrobacter intestinalis DR7 was isolated which was capable of growing on glucose and cellobiose, but impaired in its capacity to degrade cellulose. Levels of enzyme activities were determined in solubilized fractions of DR7 and DR7-M. Total endoglucanases and xylanase activity values of parent DR7 fractions were 2.84 and 1.85 folds higher than those of the mutant, and were distributed mainly in the bacterial envelope fractions, with some activity also found in the extracellular fluid. In a separate assay, measurement of the enzymatic activity bound to cellulose showed that a portion of the endoglucanase activity bound to cellulose while most xylanase activity did not bind. Notwithstanding, the wild type DR7 cells had 26-fold higher total activities of cellulose-degrading enzymes than the mutant, and 96% of its activity was exclusively located in outer membrane and periplasm fractions. In the mutant, the lower cellulose degrading enzymes activity was located only in the extracellular fluid. Most of the cellulose degrading enzymes activity of DR7 had the capability to bind to cellulose. SDS-page electrophoresis of outer membrane and periplasm cell fractions showed that DR7 and DR7-M possess similar molecular weight (MW) profiles but different quantities of 16 cellulose-binding-proteins (CBPs) in the MW range of 36 up to 225 kDa. Zymogram analysis with soluble substrates, either carboxymethylcellulose or soluble xylan, following SDS-page of DR7 and DR7-M fractions, suggested that CBPs of approximate MW 120, 110, 100, 90, 70 and 40 kDa have endoglucanase activity, and that CBPs of all fractions lack any xylanase activity.     

Nature of the inhibitory effect of collagenase on phosphodiesterase activity

The level of phosphodiesterase (PDE) activity is lower in collagenase-isolated human fat cells than in adipose tissue fragments. The inhibition is not species-specific since collagenase also inhibits PDE in rat adipose tissue and bovine heart. In subcellular fractions from isolated fat cells, the PDE activities were lowest in the plasma membrane-enriched fractions and highest in the cytosolic fractions. This is opposite to PDE in subcellular fractions obtained from adipose tissue fragments. In dose-response experiments, collagenase inhibited particulate PDE to a much larger extent than it inhibited soluble PDE. The extracellular activities of PDE were completely eliminated by collagenase. Repeated washings or reincubation of the isolated fat cells did not restore the PDE activity. A purified collagenase with low specific protease activity reduced the PDE activity in isolated fat cells to a lesser extent than did a collagenase with high specific protease activities. Collagen and several protease inhibitors were ineffective in preventing the reduction of PDE after exposure to collagenase. It is concluded that nonspecific proteases in the collagenase preparations used for fat cell isolation interact with particulate and soluble PDE causing an irreversible inhibition of PDE activity in isolated fat cells. Of the various forms of PDE, plasma membrane-associated PDE seems most sensitive to collagenase.     

Potent angiogenic inhibition effects of deacetylated chitohexaose separated from chitooligosaccharides and its mechanism of action in vitro

This study was performed to demonstrate the effects of deacetylated chitohexaose (hexamer) separated from a chitooligosaccharide (COS) mixture on tumor angiogenesis and its mechanism of action. Five fractions from dimer to hexamer were separated by a linear gradient solution of HCl on a cation-exchange resin. Then HCl was removed from the fractions by a charcoal column. The purity of the five fractions was analyzed by HPLC and the molecular masses were analyzed by MALDI-TOFMS. The hexamer expressed an inhibitory influence on CAM angiogenesis in a dose-dependent manner at concentrations of 6.25-50 μg/egg. On further investigation, we found that the hexamer had no toxic effect on normal ECV304 cells, but could inhibit the proliferation and migration of tumor-induced ECV304 cells in a dose-dependent manner. The mechanism was demonstrated through the detection of mRNA expression of VEGF, MMP-9, TIMP-1, TIMP-2, and uPA by RT-PCR, which showed that the hexamer down-regulated the VEGF and uPA mRNA expressions in ECV304 cells, but up-regulated the TIMP-1 mRNA expression.     

Increased enzymatic activities and levels of superoxide anion and phenolic compounds in cultures of basidiomycetes after temperature stress

Selected strains of basidiomycetes (Abortiporus biennis, Trametes versicolor and Cerrena unicolor) were shown to produce enhanced extracellular peroxidase (EP), superoxide dismutase (SOD) and laccase activities following the exposure of 10-day-old fungal cultures to separate high and low temperature stress. The stressful conditions also caused an increase in the concentrations of phenol compounds and superoxide anion radicals in these cultures. At first, peroxidase activity was observed at 12 hours from the moment of temperature stress application. Laccase activity appeared at 96 hours after the maximum levels of superoxide anion radicals (48 h) and SOD activity (36–72 h). The concentration of phenolic substances grew steadily during the period of cultivation. These relations between laccase, SOD and EP as well as superoxide radicals and phenol levels in the environment of ligninolytic fungi seems to be important in the course of the biosynthesis or biodegradation of lignin, as the consequence of adaptation of these basidiomycetes to environmental temperature conditions.     

忍冬木层孔菌液体培养过程中多酚含量及抗氧化活性研究

忍冬木层孔菌是木层孔菌属的一种药用真菌,目前对它的研究少有报道。以液体培养为基础,研究忍冬木层孔菌培养过程中生物量、胞内外多酚含量的变化,以及胞内外提取物抗氧化活性的变化。结果显示,菌丝体生物量在培养168–216h时达到最大值（8.33g/L）;胞内多酚和胞外多酚含量分别在216h和168h时达到最大值（15.60mg GAE/g和106.76mg GAE/L）;液体培养胞内外提取物的抗氧化活性与多酚含量正相关,多酚含量高时提取物对DPPH[1,1-Diphenyl-2-picrylhydrazyl radical 2,2-Diphenyl-1-(2,4,6-trinitrophenyl)hydrazyl]自由基的抑制率也高。     

Phosphomonoesterase activity of the cyanobacterium (blue-green alga) Calothrix parietina

Cellular and extracellular phosphomonoesterase activities were compared in Calothrix parietina D550, a strain whose original environment has been studied in detail. Activity in both fractions became detectable at about the same stage in batch culture. Differences in the influence of environmental factors between the two were slight, suggesting a common origin. The optimum temperatures for cellular and extracellular activities were 40 degrees C and 30 degrees C, respectively, and the upper limits for detectable activity were 80 degrees C and 65 degrees C. The pH optimum for both cellular and extracellular activity was 10.0-10.2. When P-limited cultures were tested with p-nitrophenyl phosphate (pNPP) as substrate, Km values for cellular and extracellular activities were 43 and 33 microM pNPP, respectively. Eleven ions were tested for their influence on activity. In most cases the effect was low or negligible at concentrations likely to be present in nature or freshwater laboratory media. Where obvious effects occurred, these were usually apparent at lower concentrations with extracellular than cellular activity. One mM Ca led to a 40% increase in extracellular activity in comparison with 0.1 mM Ca, but had no effect on cellular activity. However, inorganic phosphate, which had a marked inhibitory effect at concentrations above 10 microM, brought about a similar response with cellular and extracellular activities (approximately 60% decrease with 100 microM).     

Ischemia-Induced Translocation of Ca2+/Calmodulin-Dependent Protein Kinase II: Potential Role in Neuronal Damage

The activities of Ca2+/calmodulin (CaM)-dependent, Ca2+/phospholipid-dependent, and cyclic AMP-dependent protein kinases (CaM-KII, PKC, and PKA, respectively) were determined in rat brains after global ischemia. Both CaM-KII and PKC activities were significantly depressed in both hippocampal and cerebral cortical regions of ischemic animals, whereas no change was detected in PKA activity. The loss of CaM-KII activity was more dramatic and more sustained than the loss of PKC activity and correlated with the duration of ischemia. These decreases in enzyme activity were found in both supernatant and pellet fractions from crude homogenates. When the supernatant and pellet were analyzed for the amount of CaM-KII 50-kDa protein, a significant decrease was detected in supernatant fractions that paralleled a gain in the amount of CaM-KII in the pellet. Thus, the loss of CaM-KII activity in the supernatant can be explained by translocation of the enzyme to the pellet. Whether inactivation of CaM-KII occurs during or after the enzyme translocates from the supernatant to the pellet is unknown. Our results indicate that loss in CaM-KII activity parallels neuronal damage associated with ischemia; down-regulation of CaM-KII activity coincided with translocation of the enzyme to the particulate fraction, and it is proposed that this may be, in fact, a mechanism for controlling excessive CaM-KII phosphorylation.     

Secretion of metalloendopeptidase 24.15 (EC 3.4.24.15).

The metalloendopeptidase EP24.15 (EC3.4.24.15) is a neuropeptide-metabolizing enzyme present in neural and endocrine tissues, presumably functioning extracellularly. Because the majority of the EP24.15 activity is identified in the soluble fraction of cellular homogenates, suggesting that the enzyme is primarily an intracellular protein, we addressed the issue of how EP24.15 arrives in the extracellular environment. We utilized a model system of neuroendocrine secretion, the AtT20 cell. According to both enzymatic activity and immunologic assays, EP24.15 was synthesized in and released from AtT20 cells. Under basal conditions and after stimulation by corticotropin-releasing hormone or the calcium ionophore A23187, EP24.15 activity accumulated in the culture medium. This secretion was not attributable to cell damage, as judged by the absence of release of cytosolic enzyme markers and the ability to exclude trypan blue dye. Pulse-chase analysis and subcellular fractionation of AtT20 cell extracts suggested that the mechanism of EP24.15 secretion is not solely via classical secretory pathways. Additionally, drugs which disrupt the classical secretory pathway, such as Brefeldin A and nocodazole, blocked A23187-stimulated EP24.15 release yet had no effect on basal EP24.15 release, suggesting differences in the basal and stimulated pathways of secretion for EP24.15. In summary, EP24.15 appears to be secreted from AtT20 pituitary cells into the extracellular milieu, where the enzyme can participate in the physiologic metabolism of neuropeptides.     

Mitogenic and adjuvant activities of polysaccharides from the cellular slime mold,Dictyostelium discoideum NC-4

Cell surface and extracellular polysaccharide fractions obtained from Dictyostelium discoideum NC-4 cultured in bacteria-free medium showed strong B-cell mitogenic activities. Upon periodate treatment of the extracellular polysaccharide fraction this activity completely disappeared. The extracellular polysaccharide fraction could also enhance the antibody response in vitro against sheep red blood cells.     

心理应激对牙周炎组织氧化还原反应影响的动物实验研究

目的:明确心理应激对大鼠牙周炎组织局部病损以及抗氧化酶活性和丙二醛(MDA)含量变化的影响。方法:40只健康Sprague-Dawley大鼠随机分为对照组(C)、实验性牙周炎组(EP)、实验性牙周炎+心理应激组(EP+PS)以及实验性牙周炎+心理应激+药物组(EP+PS+DR),每组10只。分别采用丝线结扎法和慢性不可预知性应激法建立大鼠实验性牙周炎模型和心理应激模型。EP组动物仅用丝线结扎右上颌第二磨牙颈部,EP+PS组动物同时接受丝线结扎和心理应激刺激,EP+PS+DR组动物除上述处置外,按5 mg/kg/日剂量腹腔注射氟西汀,而C组大鼠无任何干预措施。4周后对所有动物进行糖水偏爱度测试、行为学观察和血清学检测,并计算牙槽骨丧失和附着丧失情况,同时测量牙龈组织中抗氧化酶活性以及MDA含量。结果:慢性不可预知性应激导致大鼠旷场实验中央区移动距离减少(P<0.05)、中央区停留时间增加(P<0.05)、糖水偏爱度降低(P<0.05)、血清皮质酮与促肾上腺皮质激素浓度升高(P<0.05);并且应激大鼠的牙槽骨丧失和附着丧失明显大于单纯牙周炎组(P<0.05);同时,心理应激状态下大鼠牙龈组织的氧化还原代谢异常,表现为超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)和过氧化氢酶(CAT)活性明显低于单纯牙周炎组(P<0.05),而MDA含量明显高于单纯牙周炎组(P<0.05)。氟西汀在拮抗心理应激的同时,可以明显改善动物牙周炎组织局部降低的抗氧化酶活性以及升高的脂质过氧化产物含量(P<0.05)。结论:心理应激可以导致牙周炎组织的氧化损伤加重,有效拮抗心理应激有助于减轻牙周炎性组织病损。     

Inhibitory effect of polyamines on the activity of endopeptidase in mung bean cotyledons

The activity of cysteine endopeptidase (EP) in the cotyledons of mung bean seeds increased with time after germination. When cotyledons were excised from the embryonic axis in the course of seedling growth, the activity of EP in the excised cotyledon markedly dropped during the following incubation of 1 d. However, the level of EP protein in excised cotyledons, as examined by immunoblotting, was similar to that in axis-attached cotyledons at the corresponding stage. Thus, it seems that the low activity of EP in excised cotyledons is not due to a decrease in the content of EP protein, but due to a loss of the activity of existing EP. Treatment of attached cotyledons with polyamines (PAs; putrescine and spermidine [Spd]) resulted in a decrease in EP activity, while the same PA-treatment brought about little alteration in the level of EP protein. This indicates that PAs somehow produce an inhibitory effect on the activity of EP. Axis-removal resulted in an accumulation of Spd in the cotyledon. The possibility is suggested that PA, especially Spd, is involved in the inhibition of EP activity in excised mung bean cotyledons.     

Mitogenic andadjuvant activities of polysaccharides from the cellular slime mold, Dictyostelium discoideum NC-4.

Cell surface and extracellular polysaccharide fractions obtained from Dictyostelium discoideum NC-4 cultured in bacteria-free medium showed strong B-cell mitogenic activities. Upon periodate treatment of the extra-cellular polysaccharide fraction this activity completely disappeared. The extracellular polysaccharide fraction could also enhance the antibody response in vitro against sheep red blood cells.