口臭症口腔微生态学的研究

目的 :研究口臭症患者口腔菌群分布。方法 :选择 3 0例口臭患者和 3 0例健康人 ,分别采集舌背舌苔和牙周袋菌斑作细菌的需氧、微需氧和厌氧菌的定量培养 ,并对牙龈卟啉单胞菌及衣氏放线菌做抑菌试验。结果 :口臭组舌背厌氧菌及微需氧菌总数 (12 .2± 1.3 2 )与健康对照组 (9.5± 1.2 4)差异有非常显著性 (P<0 .0 1) ,口臭组需氧菌总数与健康组差异有显著性 (P<0 .0 5) ;口臭组牙周袋厌氧菌及微需氧菌总数 (13 .12± 1.2 6)明显高于健康对照组 (11.3± 1.82 ) ,两者差异有非常显著性 (P<0 .0 1) ,而需氧菌差异无显著性 ;口臭症舌背以小韦荣菌为主 ,其次是黑色普氏菌 ;牙周袋菌斑以牙龈卟啉单胞菌和黑色普氏菌为主 ,其次是衣氏放线菌。并筛选出两类抗菌漱口水和一种中外合资抗口臭牙膏 ,对舌背及牙周袋菌斑的主要细菌牙龈卟啉单胞菌和衣氏放线菌有明显的抑制作用。结论 :口臭症患者口腔菌群分布和总数与正常人群存在显著的差异 ,与牙周病有关的致病菌数量明显高于正常人群 ,据抑菌试验结果采用有效的漱口液及牙膏可明显降低口腔内致病菌和改善口臭症状。     

The ubiquinone-binding site in NADH:ubiquinone oxidoreductase from Escherichia coli

An azido-ubiquinone derivative, 3-azido-2-methyl-5-methoxy[3H]-6-decyl-1,4-benzoquinone ([3H]azido-Q), was used to study the ubiquinone/protein interaction and to identify the ubiquinone-binding site in Escherichia coli NADH:ubiquinone oxidoreductase (complex I). The purified complex I showed no loss of activity after incubation with a 20-fold molar excess of [3H]azido-Q in the dark. Illumination of the incubated sample with long wavelength UV light for 10 min at 0 degrees C caused a 40% decrease of NADH:ubiquinone oxidoreductase activity. SDS-PAGE of the complex labeled with [3H]azido-Q followed by analysis of the radioactivity distribution among the subunits revealed that subunit NuoM was heavily labeled, suggesting that this protein houses the Q-binding site. When the [3H]azido-Q-labeled NuoM was purified from the labeled reductase by means of preparative SDS-PAGE, a 3-azido-2-methyl-5-methoxy-6-decyl-1,4-benzoquinone-linked peptide, with a retention time of 41.4 min, was obtained by high performance liquid chromatography of the protease K digest of the labeled subunit. This peptide had a partial NH2-terminal amino acid sequence of NH2-VMLIAILALV-, which corresponds to amino acid residues 184-193 of NuoM. The secondary structure prediction of NuoM using the Toppred hydropathy analysis showed that the Q-binding peptide overlaps with a proposed Q-binding motif located in the middle of the transmembrane helix 5 toward the cytoplasmic side of the membrane. Using the PHDhtm hydropathy plot, the labeled peptide is located in the transmembrane helix 4 toward the periplasmic side of the membrane.     

TRAIL-R2 (DR5) mediates apoptosis of synovial fibroblasts in rheumatoid arthritis

TRAIL has been proposed as an anti-inflammatory cytokine in animal models of rheumatoid arthritis (RA). Using two agonistic mAbs specific for TRAIL-R1 (DR4) and TRAIL-R2 (DR5), we examined the expression and function of these death receptors in RA synovial fibroblast cells. The synovial tissues and primary synovial fibroblast cells isolated from patients with RA, but not those isolated from patients with osteoarthritis, selectively expressed high levels of cell surface DR5 and were highly susceptible to anti-DR5 Ab (TRA-8)-mediated apoptosis. In contrast, RA synoviocytes did not show increased expression of TRAIL-R1 (DR4), nor was there any difference in expression of Fas between RA and osteoarthritis synovial cells. In vitro TRA-8 induced apoptosis of RA synovial cells and inhibited production of matrix metalloproteinases induced by pro-inflammatory cytokines. In vivo TRA-8 effectively inhibited hypercellularity of a SV40-transformed RA synovial cell line and completely prevented bone erosion and cartilage destruction induced by these cells. These results indicate that increased DR5 expression and susceptibility to DR5-mediated apoptosis are characteristic of the proliferating synovial cells in RA. As highly proliferative transformed-appearing RA synovial cells play a crucial role in bone erosion and cartilage destruction in RA, the specific targeting of DR5 on RA synovial cells with an agonistic anti-DR5 Ab may be a potential therapy for RA.     

P—选择素凝集素—EGF功能域的克隆、表达及其单克隆抗体制备

制备特异性抗人P 选择素的凝集素 表皮生长因子 (L EGF)功能域的单克隆抗体。利用特异引物 ,通过RT PCR从外周血血小板中扩增出人P 选择素的L EGF功能域基因 ,将其克隆至pET42b( )载体中 ,测序验证后转染大肠杆菌BL2 1,经诱导表达了C端融合 6×His的蛋白质。融合蛋白质经分离纯化后 ,免疫Balb/c小鼠 ,应用杂交瘤技术 ,通过间接ELISA筛选阳性克隆。获得 3株可稳定分泌抗L EGF功能域单抗的杂交瘤细胞株 (B10、F3和H5 )。其亚型分别为IgG2 、IgG1和IgG3;轻链均为κ型。所获的单抗对LPS刺激活化的人脐静脉内皮细胞均有特异性结合反应 ,并可在体外阻断经凝血酶激活的血小板与中性粒细胞间的粘附。表明所获的单抗可特异性识别结合天然P 选择素 ,具有体外抗活化血小板与中性粒细胞粘附的功能 ,为进一步应用此单抗进行P 选择素结构和功能及抗粘附治疗研究提供了实验基础。     

短期内15N标记的铵盐和硝酸盐在青藏高原高寒草甸中的运移规律

运用15N稳定性同位素示踪技术,对高寒草甸植物和土壤微生物固持沉降氮的能力及沉降氮在小嵩草(Kobresia pygaea)草甸中的运移规律进行了研究.施肥2周后,NO-3-15N和NH+4-15N的总恢复率分别为73.5%和78%.无论是NO-3-15N,还是NH+4-15N,植物所固持的15N总是比土壤有机质或者是土壤微生物固持的多.4周后,70.6%的NO-3-15N和57.4%的NH+4-15N被固持在土壤和植物中.其中,土壤有机质所固持的15N均下降了很多,而植物所固持的15N却变化很小.同前面的结果相比,较多的NO-3-15N为土壤微生物所固持.在施肥6周和8周后,NO-3-15N的总恢复率分别为58.4%和67%,而NH+4-15N的总恢复率分别为43.1%和49%.植物和土壤微生物所固持的NO-3-15N比NH+4-15N多.在整个实验期间,植物固持的NO-3N较多,而且比土壤微生物固持了较多的15N.由于无机氮的含量一直很低,无机氮库所固持的15N一般不超过1%.上述结果意味着短期内植物在高寒草甸中对沉降氮的去向起着决定作用.     

Mesenchymal Stem Cell Transplantation Enhancement in Myocardial Infarction Rat Model under Ultrasound Combined with Nitric Oxide Microbubbles

Objective

This study evaluated the effects of ultrasound combined with the homemade nitric oxide (NO) micro-bubble destruction on the in vitro proliferation, apoptosis, and migration of mesenchymal stem cells (MSCs). Furthermore, we studied whether or not irradiation of the NO micro-bubble combined with bone-marrow derived MSC infusion had a better effect on treating myocardial infarction. The possible mechanism of MSC delivery into the infarcted myocardium was also investigated.

Methods

The murine bone marrow-derived MSCs were isolated, cultured, irradiated, and combined with different concentrations of NO microbubbles. MTT proliferation assay, annexin V-FITC apoptosis detection, migration assay, and RT-PCR were performed 24 h after the irradiation. The NO micro-bubbles was a intravenously injected, followed by the infusion of MSCs, which were labeled by CM-Dil. Myocardium was harvested 48 h later and the distribution of MSCs was observed by laser scanning confocal microscope after frozen sectioning. Echocardiography, histological examination, RT-PCR, and western blotting were performed four weeks after the cell transplantation.

Results

Ultrasound combined with 1:70 NO micro-bubbles had no significant impact on the proliferation or apoptosis of MSCs. Transwell chamber findings demonstrated that MSCs migrated more efficiently in group that underwent ultrasound combined with 1:70 NO micro-bubbles. The Real-time PCR results indicated that the expression of CXCR4 was much higher in the group undergoing ultrasound combined with 1:70 NO micro-bubbles. The normalized fluorescence intensity greatly increased in the group of US+NO micro-bubbles and the cardiac function was also markedly improved. Immunohistochemical staining showed that the capillary density was much greater in the group of US+NO micro-bubbles as compared to that of the other groups. RT-PCR and western blotting also revealed a higher SDF-1 and VEGF expression in the group of US+NO micro-bubbles.

Conclusions

NO micro-bubbles could be used in the cell transplantation, which efficiently promoted the MSC homing into the infarcted myocardium.     

Dichloroacetate induces protective autophagy in LoVo cells: involvement of cathepsin D/thioredoxin-like protein 1 and Akt-mTOR-mediated signaling

Dichloroacetate (DCA) is an inhibitor of pyruvate dehydrogenase kinase (PDK), and recently it has been shown as a promising nontoxic antineoplastic agent. In this study, we demonstrated that DCA could induce autophagy in LoVo cells, which were confirmed by the formation of autophagosomes, appearance of punctate patterns of LC3 immunoreactivity and activation of autophagy associated proteins. Moreover, autophagy inhibition by 3-methyladenine (3-MA) or Atg7 siRNA treatment can significantly enhance DCA-induced apoptosis. To determine the underlying mechanism of DCA-induced autophagy, target identification using drug affinity responsive target stability (DARTS) coupled with ESI-Q-TOF MS/MS analysis were utilized to profile differentially expressed proteins between control and DCA-treated LoVo cells. As a result, Cathepsin D (CTSD) and thioredoxin-like protein 1 (TXNL1) were identified with significant alterations compared with control. Further study indicated that DCA treatment significantly promoted abnormal reactive oxygen species (ROS) production. On the other hand, DCA-triggered autophagy could be attenuated by N-acetyl cysteine (NAC), a ROS inhibitor. Finally, we demonstrated that the Akt-mTOR signaling pathway, a major negative regulator of autophagy, was suppressed by DCA treatment. To our knowledge, it was the first study to show that DCA induced protective autophagy in LoVo cells, and the potential mechanisms were involved in ROS imbalance and Akt-mTOR signaling pathway suppression.     

蕨类植物psbD基因的适应性进化和共进化分析

D2蛋白是植物光系统Ⅱ复合体（PSⅡ）核心蛋白之一,由叶绿体psbD基因编码。为了深入理解核心薄囊蕨类植物在阴生环境下的“辐射”式演化,我们对12种蕨类植物的psbD基因进行了克隆和测序,然后联合已公布的其他8种蕨类植物的psbD序列,基于ω值（非同义替换率ds和同义替换率ds的比值）探讨了该基因经受的选择压力。发现D2蛋白在大多数分支和位点受到强烈的负选择,但是树蕨类分支的psbD进化速率低且ω值较高。借助多种模型进行的共进化分析显示,树蕨类D2蛋白的168R、245H和272M两两组成具有共进化关系的氨基酸位点对。     

长潭水库铁锰含量与环境因子的季节性变化相关性分析及铁锰氧化菌群的富集培养

【背景】目前对水库水体污染原因的研究往往专注于水体的富营养化、pH值、溶解氧、氨氮、菌落总数指标的变化,而重金属含量与环境因子的季节性变化相关性分析研究较少,同时对于典型季节原位微生物种群的多样性差异研究尚未见报道。【目的】研究浙江省台州市长潭水库底部水中正二价金属离子(二价锰离子Mn²⁺;二价铁离子Fe²⁺)浓度与不同环境因子的季节性变化规律,并对其相关性进行分析;富集平水期(2月)和丰水期(8月)水库底部水体中功能微生物菌群,分析其种类和丰度的差异。【方法】分别检测12个月的水库水Mn²⁺和Fe²⁺浓度及多种环境因子(水体溶解氧浓度、pH值、总磷浓度、浊度、水库环境温度及降水量),过滤并富集培养水库底部水体的功能微生物菌群,对其16S rRNA基因V3-V4区测序并分析其菌群结构。【结果】长潭水库水中Mn²⁺和Fe²⁺浓度呈季节性变化,每年的春夏交替季节水体中铁锰含量从零开始慢慢升高,至夏秋高温季节水体中Mn²⁺和Fe²⁺浓度达到最高值,然后慢慢降低,至秋末冬初检测不到含量。在检测的多种环境因子中,水体溶解氧浓度、水库环境温度及降水量呈明显的季节性规律变化。Mn²⁺和Fe²⁺浓度与温度、降水量和浊度有正相关性,与溶解氧浓度、pH值和总磷浓度有负相关性,其中在正负相关性分析中两种金属离子的浓度与溶解氧浓度的相关性最强,其次是环境温度及降水量。丰水期和平水期中富集获得的功能微生物菌群的种类和丰度差异很大,从属水平上分类,丰水期时菌群只包含不动杆菌(Acinetobacter)和鲑色沉积物杆状菌(Sediminibacterium)这2个属的菌株,含量各占约50%;平水期时菌群则主要由杆菌属(Bacillariophyta) (47.62%)和Limnohabitans(9.52%)等9个属的菌株构成。富集获得的平水期和丰水期的两个可培养的菌群均具有去除水库水中Mn²⁺的功能,去除率分别约为35.9%和11.4%。【结论】长潭水库底部水体中Mn²⁺和Fe²⁺浓度与不同环境因子均呈季节性规律变化,它们之间呈现不同的正负相关性,丰水期和平水期的功能微生物菌群结构差异很大。本研究为利用微生物进行重金属污染水体的治理储备了微生物资源,为实现国家“美丽乡村”的建设目标提供一定的参考价值。