PPAR γ is highly expressed in F4/80 adipose tissue macrophages and dampens adipose-tissue inflammation

Macrophage infiltration into adipose tissue is a hallmark of obesity. We recently reported two phenotypically distinct subsets of adipose tissue macrophages (ATM) based on the surface expression of the glycoprotein F4/80 and responsiveness to treatment with a peroxisome proliferator-activated receptor (PPAR) γ agonist. Hence, we hypothesized that F4/80^hi and F4/80^lo ATM differentially express PPAR γ. This study phenotypically and functionally characterizes F4/80^hi and F4/80^lo ATM subsets during obesity. Changes in gene expression were also examined on sorted F4/80^lo and F4/80^hi ATM by quantitative real-time RT-PCR. We show that while F4/80^lo macrophages predominate in adipose tissue of lean mice, obesity causes accumulation of both F4/80^lo and F4/80^hi ATM. Moreover, accumulation of F4/80^hi ATM in adipose tissue is associated with impaired glucose tolerance. Phenotypically, F4/80^hi ATM express greater amounts of CD11c, MHC II, CD49b, and CX3CR1 and produce more TNF-α, MCP-1, and IL-10 than F4/80^lo ATM. Gene expression analyses of the sorted populations revealed that only the F4/80^lo population produced IL-4, whereas the F4/80^hi ATM expressed greater amounts of PPAR γ, δ, CD36 and toll-like receptor-4. In addition, the deficiency of PPAR γ in immune cells favors expression of M1 and impairs M2 macrophage marker expression in adipose tissue. Thus, PPAR γ is differentially expressed in F4/80^hi versus F4/80^low ATM subsets and its deficiency favors a predominance of M1 markers in WAT.     

Overexpression of cyclin E/CDK2 complexes overcomes FGF-induced cell cycle arrest in the presence of hypophosphorylated Rb proteins

FGF signaling inhibits chondrocyte proliferation and requires the function of the p107 and p130 members of the Rb protein family to execute growth arrest. p107 dephosphorylation plays a critical role in the chondrocyte response to FGF, as overexpression of cyclin D1/CDK4 complexes (the major p107 kinase) in rat chondrosarcoma (RCS) cells overcomes FGF-induced p107 dephosphorylation and growth arrest. In cells overexpressing cyclin D1/CDK4, FGF-induced downregulation of cyclin E/CDK2 activity was absent. To examine the role of cyclin E/CDK2 complexes in mediating FGF-induced growth arrest, this kinase was overexpressed in RCS cells. FGF-induced dephosphorylation of either p107 or p130 was not prevented by overexpressing cyclin E/CDK2 complexes. Unexpectedly, however, FGF-treated cells exhibited sustained proliferation even in the presence of hypophosphorylated p107 and p130. Both pocket proteins were able to form repressive complexes with E2F4 and E2F5 but these repressors were not translocated into the nucleus and therefore were unable to occupy their respective target DNA sites. Overexpressed cyclin E/CDK2 molecules were stably associated with p107 and p130 in FGF-treated cells in the context of E2F repressive complexes. Taken together, our data suggest a novel mechanism by which cyclin E/CDK2 complexes can promote cell cycle progression in the presence of dephosphorylated Rb proteins and provide a novel insight into the key Retinoblastoma/E2F/cyclin E pathway. Our data also highlight the importance of E2F4/p130 complexes for FGF-mediated growth arrest in chondrocytes.     

Different Binding Modes of Spermine to A-T and G-C Base Pairs Modulate the Bending and Stiffening of the DNA Double Helix

Abstract

The influence of base composition (and sequence) on the process of interaction between synthetic polynucleotides and spermine, has been investigated using ultraviolet (including second derivative) spectroscopy, and electric dichroism.

Different binding modes of spermine to poly(dG-dC) as compared to A-T containing polynucleotides, were evidenced. An interaction with the N7 and 06 of guanine is probably partially involved in the former case while simple electrostatic interaction with the phosphate groups would dominate in the latter.

In the intermediate binding range (spermine over DNA phosphate molar ratios Sp/P of the order of 0.1 to 0.2), the complexes with poly(dA) · poly(dT) and those with poly(dA-dT) displayed an important contribution of a permanent dipole moment to the orientation mechanism, as detected by the application of bipolar pulses in electric dichroism experiments. Just prior to precipitation (at Sp/P slightly larger than 0.3), these polynucleotides show electric dichroism and relaxation times characteristics corresponding to toroidal particles formation resulting from a bending of their chains. This implies asymmetric binding to phosphate sites on A-T containing polynucleotides. At low Sp/P ratios, spermine induced a stiffening of poly (dG-dC). No influence of spermine on the orientation mechanism of this polynucleotide was detected for Sp/P values ranging from zero to 0.35. The spermine-induced bending of A-T rich regions thus appears to be essential for DNA condensation into toroidal particles.     

Long noncoding RNA DLGAP1-AS1 promotes cell proliferation in hepatocellular carcinoma via sequestering miR-486-5p

Hepatocellular carcinoma (HCC) is most prevalent tumor in liver and one of the most fatal cancers in the world. Long noncoding RNAs (lncRNAs) have been accepted as important regulators in carcinomas. But there are still many lncRNAs including DLGAP1-AS1 unannotated in HCC. First of all, GEPIA suggested that DLGAP1-AS1 presented high expression in HCC tissue samples relative to the normal tissues. Besides, overexpression of DLGAP1-AS1 was also proved in HCC cell lines. Moreover, DLGAP1-AS1 knockdown efficiently suppressed cell proliferation in HCC. Interestingly, miR-486-5p was predicted and validated to interact with DLGAP1-AS1, while the level of miR-486-5p was significantly increased In HCC after DLGAP1-AS1 knockdown. Moreover, we uncovered that ectopic expression of miR-486-5p induced suppression on HCC cell proliferation and that miR-486-5p inhibition offset the effect of DLGAP1-AS1 silence on HCC cell proliferation and apoptosis. Furthermore, H3F3B was identified as target of miR-486-5p and was therefore positively regulated by DLGAP1-AS1 in HCC. Of note, H3F3B upregulation partly revived the declined cell proliferative capacity in response to DLGAP1-AS1 knockdown. To conclude, DLGAP1-AS1 exerted its oncogenic role in HCC via miR-486-5p/H3F3B axis. Our new findings provided novel theoretical basis for discovery of therapeutic targets of HCC.     

Real-time PCR genotyping and frequency of the myostatin F94L mutation in beef cattle breeds

This research developed two real-time PCR assays, employing high-resolution melt and allele-specific analysis to accurately genotype the F94L mutation in cattle. This mutation (g.433C > A) in the growth differentiation factor 8 or myostatin gene has recently been shown to be functionally associated with increased muscle mass and carcass yield in cattle. The F94L mutation is not, like other myostatin mutations, associated with reduced fertility and dystocia. It is therefore a candidate for introgression into other breeds to improve retail beef yield and the development of a simple and accurate test to genotype this specific mutation is warranted. Variations in the efficiency of enzyme cleavage compromised the accuracy of genotyping by published methods, potentially resulting in an overestimation of the frequency of the mutant allele. The frequency of the F94L mutation was determined by real-time PCR in 1140 animals from 15 breeds of cattle in Australia. The mutation was present in Simmental (0.8%), Piedmontese (2%), Droughtmaster (4%) and Limousin (94.2%) but not found in Salers, Angus, Poll Hereford, Hereford, Gelbvieh, Charolais, Jersey, Brahman, Holstein, Shorthorn or Maine Anjou. The low prevalence of F94L in all beef breeds except Limousin indicates the significant potential for this mutation to improve retail yield in Australian beef cattle.     

Behavior of tight-junction, adherens-junction and cell polarity proteins during HNF-4alpha-induced epithelial polarization

We previously reported that expression of tight-junction molecules occludin, claudin-6 and claudin-7, as well as establishment of epithelial polarity, was triggered in mouse F9 cells expressing hepatocyte nuclear factor (HNF)-4alpha [H. Chiba, T. Gotoh, T. Kojima, S. Satohisa, K. Kikuchi, M. Osanai, N. Sawada. Hepatocyte nuclear factor (HNF)-4alpha triggers formation of functional tight junctions and establishment of polarized epithelial morphology in F9 embryonal carcinoma cells, Exp. Cell Res. 286 (2003) 288-297]. Using these cells, we examined in the present study behavior of tight-junction, adherens-junction and cell polarity proteins and elucidated the molecular mechanism behind HNF-4alpha-initiated junction formation and epithelial polarization. We herein show that not only ZO-1 and ZO-2, but also ZO-3, junctional adhesion molecule (JAM)-B, JAM-C and cell polarity proteins PAR-3, PAR-6 and atypical protein kinase C (aPKC) accumulate at primordial adherens junctions in undifferentiated F9 cells. In contrast, CRB3, Pals1 and PATJ appeared to exhibit distinct subcellular localization in immature cells. Induced expression of HNF-4alpha led to translocation of these tight-junction and cell polarity proteins to beltlike tight junctions, where occludin, claudin-6 and claudin-7 were assembled, in differentiated cells. Interestingly, PAR-6, aPKC, CRB3 and Pals1, but not PAR-3 or PATJ, were also concentrated on the apical membranes in differentiated cells. These findings indicate that HNF-4alpha provokes not only expression of tight-junction adhesion molecules, but also modulation of subcellular distribution of junction and cell polarity proteins, resulting in junction formation and epithelial polarization.     

Normal‐mode‐based modeling of allosteric couplings that underlie cyclic conformational transition in F1 ATPase

F₁ ATPase, a rotary motor comprised of a central stalk ( γ subunit) enclosed by three α and β subunits alternately arranged in a hexamer, features highly cooperative binding and hydrolysis of ATP. Despite steady progress in biophysical, biochemical, and computational studies of this fascinating motor, the structural basis for cooperative ATPase involving its three catalytic sites remains not fully understood. To illuminate this key mechanistic puzzle, we have employed a coarse‐grained elastic network model to probe the allosteric couplings underlying the cyclic conformational transition in F₁ ATPase at a residue level of detail. We will elucidate how ATP binding and product (ADP and phosphate) release at two catalytic sites are coupled with the rotation of γ subunit via various domain motions in α ₃ β ₃ hexamer (including intrasubunit hinge‐bending motions in β subunits and intersubunit rigid‐body rotations between adjacent α and β subunits). To this end, we have used a normal‐mode‐based correlation analysis to quantify the allosteric couplings of these domain motions to local motions at catalytic sites and the rotation of γ subunit. We have then identified key amino acid residues involved in the above couplings, some of which have been validated against past studies of mutated and γ ‐truncated F₁ ATPase. Our finding strongly supports a binding change mechanism where ATP binding to the empty catalytic site triggers a series of intra‐ and intersubunit domain motions leading to ATP hydrolysis and product release at the other two closed catalytic sites. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

磷对铝氟交互处理下茶树主要生理生化特性的影响

通过白茶和智仁早茶在铝氟交互处理下添加外源磷的水培试验,研究了不同浓度磷(0,0.2,1.0 mmol/L)对茶树主要化学成分及抗氧化酶的影响.结果表明:(1)当施入0.2 mmol/L磷时,茶叶中的蛋白质、叶绿素和茶多酚含量及SOD、POD和CAT活性均比无磷组高,且在5.0 mmol/L铝和1.0 mmol/L氟处理下,均达到最高值(叶绿素除外),表明适量外源磷的施加对茶树的生理生化特性起促进作用,并能有效缓解铝和氟毒所致的氧化损伤,从而抑制了脂质过氧化作用;(2)随着磷浓度(1.0 mmol/L)增加,茶叶中的各指标值均比无磷组和对照低,出现最低值,表明磷对铝氟胁迫下茶树的缓解效应有限,超过一定的浓度,则表现出明显的抑制作用;(3) 在外源磷的施加下,白茶的可溶性蛋白、茶多酚和叶绿素含量及SOD、POD和CAT活性均比智仁早茶的变化幅度大.因此,外源磷对茶树幼苗生理生化作用具有一定的调控效应.