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Purpose: The purpose of this study was to examine the efficacy of an ovarian tissue transportation network for fertility preservation (FP) for cancer patients in Japan. Methods: PubMed was searched for papers on transportation of human ovarian tissue for FP. We analyzed population, area, number of cancer patients for ovarian tissue cryopreservation (OTC), quality control/assessment and safety, cost of a cryopreservation center for the building for 30 years, and medical fees of cancer patients (operation, cryopreservation, and storage of ovarian tissue). Results: More than twenty babies have been born in Denmark and Germany through a transportation system. Up to 400 new patients a year need OTC. The fees for removal, cryopreservation, and storage for 5 years, and transplantation of ovarian tissue are around €5,000, €4,000, and €5,000, respectively. It costs more than €5 million to establish and maintain one cryopreservation center for 30 years. If we have a few cryopreservation centers in Japan, we can cryopreserve 400 patients' ovarian tissue per year by safer slow freezing and maintain quality control/assessment. We need to lighten the patients' burden for easy to use FP by a government subsidy and medical insurance coverage. Conclusions: This model has been termed the Danish model ('the woman stays - the tissue moves'). This is truly patient-centered medicine. We can have maximum effects with the minimum burden. A transportation network like those of Denmark and Germany is the best strategy for FP in Japan. It may be the best system for cancer patients, medical staff, and the Ministry of Health, Labor, and Welfare. [ABSTRACT FROM AUTHOR]

We have prepared Ni- and Mg-substituted LaRhO3 and have measured the thermoelectric properties. Ni substitution decreases resistivity at room temperature, while Mg substitution decreases resistivity up to 5%, where the Mg substitution increases the carrier concentration, as confirmed by Hall coefficient measurements. [ABSTRACT FROM AUTHOR]

BACKGROUND: The relationship between patient prognosis and various tumor biological factors has been reported previously, and prognostic factors of tumor biology may improve predictions of prognosis after hepatectomy for hepatocellular carcinoma (HCC) and may contribute to a new staging classification. This study was designed to provide an immunohistochemical analysis of tumor biological factors in patients who underwent hepatectomy for HCC. METHODS: Factors analyzed included p53 overexpression, microvessel counts, proliferating cell nuclear antigen, and expression of nm23. We examined 81 HCCs from patients with chronic liver diseases. RESULTS: In patients who underwent chemoembolization before surgery, or those a who had confluent multinodular tumor, p53 expression tended to be higher than in patients without chemoembolization (33% vs 11%) or those with a simple nodular tumor (28% vs 10%), but the difference was not statistically significant ( P = 0.051 and P = 0.092, respectively). A lower tumor microvessel count and negative nm23 expression were significantly associated with poor disease-free survival by univariate analysis ( P < 0.01 and P < 0.05, respectively). A lower tumor microvessel count was found to be a significant prognostic factor for disease-free and overall survivals (risk ratios, 2.44 and 3.13, respectively; P << 0.05), in addition to tumor size, vascular invasion, and longterm ascites, by Cox's multivariate analysis. CONCLUSIONS: Tumor microvessel count appears to be a useful prognostic marker for predicting HCC recurrence and patient survival. [ABSTRACT FROM AUTHOR]

In order to construct a cell surface-engineered yeast Saccharomyces cerevisiae that facilitates adsorption and recovery of heavy metal ions, we endowed it with the ability to self-aggregate in response to binding and accumulation of copper ion. A fusion gene for the expression of GTS1, which encodes a putative zinc-finger transcription factor related to occurrence of cell-aggregation, was constructed under the control of the copper ion-inducible CUP1 promoter from the yeast metallothionein gene. The multicopy plasmid carrying the fusion gene was introduced into a cell surface-engineered yeast displaying histidine hexa-peptide, which can chelate copper ion. This transformant strain aggregated in medium only in the presence of copper ion, with aggregation induced by as little as 1 mM copper ion. The copper ion-induced aggregation did not interfere with the copper ion-adsorbing function of the cell surface-engineered yeast, indicating that this transformant strain has the twin features of enhanced cell surface adsorption of copper ion and self-aggregation in response to environmental copper ion. [ABSTRACT FROM AUTHOR]

A histidine oligopeptide (hexa-His) with the ability to chelate divalent heavy metal ions was displayed on the yeast cell surface for the purpose of enhanced adsorption of heavy metal ions. We genetically fused a hexa-His-encoding gene with the gene encoding the C-terminal half of α-agglutinin that includes a glycosylphosphatidylinositol anchor attachment signal sequence and attached the hexa-His peptide on the cell wall of Saccharomyces cerevisiae. This surface-engineered yeast adsorbed three to eight times more copper ions than the parent strain and was more resistant to copper (4 mM) than the parent (below 1 mM at pH 7.8). It was possible to recover about a half of the copper ions adsorbed by whole cells with EDTA treatment without disintegrating the cells. Thus, we succeeded in constructing a novel yeast cell with both tolerance to toxic contaminants and enhanced adsorption of metal ions onto the cell surface. [ABSTRACT FROM AUTHOR]

Saccharomyces cerevisiae strains that respond to environmental changes and transmit the information by emission of fluorescence from the cell surface were constructed. The technique of cell surface engineering enabled the yeast cells to display enhanced cyan blue fluorescent protein (ECFP) or enhanced yellow fluorescent protein (EYFP) on the surface under the control of promoters that sense environmental changes. Two model promoters were examined in this study. For monitoring the intra- and extracellular concentrations of phosphate ion, the PHO5 promoter was chosen to display ECFP. The MEP2 promoter was used to display EYFP to sense the concentrations of ammonium ion. Fluorescence was observed by fluorescence microscopy and immunofluorescence microscopy, and the intensity was measured by a flow cytometer. The relationship between ion concentration inside and outside the cells was evaluated by the change in the rate of fluorescence. This S. cerevisiae system enables environmental changes to be transmitted as intra- and extracellular information using a suitable promoter functioning at real time and in a non-invasive manner. [ABSTRACT FROM AUTHOR]

We have successfully created a novel yeast strain able to monitor changes in environmental conditions by displaying either green fluorescent protein (GFP) from Aequorea victoria or blue fluorescent protein (BFP), a variant of GFP, on its cell surface as a visible reporter. For the display of these fluorescent proteins on the cell surface of Saccharomyces cerevisiase, our cell-surface-engineering system was utilized. The GAPDH promoter, which is active in the presence of glucose, and the UPR-ICL promoter from Candida tropicalis, which starts to function in the presence of a reduced level of glucose, were employed simultaneously to express the GFP-encoding gene and the BFP-encoding gene, respectively. This cell-surface-engineered yeast strain emitted green fluorescence from the cell surface when sufficient glucose was present in the medium, and blue fluorescence from the same cell surface when the glucose in the medium was consumed. The fluorescent proteins displayed on the cell surface using the different promoters enabled us to monitor the concentrations of intra- and/or extracellular glucose that regulated activation or inactivation of the promoters. This novel yeast strain could facilitate the computerized control of various bioprocesses measuring emitted fluorescence. [ABSTRACT FROM AUTHOR]

The ZZ domain derived from Staphylococcus aureus, which binds to the Fc part of immunoglobulin G (IgG), was displayed on the cell surfaces of yeast Saccharomyces cerevisiae by cell-surface engineering using the C-terminal half of α-agglutinin under control of the 5′-upstream region of the isocitrate lyase gene from Candida tropicalis (UPR-ICL). Display of ZZ on the cell surface was confirmed by immunofluorescence microscopy. Enzyme-linked immunosorbent assay (ELISA) and sandwich ELISA using the S. cerevisiae cells displaying ZZ detected IgG and antigen (human serum albumin) down to a concentration of 1–10 ng/ml in both cases. The detection range covered by these assay systems was wide and could be varied by adjusting the amount of cells and reaction times with horseradish peroxidase (HRP) substrate. Moreover, yeast cells displaying ZZ were successfully used for repeated affinity purification of IgG from serum. These results indicate that S. cerevisiae displaying ZZ may constitute novel and genetically renewable whole-cell immunoadsorbents widely applicable to immunoassays and affinity purification. [ABSTRACT FROM AUTHOR]

The number of foreign protein molecules expressed on the cell surface of the budding yeast Saccharomyces cerevisiae by cell surface engineering was quantitatively evaluated using enhanced green fluorescent protein (EGFP). The emission from EGFP on the cell surface was affected by changes in pH. The amount of EGFP on the cell surface, displayed as α-agglutinin-fusion protein under control of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter, was determined at the optimum pH of 7.0. The fluorometric analysis and the image analysis by confocal laser scanning microscopy (CLSM) showed a similar number of molecules displayed on the cell surface, demonstrating that 104–105 molecules of α-agglutinin-fused molecules per cell were expressed. Furthermore, the amount of fluorescent protein expressed on cells harboring a multicopy plasmid was three to four times higher than that on cells harboring the gene integrated into the genome. [ABSTRACT FROM AUTHOR]

An engineered yeast with emission of fluorescence from the cell surface was constructed. Cell surface engineering was applied to display a visible reporter molecule, green fluorescent protein (GFP). A glucose-inducible promoter GAPDH as a model promoter was selected to control the expression of the reporter gene in response to environmental changes. The GFP gene was fused with the gene encoding the C-terminal half of α-agglutinin of Saccharomyces cerevisiae having a glycosylphosphatidylinositol anchor attachment signal sequence. A secretion signal sequence of the fungal glucoamylase precursor protein was connected to the N-terminal of GFP. This designed gene was integrated into the TRP1 locus of the chromosome of S. cerevisiae with homologous recombination. Fluorescence microscopy demonstrated that the transformant cells emitted green fluorescence derived from functionally expressed GFP involved in the fusion molecule. The surface display of GFP was further verified by immunofluorescence labeling with a polyclonal antibody (raised in rabbits) against GFP as the first antibody and Rhodamine Red-X-conjugated goat anti-rabbit IgG as the second antibody which cannot penetrate into the cell membrane. The display of GFP on the cell surface was confirmed using a confocal laser scanning microscope and by measuring fluorescence in each cell fraction obtained after the subcellular fractionation. As GFP was proved to be displayed as an active form on the cell surface, selection of promoters will endow yeast cells with abilities to respond to changes in environmental conditions, including nutrient concentrations in the media, through the emission of fluorescence. [ABSTRACT FROM AUTHOR]

We constructed a novel surface-engineered yeast displaying the ligand-binding domain of the rat estrogen receptor (ERLBD). ERLBD, display of which on the yeast cell surface was confirmed by immunofluorescence, possessed strong binding activity to fluorescent 17β-estradiol – an analogue of the natural ligand of the estrogen receptor – that was comparable to the activity of the native receptor. Environmental homeostasis has recently been disturbed by endocrine disruptors, which cause confusion in the hormone secretion system. It is therefore very important to identify chemical compounds with hormone-like activity and remove them from the environment. The present results demonstrate that the new arming yeast displaying ERLBD on its cell surface will be capable of screening, entrapping, and removing estradiol-like compounds from the environment. [ABSTRACT FROM AUTHOR]