The Smad3-miR-29b/miR-29c axis mediates the protective effect of macrophage migration inhibitory factor against cardiac fibrosis

Although macrophage migration inhibitory factor (MIF) is known to have antioxidant property, the role of MIF in cardiac fibrosis has not been well understood. We found that MIF was markedly increased in angiotension II (Ang-II)-infused mouse myocardium. Myocardial function was impaired and cardiac fibrosis was aggravated in Mif-knockout (Mif-KO) mice. Functionally, overexpression of MIF and MIF protein could inhibit the expression of fibrosis-associated collagen (Col) 1a1, COL3A1 and α-SMA, and Smad3 activation in mouse cardiac fibroblasts (CFs). Consistently, MIF deficiency could exacerbate the expression of COL1A1, COL3A1 and α-SMA, and Smad3 activation in Ang-II-treated CFs. Interestingly, microRNA-29b-3p (miR-29b-3p) and microRNA-29c-3p (miR-29c-3p) were down-regulated in the myocardium of Ang-II-infused Mif-KO mice but upregulated in CFs with MIF overexpression or by treatment with MIF protein. MiR-29b-3p and miR-29c-3p could suppress the expression of COL1A1, COL3A1 and α-SMA in CFs through targeting the pro-fibrosis genes of transforming growth factor beta-2 (Tgfb2) and matrix metallopeptidase 2 (Mmp2). We further demonstrated that Mif inhibited reactive oxygen species (ROS) generation and Smad3 activation, and rescued the decrease of miR-29b-3p and miR-29c-3p in Ang-II-treated CFs. Smad3 inhibitors, SIS3 and Naringenin, and Smad3 siRNA could reverse the decrease of miR-29b-3p and miR-29c-3p in Ang-II-treated CFs. Taken together, our data demonstrated that the Smad3-miR-29b/miR-29c axis mediates the inhibitory effect of macrophage migration inhibitory factor on cardiac fibrosis.     

Retracted: Curcumin ameliorates atherosclerosis through upregulation of miR-126

The potential usage of curcumin in diverse human diseases has been widely studied, including arteriosclerosis (AS). This study focused on investigating the relationship between curcumin and AS-associated microRNA, which may provide a better understanding of curcumin in a different mechanism. Human microvascular endothelial HMEC-1 cells were treated by curcumin alone or oxidized low-density lipoprotein (ox-LDL) plus curcumin, after which the following parameters were analyzed: cell viability, migration, and the expression of AS-associated factors. The regulatory effects of curcumin on miR-126 and signaling pathways involved in AS were then studied. Further, an animal model of AS was stimulated by feeding rabbits with 1% cholesterol diet. The effects of curcumin on the animal model were explored. We found that curcumin treatment significantly reduced HMEC-1 cells viability, migration, and the protein levels of MMP-2, MMP-9, and vascular endothelial growth factor (VEGF) in the presence or absence of ox-LDL. Meanwhile, the expression of VEGFR1 and VEGFR2 was repressed by curcumin. miR-126 was upregulated by curcumin. The abovementioned effects of curcumin on HMEC-1 cells were all attenuated when miR-126 was silenced. And also, VEGF was a target gene of miR-126, and curcumin could inhibit the activation of PI3K/AKT JAK2/STAT5 signaling pathways via miR-126. The effects of curcumin and its regulation on miR-126 and VEGF were confirmed in the animal model of AS. To sum up, curcumin exerted potent anti-AS property possibly via upregulating miR-126 and thereby inhibiting PI3K/AKT and JAK2/STAT5 signaling pathways.     

miR-126 在膀胱癌患者尿液中的表达及临床意义

目的:探讨miR-126在膀胱癌患者尿液中的表达与临床病理特征的关系,评估miR-126的肿瘤标志物诊断价值。方法:收集48例初发膀胱尿路上皮癌患者与32例健康对照者晨尿,提取尿液总RNA,通过实时荧光定量PCR技术检测各样本中的miR-126的表达水平,并经受试者工作曲线(ROC)分析其诊断价值。结果:膀胱癌患者尿液中的miR-126表达水平相对健康对照组明显上调(P0.01),其表达水平在不同病理级别之间存在显著差异(P均0.05),且低级别组表达水平略高于高级别组,与肿瘤大小、数目以及淋巴转移也有一定的相关性(P0.05),而与患者的年龄、性别、TNM分期等均无相关性(P0.05)。通过ROC曲线分析尿液中miR-126诊断膀胱肿瘤的曲线下面积(AUC)为0.861,当最佳切点定在7.475时,miR-126诊断膀胱肿瘤的敏感性和特异性分别为75.0%、81.2%。结论:膀胱癌患者尿液中miR-126的表达差异能够反映病情进展程度,其表达水平对膀胱肿瘤的早期诊断及病情评估具有一定的价值。     

Charge effect on the colchicine binding-site of tubulin

Poly(L-lysine) was found to enhance colchicine binding activity of brain tubulin to a several folds. Bases of biological interests that were tested and found to be inactive were spermine, spermidine and even L-lysine. Part of this enhance binding is due to the increase in the affinity of colchicine-tubulin interaction in the presence of poly(L-lysine). Moreover, poly(L-lysine) stabilized the colchicine binding site of tubulin against thermal denaturation.     

Use of the Airfuge for analysis and preparation of receptors incorporated into liposomes: Studies with the receptor for immunoglobulin E

A Beckman Airfuge has been employed for studying the interaction between lipids and the receptor for immunoglobulin E (IgE). For analytic experiments, samples were applied underneath a discontinuous sucrose gradient. After a 30-min centrifugation in a fixed-angle rotor, liposomes floated toward the top of the gradient whereas unincorporated receptor-IgE complexes remained at the bottom of the tube. Liposomes with incorporated receptors were also efficiently separated in the ACR-90 preparative rotor. These methods of "Airfuge flotation" can provide useful adjuncts to more traditional methods for density-gradient centrifugation especially when rapid analysis of small samples is desired.     

Solubilization and assay of an hepatic receptor for the haptoglobin-hemoglobin complex

Hemoglobin released into the bloodstream is tightly bound by haptoglobin. The resulting complex (HpHb) is promptly cleared from the circulation and accumulates in the liver. A binding protein with a high affinity for HpHb has been solubilized from an acetone powder of rat liver and freed from an endogenous inhibitor by passage over a column of immobilized hemoglobin. An assay procedure has been developed whereby the bound HpHb is selectively precipitated by polyethylene glycol 6000. Employing this assay, the binding reaction was shown to be linear and saturable with respect to the ligand. In contrast to several previously described receptors for glycoproteins, the carbohydrate moiety of haptoglobin did not appear to participate in the binding of HpHb by the soluble receptor.     

Primary structure of the hip gene of Escherichia coli and of its product,the β subunit of integration host factor

We describe the isolation and sequencing of the hip gene of Escherichia coli and show that it encodes the β subunit of integration host factor (IHFβ). In order to locate the coding region, we constructed a set of deletion mutants by exonucleolytic digestion of a fragment containing hip, determined which mutants were hip⁺ and which hip^? by complementation, and then sequenced the ends of the critical deletions. The 5′ end of the coding region was located precisely by comparing the deduced amino acid sequence to the actual N-terminal amino acid sequence of IHF. Our assignment of the coding region was further substantiated by the nucleotide sequences of a hip point mutant and of internal replacement mutations. We found a probable promoter for hip located about 85 base-pairs upstream from the initial AUG codon and about 75 base-pairs downstream from the 3′ end of the neighboring gene. rpsA, and we constructed an IHFβ overproducer by fusing the coding sequences to the λp_L promoter. A survey of known protein sequences revealed a close relationship between IHFβ and the type II prokaryotic DNA binding proteins (the “histone-like” proteins). This relationship is shared to a considerable extent by the other subunit of IHF, IHFα. A hip missense mutation that replaces a completely conserved glycine with aspartate has a null phenotype, suggesting that the conserved regions are functionally important.     

Structural features of cytochrome P450 1A associated with the absence of EROD activity in liver of the loricariid catfish Pterygoplichthys sp

The Amazon catfish genus Pterygoplichthys (Loricariidae, Siluriformes) is closely related to the loricariid genus Hypostomus, in which at least two species lack detectable ethoxyresorufin-O-deethylase (EROD) activity, typically catalyzed by cytochrome P450 1 (CYP1) enzymes. Pterygoplichthys sp. liver microsomes also lacked EROD, as well as activity with other substituted resorufins, but aryl hydrocarbon receptor agonists induced hepatic CYP1A mRNA and protein suggesting structural/functional differences in Pterygoplichthys CYP1s from those in other vertebrates. Comparing the sequences of CYP1As of Pterygoplichthys sp. and of two phylogenetically related siluriform species that do catalyze EROD (Ancistrus sp., Loricariidae and Corydoras sp., Callichthyidae) showed that these three proteins share amino acids at 17 positions that are not shared by any fish in a set of 24 other species. Pterygoplichthys and Ancistrus (the loricariids) have an additional 22 amino acid substitutions in common that are not shared by Corydoras or by other fish species. Pterygoplichthys has six exclusive amino acid substitutions. Molecular docking and dynamics simulations indicate that Pterygoplichthys CYP1A has a weak affinity for ER, which binds infrequently in a productive orientation, and in a less stable conformation than in CYP1As of species that catalyze EROD. ER also binds with the carbonyl moiety proximal to the heme iron. Pterygoplichthys CYP1A has amino acid substitutions that reduce the frequency of correctly oriented ER in the AS preventing the detection of EROD activity. The results indicate that loricariid CYP1As may have a peculiar substrate selectivity that differs from CYP1As of most vertebrate.     

A sensitive and specific tritium release assay for dopamine-beta-hydroxylase (DbetaH) in serum.

The release of tritium from [7-³H₂]dopamine was investigated as a possible procedure for the assay for dopamine-β-hydroxylase (DβH) in rat and human serum. The release was found to have the same characteristics as those deseribed previously for DβH in serum; for example, an optimum rate of reaction at pH 5.0 or an enhancement of release with agents such as Cu²⁺ ions and N-ethylmaleimide which are known to inactivate endogenous inhibitors of DβH in serum. Tritium release was blocked by the DβH inhibitor fusaric acid but not by inhibitors of other dopamine-metabolizing enzymes in serum. Incubation of ¹⁴C-labeled dopamine along with [7-³H₂]dopamine revealed that, under the standard assay conditions, the formation of [¹⁴C]norepinephrine was accompanied by release of one of the two tritium atoms on the 7-carbon. It was concluded that the procedure provided a simple and sensitive assay of DβH activity in serum.