Upregulation of bone-like extracellular matrix expression in human dental pulp stem cells by mechanical strain

There are many different types of periodontal diseases. One such disease causes a defect of alveolar bone that is considered serious. Hence, researchers have examined potential treatments for this type of disease using tissue engineering techniques. Periodontal tissues are exposed to mechanical stress caused by occlusion and mastication, and both the cells and extracellular matrix in these tissues undergo architectural modifications to compensate for the applied stress. Therefore, in this study we analyzed the effect of mechanical tension on the osteogenesis of human dental pulp stem cells (DPSCs). To identify osteogenesis induced by mechanical stress in dental pulp, we examined the effects of tension on DPSCs. We evaluated the effects of mechanical stimuli on the osteogenesis of human dental pulp cells grown on silk scaffolds subjected to 10% strain using a bioreactor. The tension was applied with 0.2 Hz over the course of 5 days and was then continuously applied for 10 more days. We evaluated cell differentiation by RT-PCR, Western blotting and immunohistochemistry. Applying 10% tension to the culture resulted in increases in collagen type I, fibronectin, osteoprotegerin, and bone sialoprotein expression and decreases in a-smooth muscle actin expression. These data suggest that mechanical stimulation promotes osteogenesis in DPSCs.     

Construction of a 'turn-on' fluorescent probe system for His-tagged proteins

Hexahistidine ((His)(6)) tags are used to purify genetically engineered proteins. Herein, we describe the construction of a 'turn-on' fluorescent probe system that consists of the fluorescence quencher, Dabcyl, conjugated to (His)(6), and fluorescent tetramethylrhodamine conjugated to nitrilotriacetic acid, which, in the presence of Ni(2+), can bind (His)(6). The system is turned off when Dabcyl-(His)(6) is bound to the fluorescent nitrilotriacetic acid derivative. The binding strength of this system was assessed using electrospray ionization mass spectrometry, fluorescence correlation spectroscopy, and fluorescence intensity distribution analysis-polarization. Although there was no significant enhancement in fluorescence after addition of an equimolar amount of ubiquitin, the fluorescence increased from 14% to 40% of its initial intensity when an equimolar amount of (His)(6)-ubiquitin was added. Therefore, this system should be able to specifically recognize (His)(6)-proteins with good resolution and has the additional advantage that a washing step is not required to remove fluorescent probe, that is, not bound to the (His)(6)-protein.     

Novel pyrimidines as acid pump antagonists (APAs)

A series of pyrimidine derivatives as acid pump antagonists (APAs) was synthesized and the inhibitory activities against H⁺/K⁺ ATPase isolated from hog gastric mucosa were determined. After elaborating on substituents at C2 and C4 position of the pyrimidine scaffold, we have observed that the compound 7h is a potent APA with H⁺/K⁺ ATPase, IC₅₀ = 52 nM.     

Modeling and Design of H-Infinity Controller for Piezoelectric Actuator LIPCA

<正> We proposed a dynamic model identification and design of an H-Infinity (i.e.H_∞) controller using a LightweightPiezo-Composite Actuator (LIPCA).A second-order dynamic model was obtained by using input and output data, and applyingan identification algorithm.The identified model coincides well with the real LIPCA.To reduce the resonating mode that istypical of piezoelectric actuators, a notch filter was used.A feedback controller using the H_∞ control scheme was designed basedon the identified dynamic model; thus, the LIPCA can be easily used as an actuator for biomemetic applications such as artificialmuscles or macro/micro positioning in bioengineering.The control algorithm was implemented using a microprocessor, analogfilters, and power amplifying drivers.Our simulation and experimental results demonstrate that the proposed control algorithmworks well in real environment, providing robust performance and stability with uncertain disturbances.     

Role of lymphocyte-specific protein tyrosine kinase (LCK) in the expansion of glioma-initiating cells by fractionated radiation

Lipid peroxidation products have a high reactivity against the primary amino groups of biomolecules such as aminophospholipids, proteins, and DNA. Until now, many papers have reported about the modification of biomolecules derived from lipid peroxides. Our group has also reported that aminophospholipids, such as phosphatidylethanolamine (PE), can be modified by lipid peroxidation including 13-hydroperoxyoctadecadienoic acid (13-HPODE). The aim of this study was to examine the oxidative stress in vivo by detecting the formation of N-(hexanoyl)phosphatidylethanolamine (HEPE) and N-(hexanoyl)phosphatidylserine (HEPS), a novel hexanoyl adduct, using a liquid chromatography/tandem mass spectrometry (LC/MS/MS) and a monoclonal antibody.Consequently, we observed that the formation of HEPE and HEPS occurred in the red blood cell (RBC) ghosts modified by 13-HPODE and the oxidative stress model induced by carbon tetrachloride (CCl₄) using LC/MS/MS monitoring hexanoyl ethanolamine (HEEA), a head group of HEPE, and hexanoyl serine (HESE) as a part of HEPS. Furthermore, we obtained a novel type of monoclonal antibody against HEPE. This antibody could recognize HEPE in the liver of rats with oxidative stress in vivo.     

Keratinocytes in culture accumulate phagocytosed melanosomes in the perinuclear area

There are many techniques for evaluating melanosome transfer to keratinocytes but the spectrophotometric quantification of melanosomes incorporated by keratinocyte phagocytosis has not been previously reported. Here we describe a new method that allows the spectrophotometric visualization of melanosome uptake by normal human keratinocytes in culture. Fontana-Masson staining of keratinocytes incubated with isolated melanosomes showed the accumulation of incorporated melanosomes in the perinuclear areas of keratinocytes within 48 h. Electron microscopic observations of melanosomes ingested by keratinocytes revealed that many phagosomes containing clusters of melanosomes or their fragments were localized in the perinuclear area. A known inhibitor of keratinocyte phagocytosis which inhibits protease-activated receptor-2, i.e., soybean trypsin inhibitor, decreased melanosome uptake by keratinocytes in a dose-dependent manner. These data suggest that our method is a useful model to quantitate keratinocyte phagocytosis of melanosomes visually in vitro.     

Regulation of mRNA export by nutritional status in fission yeast.

We have isolated a mutation in nup184(nup184-1) that is synthetically lethal with the mRNA export defective rae1-167 mutation in Schizosaccharomyces pombe. The consequence of the synthetic lethality is a defect in mRNA export. The predicted Nup184p is similar to Nup188p of Saccharomyces cerevisiae, and a Nup184p-GFP fusion localizes to the nuclear periphery in a punctate pattern. The Deltanup184 null mutant is viable and also is synthetically lethal with rae1-167. In a rae1(+) background, both the nup184-1 and Deltanup184 mutations confer sensitivity to growth in nutrient-rich medium (YES) that is accompanied by nuclear poly(A)+ RNA accumulation. Removal of the cAMP-dependent protein kinase, Pka1p, relieved the growth and mRNA export defects of nup184 mutants when grown in nutrient-rich medium. The activation of Pka1p is necessary, but not sufficient, to cause the severe poly(A)+ RNA export defects when nup184 mutant cells are incubated in YES, suggesting nutritional status can also regulate poly(A)+ RNA export. Our results suggest that the regulation of poly(A)+ RNA export by Pka1p kinase appears to be indirect, via a translation-dependent step, but post-translationally in response to YES.