Artificial Extracellular Matrix Proteins Containing Phenylalanine Analogues Biosynthesized in Bacteria Using T7 Expression System and the PEGylation

In vivo incorporation of phenylalanine (Phe) analogues into an artificial extracellular matrix protein (aECM-CS5-ELF) was accomplished using a bacterial expression host that harbors the mutant phenylalanyl-tRNA synthetase (PheRS) with an enlarged binding pocket. Although the Ala294Gly/Thr251Gly mutant PheRS (PheRS**) under the control of T5 promoter allows incorporation of some Phe analogues into a protein, the T5 system is not suitable for material science studies because the amount of materials produced is not sufficient due to the moderate strength of the T5 promoter. This limitation can be overcome by using a pair of T7 promoter and T7 RNA polymerase instead. In the T7 expression system, it is difficult, however, to achieve a high incorporation level of Phe analogues, due to competition of Phe analogues for incorporation with the residual Phe that is required for synthesis of active T7 RNA polymerase. In this study, we prepared the PheRS** under T7 promoter and optimized culture condition to improve both the incorporation level of recombinant aECM protein and the incorporation level of Phe analogues. Incorporation and expression levels tend to increase in the case of p-azidophenylalanine, p-iodophenylalanine, and p-acetylphenylalanine. We evaluated the lower critical transition temperature, which is dependent on the incorporation ratio and the turbidity decreased when the incorporation level increased. Circular dichromism measurement indicated that this tendency is based on conformational change from random coil to β-turn structure. We demonstrated that polyethylene glycol (PEG) can be conjugated at reaction site of Phe analogues incorporated. We also demonstrated that the increased hydrophilicity of elastin-like sequences in the aECM-CS5-ELF made by PEG conjugation could suppress nonspecific adhesion of human umbilical vein endothelial cells (HUVEC)

Selenoprotein P Controls Oxidative Stress in Cornea

The ocular surface is always attacked by oxidative stress, and cornea epithelial cells are supposed to have their own recovery system against oxidative stress. Therefore we hypothesized that tears supply key molecules for preventing oxidative stress in cornea. The potential target key molecule we focused is selenoprotein P (SeP). SeP is a carrier of selenium, which is an essential trace element for many animals, for oxidative stress metabolism in the organism, and was extremely expressed in lacrimal gland. An experiment was performed with SeP eye drops in a rat dry eye model, prepared by removing the lacrimal glands. The anticipated improvement in corneal dry eye index and the suppression of oxidative stress markers were observed in SeP eye drop group. Furthermore, the concentration of SeP was significantly higher in dry eye patients compared with normal volunteers. Collectively, we concluded that tear SeP is a key molecule to protect the ocular surface cells against environmental oxidative stress

Kidney transplantation recovers the reduction level of serum sulfatide in ESRD patients via processes correlated to oxidative stress and platelet count

Sulfatide is a major component of glycosphingolipids in lipoproteins. Recently, we reported that a low serum level of sulfatide in hemodialysis patients might be related to the high incidence of cardiovascular diseases. However, the serum kinetics of sulfatide in kidney disease patients and the function of endogenous serum sulfatide are still unclear. To obtain novel knowledge concerning these issues, we investigated the serum kinetics of sulfatide in 5 adult kidney transplant recipients. We also analyzed the correlated factors influencing the serum sulfatide level, using multiple regression analysis. Kidney transplantation caused a dramatic increase of serum sulfatide without an alteration of its composition in all recipients in a time-dependent manner; however, the recovery speed was slower than that of the improvement of kidney function and the serum sulfatide reached a nearly normal level after 1 year. Multiple regression analysis showed that the significant correlated factor influencing the serum sulfatide level was log duration (time parameter) throughout the observation period, and the correlated factors detected in the stable phase were the decrease of serum concentration of malondialdehyde (an oxidative stress marker) as well as the elevation of platelet count. The current study results demonstrated the gradual but reliable recovery of the serum sulfatide level in kidney transplant recipients for the first time, suggesting a close correlation between serum sulfatide and kidney function. The recovery of serum sulfatide might derive from the attenuation of systemic oxidative stress. The normal level of serum sulfatide in kidney transplant recipients might affect platelet function, and contribute to the reduction of cardiovascular disease incidence.ArticleGLYCOCONJUGATE JOURNAL. 28(3-4):125-135 (2011)journal articl

Design of polymeric materials for culturing human pluripotent stem cells: progress toward feeder-free and xeno-free culturing

This review describes recent developments regarding the use of natural and synthetic polymers to support the propagation of human pluripotent stem cells (hPSCs), human embryonic stem cells (hESCs), and induced pluripotent stem cells (hiPSCs) while maintaining pluripotency in feeder-free and xeno-free cultures. The development of methods for culturing these cells without using mouse embryonic fibroblasts (MEFs) as a feeder layer will enable more reproducible culture conditions and reduce the risk of xenogenic contaminants, thus increasing the potential clinical applications of differentiated hPSCs. Human or recombinant fibronectin, laminin-511, and vitronectin, which are components of the extracellular matrix (ECM), have been used instead of Matrigel for the feeder-free growth of undifferentiated hPSCs. Successful hPSC cultures have been described for the following conditions: on oligopeptide-immobilized surfaces derived from vitronectin, on microcarriers prepared from synthetic polymers, and encapsulated within three-dimensional (3D) hydrogels composed of alginate and other hydrophilic natural polymers. Recently, synthetic biomaterials that allow hPSCs to maintain pluripotency by secreting endogenous ECM components have been designed. The combination of human ECM proteins or cell adhesion molecules (e.g., oligopeptides and poly-d-lysine) and synthetic biomaterials with well-designed surfaces and/or structures (e.g., scaffolds, hydrogels, microcarriers, microcapsules, or microfibers) in the presence of a chemically defined medium containing recombinant growth factors would offer a xeno-free alternative to feeder cells for culturing hPSCs and maintaining their pluripotency

Introduction pathways and evolutionary mechanisms of alien species of Lolium spreading across sandy coasts in Japan

1. Estimating the role of specific processes in the spread of alien species necessitates the determination of introduction pathways and source populations of invaded areas. Alien grasses in the genus Lolium that have extensively invaded Japan provide a unique opportunity to estimate the expansion process through direct comparison between source and naturalised populations because the introduction pathways, contaminants in grain commodities and commercial cultivars for fodder crops or revegetation materials are well-known. Therefore, by directly comparing source and naturalised populations, we estimated the introduction pathways and whether adaptative evolution occurred in Lolium species on sandy coasts in Japan. 2. Lolium individuals sampled from naturalised populations in croplands, seaports, and sandy coasts were compared with those from two introduction sources for morphological and genetic variations based on a genome-wide single nucleotide polymorphism analysis and a common garden experiment. Furthermore, we conducted a reciprocal transplant experiment between cropland and sandy coast. 3. Populations naturalised in croplands were closely related to the cultivars, whereas those naturalised in seaports and sandy coasts were associated with contaminants. These results indicate that the cropland and sandy coast populations are derived from cultivars and contaminants, respectively. In addition, asymmetric gene flow from cropland populations to sandy coast populations was observed. The reciprocal transplant experiment clearly demonstrated the home site advantage; populations derived from croplands yielded higher floret numbers than those derived from other habitats at the cropland site; sandy coast populations had higher survival rates than those from croplands at the coastal site. Port populations exhibited a similar tendency as sandy coast populations, indicating that contaminants may be originally adapted to salty and dry environments, such as that in sandy coasts. The flowering phenology in the sandy coast populations evolved in the late flowering; therefore, late flowering alleles may have been transferred from cropland populations to sandy coast populations. 4. Synthesis. We demonstrated that two congeneric species with different ecological characteristics were introduced through multiple introduction pathways and spread across different habitats. A direct comparison between source and naturalised populations can considerably elucidate the patterns and processes of biological invasions

Impeller Stall Induced By Reverse Propagation Of Non-Uniform Flow Generated At Return Channel

LectureIn the case of centrifugal compressors, minor non-uniform flow upstream of the impeller is induced by an asymmetrical configuration in the circumferential direction at the compressor suction casing. This non-uniform flow is transmitted to the impeller discharge, but this minor non-uniform flow does not usually cause an adverse effect on the impeller stage performance. However, we found this is amplified at the return channel due to flow separation at reduced flows (depending on return channel geometry), and the amplified non-uniform flow did induce impeller stall by reverse propagation from the return channel to the impeller. These non-uniform flows caused a significant operating range reduction for a large flow coefficient impeller. The aerodynamics issues were mitigated using CFD analysis techniques, and eventually confirmed by the compressor performance during shop performance testing. The OEM conducted the CFD analyses using two (2) return channel geometries with several CFD models to verify the effect of the return channel geometry on impeller stall and to confirm the most suitable CFD modeling method for stall evaluation. Shop performance tests utilizing both return channel geometries were conducted and compared to the CFD analyses. These studies were conducted while collaborating with the end-user. The steady CFD calculation was conducted with frozen rotor interface between full annulus impeller and stator parts. The modeling of diffuser and return channel was varied as follows: 1) 1-pitch model for the return channel with mixing plane at diffuser 2) Full-annulus model for the return channel with a mixing plane at the diffuser 3) Full-annulus model for the return channel without a mixing plane at the diffuser. From the above studies and the shop performance testing, it was confirmed that the proposed CFD modeling method could simulate the measurements taken during the shop performance tests and that the CFD modeling method utilized was key to properly evaluating stall phenomena

External stimulus-responsive biomaterials designed for the culture and differentiation of ES, iPS, and adult stem cells

The physical and chemical characteristics of biomaterial surface and hydrogels can be altered by external stimuli, such as light irradiation, temperature changes, pH shifts, shear stress forces, electrical forces, and the addition of small chemical molecules. Such external stimulus-responsive biomaterials represent promising candidates that have been developed for the culture and differentiation of embryonic stem (ES) cells, induced pluripotent stem (iPS) cells, and adult stem cells. Biomaterials that are designed to respond in a reversible manner to specific external signals can be formed on micropatterned or non-micropatterned surface, in hydrogels, or on microcarriers. Stem cells and the cells differentiated from them into specific tissue lineages can be cultured and/or differentiated on dishes with immobilized external stimulus-responsive polymers. Cells can be detached from these dishes without using an enzymatic digestion method or a mechanical method when the appropriate external stimulus is generated on the surface. This review discusses the polymers and polymeric designs employed to produce surface and hydrogels for stem cell culture, differentiation, and/or cell detachment using various external stimuli