DOI： 10.1016/j.jbiosc.2023.05.005

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Publishing type：Research paper (scientific journal)   Publisher：Oxford University Press (OUP)  

Abstract

Immunoglobulin A (IgA) is involved in the maintenance of gut homeostasis. Although the oral administration of bifidobacteria increases the amount of fecal IgA, the effects of bifidobacteria on intestinal immunity remain unclear. We found and characterized membrane vesicles (MVs) derived from Bifidobacterium longum subsp. infantis towards host immune cells. B. infantis MVs consisted of a cytoplasmic membrane and extracellular solute-binding protein (ESBP) was specifically detected. In the presence of B. infantis MVs or recombinant ESBP, RAW264 cells produced the pro-inflammatory cytokine IL-6. IgA was produced by Peyer's patches cells following the addition of B. infantis MVs. Therefore, ESBP of B. infantis MVs is involved in the production of IgA by acquired immune cells via the production of IL-6 by innate immune cells.

DOI： 10.1093/bbb/zbac172

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

Abstract

We investigated the characteristics and functionalities of extracellular vesicles (EVs) from Lactiplantibacillus plantarum (previously Lactobacillus plantarum) towards host immune cells. L. plantarum produces EVs that have a cytoplasmic membrane and contain cytoplasmic metabolites, membrane and cytoplasmic proteins, and small RNAs, but not bacterial cell wall components, namely, lipoteichoic acid and peptidoglycan. In the presence of L. plantarum EVs, Raw264 cells inducibly produced the pro-inflammatory cytokines IL-1β and IL-6, the anti-inflammatory cytokine IL-10, and IF-γ and IL-12, which are involved in the differentiation of naive T-helper cells into T-helper type 1 cells. IgA was produced by PP cells following the addition of EVs. Therefore, L. plantarum EVs activated innate and acquired immune responses. L. plantarum EVs are recognized by Toll-like receptor 2 (TLR2), which activates NF-κB, but not by other TLRs or NOD-like receptors. N-acylated peptides from lipoprotein19180 (Lp19180) in L. plantarum EVs were identified as novel TLR2 ligands. Therefore, L. plantarum induces an immunostimulation though the TLR2 recognition of the N-acylated amino acid moiety of Lp19180 in EVs. Additionally, we detected a large amount of EVs in the rat gastrointestinal tract for the first time, suggesting that EVs released by probiotics function as a modulator of intestinal immunity.

DOI： 10.1038/s41598-022-17629-7

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Other Link： https://www.nature.com/articles/s41598-022-17629-7

Language：English   Publishing type：Research paper (scientific journal)  

Transforming growth factor (TGF)-β1 and prostaglandin E2 (PGE2) are humoral factors critically involved in the induction of immunosuppression in the microenvironment of various types of tumors, including melanoma. In this study, we identified a natural compound that attenuated TGF-β1- and PGE2-induced immunosuppression and examined its effect on B16 melanoma growth in mice. By screening 502 natural compounds for attenuating activity against TGF-β1- or PGE2-induced suppression of cytolysis in poly(I:C)-stimulated murine splenocytes, we found that betulin was the most potent compound. Betulin also reduced TGF-β1- and PGE2-induced downregulation of perforin and granzyme B mRNA expression and cell surface expression of NKG2D and CD69 in natural killer (NK) cells. Cell depletion and coculture experiments showed that NK cells, dendritic cells, B cells, and T cells were necessary for the attenuating effects of betulin. Structure-activity relationship analysis revealed that two hydroxyl groups at positions C3 and C28 of betulin, their cis-configuration, and methyl group at C30 played crucial roles in its attenuating activity. In a subcutaneous implantation model of B16 melanoma in mice, intratumor administration of betulin and LY2157299, a TGF-β1 type I receptor kinase inhibitor, significantly retarded the growth of B16 melanoma. Notably, betulin increased significantly the number of CD69 positive NK cells in tumor sites at early stages of post-tumor cell injection. Our data suggest that betulin inhibits the growth of B16 melanoma by enhancing NK cell activity through attenuating the immunosuppressive tumor microenvironment.

DOI： 10.1248/bpb.b21-00921

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

We analyzed the mechanisms underlying enhanced IgA production in the cells of Peyer's patch cells via membrane vesicles derived from Lactobacillus sakei subsp. sakei NBRC 15893. Depletion of CD11c+ cells from Peyer's patch cells suppressed the enhanced IgA production mediated by membrane vesicles. Meanwhile, the stimulation of bone-marrow-derived dendritic cells with membrane vesicles increased gene expression of inducible nitric oxide synthase, retinaldehyde dehydrogenase 2, and several inflammatory cytokines. The production of nitric oxide and interleukin (IL)-6 by membrane vesicle stimulation was induced via Toll-like receptor 2 on bone marrow-derived dendritic cells. Inhibition of inducible nitric oxide synthase and retinaldehyde dehydrogenase 2, as well as the neutralization of IL-6 in Peyer's patch cells, suppressed the enhanced IgA production by membrane vesicle stimulation. Hence, nitric oxide, retinoic acid, and IL-6 induced by membrane vesicles play crucial roles in the enhanced IgA production elicited by membrane vesicles in Peyer's patch cells.

DOI： 10.1093/bbb/zbab065

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Authorship：Last author,　Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：BMFH Press  

DOI： 10.12938/bmfh.2020-003

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Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.jbiosc.2020.06.004

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Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.jbiosc.2019.11.009

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Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.jbiosc.2019.07.004

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Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.jbiosc.2019.06.017

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Language：Japanese   Publisher：(公社)日本生物工学会  

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Language：Japanese   Publisher：(公社)日本生物工学会  

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：BMFH Press  

DOI： 10.12938/bmfh.18-015

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Authorship：Lead author   Publishing type：Research paper (scientific journal)   Publisher：Informa UK Limited  

DOI： 10.1080/09168451.2017.1320518

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Language：English   Publisher：BMFH Press  

<p>Co-culture of lactic acid bacteria (LAB) and yeast induces specific responses that are not observed in pure culture. Gene expression profiles of <i>Lactobacillus paracasei</i> ATCC 334 co-cultured with <i>Saccharomyces cerevisiae</i> IFO 0216 were analyzed by DNA microarray, and the responses induced by direct contact with the yeast cells were investigated. Coating the LAB cells with recombinant DnaK, which acts as an adhesive protein between LAB and yeast cells, enhanced the ratio of adhesion of the LAB cells to the yeast cells. The signals induced by direct contact were clarified by removal of the LAB cells unbound to the yeast cells. The genes induced by direct contact with heat-inactivated yeast cells were very similar to both those induced by the intact yeast cells and those induced by a soluble mannan. The top 20 genes upregulated by direct contact with the heat-inactivated yeast cells mainly encoded proteins related to exopolysaccharide synthesis, modification of surface proteins, and transport systems. In the case of the most upregulated gene, LSEI_0669, encoding a protein that has a region homologous to polyprenyl glycosylphosphotransferase, the expression level was upregulated 7.6-, 11.0-, and 8.8-fold by the heat-inactivated yeast cells, the intact yeast cells, and the soluble mannan, respectively, whereas it was only upregulated 1.8-fold when the non-adherent LAB cells were not removed before RNA extraction. Our results indicated that the LAB responded to direct contact with the yeast cells through recognition of mannan on the surface of the yeast.</p>

DOI： 10.12938/bmfh.BMFH-2016-015

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

Rhizomucor pusillus NBRC 4578 efficiently produces ethanol from lignocellulosic biomass because of its ability to ferment not only D-glucose, but also D-xylose. When the strain was cultivated on D-xylose, ethanol was gradually formed in the culture medium with a decrease in D-xylose and the simultaneous accumulation of xylitol, which suggested that the strain catabolized D-xylose with D-xylose reductase (XR) and xylitol dehydrogenase (XDH). XR (RpXR) was purified to homogeneity from the crude extract prepared from the mycelia of the strain grown on D-xylose. The purified enzyme was found to be NADPH-dependent and prefer pentoses such as D-xylose, D-ribose, and L-arabinose as substrates. Isolation of the genomic DNA and cDNA of the xyl1 gene encoding RpXR revealed that the gene was interrupted by two introns and the exon of the gene encoded a protein composed of 322 amino acids with a M_r of 36,724. Phylogenetic analysis showed that RpXR is more related to 4-dihydromethyltrisporate dehydrogenases from Mucoraseae fungi rather than the previously reported fungal XRs. Quantitative real-time PCR indicated that transcription of the xyl1 gene was marked in the presence of D-xylose and L-arabinose, but was week in the presence of D-glucose. These biochemical and expression analyses suggest that RpXR is involved in the catabolism of L-arabinose as well as D-xylose. This is the first report of the purification, characterization, and gene cloning of XR from zygomycetous fungi.

DOI： 10.1016/j.jbiosc.2014.06.012

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CiNii Books

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Authorship：Lead author   Publishing type：Research paper (scientific journal)   Publisher：Oxford University Press (OUP)  

DOI： 10.1080/09168451.2014.943646

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Publishing type：Research paper (scientific journal)   Publisher：Oxford University Press (OUP)  

DOI： 10.1111/1574-6968.12589

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Language：English   Publishing type：Research paper (scientific journal)  

To determine the L-methionine (L-Met) concentration in an extract from dried blood spots (DBSs) for newborn mass screening for homocystinuria (HCU) due to cystathionine β-synthase (CBS) deficiency, a new fluorometric microplate assay using a methionine-specific dehydrogenase (MetDH) and the diaphorase/reazusrin system was established. We created by directed mutagenesis an NAD⁺-dependent MetDH from phenylalanine dehydrogenase (PheDH) showing higher substrate specificity toward L-Met than L-phenylalanine (L-Phe). However, it also exhibited notable activity for branched-chain amino acids (BCAAs). BCAAs in blood clearly interfered with the determination of L-Met in the DBS specimens using a single application of MetDH. To measure L-Met selectively, we used a branched-chain amino acid transaminase (BCAT) to eliminate the BCAAs in the specimens and screened for a BCAT with low activity toward L-Met. In microplate assays using MetDH, pretreatment of specimens with the BCAT from Lactobacillus delbrueckii subsp. bulgaricus coupled with L-glutamate oxidase minimized the effects of BCAAs, and L-Met concentrations were determined with high accuracy even at elevated BCAA concentrations. This enzymatic end-point assay is suitable for determining L-Met concentrations in DBSs for neonatal screening for HCU due to CBS deficiency.

DOI： 10.1016/j.ab.2012.06.019

PubMed

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Language：English   Publishing type：Research paper (scientific journal)  

To evaluate the degree of cellular dedifferentiation, subculture of chondrocytes was conducted on a surface coated with collagen type I at a density of 1.05 mg/cm2. In the primary culture, most of the cells were round in shape on the collagen (CL) substrate, whereas fibroblastic and partially extended cells were dominant on the polystyrene plastic (PS) substrate. Stereoscopic observation revealed that the round-shaped cells on the CL substrate were hemispherical with nebulous and punctuated F-actin filaments, whereas the fibroblastic cells on the PS substrate were flattened with fully developed stress fibers. This suggested that cell polarization was suppressed during culture on the former substrate. Although serial passages of chondrocytes through subcultures on the CL and PS substrates caused a decrease in the number of round-shaped cells, the morphological change was appreciably suppressed on the CL substrate, as compared with that on the PS substrate. It was found that only round-shaped cells formed collagen type II, which supports the view that cellular dedifferentiation can be suppressed to some extent on the CL substrate. Three-dimensional cultures in collagen gel were performed with cells isolated freshly and passaged on the CL or PS substrate. Cell density at 21 days in the culture of cells passaged on the CL substrate was comparable to that in the culture of freshly isolated cells, in spite of a significant reduction in cell density observed in the culture of cells passaged on the PS substrate. In addition, histological analysis revealed that the expression of glycosaminoglycans and collagen type II was of significance in the collagen gel with cells passaged on the CL substrate, and likewise in the gel with freshly isolated cells. This indicated that the CL substrate could offer a monolayer culture system for expanding chondrocyte cells. © Mary Ann Liebert, Inc.

DOI： 10.1089/ten.2005.11.597

Scopus

PubMed

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Language：English   Publishing type：Research paper (scientific journal)  

The subculture of rabbit chondrocytes with serial passaging was carried out for cell expansion on a collagen-coated surface, and the morphological transition of round-shaped cells to spindle-shaped ones was examined. The observation of cytoskeletal formation by staining F-actin and vinculin supported the view that the round-shaped cells were in the process of differentiation with immature stress fibers relating to less cellular polarity. The cellular morphology was estimated in terms of the distribution of roundness, R C, during the subculturing on the collagen substrate. The frequency of the number of round-shaped cells, which was defined as the ratio of the number of cells with RC>0.9 against the total cell number, was correlated in a logarithmic formula with the number of population doublings during the subcultures. Kinetic models were adopted for the process design of the combined culture of chondrocytes with monolayer growth on the collagen substrate and subsequent three-dimensional growth in Atelocollagen gel, employing the boundary conditions based on the population balance between differentiated and dedifferentiated cells. The combined culture was performed successfully according to the process design scheduled as monolayer growth for 240 h and three-dimensional growth for 264 h, the number of seed cells being 68% of that in the conventional culture for 504 h where monolayer growth for cell expansion was not included. © 2005, The Society for Biotechnology.

DOI： 10.1263/jbb.100.67

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Language：Japanese   Publishing type：Research paper (scientific journal)  

A three-dimensional growth model with cell placement was proposed to express the growth manner of rabbit chondrocytes embedded in collagen gel. This model allows the description of a growth profile consisting of lag and exponential growth phases as well as a stationary phase caused by spatial restriction of cell division (spatial contact inhibition), by considering that the gel matrix is compartmentalized into a set of unit cubes equivalent to the average cell volume, and that the spatial distribution of dissolved oxygen is a limiting factor for growth rate (generation time) of the respective cells. In the cultures conducted at initial cell densities of X 0=1.1×105 and 6.8×105cells/cm 3-gel, the calculated values of cell density based on the model were in fair agreement with the experimental data. Further model analysis was conducted to evaluate the contributions of dissolved oxygen concentration and local cell density to cell division, and it was demonstrated that the effect of spatial contact inhibition become of significance at low cell density in the case of X0=1.1×105 cells/cm3-gel as compared with X0=6.8×105cells/cm3-gel. It was also found that relatively small cell aggregates were interspersed inside the gel matrix when X0=6.8×105 cells/cm 3-gel, whereas the chondrocytes formed larger cell aggregates in the case of X0=1.1×105 cells/cm3-gel. The proposed model can be a useful tool to estimate the overall cell propagation and spatial cell distribution in the collagen-gel embedded culture of chondrocytes.

DOI： 10.1252/kakoronbunshu.30.515

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Language：English   Publishing type：Research paper (scientific journal)  

The effects of coating the culture surface with bovine type I collagen on the culture properties of anchorage-dependent cells were investigated. When human fibroblasts were cultured on a surface coated with collagen at 5.8×10-3 mg/cm2, cell attachment and subsequent cell growth were both enhanced compared to the culture on an uncoated surface. The degrees of cell attachment and growth enhancement were numerically characterized using the time constant of cell adhesion (τ) and doubling time (td) as kinetic parameters. These parameters applied to cultures of human keratinocytes and rabbit chondrocytes allowed the effects of collagen coating on the respective culture properties of both types of cells to be evaluated. In addition, the relative parameters Rτ and Rtd (defined as the ratios of the τ and td values at a given collagen concentration against those without collagen coating, respectively) were employed to estimate the effects of collagen based on a standardized criterion. Similar Rτ and Rtd profiles were obtained for collagen concentrations ranging from 5.8×10-13 to 5.8×10-3 mg/cm2, whether the cells were fibroblasts, keratinocytes or chondrocytes. It was also revealed that coating the surface with collagen at a concentration over 5.8×10-7 mg/cm2 led to reductions in both the Rτ and Rtd values, i.e. the promotion of cell attachment and growth, in the culture of each type of cells examined.

DOI： 10.1016/S1389-1723(01)80244-9

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Language：Japanese  

CiNii Books

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Language：Japanese  

CiNii Books

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