Vimentin is important in the neural differentiation of PC12 cells promoted by sialylation

Sialic acid modification is a kind of post-translational modification. To investigate the regulation effect of sialic acid on neural differentiation, we used CycloManN propanyl perac (CycloManN pro), a metabolic precursor of sialic acid, to treat PC12 cells. We noted that CycloManN pro indeed robustly promoted global sialylation detected by MAL II lectin blot in PC12 cells. Simultaneously, we interestingly found that the neurite outgrowth of PC12 cells was significantly promoted by the CycloManN pro treatment. The profile analysis of sialylated proteins showed that a protein band at 55KD was greatly enhanced especially in PC12L cells after CycloManN pro treatment. After enrichment with lectin MAL II, the proteins in this band were analyzed by mass spectrometry. The results showed that 23 proteins were in the band, but the score of vimentin was the highest among them. To investigate further the role of vimentin in the process of neurite differentiation, vimentin construct was transfected into PC12 cells. We interestingly observed that ectopic expression of vimentin significantly enhanced the neurite outgrowth induced by CycloManN pro. However, after three potential glycosylation sites (Ser-7, Thr-33, Ser-34:) of vimentin were mutated to alanine, overexpression of the mutated vimentin completely lost the enhancement activity for the neural differentiation even in the presence of CycloManN pro. Taken together, our study demonstrated that vimentin was important in the induction of neural differentiation by CycloManN pro.     

Induction of hepatocyte‐like cells from human umbilical cord‐derived mesenchymal stem cells by defined microRNAs

Generating functional hepatocyte‐like cells (HLCs) from mesenchymal stem cells (MSCs) is of great urgency for bio‐artificial liver support system (BALSS). Previously, we obtained HLCs from human umbilical cord‐derived MSCs by overexpressing seven microRNAs (HLC‐7) and characterized their liver functions in vitro and in vivo. Here, we aimed to screen out the optimal miRNA candidates for hepatic differentiation. We sequentially removed individual miRNAs from the pool and examined the effect of transfection with remainder using RT‐PCR, periodic acid—Schiff (PAS) staining and low‐density lipoprotein (LDL) uptake assays and by assessing their function in liver injury models. Surprisingly, miR‐30a and miR‐1290 were dispensable for hepatic differentiation. The remaining five miRNAs (miR‐122, miR‐148a, miR‐424, miR‐542‐5p and miR‐1246) are essential for this process, because omitting any one from the five‐miRNA combination prevented hepatic trans‐differentiation. We found that HLCs trans‐differentiated from five microRNAs (HLC‐5) expressed high level of hepatic markers and functioned similar to hepatocytes. Intravenous transplantation of HLC‐5 into nude mice with CCl₄‐induced fulminant liver failure and acute liver injury not only improved serum parameters and their liver histology, but also improved survival rate of mice in severe hepatic failure. These data indicated that HLC‐5 functioned similar to HLC‐7 in vitro and in vivo, which have been shown to resemble hepatocytes. Instead of using seven‐miRNA combination, a simplified five‐miRNA combination can be used to obtain functional HLCs in only 7 days. Our study demonstrated an optimized and efficient method for generating functional MSC‐derived HLCs that may serve as an attractive cell alternative for BALSS.     

Saitohin Q7R polymorphism is associated with late‐onset Alzheimer's disease susceptibility among caucasian populations: a meta‐analysis

Saitohin (STH) Q7R polymorphism has been reported to influence the individual's susceptibility to Alzheimer's disease (AD); however, conclusions remain controversial. Therefore, we performed this meta‐analysis to explore the association between STH Q7R polymorphism and AD risk. Systematic literature searches were performed in the PubMed, Embase, Cochrane Library and Web of Science for studies published before 31 August 2016. Pooled odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to assess the strength of the association using a fixed‐ or random‐effects model. Subgroup analyses, Galbraith plot and sensitivity analyses were also performed. All statistical analyses were performed with STATA Version 12.0. A total of 19 case–control studies from 17 publications with 4387 cases and 3972 controls were included in our meta‐analysis. The results showed that the Q7R polymorphism was significantly associated with an increased risk of AD in a recessive model (RR versus QQ+QR, OR = 1.27, 95% CI = 1.01–1.60, P = 0.040). After excluding the four studies not carried out in caucasians, the overall association was unchanged in all comparison models. Further subgroup analyses stratified by the time of AD onset, and the quality of included studies provided statistical evidence of significant increased risk of AD in RR versus QQ+QR model only in late‐onset subjects (OR = 1.56, 95% CI = 1.07–2.26, P = 0.021) and in studies with high quality (OR = 1.37, 95% CI = 1.01–1.86, P = 0.043). This meta‐analysis suggests that the RR genotype in saitohin Q7R polymorphism may be a human‐specific risk factor for AD, especially among late‐onset AD subjects and caucasian populations.     

Selected contribution: effects of ischemia-reperfusion on vascular contractility and alpha(1)-adrenergic-receptor signaling in the rat tail artery.

To determine the effects of ischemia-reperfusion (I/R) on alpha(1)-adrenergic-receptor (alpha(1)-AR) functions, alpha(1)-AR-mediated contraction, inositol phosphate (IP) accumulation, and alpha(1)-AR-G protein coupling were examined in the tail arteries of anesthetized rats after 60 min of ischemia and 60 min of reperfusion. The contractile response to norepinephrine (NE) was significantly increased after I/R, whereas the contractile response to KCl remained unchanged. This was accompanied by a 69% increase in NE-stimulated IP accumulation. Furthermore, receptor-stimulated coupling of alpha(1a)-AR to G alpha(q/11) proteins was increased, whereas the coupling of alpha(1b)-AR or alpha(1d)-AR to their G proteins was not altered by I/R. These changes in vascular alpha(1)-AR function occurred without concurrent alteration in expression levels of membrane alpha(1)-AR subtypes or in the associated G proteins. These data demonstrate that I/R increases alpha(1a)-AR-G(q/11) protein coupling and alpha(1)-AR-stimulated IP accumulation in the tail artery. The alterations in alpha(1)-AR signaling are associated with and may underlie the enhanced contractile response of the tail artery to adrenergic stimulation after I/R.     

Comparison of population genetic structures of common wild rice (Oryza rufipogon Griff.), as revealed by analyses of quantitative traits, allozymes, and RFLPs

We investigated genetic diversity among and within natural populations of Asian common wild rice, Oryza rufipogon, from three different classes of data: quantitative traits, allozymes, and restriction fragment length polymorphisms (RFLPs). The seven populations examined showed polymorphism to varying degrees. The amount of intrapopulation variability appeared to be influenced not only by breeding system but also by the evolutionary history of each population. Interpopulation differentiation was clear, but different classes of data elucidated different patterns. Quantitative traits revealed ecotype differentiation into perennial and annual types of population, whereas allozyme and RFLP analyses revealed geographical differentiation among populations. These results suggest that the diversity patterns shown by quantitative trait analysis reflect mainly the occurrence of adaptive differentiation in response to habitat conditions and that those shown by allozyme and RFLP analyses reflect mainly the effect of isolation by distance. Population differentiation parameters (F(ST)) were highly variable among loci in allozymes as well as in RFLPs.     

Maohuoside A promotes osteogenesis of rat mesenchymal stem cells via BMP and MAPK signaling pathways

Osteoporosis is becoming a more prevalent health problem with the aging of the population around the world. Epimedium koreanum Nakai is one of the most used herbs in East Asia for curing osteoporosis, with its major ingredient, icariin, mostly explored by researchers. In this article, maohuoside A (MHA), a single isolated compound from the herb, was identified to be more potent than icariin in promoting osteogenesis of rat bone marrow-derived mesenchymal stem cells (rMSCs) (increasing by 16.6, 33.3, and 15.8% on D3, D7, and D11, respectively). Alkaline phosphatase (ALP) assay and calcium content measurement were assigned to quantify the promoted osteogenesis and alizarin red S (ARS) staining was conducted to visualize it. Quantitative real-time PCR (Q-PCR) was assayed to evaluate the mRNA expression of marker genes in osteogenesis and master regulators in BMP pathway. Moreover, PD98059 (PD) and SB203580 (SB), inhibitor of ERK1/2 and p38 MAPK pathway, were administered to assess the involvement of MAPK pathway in the promotion process. In conclusion, MHA pronouncedly enhanced the osteogenesis of rMSC, plausibly via the BMP and MAPK signaling pathways.     

Purification and characterization of a highly selective sucrose isomerase from Erwinia rhapontici NX-5

A highly selective sucrose isomerase (SIase) was purified to homogeneity from the cell-free extract of Erwinia rhapontici NX-5 with a recovery of 27.7% and a fold purification of 213.6. The purified SIase showed a high specific activity of 427.1 U mg⁻¹ with molecular weight of 65.6 kDa. The K _m for sucrose was 222 mM while V _max was 546 U mg⁻¹. The optimum pH and temperature for SIase activity were 6.0 and 30 °C, respectively. The purified SIase was stable in the temperature range of 10–40 °C and retained 65% of the enzyme activity after 2 weeks’ storage at 30 °C. The SIase activity was enhanced by Mg²⁺ and Mn²⁺, inhibited by Ca²⁺, Cu²⁺, Zn²⁺, and Co²⁺, completely inhibited by Hg²⁺ and Ag²⁺. The purified SIase was strongly inhibited by SDS, while partially inhibited by dimethylformamide, tetrahydrofuran, and PMSF. Additionally, glucose and fructose acted as competitive inhibitors for purified SIase.     

Production of bioactive peptides in an in vitro system

An in vitro system for the preparation of bioactive peptides is described. This system couples three different posttranslational modification enzymes, prohormone convertases (PCs), carboxypeptidase E, and peptidyl alpha-amidating enzyme, to transform recombinant precursors into bioactive peptides. Three different precursors, mouse proopiomelanocortin (mPOMC), rat proenkephalin (rPE), and human proghrelin, were used as model systems. The conversion of mPOMC and rPE to smaller peptide products was measured by radioimmunoassay. After optimization of the system, excellent efficiency was obtained: about 85% of starting mPOMC was converted to des-acetyl alpha-melanocyte-stimulating hormone (alpha-MSH). For proenkephalin, 75 and 96% yields were obtained for the opioid peptides Met-RGL and Met-enk, respectively. Cell-based assays demonstrated that in-vitro-generated des-acetyl alpha-MSH successfully activated the melanocortin 4 receptor. Proghrelin digestion was used to screen the specificity of PC cleavage and to confirm the cleavage site by mass spectroscopy. Mature ghrelin was produced by human furin, mouse prohormone convertase 1, and human prohormone convertase 7 but not by mouse prohormone convertase 2. These results demonstrate that our in vitro system (1) can produce peptides in quantities sufficient to carry out functional analyses, (2) can be used to determine the specificity of proprotein convertases on recombinant precursors, and (3) has the potential to identify novel peptide functions on both known and orphan G-protein-coupled receptors.     

Binding of the environmental pollutant naphthol to bovine serum albumin

The interactions between naphthol and bovine serum albumin (BSA) were investigated by spectroscopy. Our results prove the formation of complex between naphthol and BSA. Hydrophobic interaction dominates in the association reaction. The isomers stack with the aromatic residues in their binding sites with different geometries. Effects of BSA on the excited-state proton transfer and fluorescence spectra of the isomers indicate the different characters of their binding sites. 1-Naphthol inserts deeply into a hydrophobic cavity whereas 2-naphthol is in a basic environment on the surface of BSA. Naphthol statically quenches the fluorescence of BSA in a concentration-dependent manner positively deviating from the linear Stern-Volmer equation. Naphthol binds near Trp-134 in the subdomain IA of the native BSA and is accessible to Trp-212 when BSA is unfolded by naphthol. The folding pattern of the main chain is altered at high naphthol concentration as revealed by the change in the secondary structure. The binding of 1-naphthol is more cooperative than that of 2-naphthol. The extent of cooperativity was estimated by the Hill equation.