Acceleration of matrix metalloproteinase-1 production and activation of platelet-derived growth factor receptor beta in human coronary smooth muscle cells by oxidized LDL and 4-hydroxynonenal

Increases in matrix metalloproteinases (MMPs) at atherosclerotic lesions are involved in the migration of smooth muscle cells (SMCs) into the intima and to the rupture of plaques, being implicated in the progression of atherosclerosis. The present study examined the mechanisms underlying the production of MMP-1, interstitial collagenase-1, induced by oxidized low-density lipoprotein (oxLDL) and 4-hydroxynonenal (4-HNE), factors proposed to play a pivotal role in atherogenesis, in human coronary SMCs. oxLDL promoted the production of MMP-1 with the preceding phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. Immunoprecipitation of platelet-derived growth factor receptor beta (PDGFR-beta) revealed that oxLDL induced tyrosine phosphorylation of the receptor. Inhibition of the activation of PDGFR-beta and ERK1/2 resulted in a suppression of the production of MMP-1. Consistently, 4-HNE also elicited the production of MMP-1 with the preceding phosphorylation of PDGFR-beta and ERK1/2. The 4-HNE-induced production of MMP-1 was prevented when the activation of PDGFR-beta and ERK1/2 was inhibited. The present results suggest that the activation of PDGFR-beta and ERK1/2 is involved in the production of MMP-1 in oxLDL- and 4-HNE-stimulated human coronary SMCs.     

Presentation of Various Tactile Sensations Using Micro-Needle Electrotactile Display

Tactile displays provoke tactile sensations by artificially stimulating tactile receptors. While many types of tactile displays have been developed, electrotactile displays that exploit electric stimulation can be designed to be thin, light, flexible and thus, wearable. However, the high voltages required to stimulate tactile receptors and limited varieties of possible sensations pose problems. In our previous work, we developed an electrotactile display using a micro-needle electrode array that can drastically reduce the required voltage by penetrating through the high-impedance stratum corneum painlessly, but displaying various tactile sensations was still a challenge. In this work, we demonstrate presentation of tactile sensation of different roughness to the subjects, which is enabled by the arrangement of the electrodes; the needle electrodes are on the fingertip and the ground electrode is on the fingernail. With this arrangement, the display can stimulate the tactile receptors that are located not only in the shallow regions of the finger but also those in the deep regions. It was experimentally revealed that the required voltage was further reduced compared to previous devices and that the roughness presented by the display was controlled by the pulse frequency and the switching time, or the stimulation flow rate. The proposed electrotactile display is readily applicable as a new wearable haptic device for advanced information communication technology.     

Fatty liver induced by free radicals and lipid peroxidation

An excessive accumulation of fat in the liver leads to chronic liver injury such as non-alcoholic fatty liver disease (NAFLD), which is an important medical problem affecting many populations worldwide. Oxidative stress has been implicated in the pathogenesis of NAFLD, but the exact nature of active species and the underlying mechanisms have not been elucidated. It was previously found that the administration of free radical-generating azo compound to mice induced accumulation of fat droplet in the liver. The present study was performed aiming at elucidating the changes of lipid classes and fatty acid composition and also measuring the levels of lipid peroxidation products in the liver induced by azo compound administration to mouse. The effects of azo compound on the liver were compared with those induced by high fat diet, a well-established cause of NAFLD. Azo compounds given to mice either by intraperitoneal administration or by dissolving to drinking water induced triacylglycerol (TG) increase and concomitant phospholipid decrease in the liver, whose pattern was quite similar to that induced by high fat diet. Lipid peroxidation products such as hydroxyoctadecadienoic acid and hydroxyeicosatetraenoic acid were increased in the liver in association with the increase in TG. These results show that free radicals as well as high fat diet induce fatty liver by similar mechanisms, in which lipid peroxidation may be involved.     

Cryopreservation of porcine embryos derived from in vitro-matured oocytes

This study describes a cryopreservation method for porcine in vitro-produced (IVP) embryos using as a model parthenogenetic embryos derived from in vitro-matured (IVM) oocytes. IVP embryos at the expanded blastocyst stage were cryopreserved by vitrification using the minimum volume cooling (MVC) method and exhibited an embryo survival rate of 41.2%. Survival was then significantly improved (83.3%, P < 0.05) by decreasing the amount of cytoplasmic lipid droplets (delipation) prior to vitrification. IVP embryos at the 4-cell stage also survived cryopreservation when vitrified after delipation (survival rate, 36.0%), whereas post-thaw survival of nondelipated embryos was quite low (9.7%). Furthermore, it was demonstrated that porcine IVP morulae can be cryopreserved by vitrification following delipation by a noninvasive method (survival rate, 82.5%). These results clearly confirm that porcine embryos derived from IVM oocytes can be effectively cryopreserved with high embryo survival using the MVC method in conjunction with delipation.     

A protocol for the construction of microsatellite enriched genomic library

An improved protocol for constructing microsatellite-enriched libraries was developed. The procedure depends on digesting genomic DNA with a restriction enzyme that generates blunt-ends, and on ligating linkers that, when dimerized, create a restriction site for a different blunt-end producing restriction enzyme. Efficient ligation of linkers to the genomic DNA fragments is achieved by including restriction enzymes in the ligation reaction that eliminate unwanted ligation products. After ligation, the reaction mixture is subjected to subtractive hybridization without purification. DNA fragments containing microsatellites are captured by biotin-labeled oligonucleotide repeats and recovered using streptavidin-coated beads. The recovered fragments are amplified by PCR using the linker sequence as primer, and cloned directly into a plasmid vector. The linker has the sequence GTTT on the 5′ end, which promotes efficient adenylation of the 3′ ends of the PCR products. Consequently, the amplified fragments could be cloned into vectors without purification. This procedure enables efficient enrichment and cloning of microsatellite sequences, resulting in a library with a low level of redundancy.