Phenotypic Diagnosis of Lineage and Differentiation During Sake Yeast Breeding.

Sake yeast was developed exclusively in Japan. Its diversification during breeding remains largely uncharacterized. To evaluate the breeding processes of the sake lineage, we thoroughly investigated the phenotypes and differentiation of 27 sake yeast strains using high-dimensional, single-cell, morphological phenotyping. Although the genetic diversity of the sake yeast lineage is relatively low, its morphological diversity has expanded substantially compared to that of the Saccharomycescerevisiae species as a whole. Evaluation of the different types of breeding processes showed that the generation of hybrids (crossbreeding) has more profound effects on cell morphology than the isolation of mutants (mutation breeding). Analysis of phenotypic robustness revealed that some sake yeast strains are more morphologically heterogeneous, possibly due to impairment of cellular network hubs. This study provides a new perspective for studying yeast breeding genetics and micro-organism breeding strategies.

Identification of a mutation causing a defective spindle assembly checkpoint in high ethyl caproate-producing sake yeast strain K1801.

In high-quality sake brewing, the cerulenin-resistant sake yeast K1801 with high ethyl caproate-producing ability has been used widely; however, K1801 has a defective spindle assembly checkpoint (SAC). To identify the mutation causing this defect, we first searched for sake yeasts with a SAC-defect like K1801 and found that K13 had such a defect. Then, we searched for a common SNP in only K1801 and K13 by examining 15 checkpoint-related genes in 23 sake yeasts, and found 1 mutation, R48P of Cdc55, the PP2A regulatory B subunit that is important for the SAC. Furthermore, we confirmed that the Cdc55-R48P mutation was responsible for the SAC-defect in K1801 by molecular genetic analyses. Morphological analysis indicated that this mutation caused a high cell morphological variation. But this mutation did not affect the excellent brewing properties of K1801. Thus, this mutation is a target for breeding of a new risk-free K1801 with normal checkpoint integrity.

A chimeric epidermal growth factor with fibrin affinity promotes repair of injured keratinocyte sheets.

The aim of the present study is to create a novel chimeric protein of epidermal growth factor (EGF) with fibrin affinity and demonstrate its potential for repairing injured tissues by immobilization to fibrin. The chimeric protein (FBD-EGF) was produced by the fusion of the fibronectin fibrin-binding domain (FBD) to EGF. It showed dose-dependent binding to fibrin and its binding was stable for at least 7days, while native EGF showed little affinity. FBD-EGF promoted the growth of fibroblasts and keratinocytes in the fibrin-bound state as well as in the soluble state. Its activity was further studied in a keratinocyte culture system in which fibrin was exposed upon injury of cell sheets. Fibrin-bound FBD-EGF promoted growth of the sheets over the injured area at a significantly faster rate (approximately eightfold) than native EGF (p<0.01). Wounds 2mm wide were closed in 7-9days. This repair process was inhibited by anti-EGF. Keratinocytes proliferated more extensively in the leading edges of sheets contacting fibrin with FBD-EGF, approximately 1.7-fold more than in the adjacent regions. These results imply that the stable binding of chimeric EGF to fibrin is effective for the repair of injured keratinocyte sheets, suggesting a potential use in tissue engineering.

Resorbable scaffolds from three different techniques: electrospun fabrics, salt-leaching porous films, and smooth flat surfaces.

Nanofibrous scaffolds of poly[(L-lactide)-co-(1,5-dioxepan-2-one)] generated by electrospinning have been compared with porous films obtained by solvent cast/salt leaching and homogeneous films. A comparison between the fibrous materials and the homogeneous solvent-cast films revealed that the surface of the nanofibers was more hydrophobic and that the nanofibers were degraded more rapidly in the presence of proteinase. It was obvious that the strain-to-break was reduced by the nanofiber formation, it decreased from 370% to 130% independent of fiber diameter. These values were however considerably higher than the strain-to-break of the solvent-cast/salt leaching scaffold. In addition, the nanofibrous material accelerated the adhesion and growth of the mesenchymal stem cell compared to the smooth material.

Surface modification of plastic, glass and titanium by photoimmobilization of polyethylene glycol for antibiofouling.

Photoreactive poly(ethylene glycol) (PEG) was prepared and the polymer was photoimmobilized on organic, inorganic and metal surfaces to reduce their interaction with proteins and cells. The photoreactive PEG was synthesized by co-polymerization of methacrylate-PEG and acryloyl 4-azidobenzene. Surface modification was carried in the presence and the absence of a micropatterned photomask. It was then straightforward to confirm the immobilization using the micropatterning. Using the micropatterning method, immobilization of the photoreactive PEG on plastic (Thermanox), glass and titanium was confirmed by time-of-flight secondary ion mass spectroscopy and atomic force microscopy observations. The contact angle on an unpatterned surface was measured. Although the original surfaces have different contact angles, the contact angle on PEG-immobilized surfaces was the same on all surfaces. This result demonstrated that the surface was completely covered with PEG by the photoimmobilization. To assess non-specific protein adsorption on the micropatterned surface, horseradish peroxidase (HRP)-conjugated proteins were adsorbed. Reduced protein adsorption was confirmed by vanishingly small staining of HRP substrates on the immobilized regions. COS-7 cells were cultured on the micropatterned surface. The cells did not adhere to the PEG-coated regions. In conclusion, photoreactive PEG was immobilized on various surfaces and tended to reduce interactions with proteins and cells.

Modification of the titan surface with photoreactive gelatin to regulate cell attachment.

Titan (TiO2) was modified with photoreactive gelatin in order to regulate the attachment of cells. Photoreactive gelatin, which was synthesized by the coupling reaction of gelatin with N-(4-azidobenzoyloxy) succinimide, was immobilized onto the n-octadecyltrimethoxysilane (ODS)-TiO2 or TiO2 surface by ultraviolet irradiation both in the absence and presence of a photo mask. In the absence of a photo mask, the modified titan surface was analyzed by measuring water contact angles and X-ray photoelectron spectroscopy (XPS). The result showed that ODS hydrophobilized the titan surface, and that the immobilization of gelatin affected the surface's hydrophilicity. XPS shows that titan was covered with organic material, including ODS and gelatin. With the photo mask in place, micropatterning of the gelatin was performed. This pattern was confirmed by optical microscopy and time-of-flight secondary ion-mass spectroscopy (TOF-SIMS). Monkey COS-7 epithelial cells were cultured on the unpattern- and pattern-immobilized plate. A significantly higher degree of cell attachment was found on the photoreactive gelatin-immobilized regions than on those that were not immobilized. It was concluded that the cellular pattern on titan was regulated by immobilized photoreactive gelatin.

AFM observation of immobilized self-oscillating polymer.

Various types of nanocomponents have been developed to construct a nanodevice or nanomachine. Here, we add a new nanocomponent that has the function of self-oscillation. A thermoresponsive polymer carrying a Ru complex, a catalyst of the Belousov-Zhabotinsky reaction, was synthesized and immobilized on a glass plate. Periodic turbidity changes in the aqueous solution of the polymer were observed, and nanoscale self-oscillation of the immobilized polymer was observed by a scanning probe microscope.

A composite of hydroxyapatite with electrospun biodegradable nanofibers as a tissue engineering material.

Biodegradable and biocompatible poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), a copolymer of microbial polyester, was fabricated as a nanofibrous film by electrospinning and composited with hydroxyapatite (HAp) by soaking in simulated body fluid. Compared with a PHBV cast (flat) film, the electrospun PHBV nanofibrous film was hydrophobic. However, after HAp deposition, both of the surfaces were extremely hydrophilic. The degradation rate of HAp/PHBV nanofibrous films in the presence of polyhydroxybutyrate depolymerase was very fast. Nanofiber formation increased the specific surface area and HAp enhanced the invasion of enzyme into the film by increasing surface hydrophilicity. The surface of the nanofibrous film showed enhanced cell adhesion over that of the flat film, although cell adhesion was not significantly affected by the combination with HAp.

A microfabrication method of a biodegradable polymer chip for a controlled release system.

A simple microfabrication method for a controlled-release drug-delivery system has been designed using biodegradable polymeric microchips. Microholes were made in a poly(L-lactic acid) plate and dyes were cast in each well. After drying, the wells were sealed with polymers having different biodegradation rates using a mold that had hollows corresponding to the wells. The polymers were prepared by mixing polylactides with the co-polymers. The sealing was confirmed by ultrasonication. The plate was incubated in phosphate-buffered saline and the dye released from the plate as the degradation proceeded was detected spectrophotometrically. The higher the degradation rate of the polymer sealing, the faster the sealed dye was released. This biodegradable biochip is useful for the design of controlled-release drug-delivery systems.

Culture of human umbilical vein endothelial cells on immobilized vascular endothelial growth factor.

Vascular endothelial growth factor (VEGF) was immobilized on substrata in photoreactive gelatin to control the adhesion and growth of vascular endothelial cells. The gelatin and VEGF were mixed in water and cast on a polystyrene dish or a silane-coated glass plate. The surface was then photoirradiated in the presence or absence of a photomask and washed. Toughness of the immobilized material was confirmed by ethanol treatment. Human umbilical vein endothelial cells (HUVECs) grew on the immobilized VEGF but not on a nontreated surface. Growth of HUVEC increased significantly with an increase in the amount of immobilized VEGF, and the effects were inhibited by treatment with anti-VEGF antibody. Thus, immobilized VEGF specifically interacted with HUVECs to permit growth in culture. Micropatterning of HUVEC cultures was also achieved using micropattern-immobilized VEGF. This patterning technique may be useful for the formation of blood vessel networks in vitro.

Photo-immobilization of a phospholipid polymer for surface modification.

A photo-reactive polymer having a phospholipid polar group was prepared, and the polymer was photo-immobilized on polymeric surfaces, where its interactions with biocomponents were investigated. By using a photo-immobilization method, the polymer was used for surface modification of polyethylene and polypropylene, polymers whose surfaces were not treated in our previous development of the phosphorylcholine-derived polymer. The photo-reactive polymer was synthesized by a coupling reaction involving copolymer consisting of 2-methacryloyloxyethyl phosphorylcholine and methacrylic acid with 4-azidoaniline. When the polymer was unpattern immobilized on the surface, X-ray photo-electron spectroscopic analysis and static contact angle measurements were performed. It was shown that the surface was covered with phospholipid polar groups. Micropattern immobilization was carried out using a micropatterned photo-mask. Measurements using atomic force microscopy showed that the swelled micropatterned polymer was five times as thick as the dried one. Protein adsorption and platelet adhesion were reduced on the polymer-immobilized regions. Mammalian cells did not adhere, and formed aggregates on the immobilized regions. In conclusion, the photo-reactive phospholipid polymer was covalently immobilized on the conventional polymer surfaces and it tended to reduce interactions with proteins and cells.

Synthesis of photoreactive pullulan for surface modification.

Photoreactive pullulan was prepared, the polymer was photoimmobilized on polymeric or organic surfaces, and its interactions with a protein and a cell type were investigated. The photoreactive pullulan was synthesized by a coupling reaction with 4-azidobenzonic acid. Surface modification was carried out in the presence or absence of a micropatterned photomask containing 100 microm transparent stripes with 150 microm gaps, making it easy to confirm the immobilization. By the micropatterning method, immobilization of the photoreactive pullulan on polystyrene, polyethylene, and silane-coupled glass was confirmed. Contact angles were measured on the unpatterned surfaces. Although the original surfaces have different contact angles, the contact angle on Az-pullulan-immobilized surface was the same on all surfaces. This result demonstrated that photoimmobilization completely covered the surface with Az-pullulan. Protein adsorption was investigated using fluorescently labeled albumin applied to the micropatterned surface: fluorescence microscopy demonstrated that adsorption was reduced on the pullulan-immobilized regions. Culture of RAW264 cells, derived from mouse leukemic monocytes, on the micropatterned surface for 22 h showed that cells did not adhere to the immobilized pullulan regions. In conclusion, photoreactive pullulan was covalently immobilized on various surfaces and tended to reduce interactions with proteins and cells.

Immobilization of DNAzyme as a thermostable biocatalyst.

Deoxyribozyme (DNAzyme) carrying peroxidase activity was immobilized on two types of particles and the enzymatic activity was measured. The DNA recognizing porphyrin were prepared according to Travascio et al. ([1998] Chem Biol 5:505-517) and the interactions with hemin were investigated by ultraviolet absorbance and circular dichroism spectroscopies. The DNA interacted with hemin and significant conformational change was induced by the interaction. Therefore, the end of this DNA was modified with a thiol group and it was immobilized on thiol-containing polysaccharide beads or on gold particles. The DNA immobilized on the gold particle showed activity catalyzing the peroxidation reaction. No significant reduction of activity was observed even after immobilization. The immobilized DNAzyme could be repeatedly utilized without significant loss of activity. In addition, heat treatment did not reduce the activity, although a protein enzyme, horseradish peroxidase, lost its activity after the heat treatment. The repertoire of DNAzyme is still currently limited. However, in the future the utilization of DNAzyme in the field of biotechnology will be important with the increase of discoveries of new functional DNAzymes.

Immobilization of erythropoietin to culture erythropoietin-dependent human leukemia cell line.

To investigate the effect of immobilized cytokine, erythropoietin (Epo) was immobilized on a culture plate and the Epo-dependent human leukemia cell line UT-7/Epo then was cultured upon the plate. A photo-reactive gelatin was mixed with Epo and the mixture was cast on a plate. The plate was then irradiated with ultraviolet light in the presence or absence of a photo-mask. After washing with water, a micropatterned or unpatterned surface was formed. A leukemia cell line dependent on Epo, UT-7/Epo, was cultured on the sample plate. On the micropatterned surface, apoptosis of cells was induced on the surface without Epo, but was not observed on the Epo-immobilized surface. This result demonstrated that Epo stimulated the cells even after immobilization. Although the activity of immobilized Epo was low, the activity was slightly higher than that achieved by soluble Epo at higher concentration. In addition, the immobilized Epo could be repeatedly used for culture of UT-7/Epo cell. The present study provided a convenient immobilization method and indicated that immobilization of cytokines will be useful for creating an artificial cell culture device.

Immobilization of leukemia inhibitory factor (LIF) to culture murine embryonic stem cells.

Murine embryonic stem (ES) cells were cultured on a material containing immobilized leukemia inhibitory factor (LIF). To immobilize LIF, we synthesized photoreactive gelatin mixed with LIF and cast the mixture on a polystyrene plate, which was then dried. LIF was immobilized by photoirradiation in the presence or absence of a photo mask. The plate was washed until LIF was no longer released. Murine ES cells were cultured on the immobilized LIF. Activation of STAT3 was maintained on the immobilized LIF for 6 d even after removing soluble LIF. Oct-3/4 was also expressed in the cells cultured on the immobilized LIF. As a result, the mouse ES cells were cultured without differentiating on the immobilized LIF for 6 d. It was possible to culture murine ES cells without adding soluble LIF at each medium change. We conclude that our material containing immobilized LIF might be useful in the culture of murine ES cells.