Akiko Ogawa

Improvement of islet culture with sericin.

Islet transplantation is a promising treatment for diabetes. Serum is a necessary supplement in islet cultures, but it has various disadvantages including the risk of contamination by several pathogens. Results of this study suggest that sericin is a useful alternative supplement. Sericin accelerated the proliferation of the rat insulinoma cell line RIN-5F and improved the serum-free culture of rat islets.

Development of peptide arrays for detection of IgE-binding epitopes in cow's milk allergens

Peptide arrays have become versatile tools for high throughput screening assays in biomedical and pharmaceutical research. In this study, we constructed a peptide array that contained linear peptide fragments reported as IgE-binding epitopes for cows milk allergy (CMA). Various peptides with different solubility in aqueous solutions were dissolved in the buffer solutions containing sodium dodecyl sulfate, and we achieved a consistent spotting of peptide solutions using a piezoelectric ceramic micropump. The IgE-binding patterns were successfully detected by observing the binding of Alexa 647-labeled anti-human IgE using sera from CMA patients. Our technique in this study will provide a potent capability for the development of a peptide array for mapping IgE-epitopes in milk proteins, and it will help researchers better understand the IgE-epitopes associated with the clinical outcome of CMA.

Generation of a novel apoptosis-resistant hepatoma cell line

The expansionable human hepatoma cell lines have potential for use in a bio-artificial liver (BAL) system for liver disease due to the shortage of donation. However, at present, bioartificial livers are incomplete and the functions need to be improved or at least maintained for a longer period. In the present study, the authors aimed to establish a novel hepatoma cell line for a longer-term or permanent artificial liver. For this purpose, bcl-2, an anti-apoptosis gene, was introduced into hepatoma HepG2 cells. Over-expression of Bcl-2 significantly inhibited apoptosis. After 15 d of serum-deprived culture, the viability of HepG2-Bcl2 was 51% while that of mock transfectant (HepG2-mock) was decreased to 14%. In the presence of hygromycin B, HepG2-mock were dead by day 6, while the HepG2-Bcl2 viability at day 9 was 65%. Over-expression of Bcl-2 prolonged the period of the stationary phase in the growth curve and did not affect the growth rate during the exponential phase. To test the liver function, albumin production was measured. After 10 d of culture, the albumin concentration in the culture supernatant of HepG2-Bcl2 was 30 ng ml(-1), while that of HepG2-mock was 23 ng ml(-1). The cytochrome P-450 activity per culture of 3-methyl-cholanthrene-treated HepG2-Bcl2 was double that of treated HepG2-mock. Introduction of Bcl-2 was effective for the generation of a novel hepatoma cell line for artificial livers.

Progesterone, but not 17β-estradiol, up-regulates erythropoietin (EPO) production in human amniotic epithelial cells

Human amniotic epithelial (HAE) cells have great potential for successful use in cell therapy, since they do not cause acute rejection upon allotransplantation. However, to date, HAE cells have not well been studied. We previously reported that HAE cells produce erythropoietin (EPO), which is known to be a regulator of hematopoiesis, and that the induction mechanism of HAE cells is unknown, although EPO production from HAE cells is not increased by hypoxia which induces several cell types to produce EPO. In this study, we determined whether female sex hormones, including progesterone and 17beta-estradiol, affect the EPO production of HAE cells. Bioactive measurement of EPO activity in the culture supernatants of HAE-SV40 cells, which were immortalized by transfection with a simian virus 40 large T antigen, revealed that EPO bioactivity was significantly increased by treatment with progesterone, but not 17beta-estradiol. Treatment of HAE-SV40 cells with progesterone transiently increased the EPO mRNA level by fivefold, while there was no change in response to 17beta-estradiol. Furthermore, the progesterone receptor (PR)-B was detected in both HAE cells and HAE-SV40 cells by Western blotting. These results suggest that EPO synthesis in HAE-SV40 cells is stimulated by progesterone, but not by 17beta-estradiol, and thus it is highly likely that the EPO synthesis of HAE cells is also regulated by progesterone.

Epilithic and endolithic microorganisms and deterioration on stone church facades subject to urban pollution in a sub-tropical climate

Abstract Weathering of two church facades in Rio de Janeiro was caused substantially by salts, mainly halite and gypsum, detected by SEM and chemical analyses, which cause physical stresses by deposition within the rock. Biofilm populations, determined by SEM and as operational taxonomic units (OTUs), degraded stone by penetration, solubilization and redeposition of minerals on their surfaces. Endolithic cyanobacteria were associated with gypsum deposits. Microbiomes were typical for high-stress environments, high salt, intense insolation, low water and low nutrients (eg halophilic Rubrobacter, Salinicola, Sterigmatomyces). The main colonizers on the church most affected by traffic (Nossa Senhora da Candelária – CA) were Actinobacteria; Gammaproteobacteria (chiefly Pseudomonas) were predominant on the site situated in a leafy square (São Francisco de Paula – SF). Major Gammaproteobacteria on CA were halophilic Halomonas and Rhodobacteriaceae. Fungal OTUs on both churches were principally dimorphic, yeast-like basidiomycetes. Many OTUs of thermophilic microorganisms (eg the Thermomicrobia class, Chloroflexi) were present. This is the first use of next generation sequencing (NGS) to study microbial biofilm interactions with metamorphic and granite buildings in an intensely urban, sub-tropical climate.

Effects of Sericin on Promoting Proliferation and Inhibiting Apoptosis of Mammalian Cells

We have previously reported that sericin improves proliferation of all cells tested, as well suppressing cell death induced by heat stress, implying that sericin inhibits apoptosis. In sericin treatment, caspase7/3 activity, executioners for inducing apoptosis, was decreased, while caspase9 activity was not. In order to analyze the mode of action of sericin in detail, the gene expression pattern was studied by transcriptome analysis using cDNA microarrays. The expression of several genes was significantly upregulated, and others were downregulated by sericin treatment. Among the detected genes, some growth- and apoptosis-related genes may be involved in the proliferative and survival effects of sericin.

Novel Serum-Free Cryopreservation of Mammalian Cells Using Seric

Cryopreservation is a pivotal process for obtaining a continuous source of functional cell lines. Currently, fetal bovine serum (FBS) supplemented with 10% dimethyl sulfoxide (DMSO) is extensively used as a cryopreservative solution. However, FBS carries the risk of infection by abnormal prion proteins and viruses. Therefore, development of serum-free cryopreservative solutions is desirable. We have previously developed a novel serum-free cryopreservative solution using sericin. Sericin cryopreservative solution consists of phosphate-buffered saline, 1% sericin, 0.5% maltose, 0.3% proline, 0.3% glutamine and 10% DMSO. In this study, we evaluated sericin cryopreservative solution using the rat insulinoma cell line RIN5F and the mouse hybridoma cell line 2E3-O. After thawing, proliferation and production of biological substances by cells cryopreserved in sericin solution were similar to those of cells cryopreserved in FBS solution. These results indicate that sericin solution is suitable as a serum-free cryopreservative.

Microbiomes of Biofilms on Decorative Siliceous Stone: Drawbacks and Advantages of Next Generation Sequencing

Next Generation Sequencing (NGS), using the Illumina® metabarcoding system, showed differences between biofilm communities on three degraded siliceous stone church façades in central Rio de Janeiro. Two church biofilms (on granite and augen gneiss) were dominated by Actinobacteria; the third (granite), surrounded by trees and further from intense vehicular traffic, by Gammaproteobacteria. Yeast-like forms of Basidiomycetes and Ascomycetes were major fungi on all facades, but 22.8% of Operational Taxonomic Units could not be assigned to any fungal taxon after DNA amplification with ITS primers and analysis with the UNITE database, indicating the need for more fungal NGS studies. The pipeline used in analysis of the V4 region of rRNA bacterial gene sequences influenced the taxa detected, with two major classes and many genera identified only by the pipeline using the Greengenes, and not the Silva, database. Principal Components Analysis separated façade biofilms into the appropriate three groups and indicated greater dissimilarity of the tree-surrounded church biofilm from the others, confirmed by Jaccard Similarity coefficients, suggesting that local environment influences community composition more than stone type. NGS allows rapid and detailed analysis of microbiomes, but results must be carefully assessed and must not be used as the sole indication of community composition.

Effective Antibody Production by Reusing Culture Medium Previously Used in Antibody Purification

Culture medium used in antibody purification contains growth factors, but is usually discarded after antibody purification. We examined to determine whether the growth factors can be (i) recovered and (ii) used in subsequent cell culture. Using this medium enhanced the survival of hybridoma cells and improved antibody production. Culture medium previously used in antibody purification offers an attractive resource for mammalian cell culture.

Effect of Silver or Copper Nanoparticles-Dispersed Silane Coatings on Biofilm Formation in Cooling Water Systems

Biofouling often occurs in cooling water systems, resulting in the reduction of heat exchange efficiency and corrosion of the cooling pipes, which raises the running costs. Therefore, controlling biofouling is very important. To regulate biofouling, we focus on the formation of biofilm, which is the early step of biofouling. In this study, we investigated whether silver or copper nanoparticles-dispersed silane coatings inhibited biofilm formation in cooling systems. We developed a closed laboratory biofilm reactor as a model of a cooling pipe and used seawater as a model for cooling water. Silver or copper nanoparticles-dispersed silane coating (Ag coating and Cu coating) coupons were soaked in seawater, and the seawater was circulated in the laboratory biofilm reactor for several days to create biofilms. Three-dimensional images of the surface showed that sea-island-like structures were formed on silane coatings and low concentration Cu coating, whereas nothing was formed on high concentration Cu coatings and low concentration Ag coating. The sea-island-like structures were analyzed by Raman spectroscopy to estimate the components of the biofilm. We found that both the Cu coating and Ag coating were effective methods to inhibit biofilm formation in cooling pipes.