Epoxyeicosatrienoic acids protect rat hearts against tumor necrosis factor-α-induced injury

Epoxyeicosatrienoic acids (EET), the primary arachidonic acid metabolites of cytochrome P450 2J (CYP2J) epoxygenases, possess potent vasodilatory, anti-inflammatory, antiapoptotic, and mitogenic effects. To date, little is known about the role of CYP2J2 and EETs in tumor necrosis factor (TNF)-α-induced cardiac injury. We utilized cell culture and in vivo models to examine the effects of exogenously applied EETs or CYP2J2 overexpression on TNF-α-induced cardiac apoptosis and cardiac dysfunction. In neonatal rat cardiomyocytes, TNF-α-induced apoptosis was markedly attenuated by EETs or CYP2J2 overexpression, leading to significantly improved cell survival. Further studies showed that TNF-α decreased expression of the antiapoptotic proteins Bcl-2 and Bcl-xL, decreased IκBα and PPARγ, and also inhibited PI3K-dependent Akt and EGFR signaling. Both EETs and CYP2J2 overexpression reversed the effects of TNF-α on these pathways. Furthermore, overexpression of CYP2J2 in rats prevented the decline in cardiac function that is normally observed in TNF-α-challenged animals. These results demonstrate that EETs or CYP2J2 overexpression can prevent TNF-α-induced cardiac cell injury and cardiac dysfunction by inhibiting apoptosis, reducing inflammation, and enhancing PPARγ expression. Targeting the CYP2J2 epoxygenase pathway may represent a novel approach to mitigate cardiac injury in diseases such as heart failure, where increased TNF-α levels are known to occur.     

A novel biomimetic olfactory-based biosensor for single olfactory sensory neuron monitoring

This paper presents a novel biomimetic olfactory biosensor for the study of olfactory transduction mechanisms on the basis of light addressable potentiometric sensor (LAPS), in which rat olfactory sensory neurons (OSNs) are used as sensing elements. Rat OSNs are cultured on the surface of LAPS chip. To validate the origin of the electrical signals recorded by LAPS, the inhibitory effect of MDL12330A to the olfactory signals of OSNs is tested, which is the specific inhibitor of adenylyl cyclase. The enhancive effect of LY294002 to the responses of OSNs is also investigated, which is the specific inhibitor of phosphatidylinositol 3-kinase (PI3K). The results show that this hybrid biosensor can record the responses of OSNs to odours efficiently in a non-invasive way for a long term, and the responses can be inhibited by MDL12330A and enhanced by LY294002. All these results demonstrate that this hybrid biosensor can be used to monitor electrophysiology of OSNs in a non-invasive way and suggest it could be a promising tool for the study of olfactory transduction mechanisms.     

The cysteine-rich region of T1R3 determines responses to intensely sweet proteins

A wide variety of chemically diverse compounds taste sweet, including natural sugars such as glucose, fructose, sucrose, and sugar alcohols, small molecule artificial sweeteners such as saccharin and acesulfame K, and proteins such as monellin and thaumatin. Brazzein, like monellin and thaumatin, is a naturally occurring plant protein that humans, apes, and Old World monkeys perceive as tasting sweet but that is not perceived as sweet by other species including New World monkeys, mouse, and rat. It has been shown that heterologous expression of T1R2 plus T1R3 together yields a receptor responsive to many of the above-mentioned sweet tasting ligands. We have determined that the molecular basis for species-specific sensitivity to brazzein sweetness depends on a site within the cysteine-rich region of human T1R3. Other mutations in this region of T1R3 affected receptor activity toward monellin, and in some cases, overall efficacy to multiple sweet compounds, implicating this region as a previously unrecognized important determinant of sweet receptor function.     

Low-temperature effect on the sterol-dependent processing of SREBPs and transcription of related genes in HepG2 cells

Lowering the growth temperature of HepG2 cells from 37 degrees C to 20 degrees C results in a 73% reduction in human squalene synthase (HSS) protein, a 76% reduction in HSS mRNA, and a 96% reduction in promoter activity of a secreted alkaline phosphatase-HSS reporter gene. A similar decrease in either mRNA or protein levels is observed for 3-hydroxy-3-methylglutaryl CoA reductase, farnesyl diphosphate synthase, the LDL receptor, and fatty acid synthase. All these proteins and mRNAs show either a decrease or a complete loss of sterol-dependent regulation in cells grown at 20 degrees C. In contrast, sterol regulatory element binding proteins (SREBPs)-1 and -2 exhibit a 2- to 3-fold increase in mRNA levels at 20 degrees C. The membrane-bound form of the SREBPs is dramatically increased, but the proteolytic processing to the nuclear (N-SREBP) form is inhibited under these conditions. Overexpression of the N-SREBP or SREBP cleavage-activating protein (SCAP), but not site-1 or site-2 proteases, restores the activation of the HSS promoter at 20 degrees C, most likely by liberating the SCAP-SREBP complex so that it can move to the Golgi for processing. These results indicate that the cholesterol synthesizing machinery is down-regulated at low temperatures, and points to the transport of the SCAP-SREBP complex to the Golgi as the specific down-regulated step.     

A novel,simplified, and reproducible porcine model of acute ischemic liver failure with portal vein preservation

The current ischemic models of liver failure are difficult and usually time-consuming to produce. The aim of this study was to develop a simplified and reproducible porcine model of acute liver failure for use in preclinical research. Eighteen Bama miniature pigs were randomly divided into Groups A, B, and C. The hepatic artery and common bile duct were ligated in all groups. While the portal vein was completely preserved in Group A, it was narrowed by 1/3 and 1/2 in Groups B and C, respectively. Results of biochemical analyses, encephalopathy scores, and survival times were compared among the groups. Results of hematoxylin-eosin staining, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling, Masson staining, and Ki-67 analyses were recorded. Survival times in Groups B and C were 11.67 ± 1.86 and 2.16 ± 0.75 days, respectively, shorter than that in Group A (>15 days). Following surgery, alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, alkaline phosphatase, total bilirubin, and direct bilirubin levels significantly increased relative to baseline values in all groups (P<0.05). Groups B and C exhibited a significant decrease in encephalopathy scores and a significant increase in ammonia levels, which were negatively correlated with one another. Pathological analysis revealed obvious necrosis of liver cells, which correlated closely with the degree of portal vein constriction. Our simple, highly reproducible model effectively mimics the clinical characteristics of acute liver failure in humans and provides a foundation for further research on artificial liver support system development.     

A rapid and efficient method for primary culture of human adipose-derived stem cells

Adipose tissue contains some populations, adipose-derived stem cells (ADSCs) which can differentiate into adipogenic, chondrogenic, osteogenic, myogenic, and endothelial cells. Furthermore, adipose tissue can be easily obtained in large quantities through a simple liposuction. ADSCs are thought to be an alternate source of autologous adult stem cells for cell-based therapy. However, it is time-consuming and inefficient to harvest ADSCs by using a traditional collagenase-digestion method. To meet the demand of large quantities of ADSCs in the basic and applied research of regenerative medicine, we developed a rapid and efficient method for isolation and culture of primary ADSCs. The results indicated that the ADSCs obtained with our method possessed strong abilities of proliferation and colony formation in vitro, and could keep low level of cell senescence with stable population doubling during long-term culture in vitro. Furthermore, these harvested ADSCs were capable to differentiate into osteogenic and adipogenic lineages in the specific induction medium. In addition, the results of flow cytometry analysis indicated that these ADSCs could positively express multiple CD markers, such as CD44, CD105, CD29, CD90, and CD13, and hardly expressed CD31, CD34, CD45, and CD106, which was homologous to the mesenchymal stem cells. Therefore, the ADSCs isolated with our method are consistent with previously reported characteristics of the ADSCs. This new method that we established in this study is an efficient tool to isolate and culture the stem cells from adipose tissue.     

Resurrection of a functional phosphatidylinositol transfer protein from a pseudo-Sec14 scaffold by directed evolution

Sec14-superfamily proteins integrate the lipid metabolome with phosphoinositide synthesis and signaling via primed presentation of phosphatidylinositol (PtdIns) to PtdIns kinases. Sec14 action as a PtdIns-presentation scaffold requires heterotypic exchange of phosphatidylcholine (PtdCho) for PtdIns, or vice versa, in a poorly understood progression of regulated conformational transitions. We identify mutations that confer Sec14-like activities to a functionally inert pseudo-Sec14 (Sfh1), which seemingly conserves all of the structural requirements for Sec14 function. Unexpectedly, the "activation" phenotype results from alteration of residues conserved between Sfh1 and Sec14. Using biochemical and biophysical, structural, and computational approaches, we find the activation mechanism reconfigures atomic interactions between amino acid side chains and internal water in an unusual hydrophilic microenvironment within the hydrophobic Sfh1 ligand-binding cavity. These altered dynamics reconstitute a functional "gating module" that propagates conformational energy from within the hydrophobic pocket to the helical unit that gates pocket access. The net effect is enhanced rates of phospholipid-cycling into and out of the Sfh1* hydrophobic pocket. Taken together, the directed evolution approach reveals an unexpectedly flexible functional engineering of a Sec14-like PtdIns transfer protein-an engineering invisible to standard bioinformatic, crystallographic, and rational mutagenesis approaches.