Resveratrol transcriptionally regulates miRNA-18a-5p expression ameliorating diabetic nephropathy via increasing autophagy.

OBJECTIVE: To investigate the effects of resveratrol on autophagy in the chronically diabetic nephropathy and to study the effects of the different expression of microRNAs after resveratrol (RSV) treated in db/db mice (diabetic mice). MATERIALS AND METHODS: Db/m (non- diabetic) and db/db mice were randomly divided into intra gastric RSV treatment group or control group. Renal tissues were prepared for HE/PAS staining. In vitro, mouse podocytes cell lines were grown in different mediums with different dose of resveratrol treatment. microRNA (miRNA) gene chips assay was performed for differentially expressed miRNAs screening. Western blot was used to detect protein levels. RESULTS: In vivo, RSV significantly decreased urinary albumin, serum creatinine, mesangial area and glomerular size in db/db mice. After RSV treatment, LC3-II/LC3-I and synaptopodin were increased while cleaved-caspase 3 was decreased in kidney tissues. In vitro, podocytes treated with RSV exhibited significantly increased LC3-II/LC3-I and decreased cleaved caspase 3. Moreover, this effect of RSV can be enhanced by rapamycin (RAPA, an activator of autophagy) but partially reversed by 3-MA (an autophagy inhibitor). Further, we found that miR-18a-5p was significantly upregulated after RSV treatment in db/db mice. Overexpression of miR-18a-5p in podocytes resulted in significant inhibition of cleaved-caspase 3 protein, and increased the ratio of LC3-II/LC3-I. Dual luciferase report assay validated that Atactic telangiectasis mutation (ATM) was a target of miR-18a-5p. In podocytes, downregulation of cleaved caspase 3 and the enhanced ratio of protein LC3-II/LC3-I were detected in cells transfected with ATM siRNA. CONCLUSIONS: Role of miRNA-18a-5p in the regulation of autophagy via targeting ATM may represent a promising therapeutic target for preventing and attenuating diabetic nephropathy.

Neutral Ceramidase Secreted Via Exosome Protects Against Palmitate-Induced Apoptosis in INS-1 Cells.

Aims: To investigate the effects of neutral ceramidase (NCDase) packaged in exosomes that are secreted from ß-cells on free fatty acid (FFA)-induced ß-cells apoptosis and its role in regulation of sphingolipid-mediated signaling pathway. Methods: HPLC and Western blotting were performed to determine NCDase activity and expression. Annexin V-fluorescein-isothiocyanate/propidium iodide flow cytometry was used to assess apoptosis. Electrospray ionization tandem mass spectrometry was used for ceramide (Cer), sphingosine-1-phosphate (S1P), and sphingosine (SPH) determination. Results: INS-1 cells over-expressed NCDase secreted active NCDase via exosomes. Exosomes isolated from the cultured medium of INS-1 cells that oxpressed NCDase could ameliorate palmitate-induced apoptosis. Furthermore, the results showed that exosome-derived NCDase treatment reduced intracellular Cer/S1P ratio. Conclusions: ß-cell secreted active NCDase via exosome, the exosome-packaged-NCDase protected ß-cells from FFA-induced apoptosis through regulating sphingolipid metabolites and it might be a potential treatment for ß-cell lipotoxicity and type 2 diabetes.

The Inhibitory Activities of Recombinant Eglin C Mutants on Kexin and Furin, Using Site-directed Mutagenesis and Molecular Modeling.

Mammalian furin and yeast kexin are members of the proprotein convertase family involved in the proteolytic processing of many important precursor proteins. Here the gene coding for the subtilisin inhibitor eglin C was totally synthesized and expressed in E.coli. Substitution of residues at each position P(1), P(2) and P(4) of eglin C with a basic residue using protein engineering could make eglin C a very strong inhibitor for furin (K(i) around 10(-9) mol/L),and even more strong for kexin (K( i ) around 10(-11) mol/ L). Results indicated that (1) A basic residue Lys or Arg at P(1) site is prerequisite for the inhibitor. (2) The second mutation with basic residue at P(4) site drastically increase the inhibitory activity by two orders of magnitude. (3) A basic residue at P(2) site is favorable for the binding to the enzyme, but unfavorable for the stability of the inhibitor, resulting in a temporary inhibition. (4) A hydrophobic residue is preferential at P(3) site. Based on the known crystal structures of subtilisin and eglin C, the interaction between the enzyme and inhibitor was modeled, and their involved residues were predicted which gave a good explanation to the experimental results.

A differential geometric treatment of protein structure comparison.

The technique of model-building a protein of known sequence but unknown tertiary structure from the structures of homologous proteins is probably so far the most reliable means of mapping from primary to tertiary structure. A key step towards the realization of the aim is to develop ways of aligning three-dimensional structures of homologous proteins, thereby deriving the rules useful for protein modelling. We have developed a generalized differential-geometric representation of protein local conformation for use in a protein comparison program which aligns protein sequences on the basis of their sequence and conformational knowledge. Because the differential-geometric distance measure between local conformations is independent of the coordinate frame and remains chirality information, the comparison program is easily implemented, relatively rational and reasonably fast. The utility of this program for aligning closely and distantly related homologous proteins is demonstrated by multiple alignment of globins, serine proteinases and aspartic proteinase domains. Particularly, the method has reached the rational alignment between the mammalian and microbial serine proteinases as compared with many published alignment programs.

Morphological and chiral symmetry breaking in reaction-diffusion systems.

Morphological and chiral symmetry breaking in reaction-diffusion systems is considered on the basis of the theory of imperfect codimension-two bifurcations. A new type of pattern selection with two triggers is elucidated: (1) morphologically asymmetric structures displaying optical activity can probably be originated from initially racemic and homogeneous conditions when chiral interaction, having the characteristic strength delta (such as electroweak interaction and circularly polarized light) as well as external field, having the characteristic strength eta (such as gravitational field and electrostatic field) are considered; (2) the selective sensitivity of molecular chirality and morphological asymmetry is omicron(delta 1/3) and omicron(eta 1/3), respectively; the sensitivity of mode-mode interaction between chiral polarization and concentration vector is omicron(delta 2/3) or omicron(eta 2/3), respectively. The relation of these conclusions to the life problem is discussed briefly.