镉对一株深色有隔内生真菌生长、矿质营养与草酸分泌的影响

【背景】深色有隔内生真菌(dark septate endophyte,DSE)广泛定殖于镉(Cd)污染生境的植物根系,具有增强植物镉耐性的重要生态功能,但人们关于DSE对镉胁迫的生理响应的了解有限。【目的】研究一株DSE嗜鱼外瓶霉(Exophiala pisciphila)对镉胁迫的矿质营养与低分子量有机酸分泌的响应。【方法】采用液体培养法,研究不同浓度(0、25、50、100、200、400 mg/L)镉胁迫对DSE菌丝生长、矿质元素(氮、磷、钾、硫、镁、铁、钙)与镉含量、草酸分泌的影响。【结果】随着镉胁迫浓度增加,菌丝生物量显著下降,降幅为22.8%−90.6%,菌丝的氮、钾和铁含量分别减少26.0%−52.8%、53.8%−92.9%和12.8%−34.3%,而磷、镁和钙含量分别增加15.4%−111.4%、20.4%−31.4%和35.1%−62.5%,硫含量在100 mg/L镉胁迫时增加25.1%。镉胁迫还导致培养液pH值下降,草酸浓度及单位菌丝草酸分泌量显著增加。相关分析发现,菌丝镉含量与硫呈显著负相关(P<0.05),与菌丝钾含量呈极显著负相关(P<0.01),与草酸分泌量呈极显著正相关(P<0.01)。【结论】镉胁迫显著抑制DSE的生长,改变矿质元素的吸收,促进草酸分泌。     

棘孢木霉菌对钠胁迫的生理响应机制

【背景】棘孢木霉菌制剂被广泛应用于生物防治和次生盐渍化土壤的微生物修复,但是关于棘孢木霉在盐渍化胁迫条件下生长的耐盐机理及其富集盐离子的能力尚缺乏深入研究。【目的】揭示一株耐盐棘孢木霉菌(Trichodermaasperellum)CTCCSJ-W-SBW10264(T264)对钠胁迫的生理响应机制及其对钠离子的吸附和累积特性。【方法】设计梯度浓度钠胁迫培养实验,采集不同培养时期的菌丝样本,测定细胞氧化损伤相关指标H_2O_2和丙二醛(Malondialdehyde,MDA)的含量及细胞抗氧化相关酶的活性变化。【结果】钠胁迫实验表明,棘孢木霉T264能够在1.22mol/L的钠胁迫环境中生存,在低于0.25 mol/L的钠胁迫下其生长不会被明显抑制。细胞氧化损伤及氧化损伤响应相关指标的研究结果表明,培养液中钠离子浓度越高,棘孢木霉的膜系统氧化水平(MDA含量)越高,而且随着细胞中MDA和H_2O_2的累积,细胞抵御氧化损伤相关酶的活性也有明显提高,在钠盐处理24 h后,0.5、1.0和1.22 mol/L的钠离子胁迫分别使过氧化物酶(Peroxidase,POD)、超氧化物歧化酶(SuperoxideDismutase,SOD)和过氧化氢酶(Catalase,CAT)活性达到峰值,依次为36.66、3.34和233.3 U/mg。钠离子吸附和累积特性实验结果表明,棘孢木霉T264的菌丝对钠离子有强的吸附能力。在0.05 mol/L的钠离子环境中培养72 h后,菌丝表面钠离子吸附量为1 347.6 mg/g,菌丝内部钠离子累积量为218.6 mg/g,木霉菌菌丝通过菌丝表面吸附和菌丝内部累积对培养液中钠离子的去除率达到32%。【结论】T264的抗氧化损伤相关酶在其耐受钠离子胁迫过程中发挥重要作用,菌株T264对高浓度钠离子有强适应性,而且对环境中钠离子有高效的吸附和累积作用。     

盐碱胁迫对赤霞珠葡萄幼苗生长及相关生理指标的影响

【目的】了解不同盐分配比和不同浓度梯度复合盐碱胁迫对‘赤霞珠’葡萄（Vitis vinfera ‘Cabernet Sauvignon’）幼苗生长和抗逆生理指标的影响，确认其耐盐碱性范围及其耐盐碱能力。【方法】 以1年生‘赤霞珠’自根苗为试材，用NaCl、Na2SO4、NaHCO3、Na2CO3按不同比例混合配置中性盐、弱碱性盐、强碱性盐3组复合盐溶液，每组各设置50、100、150 mmol/L3个浓度梯度，以不做处理的溶液作为对照，于幼苗生长期间进行定期浇灌处理，通过室内盆栽试验来模拟不同类型、不同程度盐碱胁迫，在处理后不同时期测定幼苗生长形态、生理及光合指标。【结果】（1）茎粗和叶面积随盐胁迫浓度增大，呈现先增大后减小的趋势， 而‘赤霞珠’幼苗的株高基本呈现下降趋势。其中，中性、弱碱性、强碱性盐胁迫组间相比，‘赤霞珠’幼苗株高在强碱性盐胁迫组150 mmol/L处理10 d、150 mmol/L处理40 d都较同期中性、弱碱性组相应浓度显著降低；（2）随着盐浓度的增大，SOD活性及MDA含量呈先增大后减小的趋势，而‘赤霞珠’幼苗叶片的POD活性呈现缓慢增加的趋势，强碱性盐胁迫组叶片POD活性在处理30-50d时均高于相同浓度的中性、弱碱性盐胁迫处理组，但随着时间处理延长，到处理60 d后逐渐低于同浓度其他两处理组，其中150 mmol/L浓度表现得更明显。；（3）‘赤霞珠’葡萄幼苗胞间CO2浓度、净光合速率、蒸腾速率随着盐碱浓度的增加主要呈下降趋势，其胞间CO2浓度与净光合速率均为50 mmol/L浓度处理时较高。在同期相同盐浓度处理下，叶片净光合速率在强碱性盐胁迫150 mmol/L浓度处理75 d时显著低于同期中性、弱碱性处理组。（4）在相同浓度条件下，强碱性盐胁迫处理的幼苗株高及最大叶面积都显著低于中性盐和弱碱性盐处理组；叶片SOD活性在50 mmol/L中性盐胁迫处理下较CK显著提高了27%；叶片MDA含量在150 mmol/L强碱性盐胁迫下随着时间延长显著逐渐增加。【结论】‘赤霞珠’幼苗生长在盐碱胁迫下受到一定限制，但其株高、叶面积、胞间CO2浓度、净光合速率、蒸腾速率在50 mmol/L浓度处理下均呈现较好的增长趋势，而其株高及茎粗在强碱性盐胁迫150 mmol/L盐碱溶液处理下均增长最不明显；在一定低浓度浓度范围内50 mmol/L盐碱处理有利于赤霞珠葡萄幼苗生长。     

磷与信号抑制剂对外生菌根真菌分泌草酸的调控作用

摘要:【目的】磷是树木生长的必需营养元素之一,磷素营养丰缺条件下,研究外生菌根真菌的草酸分泌及调控有益于揭示它们活化利用土壤无机磷的机理。【方法】试验设置低、正常、高3种不同磷浓度,液体培养外生菌根真菌,研究了磷和Ca2+信号/阴离子通道抑制剂对草酸分泌的调控作用。【结果】外生菌根真菌能分泌大量的氢离子和草酸、乙酸、苹果酸、柠檬酸和丁二酸等多种有机酸,对溶解难溶性无机磷有重要作用。在外生菌根真菌分泌的有机酸中,草酸占15.14%－36.01%;低磷促进草酸分泌,正常和高磷则产生抑制作用;培养液磷浓度和菌丝含磷量分别与供试菌种的草酸分泌速率呈显著或极显著负相关,相关系数依次为r=－0.264*和r=－0.349＊＊,n=60,*表示显著(P0.05),＊＊表示极显著(P 0.01),说明磷能调控外生菌根真菌分泌草酸。在低磷胁迫下,钙调蛋白抑制剂、Ca2+通道抑制剂、Ca2+内膜通道抑制剂和阴离子通道抑制剂显著抑制外生菌根真菌分泌草酸。但是,在正常和高磷条件下,草酸分泌速率低,未响应Ca2+信号/阴离子通道抑制剂。【结论】供试外生菌根真菌能分泌大量的氢离子和有机酸(尤其是草酸),有益于溶解土壤无机磷,改善寄主植物的磷营养;供磷水平调控草酸分泌速率;在低磷胁迫下,Ca2+信号是介导外生菌根真菌分泌草酸的信号因子。     

米根霉在氮源限制下的木糖代谢和关键酶特性北大核心CSCD

【目的】解析氮源浓度对米根霉木糖代谢途径及产物的影响,提高木糖利用率。【方法】以木糖为碳源,考察不同氮源浓度下米根霉的生物量、有机酸积累量、木糖代谢关键酶(木糖还原酶、葡萄糖-6-磷酸脱氢酶)活力以及胞内还原力(NADH/NAD+、NADPH/NADP+)的差异。【结果】富氮条件下(2.4 g/L尿素),木糖代谢速率达2.03 g/(L·h),木糖还原酶、葡萄糖-6-磷酸脱氢酶的活力以及胞内还原力较高,生物量达18.01g/L,几乎不积累有机酸;限氮条件下(0.15 g/L尿素),木糖还原酶、葡萄糖-6-磷酸脱氢酶的活力以及胞内还原力水平降低,生物量仅4.02 g/L,富马酸积累量为6.55 g/L,残余木糖量较高;氮源浓度为0.6 g/L时,木糖还原酶和葡萄糖-6-磷酸脱氢酶的活力以及NADPH/NADP+处于前二者之间,此时生物量9.11 g/L,有机酸积累量较大,其中富马酸为12.28 g/L。【结论】充足的氮源可使米根霉通过木糖代谢关键酶与胞内还原力的协同效应强化木糖代谢活力,通过优化氮源浓度后,米根霉可积累更多有机酸。     

氮源类型和水平对3株球状绿藻生长、油脂和花生四烯酸积累的影响

【背景】缺刻叶球藻(Lobosphaera incisa Reisigl)是一种单细胞球状绿藻,是已知花生四烯酸(Arachidonic acid,AA)含量最丰富的植物资源之一。然而目前其分类和命名仍然较为混乱。【目的】明确3株球状绿藻(SAG2468、SAG2043、H4301)的分类地位及在不同氮源(硝酸钠、尿素、碳酸氢铵、碳酸铵、硝酸铵、氯化铵和硫酸铵)和氮浓度(18mmol/L,3mmol/L)条件下油脂和AA积累的特性。【方法】通过分子系统学和形态观察的方法对3株球状绿藻进行分类界定;采用干重法、重量法和气相色谱分析的方法分别对其生物量、油脂含量、脂肪酸组成及AA含量进行测定。【结果】3株球状绿藻均隶属于叶球藻属(Lobosphaera),SAG2468原定名为缺刻缘绿藻(Parietochloris incisa),现修订为缺刻叶球藻(Lobosphaera incisa),与H4301为缺刻叶球藻的不同地理株系。SAG2043原定名为双隔蚁形藻(Myrmecia bisecta),现修订为双隔叶球藻(Lobosphaera bisecta)。3株微藻在高氮(18 mmol/L)和低氮(3 mmol/L)浓度的硝酸钠和尿素培养条件下均可良好生长,铵盐对藻细胞生长普遍有抑制作用,且浓度越高抑制作用越显著。低氮胁迫能显著促进油脂和AA的积累(P0.05),SAG2043在3 mmol/L硝酸钠条件下油脂和AA产率最高,分别为142.15mg/(L·d)和35.51mg/(L·d),明显高于另外2株微藻(P0.05)。此时SAG2043对应获得的生物量为4.9 g/L,油脂含量为43.49%,AA含量高达干重的10.86%,占总脂肪酸含量的31.75%。【结论】SAG2043是一株更具AA开发潜力的微藻。     

两株内生芽孢杆菌对盐胁迫下大豆幼苗超氧化物歧化酶和过氧化物酶活性影响

【背景】盐胁迫环境严重影响大豆幼苗生长,内生菌可提高作物的抗逆性。【目的】探究接种内生枯草芽孢杆菌127和解蛋白芽孢杆菌133对盐胁迫下大豆幼苗体内超氧化物歧化酶(superoxide dismutase,SOD)和过氧化物酶(peroxidase,POD)活性的影响。【方法】以“徐豆20”为实验材料,采用盆栽实验法,设置对照组、盐胁迫组和盐胁迫接菌组,在人工气候培养条件下,用不同NaCl浓度(50、100、150、200、250和300 mmol/L)处理大豆幼苗,并接种不同OD600值(OD0.33、OD0.50和OD0.75)的菌悬液。【结果】培养14 d,接种枯草芽孢杆菌127的菌悬液OD0.33和OD0.75分别在盐浓度300 mmol/L和100 mmol/L时,SOD活性均为1.04 U/g-FW;接种解蛋白芽孢杆菌133的菌悬液OD0.50在盐浓度300 mmol/L胁迫下POD活性最高为7 820 U/(g·min),对大豆幼苗修复效果较显著。培养28 d,接种枯草芽孢杆菌127的菌悬液OD0.50,在150 mmol/L时SOD活性最高(0.88 U/g-FW);接...     

硝酸盐对牛粪梭菌甲酸脱氢酶缺失型Wood-Ljungdahl途径固碳的影响

【背景】异于同型产乙酸菌通常利用Wood-Ljungdahl途径将2分子CO₂还原为1分子乙酰辅酶A,Clostridium bovifaecis缺失Wood-Ljungdahl途径甲基支路第1步将CO₂还原为甲酸的甲酸脱氢酶,需甲酸存在时将1分子甲酸和1分子CO₂还原为乙酰辅酶A发生葡萄糖的同型产乙酸型发酵。已有报道显示,硝酸盐也可作为同型产乙酸菌的电子受体,而且对不同同型产乙酸菌的代谢影响有所不同,然而硝酸盐对这种独特的甲酸脱氢酶缺失型Wood-Ljungdahl途径固碳的影响尚不清楚。【目的】探究硝酸盐对C.bovifaecis甲酸脱氢酶缺失型Wood-Ljungdahl途径固碳的影响。【方法】硝酸盐浓度分别为10 mmol/L和30 mmol/L时,以未添加硝酸盐为对照实验,研究C.bovifaecis在葡萄糖+甲酸+CO₂为基质条件下的细菌生长、底物消耗和产物生成情况。【结果】10 mmol/L和30 mmol/L硝酸盐存在时,主要产物乙醇浓度分别为5.80 mmol/L和1.66 mmo...     

草酸对氧化胁迫-水稻叶片中核酮糖-1，5-二磷酸羧化酶/加氧酶降解的保护作用

以杂交稻(汕优63)为试验材料,在木村B营养液中培养至三叶期,用草酸5mmol/L预处理水稻2d,再处以氧化胁迫(用0．1mmol／L浓度的活性氧诱发剂甲基紫精处理)。结果表明MV诱发的氧化胁迫下,Rubisco及其它可溶性蛋白快速降解。草酸预处理可明显缓解Rubisco及其它可溶性蛋白的降解,降解速率分别降低1／3和1／2左右。植株经草酸处理后其叶片中几种抗氧化酶如AsA-POD、SOD、CAT活性大大提高,这可能是草酸预处理可缓解氧化胁迫下Rubisco和其它可溶性蛋白降解的重要原因。既然草酸能有效地诱导植物的抗氧化防卫反应,它可能作为一种诱抗剂来提高植物的抗逆性。     

RP-HPLC法测定长期盐胁迫下盐爪爪和盐穗木中的甜菜碱

通过对影响甜菜碱提取和测定的各种因素进行研究,建立了一种准确、快速的测定长期盐胁迫下盐爪爪和盐穗木中甜菜碱的HPLC方法。样品采用超声波甲醇提取,并依次通过强阴阳离子交换树脂(Dowex 50×8和Dowex 50×2)进行纯化,得到的样品采用反相C18柱进行分离,色谱条件:流动相为50 mmol/L、pH 4.5的KH2PO4溶液;波长200 nm下紫外测定,流速0.7 mL/min;此方法的检出限为0.02μg/mL,平均回收率为97.2%。通过对0~500 mmol/L NaCl胁迫下的盐爪爪和盐穗木中甜菜碱进行测定,甜菜碱的含量均呈现先增加后减少的趋势,盐爪爪在NaCl浓度为200 mmol/L时达到最大,而盐穗木在NaCl浓度为300 mmol/L时达到最大。     

TMJ osteoarthritis: a new approach to diagnosis

Disorders of the temporomandibular joint (TMJ), including TMJ osteoarthritis (TMJ OA), are the topic of intensive clinical research; however, this is not the case in the archaeological literature, with the majority of work on the subject ceasing with the early 1990s. The methods employed in the diagnosis of TMJ OA within the archaeological work appear nonrepresentative of the disease and may have led to erroneous assumptions about the pattern and prevalence of OA. This current work presents a new method for evaluating OA specifically for the TMJ, considering both the biomechanics of the joint and the mechanisms of the disease. Totally, 496 specimens (including a group of modern documented specimens) were analyzed for the presence of TMJ OA using the following criteria: eburnation, osteophytes (marginal and new bone on joint surface), porosity, and alteration to joint contour. The results suggest that eburnation occurs rarely in the TMJ, so should not be used as an exclusive criterion. Rather a combination of at least two of the other criteria should be used, with osteophytes and porosity occurring the most frequently on both the mandibular condyle and articular eminence. Additionally, the prevalence of TMJ OA in the modern assemblage was similar to that observed in current clinical research, suggesting that the method employed here was able to produce a reasonable approximation of what is found in contemporary living populations.     

Chondroprotection of PPARα activation by WY14643 via autophagy involving Akt and ERK in LPS‐treated mouse chondrocytes and osteoarthritis model

Autophagy maintains cellular homoeostasis. The enhancement of autophagy in chondrocytes could prevent osteoarthritis (OA) progression in articular cartilage. Peroxisome proliferator‐activated receptor α (PPARα) activation may also protect articular chondrocytes against cartilage degradation in OA. However, whether the protective effect of activated PPARα is associated with autophagy induction in chondrocytes is not determined. In this study, we investigated the effect of PPARα activation by its agonist, WY14643, on the protein expression level of Aggrecan and ADAMTS5, and the protein expression level of autophagy biomarkers, including LC3B and P62, using Western blotting analysis in isolated mouse chondrocytes pre‐treated with lipopolysaccharides (LPS, mimicking OA chondrocytes) with or without the autophagy inhibitor chloroquine diphosphate salt. Furthermore, Akt and ERK phosphorylation was detected in LPS‐treated chondrocytes in response to WY14643. In addition, the effect of intra‐articularly injected WY14643 on articular cartilage in a mouse OA model established by the destabilization of the medial meniscus was assessed using the Osteoarthritis Research Society International (OARSI) histopathology assessment system, along with the detection of Aggrecan, ADAMTS5, LC3B and P62 protein levels using immunohistochemistry assay. The results indicated that PPARα activation by WY14643 promoted proteoglycan synthesis by autophagy enhancement in OA chondrocytes in vivo and in vitro concomitant with the elevation of Akt and ERK phosphorylation. Therefore, autophagy could contribute to the chondroprotection of PPARα activation by WY14643, with the implication that PPARα activation by WY14643 may be a potential approach for OA therapy.     

骨关节炎相关细胞因子及蛋白的研究进展

骨关节炎（osteoarthritis,OA）是人体中轴关节及外周运动关节最常见的退行性疾病,该病的临床症状表现为疼痛、关节僵硬和关节活动受限,罹患该病非常痛苦,严重影响生活质量,是成年人疼痛、运动功能障碍和致残的常见原因之一。OA是临床上常见的骨关节疾病,老年人多发,是老年人丧失劳动力的重要原因,对社会经济影响很大。国内外对OA的发病机制做了大量的研究,但其确切的病因及病理机制尚未完全阐明。目前,OA没有特效治愈方法,治疗目标局限于使用止痛药来缓解疼痛,并最终行人工关节置换手术。即使人工关节置换手术成功,人造关节寿命也是有限的,其他严重问题还有植入物的松动和失败等。因此,对该病病因、病理机制的探索成为目前的热点和焦点,也是解决OA这个问题的关键。本文就近年来研究较热门的骨关节炎相关细胞因子及蛋白做一综述。     

半胱胺对肝细胞代谢油酸过程中ROS生成和ATPase活性的影响

目的 研究了半胱胺（CS）对油酸（0A）代谢过程中自由基（ROS）生成和ATP酶（ATPase）活性的影响。方法 通过培养体外小鼠原代肝细胞,添加不同浓度的OA作用24h,通过luminol化学发光法,TBA法、DTNB法、无机磷比色法分别测定ROS、丙二醛（MDA）、谷胱甘肽（GSH）和Na^＋-K＋-ATP酶活性;选取OA（0,150,250taM／L）三个梯度,添加CS（-,50,100,150,200,250,300,350,400,450,500taM／L）,测定相同的指标。结果 添加OA大于150μM／L时,ROS、MDA生成量急剧升高,ATPase活性显著降低（P〈0．05）。添加CS能提高GSH,减少ROS和MDA的生成,提高ATPase的活性,且作用的效果显著（P〈0．05）。但大量添加CS（350-500μM／L）则效果相反。结论 大量添加OA会引起肝细胞氧化应激;添加CS能清除ROS,提高ATPase活性;并伴随OA添加量的增加,CS的最佳需要量也增加。     

High glucose induces vascular endothelial growth factor production in human synovial fibroblasts through reactive oxygen species generation

Background

Diabetes is an independent risk factor of osteoarthritis (OA). Angiogenesis is essential for the progression of OA. Here, we investigated the intracellular signaling pathways involved in high glucose (HG)-induced vascular endothelial growth factor (VEGF) expression in human synovial fibroblast cells.

Methods

HG-mediated VEGF expression was assessed with qPCR and ELISA. The mechanisms of action of HG in different signaling pathways were studied using Western blotting. Knockdown of proteins was achieved by transfection with siRNA. Chromatin immunoprecipitation assays were used to study in vivo binding of c-Jun to the VEGF promoter.

Results

Stimulation of OA synovial fibroblasts (OASF) with HG induced concentration- and time-dependent increases in VEGF expression. Treatment of OASF with HG increased reactive oxygen species (ROS) generation. Pretreatment with NADPH oxidase inhibitor (APO or DPI), ROS scavenger (NAC), PI3K inhibitor (Ly294002 or wortmannin), Akt inhibitor, or AP-1 inhibitor (curcumin or tanshinone IIA) blocked the HG-induced VEGF production. HG also increased PI3K and Akt activation. Treatment of OASF with HG increased the accumulation of phosphorylated c-Jun in the nucleus, AP-1-luciferase activity, and c-Jun binding to the AP-1 element on the VEGF promoter.

Conclusions

Our results suggest that the HG increases VEGF expression in human synovial fibroblasts via the ROS, PI3K, Akt, c-Jun and AP-1 signaling pathway.

General significance

We link high glucose on VEGF expression in osteoarthritis.     

The role of chondrocyte senescence in osteoarthritis

Replicative senescence occurs when normal somatic cells stop dividing. Senescent cells remain viable, but show alterations in phenotype, e.g. altered expression of matrix metalloproteinases (MMPs); these enzymes are known to be involved in cartilage destruction. It is assumed that cells deplete their replicative potential during aging, and age is a major risk factor for osteoarthritis (OA). Therefore, we hypothesized that chondrocytes in aging or diseased cartilage become senescent with associated phenotypic changes contributing to development or progression of OA. Articular cartilage was obtained from OA patients undergoing arthroplasty, with 'normal' cartilage from trauma surgery for hip fracture. Senescent cells were identified using the senescence-associated beta-galactosidase (SA-beta-gal) marker. Telomere length was assessed using Southern blot. MMP expression was measured at the mRNA level using Taqman RT-PCR. No SA-beta-gal staining was observed in control cartilage regardless of patient age. In contrast, SA-beta-gal staining was observed in damaged OA cartilage adjacent to the lesion. Cultured chondrocytes isolated from sites near a lesion contained a greater percentage of SA-beta-gal positive cells than cultures isolated from distal sites or normal cartilage. Mean telomere length was shorter in cells near the lesion compared to distal sites in the same joint; thus the former population has undergone cell division. The expression of collagenases MMP-1, -8 and -13 and tissue inhibitor of metalloproteinases (TIMP)-1 was altered in OA cartilage, but no difference was detected between lesion and distal sites in the same joint (i.e. no correlation was found between senescent cells and proteinase/ inhibitor expression).     

Protein serine/threonine phosphatases as binding proteins for okadaic acid

Recently, many potent inhibitors of protein serine/threonine phosphatases (PPs) have been found. Some of them have proven to be tumor promoters in mouse skin two-step carcinogenesis and rat liver medium-term tests. Among these inhibitors, okadaic acid (OA) selectively inhibits PP2A, and its use has therefore been proposed to facilitate analysis of biological roles of this phosphatase. OA shows bimodal effects on in vitro transformation and, in addition to such epigenetic changes, also induces marked genetic changes. OA treatment for more than 1 week flattened NIH 3T3 transformants irreversibly, with loss of the transfected genes. It is also known to induce diphtheria toxin-resistant mutations in Chinese hamster lung cells and sister chromatid exchanges (SCEs) in Chinese hamster ovary cells and human lymphocytes. To analyze roles of protein phosphatases in gene stability, we isolated OA-resistant mutants. They were proven to have a mutation in the PP2A catalytic subunit, in which cysteine 269 had beensubstituted for glycine; and it was demonstrated that this region interacts with OA. The recombinant mutant protein was 4 9-fold more resistant to OA than the wild type. Although the OA resistant mutants of CHO cells expressed high levels of P-glycoprotein, inhibition of PP2A itself was suggested to lead to SCE induction. However, the number of molecular species of PP which are known to be sensitive to OA continues to increase, and we have isolated cDNA for a novel type of OA sensitive PP. Our studies indicate that the fact that the roles of PP2A cannot be elucidated using only OA is of crucial importance.     

拟南芥草酸不敏感突变体的筛选与分析

草酸是多种真菌的致病因子。在含1.2 mmol/L 草酸和10 mmol/L 雌二醇的MS缺钙培养基上, 从大约含6000个独立株系的拟南芥化学诱导突变体库中筛选草酸不敏感的突变体。初筛获得的可能的草酸不敏感突变体单株收种后, 进一步复筛获得5株较抗草酸的突变体D33、D74、D154、D282和D630。对它们的TAIL-PCR的第三步产物回收、测序、比对的结果表明：D33的T-DNA插入位点位于At2g39720 (Zinc finger ) and At2g39730 (Rubisco activase) 之间, D74、D154、D282和D630都插在At5g10450 (14-3-3 protein GF14 lambda) 的第一个内含子上。突变体后继的遗传分析与分子分析正在进行中。     

拟南芥草酸不敏感突变体的筛选与分析

草酸是多种真菌的致病因子.在含 1.2 mmol/L 草酸和 10 μmol/L 雌二醇的 MS 缺钙培养基上,从大约含 6000个独立株系的拟南芥化学诱导突变体库中筛选草酸不敏感的突变体.初筛获得的可能的草酸不敏感突变体单株收种后,进一步复筛获得 5 株较抗草酸的突变体 D33、D74、D154、D282 和 D630.对它们的 TAIL-PCR 的第三步产物回收、测序、比对的结果表明:D33 的 T-DNA 插入位点位于 At2g39720(Zinc finger)and At2g39730 (Rubisco activase) 之间,D74、D154、D282 和 D630 都插在 At5g10450 (14-3-3 protein GF14 lambda) 的第一个内含子上.突变体后继的遗传分析与分子分析正在进行中.     

Gene expression,protein profiling,and chemotactic activity of infrapatellar fat pad mesenchymal stem cells in pathologies of the knee joint

The infrapatellar fat pad (IPFP) is a periarticular adipose knee tissue. This tissue contains a large number of mesenchymal stem cells (MSCs). In the present work, we wanted to study the IPFP MSCs and their relationship and differences in two groups, anterior cruciate ligament (ACL) ruptures knees and ostheoarthrosis (OA). The IPFP of 42 patients with OA or ACL rupture were analyzed. Isolation, primary culture, and a genetic and proteomic study of MSCs from IPFP were performed. Gene expression of IL-6, tumor necrosis factor (TNF), IL-8, HSPA1A (Hsp70), CXCL10, RANTES, MMP1, MMP3, TIMP1, and BMP7 was analyzed by real-time quantitative polymerase chain reaction (RT-qPCR). We analyzed MSCs from from 12 diferents patients in two cellular pools (6 from AO disease and 6 from ALC rupture to form two cell pool), for the iTRAQ Proteomic Assay. The conditional media were used in quantitative analysis of MSC soluble factors by Luminex and for de migration assay. A higher gene expression of IL-6, TNF, CXCL10, RANTES, and MMP1 and OPG in MSCs from OA versus ACL (p < 0.05) was observed. Conversely HSPA1A, TIMP1, and RANKL showed a significant lower expression in OA-MSCs (p < 0.05). In the secretome analysis, adipsin and visfantin levels in the supernatants from OA-MSCs were lower (p < 0.05) respect to ACL-MSCs. Also, the monocytic cells migrated two-folds in the presence of conditioned media from OA-MSCs patients versus patients with ACL-MSC. The infrapatellar pad should be considered as an adipose tissue capable of producing and excreting inflammatory mediators directly in the knee joint, influencing the development and progression of knee joint pathologies.