The photosynthetic F1-α3β3 and α1β1 catalytic core complexes

Minimal photosynthetic catalytic F₁() core complexes, containing equimolar ratios of the and subunits, were isolated from membrane-bound spinach chloroplast CF₁ and Rhodospirillum rubrum chromatophore RrF₁. A CF₁-₃₃ hexamer and RrF₁-₁₁ dimer, which were purified from the respective F₁() complexes, exhibit lower rates and different properties from their parent F₁-ATPases. Most interesting is their complete resistance to inhibition by the general F₁ inhibitor azide and the specific CF₁ inhibitor tentoxin. These inhibitors were earlier reported to inhibit multisite, but not unisite, catalysis in all sensitive F₁-ATPases and were therefore suggested to block catalytic site cooperativity. The absence of this typical property of all F₁-ATPases in the ₁₁ dimer is consistant with the view that the dimer contains only a single catalytic site. The ₃₃ hexamer contains however all F₁ catalytic sites. Therefore the observation that CF₁-₃₃ can bind tentoxin and is stimulated by it suggests that the F₁ subunit, which is required for obtaining inhibition by tentoxin as well as azide, plays an important role in the cooperative interactions between the F₁-catalytic sites.Abbreviations CF₀F₁ chloroplast F₀F₁ - CF₁ chloroplast F₁ - CF₁ chloroplast F₁ subunit - CF₁ chloroplast F₁ subunit - CF₁() a complex containing equal amounts of the CF₁ and subunits - MF₁ mitochondrial F₁ - RrF₀F₁ Rhodospirillum rubrum F₀F₁ - RrF₁ R. rubrum F₁ - RrF₁ R. rubrum F₁ subunit - RrF₁ R. rubrum F₁ subunit - RrF₁() a complex containing equal amounts of the RrF₁ and subunits - Rubisco Ribulose-1,5-bisphosphate carboxylase - TF₁ thermophilic bacterium PS3 F₁     

SUMO-1共价修饰ataxin-3

为了探讨ataxin-3的正常生理功能以及脊髓小脑型共济失调Ⅲ型/马查多-约瑟夫病的发病机理,采用酵母双杂交技术,选择polyQ扩展突变型ataxin-3全长构建诱饵质粒,筛选成人脑cDNA文库,寻找与之相互作用的蛋白质,筛选到互作蛋白smallubiquitin-likemodifier1(SUMO-1).进一步运用免疫共沉淀技术证实,SUMO-1在哺乳动物细胞中共价修饰野生型和polyQ扩展突变型ataxin-3.免疫荧光共定位实验发现,polyQ扩展突变型ataxin-3形成的核内蛋白聚合体与SUMO-1共定位.研究提示,ataxin-3的正常生理功能可能受SUMO-1的调节,SUMO-1可能参与了脊髓小脑型共济失调Ⅲ型/马查多-约瑟夫病的发病机制.     

下调Perilipin 1基因表达对3T3-L1细胞脂解的影响

目的:研究下调围脂滴蛋白基因(PLIN1)表达对3T3-L1细胞脂解的影响。方法:采用RNA干扰技术,构建3组阳性及1组阴性sh-PLIN1重组载体,并进行菌液PCR和DNA测序鉴定。Western blot测定PLIN1A蛋白表达,评价载体下调效果。细胞转染有效载体2天后,Bodipy 493/503染色脂滴;酶学方法测定细胞中甘油三酯和甘油含量;Western blot检测甘油三酯脂肪酶(ATGL)、激素敏感性脂肪酶(HSL)及其磷酸化蛋白(p-HSL)的表达。酶联免疫吸附法(ELISA)测定细胞中环磷酸腺苷(c AMP)和蛋白激酶A(PKA)的浓度。结果:各sh-PLIN1干扰载体构建成功,且3组阳性载体均能显著下调PLIN1A蛋白的表达(P0.05)。转染有效载体后,与阴性转染组相比,sh-PLIN1转染组细胞中脂滴减小,甘油三酯含量降低,甘油含量升高,ATGL和HSL相对表达量显著升高(P0.05),p-HSL相对表达量及c AMP、PKA的浓度无显著性差异(P0.05)。结论:下调PLIN1基因表达可加快3T3-L1细胞脂解速率,其可能通过上调ATGL和HSL的表达而实现,c AMP/PKA信号通路对其无明显调节作用。     

Lnc-RAP3对小鼠3T3-L1前脂肪细胞分化的影响

长链非编码RNA (long non-coding RNA, lncRNA)是一类长度大于200nt、没有长开放阅读框架但往往具有mRNA结构特征的RNA,可以在转录及转录后水平参与基因的表达调控。近年来,有研究证实lncRNA对脂肪生成具有重要作用。Lnc-RAP3位于小鼠(Mus musculus)17号染色体,其表达量在小鼠脂肪细胞分化前后呈现显著差异,但其具体的生物学功能尚不清楚。为探讨lnc-RAP3在小鼠3T3-L1前脂肪细胞成脂分化中的作用,本文首先构建了lnc-RAP3的真核表达载体pcDNA3.1-RAP3,利用脂质体将pcDNA3.1-RAP3和人工合成的lnc-RAP3的siRNAs分别转染3T3-L1前脂肪细胞,并对转染后的细胞进行诱导分化,并通过油红O染色、qRT-PCR检测成脂分化相关基因表达等方法比较过表达和敲降lnc-RAP3对3T3-L1前脂肪细胞成脂分化的影响。结果显示,过表达lnc-RAP3后,细胞内脂滴聚集显著减少(P<0.05),在诱导分化第0 d、2 d和4 d时C/EBPα、Glut4、PPARγ、LPL和FAS的表达水平均呈显著(P<0.05)或极显著(P<0.01)下降;敲降lnc-RAP3后,细胞内脂滴聚集显著增多(P<0.05),同时在诱导分化第0 d、2 d时PPARγ、LPL、C/EBPα、FAS和Glut4的表达水平呈显著(P<0.05)或极显著(P<0.01)升高。本研究结果表明,lnc-RAP3可能通过影响成脂分化相关基因的表达来抑制3T3-L1前脂肪细胞的成脂分化。     

游离脂肪酸对3T3-L1脂肪细胞糖代谢的影响

目的:通过培养3T3-L1前脂肪细胞,并诱导其分化至成熟,研究游离脂肪酸对脂肪细胞糖代谢的影响。方法:培养诱导3T3-L1脂肪细胞,用油红O染色鉴定并比较其形态结构的变化。LPS、EPA、SA、PA干预成熟脂肪细胞,收集不同时间的培养基,葡萄糖氧化酶法算出各组脂肪细胞的葡萄糖消耗量。用Western blot检测不同时间各组干预后细胞AMPK、GLUT4蛋白含量。结果:油红O染色鉴定成熟脂肪细胞胞浆中的脂滴染成红色,并出现戒环样结构;诱导分化第8天,90%以上细胞均分化成熟。含LPS、EPA、SA、PA的培养基作用于成熟脂肪细胞,随着时间的延长,显著抑制脂肪细胞对葡萄糖的吸收(P<0.05),同时,脂肪细胞AMPK、GLUT4蛋白含量在减少(P<0.05)。结论:游离脂肪酸可以诱导胰岛素抵抗的分子机制可能是通过胰岛素信号通路激活蛋白激酶(AMPK),进而影响GLUT4的蛋白表达,使脂肪细胞的葡萄糖吸收率减低,影响脂肪细胞的糖代谢。     

舒林酸调节IKK通路对3T3-L1细胞IRS-1酪氨酸磷酸化的影响

目的探讨舒林酸通过调节IKK通路对分化成熟3T3-L1细胞胰岛素受体后信号转导蛋白胰岛素受体底物1（IRS-1）蛋白酪氨酸/丝氨酸（Tyr/Ser）残基磷酸化表达的影响。 方法用地塞米松、IBMX和胰岛素三联培养诱导3T3-L1前脂肪细胞分化为成熟脂肪细胞,油红O染色观察脂肪细胞形态。诱导分化成熟的脂肪细胞如下分组干预,实时荧光定量PCR检测不同浓度炎症因子IL-1 β（0,1,10,100 ng/ml）和（或）不同浓度IKK特异阻断剂舒林酸（0,0.1,1,10 mmol/L）对诱导分化成熟的脂肪细胞IKK通路激活状态的影响。Western Blot检测IL-1β和（或）舒林酸对诱导分化成熟的脂肪细胞IRS-1酪氨酸/丝氨酸残基磷酸化状态的影响。采用单因素方差分析进行统计学分析。 结果实时荧光定量PCR和Western Blot结果显示,IL-1β 10 ng/ml组诱导成熟脂肪细胞IKKβ mRNA较对照组相对表达水平增加,分别为[（2.85±0.16）﹪,（1.00±0.12）﹪,P < 0.01];而IRS-1酪氨酸的磷酸化相对表达量较对照组下降,分别为[（0.72±0.26）﹪,（1.00±0.24）﹪,P < 0.01]。进一步予舒林酸（1?mmol/?L、10?mmol/L）干预后较对照组显著逆转IL-1β诱导脂肪细胞IRS-1酪氨酸磷酸化的表达水平,分别为[（1.72±0.16）﹪,（1.90±0.08）﹪,（1.00±0.13）﹪,P < 0.01],同时下调IRS-1丝氨酸磷酸化的表达水平[（0.79±0.16）﹪,（0.66±0.08）﹪,（1.00±0.10）﹪,P < 0.05]。 结?论IL-1β通过促进诱导分化成熟脂肪细胞IKKβ的表达,激活脂肪细胞IKK炎症通路,抑制脂肪细胞IRS-1酪氨酸残基磷酸化的表达,舒林酸通过调节脂肪细胞IRS-1酪氨酸/丝氨酸残基磷酸化的表达,改善脂肪细胞胰岛素受体后信号转导。     

Troglitazone activates TRPV1 and causes deacetylation of PPARγ in 3T3-L1 cells

Published research suggests that activation of transient receptor potential vanilloid subfamily 1 (TRPV1) enhances the expression and deacetylation of peroxisome proliferator-activated receptor gamma (PPARγ) to cause browning of white adipose tissue. Here, we show that TRPV1 activation by capsaicin significantly prevents high fat diet-induced obesity in mice. This is associated with an increase in the expression and deacetylation of PPARγ in the epididymal fat of these mice. Consistent with the TRPV1 activation in vivo, overexpression of TRPV1 enhanced the PPARγ and other thermogenic genes in cultured 3T3-L1 preadipocytes. To determine the interaction between TRPV1 and PPARγ signaling, we analyzed the effect of Troglitazone (Trog; a thiazolidinedione derivative and an agonist of PAARγ) treatment on cultured 3T3-L1 cells. Trog enhanced the expression of TRPV1, PPARγ and thermogenic proteins in undifferentiated 3T3-L1 cells but not in differentiated cells. Acute application of Trog stimulated a robust Ca²⁺ influx into 3T3-L1 cells and TRPV1 inhibition by capsazepine prevented this. More interestingly, Trog or capsaicin treatment caused the deacetylation of PPARγ in 3T3-L1 cells and inhibition of TRPV1 or Sirtuin 1 - prevented this. Our data suggest a novel effect of Trog to induce PPARγ deacetylation by activating TRPV1. This research has a significant implication on the role of TRPV1 and PPARγ signaling in the browning of white adipose tissue.     

槟榔碱对3T3-L1脂肪细胞脂代谢的影响

目的：观察槟榔碱对3T3-L1脂肪细胞脂代谢的影响并探讨其可能机制。方法：采用经典的"鸡尾酒"法诱导3T3-L1前脂肪细胞分化成熟,随后用不同浓度的槟榔碱（0、25、50、100 μmol/L）处理成熟脂肪细胞72 h。72 h后,四甲基偶氮唑盐（MTT）法检测细胞的活性;油红O染色观察胞浆内脂滴情况;Western blot检测脂肪酸合成酶（FAS）、甘油三酯脂肪酶（ATGL）、激素敏感性脂肪酶（HSL）蛋白表达。结果：诱导分化成熟的脂肪细胞胞浆内可见大量脂滴;MTT显示：0~100 μmol/L槟榔碱对脂肪细胞活力无显著影响;油红O染色后脂质含量测定结果表明槟榔碱能减少成熟脂肪细胞中脂质含量;Western blot结果显示：与0 μmol/L组（对照组）相比,槟榔碱可显著降低脂肪细胞内FAS的蛋白表达,增加ATGL和HSL的蛋白表达;其中以50 μmol/L组最为显著。结论：槟榔碱使脂肪细胞脂解增强,可能与降低脂质合成关键酶FAS的表达,增加脂质分解代谢关键酶ATGL和HSL的表达有关。     

Galectin-3 binds to MUC1-N-terminal domain and triggers recruitment of β-catenin in MUC1-expressing mouse 3T3 cells

Background

Galectin-3 is expressed in a variety of tumors and its expression level is related with tumor progression. Aberrant expression of MUC1 in various tumors is also associated with a poor prognosis. It has been reported that MUC1 is a natural ligand of galectin-3.

Methods

A stable MUC1 transfectant was produced by introducing MUC1 cDNA into mouse 3T3 fibroblasts (MUC1/3T3 cells). MUC1 was prepared from MUC1/3T3 cells; MUC1-N-terminal domain (MUC1-ND) and -C-terminal domain (MUC1-CD) were separated by CsCl ultracentrifugation, and then the galectin-3-binding domain was determined by co-immuniprecipitation assay. After ligation of galectin-3 to 3T3/MUC1 cells, MUC1-CD was immunoprecipitated from the cell lysate. The immunoprecipitate was subjected to SDS-PAGE and Western blotting, followed by detection of co-immunoprecipitated β-catenin.

Results

Galectin-3 binds to the N-terminal domain of MUC1 but not to the C-terminal one. Galectin-3 present on the cell surface increased with the expression of MUC1 and is colocalized with MUC1. It should be noted that β-catenin was detected in the immunoprecipitate with anti-MUC1-CD Ab from a lysate of galectin-3-treated 3T3/MUC1 cells.

Conclusions

Galectin-3 binds to MUC1-ND and triggers MUC1-mediated signaling in 3T3/MUC1 cells, leading to recruitment of β-catenin to MUC1-CD.

General significance

This signaling may be another MUC1-mediated pathway and function in parallel with a growth factor-dependent MUC1-mediated pathway.     

Methylation of elongation factor 1α in mouse 3T3B and 3T3BSV40 cells

Two-dimensional gel electrophoretic (NEPHGE) analysis of proteins from mouse 3T3B and 3T3B/SV40 cells labelled with [methyl-³H]methionine in the presence of cycloheximide have revealed that the elongation factor 1α (EF-1α) in these cells is methylated and that the extent of methylation is higher in the SV40 transformed cell type. It is suggested that methylation may account for differences in growth properties for the different cell types.     

Nucleosides. 124. 3-Amino-3-deoxyhexopyranosyl Nucleosides. 7. 1-(3-Deoxy-3-Nitro-β-D-glucopyranosyl) Uracil and Its Galactopyranosyl Isomer1

Abstract

Crystalline 1-(3-deoxy-3-nitro-β-D-glucopyranosyl) uracil (3), originally prepared by nitromethane condensation of “uridine dialdehyde,” was found to contain the galactosyl isomer (4). Each isomer was obtained in pure form by 4′,6′-O-benzylidenation of the mixture of 3 and 4, followed by chromatographic separation and subsequent O-debenzylidenation. The structure of each isomer was established by chemical conversion of the isomer into the corresponding known 3′-acetamido-2′,4′,6′-tri-O-acetyl derivative.     

JNK1基因沉默对3T3-L1脂肪细胞高分子量脂联素表达的影响

利用短发夹RNA(short hairpin RNA,sh RNA)构建慢病毒载体,利用慢病毒感染3T3-L1细胞,沉默3T3-L1脂肪细胞的JNK1基因,观察其受抑制后二硫键A氧化还原酶样蛋白(disulfide-bond A oxidoreductase-like protein,Dsb A-L)和高分子量(high molecular weight,HMW)脂联素表达的影响。建立携带sh RNA-JNK1的慢病毒载体,用q PCR和Western blotting从基因和蛋白水平筛选出最佳干扰效果的sh RNA-JNK1慢病毒。利用筛选出的sh RNA-JNK1慢病毒沉默3T3-L1脂肪细胞JNK1基因,通过Western blotting检测Dsb A-L、HMW脂联素的表达,并探讨了JNK1基因沉默是否干扰了AMPK通路的活化。在3T3-L1细胞中筛选出对JNK1沉默效果最佳的sh RNA-JNK1组,抑制率达到60%以上(p0.001)。用sh RNA-JNK1慢病毒感染3T3-L1脂肪细胞,用Western blotting检测,结果 AMPK、P-AMPK、Dsb A-L和HMW表达明显增加(p0.001);在3T3-L1脂肪细胞中sh RNA-JNK1慢病毒逆转了AMPK抑制剂Compound C对AMPK、P-AMPK、Dsb A-L和HMW脂联素表达的抑制。3T3-L1脂肪细胞中,沉默JNK1基因后可促进Dsb A-L和HMW脂联素的表达,且JNK1有可能通过AMPK通路实现对脂联素多聚化的调控。     

The αβ complexes of ATP synthase: the α3β3 oligomer and α1β1 protomer

The basic structures of the catalytic portion (F₁, ₃₃) of ATP synthase are the ₃₃ hexamer (oligomer with cooperativity) and ₁₁ heterodimer (protomer). These were reconstituted from the and subunits of thermophilic F₁ (TF₁), and the ₃₃ hexamer was crystallized. On electrophoresis, both the dimer and hexamer showed bands with ATPase activity. Using the dimer and hexamer, we studied the nucleotide-dependent rapid molecular dynamics. The formation of the hexamer required neither nucleotide nor Mg. The hexamer was dissociated into the dimer in the presence of MgADP, while the dimer was associated into the hexamer in the presence of MgATP. The hexamer, like mitochondrial F₁ and TF₁, showed two kinds of ATPase activity: one was cooperative and was inhibited by only one BzADP per hexamer, and the other was inhibited by three BzADP per hexamer.     

“de novo” trisomy 1q32→1qter and monosomy 3p25→3pter

Summary The syndrome of familial lymphedema (type Meige) with distichiasis was observed in father and son. The association with uvula bifida and submucous cleft of the palate is described for the first time.