Publications by authors named "Naoki Inoue"

146 Publications

The Guinea pig cytomegalovirus GP119.1 gene encodes an IgG-binding glycoprotein that is incorporated into the virion.

Microbiol Immunol 2021 Jan 7;65(1):28-39. Epub 2021 Jan 7.

Microbiology and Immunology, Gifu Pharmaceutical University, Japan.

Cytomegaloviruses (CMVs) encode various immunoevasins, including viral receptors for the Fc domain of host IgG (vFcγR), to evade host immune responses. Although guinea pig CMV (GPCMV) provides a useful animal model for congenital CMV infection, the GPCMV genes encoding such receptors have not yet been characterized. In this study, we analyzed a locus that may encode gene products for the GPCMV immune evasion mechanisms and identified the following. (a) RACE analyses identified four transcripts in the GP117 to GP122 locus. One of the transcripts contained the GP119.1 ORF, which has weak homologies with human CMV UL119/UL118 encoding a viral FcγR and with guinea pig FcγR. (b) A transient transfection assay with plasmids expressing EGFP-tagged GP119.1 or its mutated forms identified its true translational initiation site, localization mainly in the endoplasmic reticulum, and N-glycosylation. (c) Importantly, GP119.1 bound to guinea pig IgG or the IgG-Fc fragment. (d) GP119.1 is present in the virion with a molecular mass of 15 and 23~30 kDa, and a portion of the GP119.1 products are N-glycosylated. (e) GP119.1 was dispensable for viral growth on guinea pig fibroblasts and epithelial cells in vitro. Taken together, our findings indicate that GP119.1 is an IgG-Fc binding glycoprotein incorporated into the virion, and this finding warrants further studies on the functions of GP119.1 in animal models.
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http://dx.doi.org/10.1111/1348-0421.12867DOI Listing
January 2021

The Carboxyl-Terminal Penta-Peptide Repeats of Major Royal Jelly Protein 3 Enhance Cell Proliferation.

Biol Pharm Bull 2020 ;43(12):1911-1916

Microbiology and Immunology, Gifu Pharmaceutical University.

Royal jelly (RJ) is known as an important functional foodstuff that promotes several health benefits and contains various bioactive substances, including major royal jelly proteins (MRJPs). Among the MRJPs, MRJP3 possesses both cell proliferation and wound healing effects. As the carboxyl domain of MRJP3 contains tandem penta-peptide repeat (TPR) sequences unique to MRJP3 among the MRJPs, we purified the TPRs as glutathione-S-transferase (GST)-fusion proteins and demonstrated their dose-dependent effects on THP-1 and Vero cell proliferation. The GST-TPR protein with 19 repeats (GST-TPR19) showed cell proliferative activity equivalent to MRJP3 and higher than GST-TPR6. GST-TPR19 also exhibited wound healing activity at a level similar to MRJP3. Digestion of GST-TPR19 with trypsin had no effect on its cell proliferative activity, suggesting that the main digested products; i.e., penta-peptides (Q-N-x-N-[K/R]), maintain the cell proliferative ability of MRJP3. In conclusion, the TPRs of MRJP3 are critical to the beneficial effect(s) of RJ.
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http://dx.doi.org/10.1248/bpb.b20-00607DOI Listing
January 2020

Identification and functional analyses of a cell-death inhibitor encoded by guinea pig cytomegalovirus gp38.1 in cell culture and in animals.

J Gen Virol 2020 12 11;101(12):1270-1279. Epub 2020 Sep 11.

Microbiology and Immunology, Gifu Pharmaceutical University, Gifu, Japan.

Cytomegaloviruses (CMVs) employ an array of strategies designed to interfere with host defence responses against pathogens. Studies on such evasion mechanisms are important for understanding the pathogenesis of CMV diseases. Although guinea pig CMV (GPCMV) provides a useful animal model for congenital CMV infection, its evasion strategies are not fully elucidated. Here, we analysed a genome locus that may encode gene products for the GPCMV evasion mechanisms and found the following. (1) RACE analyses identified five transcripts in the GP38-gp38.4 locus, one of which was a spliced product encoding gp38.1. Similarities in the splicing pattern and gene position of gp38.1 to human CMV UL37 and its exon 1 encoding vMIA (viral mitochondria-localized inhibitor of apoptosis) suggest that the gp38.1 gene encodes an apoptosis inhibitor. (2) In a transient transfection assay, gp38.1 localized in the mitochondria and relocated BAX from the cytoplasm to the mitochondria, although its co-localization with BAK was not evident. Further, the expression of gp38.1 partially reduced staurosporine-induced apoptosis. (3) GPCMV defective in the gp38.1 ORF (Δ38.1) and the virus that rescues the defect (r38.1) were generated. Guinea pig fibroblast cells infected with Δ38.1 died earlier than r38.1-infected cells, which resulted in the lower yields of Δ38.1. (4) In animals, viral loads in the spleens of r38.1-infected guinea pigs were higher than those in the spleens of Δ38.1-infected animals. In conclusion, although GPCMV gp38.1 exerts a vMIA-like function, its inhibitory effect was not robust, suggesting the presence of additional inhibitory molecule(s), such as a BAK-specific inhibitor.
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http://dx.doi.org/10.1099/jgv.0.001493DOI Listing
December 2020

[Fall-related efficacy is associated with the progression of frailty in community-dwelling older people].

Nihon Ronen Igakkai Zasshi 2020 ;57(3):308-315

School of Allied Health Science, Kitasato University.

Aim: The goal of this study was to verify the association between frailty and fall-related efficacy in community-dwelling older people by performing a cross-sectional and longitudinal data analysis.

Methods: In this study, 339 people aged 65 years and older participated in a baseline survey. Furthermore, people who were not identified as frail in the baseline survey participated in a follow-up survey 6 months later. Frailty was assessed in the baseline and follow-up surveys after 6 months using the Kihon checklist. Fall-related efficacy was assessed at baseline using the short Falls Efficacy Scale International (short FES-I). Potential confounding factors, such as the lower limb functions and psychological functions, were also investigated at baseline. The association between frailty and short FES-I was analyzed using a logistic regression analysis adjusted for potential confounding factors.

Results: At baseline and the follow-up survey, 10.1% and 6.3% of the participants were judged to demonstrate frailty, respectively. The results of the baseline and follow-up data analysis showed that even if potential confounding factors were adjusted for, the short FES-I was significantly associated with frailty. Furthermore, the ability to distinguish the onset of frailty using the short FES-I was analyzed using a receiver operating characteristic curve, and the area under curve, sensitivity, and specificity values were 0.78, 0.92 and 0.56, respectively.

Conclusions: A clear association between frailty and fall-related efficacy was thus observed, as indicated in the cross-sectional and longitudinal data analysis. Furthermore, based on the results of the longitudinal data analysis, the short FES-I was found to be able to predict the progression of frailty and it can thus be a useful screening tool for assessing frailty.
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http://dx.doi.org/10.3143/geriatrics.57.308DOI Listing
December 2020

Enhancement of guinea pig cytomegalovirus infection by two endogenously expressed components of the pentameric glycoprotein complex in epithelial cells.

Sci Rep 2020 05 22;10(1):8530. Epub 2020 May 22.

Microbiology and Immunology, Gifu Pharmaceutical University, Gifu, Japan.

A better understanding of the mechanisms underlying cell tropisms and the efficiency of viral infection is critical for the development of vaccines and antiviral drugs for viral diseases. In this study, we worked on the entry mechanisms of guinea pig cytomegalovirus and found that endogenous expression of a combination of two components (GP131 and GP133) of the pentameric glycoprotein complex, which is required for non-fibroblast cell tropisms, enhanced viral infection more than 10-fold. In addition, D138A alteration in GP131 increased this enhancement by an additional 10-fold. Although differences in the efficiency of viral infection among various cell types are usually explained by differences in viral entry or traffic processes, our experimental evidences dismissed such possibilities. Instead, our findings that i) endogenous expression of GP131 and GP133 after nuclear delivery of viral DNA still enhanced infection and ii) an HDAC inhibitor overcame the need of the endogenous expression led us to hypothesize a novel mechanism that controls the efficiency of viral infection through the activation of gene expression from viral DNA delivered to the nuclei. Further studies of this unexpected phenomena warrant to understand novel but also general mechanisms for cell tropisms of viral infection and determinants that control infection efficiency.
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http://dx.doi.org/10.1038/s41598-020-65545-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7244513PMC
May 2020

Activation of c-Jun by human cytomegalovirus UL42 through JNK activation.

PLoS One 2020 5;15(5):e0232635. Epub 2020 May 5.

Microbiology and Immunology, Gifu Pharmaceutical University, Gifu, Japan.

c-Jun is a major component of the AP-1 transactivator complex. In this report, we demonstrated that AP-1 was activated by the expression of UL42, a human cytomegalovirus-encoded membrane protein that has two PPXY (PY) motifs and a C-terminal transmembrane domain (TMD). Although UL42 interacts with Itch, an ubiquitin E3 ligase, through the PY motifs, UL42 phosphorylated c-Jun and c-Jun N-terminal kinase (JNK) in the absence of any interaction with Itch. Experiments using mutated versions of UL42 suggest the importance of the carboxyl half (a.a. 52-124) of UL42 for the activation of the JNK signaling, while C-terminal TMD alone is not sufficient. Thus, we hypothesize that UL42 plays a role in the activation of JNK signaling in HCMV-infected cells. (118 words).
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0232635PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7199950PMC
August 2020

Collagen synthesis-promoting and collagenase inhibitory activities of constituents isolated from the rhizomes of Picrorhiza kurroa Royle ex Benth.

Fitoterapia 2020 Jun 1;143:104584. Epub 2020 Apr 1.

Pharmaceutical Research and Technology Institute, Kindai University, 3-4-1 Kowakae, Higashi-osaka, Osaka 577-8502, Japan; Antiaging Center, Kindai University, 3-4-1 Kowakae, Higashi-osaka, Osaka 577-8502, Japan.

Three new acylated phenylethanoid glycosides, kurroaosides A (14), B (15), and C (16), and a new acylated cucurbitane-type triterpene glycoside, kurroaoside D (17), were isolated from a methanol extract of the rhizomes of Picrorhiza kurroa Royle ex Benth. (Plantaginaceae) along with 29 known isolates including 10 acylated phenylethanoid glycosides (18-27), three cucurbitane-type triterpene glycosides (32-34), and a nortriterpene glycoside (35). The structures of these new compounds (14-17), including their stereochemistry, were determined based on chemical and physicochemical evidence derived from NMR and MS analysis. Among the isolates, acylated iridoid glycosides, picrosides I (8), II (9), III (10), and IV (11) and 6-feruloylcatalpol (12), phenylethanoid glycosides (14-16), triterpene glycosides, cucurbitacin B 2-O-β-D-glucopyranoside (32) and 25-acetoxy-2-β-D-glucopyranosyloxy-3,16,20-trihydroxy-9-methyl-19-norlanosta-5-en-22-one (35), and an acetophenone glycoside, picein (36), significantly promoted collagen synthesis at 10-30 μM, with no cytotoxicity being observed at the effective concentrations. Furthermore, acylated phenylethanoid glycosides, calceolarioside A (19, IC = 69.2 μM), plantamajoside (20, 51.8 μM), isoplantamajoside (21, 76.8 μM), and scroside E (23, 65.5 μM), exhibited collagenase inhibitory activity equivalent to that of positive agents caffeic acid (75.6 μM) and epigallocatechin 3-O-gallate (75.4 μM).
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http://dx.doi.org/10.1016/j.fitote.2020.104584DOI Listing
June 2020

Eosinophils are the main cellular targets for oral gene delivery using Lactic acid bacteria.

Vaccine 2020 04 17;38(17):3330-3338. Epub 2020 Mar 17.

Department of Microbiology and Immunology, Gifu Pharmaceutical University, 1-25-4 Daigaku Nishi, Gifu 501-1196, Japan.

Lactic acid bacteria have been studied as a vehicle for the delivery of plasmid DNA to the gastrointestinal tract. However, low levels of gene expression in vivo limit their practical use. Furthermore, it is still unclear how the orally administrated bacteria transfer their harbored plasmid DNA to host intestinal cells. To more easily track the delivery of plasmid DNA for eukaryotic expression in the intestine, we constructed an L. lactis-E. coli shuttle plasmid (pLEC) that allowed significantly elevated expression of the target protein of interest in eukaryotic cells. We first demonstrated its usefulness for delivery from L. lactis to Caco-2 cells in vitro. We then investigated the cellular target for the L. lactis DNA delivery system in vivo. Mice were orally administrated with LL/pLEC:EGFP, an L. lactis strain carrying pLEC for EGFP expression, and immunofluorescent analyses of frozen sections prepared from their small intestines identified a number of EGFP-expressing cells in the lamina propria and some in the sub-epithelial dome of the Peyer's patches. Flow cytometric analysis revealed that these EGFP-expressing cells were both CD11c- and F4/80-positive but CXCR1-negative, suggesting that they are eosinophils. Immunostaining of the sections with an antibody against Siglec-F, a marker protein of eosinophils, confirmed the flow cytometric findings. Thus, the target cells of DNA delivery from L. lactis in the intestines are mainly eosinophils in the lamina propria and Peyer's patches. This finding may open a new approach to the development of DNA vaccines for oral administration.
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http://dx.doi.org/10.1016/j.vaccine.2020.02.084DOI Listing
April 2020

Clinical Diagnostic Testing for Human Cytomegalovirus Infections.

J Infect Dis 2020 03;221(Suppl 1):S74-S85

Viral Vaccine Preventable Diseases Branch, Division of Viral Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA.

Human cytomegalovirus (HCMV) infections are among the most common complications arising in transplant patients, elevating the risk of various complications including loss of graft and death. HCMV infections are also responsible for more congenital infections worldwide than any other agent. Congenital HCMV (cCMV) infections are the leading nongenetic cause of sensorineural hearing loss and a source of significant neurological disabilities in children. While there is overlap in the clinical and laboratory approaches to diagnosis of HCMV infections in these settings, the management, follow-up, treatment, and diagnostic strategies differ considerably. As yet, no country has implemented a universal screening program for cCMV. Here, we summarize the issues, limitations, and application of diagnostic strategies for transplant recipients and congenital infection, including examples of screening programs for congenital HCMV that have been implemented at several centers in Japan, Italy, and the United States.
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http://dx.doi.org/10.1093/infdis/jiz601DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7057790PMC
March 2020

Food-Derived Collagen Peptides, Prolyl-Hydroxyproline (Pro-Hyp), and Hydroxyprolyl-Glycine (Hyp-Gly) Enhance Growth of Primary Cultured Mouse Skin Fibroblast Using Fetal Bovine Serum Free from Hydroxyprolyl Peptide.

Int J Mol Sci 2019 Dec 28;21(1). Epub 2019 Dec 28.

Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Kyoto 606 8502, Japan.

Prolyl-hydroxyproline (Pro-Hyp) and hydroxyprolyl-glycine (Hyp-Gly) appear in human blood after ingestion of collagen hydrolysate and trigger growth of fibroblasts attached on collagen gel, which has been associated with beneficial effects upon ingestion of collagen hydrolysate, such as improvement of skin and joint conditions. In the present study, inconsistent results were obtained by using different lots of fetal bovine serum (FBS). Fibroblasts proliferated in collagen gel without adding Pro-Hyp and Hyp-Gly and did not respond to addition of Pro-Hyp and Hyp-Gly, which raises doubts about conclusions from prior research. Unexpectedly high levels of hydroxyprolyl peptides, including Pro-Hyp, however, were present in the FBS (approximately 100 µM), and also in other commercially available forms of FBS (70-80 µM). After removal of low molecular weight (LMW, < 6000 Da) compounds from the FBS by size exclusion chromatography, Pro-Hyp and Hyp-Gly again triggered growth of fibroblasts attached on collagen and increased the number of fibroblasts migrated from mouse skin. These results indicate the presence of bioactive hydroxyprolyl peptides in commercially available FBS, which can mask effects of Pro-Hyp and Hyp-Gly supplementation; our work confirms that Pro-Hyp and Hyp-Gly do play crucial roles in proliferation of fibroblasts.
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http://dx.doi.org/10.3390/ijms21010229DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6982277PMC
December 2019

Effects of Collagen Hydrolysates on Human Brain Structure and Cognitive Function: A Pilot Clinical Study.

Nutrients 2019 Dec 23;12(1). Epub 2019 Dec 23.

Department of Anti-aging Medicine, Ehime University Graduate School of Medicine, Ehime 791-0295, Japan.

This study investigated the effects of collagen hydrolysates (CH) on language cognitive function and brain structure. In this open-label study, 5 g CH was administered once a day for 4 weeks to 30 healthy participants aged 49-63 years. The primary outcome measures were the brain healthcare quotients based on gray matter volume (GM-BHQ) and fractional anisotropy (FA-BHQ). The secondary outcome measures were changes in scores between week 0 and week 4 for word list memory (WLM) and standard verbal paired associate learning (S-PA) tests as well as changes in the physical, mental, and role/social component summary scores of the Short Form-36(SF-36) quality of life instrument. CH ingestion resulted in significant improvements in FA-BHQ ( = 0.0095), a measure of brain structure, as well in scores for the WLM ( = 0.0046) and S-PA ( = 0.0007) tests, which measure cognitive function. There were moderate correlations between the change in WLM score and the change in GM-BHQ ( = 0.4448; Spearman's rank correlation) and between the change in S-PA score and the change in FA-BHQ ( = 0.4645). Daily ingestion of CH changed brain structure and improved language cognitive function.
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http://dx.doi.org/10.3390/nu12010050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7019356PMC
December 2019

Acylated iridoid glycosides with hyaluronidase inhibitory activity from the rhizomes of Picrorhiza kurroa Royle ex Benth.

Phytochemistry 2020 Jan 31;169:112185. Epub 2019 Oct 31.

Pharmaceutical Research and Technology Institute, 3-4-1 Kowakae, Higashi-osaka, Osaka, 577-8502, Japan; Antiaging Center, Kindai University, 3-4-1 Kowakae, Higashi-osaka, Osaka, 577-8502, Japan.

Seven new acylated iridoid glycosides, picrorhizaosides A-G (1-7), were isolated from the methanol extract of the rhizomes of Picrorhiza kurroa Royle ex Benth. (Plantaginaceae), in addition to six known iridoid glycosides (8-13). The structures of these new iridoids, including their stereochemistry, were determined based on chemical and physicochemical evidence derived from NMR and MS analysis. Of the isolates, picrorhizaosides D (4, IC = 43.4 μM) and E (5, 35.8 μM); picrosides I (8, 60.7 μM), II (9, 22.3 μM), and IV (11, 59.2 μM); and minecoside (13, 57.2 μM), exhibited a similar or stronger hyaluronidase inhibitory activity than those of the antiallergic medicines disodium cromoglycate (64.8 μM), ketotifen fumarate (76.5 μM), and tranilast (227 μM).
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http://dx.doi.org/10.1016/j.phytochem.2019.112185DOI Listing
January 2020

Regional differences in Ca entry along the proximal-middle-distal muscle axis during eccentric contractions in rat skeletal muscle.

J Appl Physiol (1985) 2019 09 1;127(3):828-837. Epub 2019 Aug 1.

Department of Engineering Science, Bioscience and Technology Program, University of Electro-Communications, Chofu, Tokyo, Japan.

Eccentric (ECC) contraction-induced muscle damage is associated with calcium ion (Ca) influx from the extracellular milieu through stretch-activated channels. It remains unknown whether Ca influx consequent to repetitive ECC contractions is nonuniform across different muscle regions. We tested the hypothesis that there are regional differences in Ca entry along the proximal-middle-distal muscle axis. Tibialis anterior (TA) muscles of adult male Wistar rats were exposed by reflecting the overlying skin and fasciae and ECC contractions evoked by peroneal nerve stimulation paired with simultaneous ankle extension (50 times/set, 2 protocols: 1 set and 10 sets). During ECC in the proximal, middle, and distal TA, we determined ) muscle fiber extension by high-speed camera (200 frames/s) and ) Ca accumulation by in vivo bioimaging (Ca-sensitive probe Fura-2-acetoxymethyl ester). Muscle fiber extension from resting was significantly different among regions (i.e., proximal, 4.0%: < middle, 11.2%: < distal, 17.0%; ECC phase length at 500th contraction). Intracellular Ca accumulation after 1 set of ECC was higher in the distal (1.46 ± 0.04, < 0.05) than the proximal (1.27 ± 0.04) or middle (1.26 ± 0.05) regions. However, this regional Ca accumulation difference disappeared by 32.5 min after the 1 set protocol when the muscle was quiescent and by contraction set 5 for the 10-set protocol. The initial preferential ECC-induced Ca accumulation observed distally was associated spatially with the greater muscle extension compared with that of the proximal and middle regions. Disappearance of the regional Ca accumulation disparity in quiescent and ECC-contracting muscle might be explained, in part, by axial Ca propagation and account for the uniformity of muscle damage across regions evident 3 days post-ECC. After 1 set of 50 eccentric (ECC) contractions in the anterior tibialis muscle, intracellular Ca ([Ca]i) accumulation evinces substantial regional heterogeneity that is spatially coherent with muscle length changes (i.e., distal [Ca]i > middle, proximal). However, irrespective of whether 50 or 500 ECC contractions are performed, this heterogeneity is subsequently abolished, at least in part, by axial intracellular Ca propagation. This Ca homogenization across regions is consistent with the absence of any interregional difference in muscle damage 3 days post-ECC.
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http://dx.doi.org/10.1152/japplphysiol.01005.2018DOI Listing
September 2019

Current issues regarding the application of recombinant lactic acid bacteria to mucosal vaccine carriers.

Appl Microbiol Biotechnol 2019 Aug 7;103(15):5947-5955. Epub 2019 Jun 7.

Department of Microbiology and Immunology, Gifu Pharmaceutical University, 1-25-4 Daigaku Nishi, Gifu, 501-1196, Japan.

Over the past two decades, lactic acid bacteria (LAB) have been intensively studied as potential bacterial carriers for therapeutic materials, such as vaccine antigens, to the mucosal tissues. LAB have several attractive advantages as carriers of mucosal vaccines, and the effectiveness of LAB vaccines has been demonstrated in numerous studies. Research on LAB vaccines to date has focused on whether antigen-specific immunity, particularly antibody responses, can be induced. However, with recent developments in immunology, microbiology, and vaccinology, more detailed analyses of the underlying mechanisms, especially, of the induction of cell-mediated immunity and memory cells, have been required for vaccine development and licensure. In this mini-review, we will discuss the issues, including (i) immune responses other than antibody production, (ii) persistence of LAB vaccine immunity, (iii) comparative evaluation of LAB vaccines with any existing or reference vaccines, (iv) strategies for increasing the effectiveness of LAB vaccines, and (iv) effects of microbiota on the efficacy of LAB vaccines. Although these issues have been rarely studied or discussed to date in relation to LAB vaccine research, further understanding of them is critical for the practical application of LAB vaccine systems.
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http://dx.doi.org/10.1007/s00253-019-09912-xDOI Listing
August 2019

Evaluation of the indirect and IgM-capture anti-human cytomegalovirus IgM ELISA methods as confirmed by cytomegalovirus IgG avidity.

Microbiol Immunol 2019 May 22;63(5):172-178. Epub 2019 May 22.

Department of Microbiology, Fukushima Medical University School of Medicine, Fukushima, Japan.

Primary cytomegalovirus (CMV) infection during pregnancy often results in congenital CMV infection with severe clinical complications. IgM antibodies are one of the indices of primary infection. The IgG avidity index (AI) is also known to remain low for 3 months after primary infection. Here, we evaluated and compared the performance of CMV IgM and IgG avidity assays. Because sensitivity and specificity reportedly differ between CMV IgM kits, CMV IgM detection was compared between the two commercially available ELISA kits that are most commonly used in Japan. Sera for CMV IgM were first screened using a traditional indirect ELISA kit. Selected samples were then tested for CMV IgM and CMV AI using a CMV IgM-capture ELISA kit and a CMV IgG avidity assay, respectively. The rate of concordance between the IgM kits was 89% (42/47), indicating the absence of any significant difference. Most of the CMV IgM-positive plasma samples showed high CMV IgG AI; however, 18 commercially available plasma samples with low CMV IgG AI were all CMV IgM-positive. One plausible explanation for this discrepancy is that the duration of low IgG AI is shorter than that of IgM positivity. Alternatively, CMV IgM tests may generate pseudo-positive readouts in cases of congenital infection. Nevertheless, our study confirms that CMV IgG AI can be a reliable indicator of CMV primary infection.
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http://dx.doi.org/10.1111/1348-0421.12683DOI Listing
May 2019

Protective effects of oral immunization with formalin-inactivated whole-cell Citrobacter rodentium on Citrobacter rodentium infection in mice.

J Microbiol Methods 2019 04 25;159:62-68. Epub 2019 Feb 25.

Department of Pharmacy, Laboratory of Microbiology and Immunology, Gifu Pharmaceutical University, Gifu 501-1196, Japan. Electronic address:

Evaluation of the efficacy of vaccine candidates that prevent enteropathogenic and enterohemorrhagic Escherichia coli (EPEC/EHEC) infection in mouse models is difficult due to their limited pathogenicity in mice. Citrobacter rodentium, a murine pathogenic bacterium that shares its infection strategy and virulence genes with EPEC/EHEC, has been used as a model pathogen to develop novel vaccine strategies or platforms for these bacteria. However, there are few reports on the comparative effectiveness of novel vaccine platforms as no C. rodentium vaccines have yet been prepared by standard methods such as bacteria attenuation or inactivation. In this study, we investigated the protective effect of the oral administration of formalin-inactivated C. rodentium (Fo-CR) on C. rodentium infection in two mouse strains, C57BL/6 and C3H/HeN, as these strains have different degrees of susceptibility to infection. In C57BL/6 mice, administration of Fo-CR induced significant C. rodentium-specific mucosal and systemic antibody responses, promoted bacterial clearance from the gut and inhibited colonic hyperplasia. Furthermore, in C3H/HeN mice, the administration followed by lethal C. rodentium infection induced significantly high avidity serum IgG specific to C. rodentium and inhibited death, body weight loss, and bacterial invasion to visceral organs. In conclusion, the oral administration of Fo-CR resulted in the protection of mice from C. rodentium infection, indicating that it serves as a reference method for evaluating the efficacy of novel oral vaccine candidates or platforms.
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http://dx.doi.org/10.1016/j.mimet.2019.02.016DOI Listing
April 2019

Roles of GP33, a guinea pig cytomegalovirus-encoded G protein-coupled receptor homolog, in cellular signaling, viral growth and inflammation in vitro and in vivo.

PLoS Pathog 2018 12 20;14(12):e1007487. Epub 2018 Dec 20.

Laboratory of Microbiology and Immunology, Gifu Pharmaceutical University, Gifu, Japan.

Cytomegaloviruses (CMVs) encode cellular homologs to evade host immune functions. In this study, we analyzed the roles of GP33, a guinea pig CMV (GPCMV)-encoded G protein-coupled receptor (GPCR) homolog, in cellular signaling, viral growth and pathogenesis. The cDNA structure of GP33 was determined by RACE. The effects of GP33 on some signaling pathways were analyzed in transient transfection assays. The redET two-step recombination system for a BAC containing the GPCMV genome was used to construct a mutant GPCMV containing an early stop codon in the GP33 gene (Δ33) and a rescued GPCMV (r33). We found the following: 1) GP33 activated the CRE- and NFAT-, but not the NFκB-mediated signaling pathway. 2) GP33 was dispensable for infection in tissue cultures and in normal animals. 3) In pregnant animals, viral loads of r33 in the livers, lungs, spleens, and placentas at 6 days post-infection were higher than those of Δ33, although the viruses were cleared by 3 weeks post-infection. 4) The presence of GP33 was associated with frequent lesions, including alveolar hemorrhage in the lungs, and inflammation in the lungs, livers, and spleens of the dams. Our findings suggest that GP33 has critical roles in the pathogenesis of GPCMV during pregnancy. We hypothesize that GP33-mediated signaling activates cytokine secretion from the infected cells, which results in inflammation in some of the maternal organs and the placentas. Alternatively, GP33 may facilitate transient inflammation that is induced by the chemokine network specific to the pregnancy.
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http://dx.doi.org/10.1371/journal.ppat.1007487DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6319746PMC
December 2018

M cell-targeting strategy enhances systemic and mucosal immune responses induced by oral administration of nuclease-producing L. lactis.

Appl Microbiol Biotechnol 2018 Dec 11;102(24):10703-10711. Epub 2018 Oct 11.

Department of Microbiology and Immunology, Gifu Pharmaceutical University, 1-25-4 Daigaku Nishi, Gifu, 501-1196, Japan.

Efficient delivery of antigens to the gut-associated lymphoid tissue (GALT) is the most critical step for the induction of mucosal immunity by oral vaccines. As M cells are the main portal for luminal antigens into the GALT, the M cell-targeting of antigens affords a promising strategy toward the development of effective oral vaccines. Lactococcus lactis is a fascinating recombinant host for oral vaccines, as they survive and produce antigens in the gut and have a particularly safe profile for human use. In this study, we developed and evaluated an M cell-targeting oral immunization system using recombinant L. lactis strains. For the purpose, we generated an L. lactis strain that secretes a model antigen fused with the OmpH β1α1 domain of Yersinia enterocolitica, which has been shown to bind to a complement C5a receptor on the M cell surface. As the model antigen, Staphylococcus aureus nuclease was used for fusion, resulting in L. lactis-expressing Nuc-OmpH (LL/Nuc-OmpH). Ex vivo intestinal loop assays showed that the amount of Nuc-OmpH taken up into Peyer's patches was more than that of the unfused nuclease (Nuc). In addition, oral administration of the recombinant L. lactis strains to mice demonstrated that LL/Nuc-OmpH-induced nuclease-specific fecal IgA and serum IgG titers were significantly higher than those induced by LL/Nuc. These results indicate that OmpH works as an M cell-targeting molecule when fused with antigens secreted from L. lactis and that the M cell-targeting strategy affords a promising platform for L. lactis-based mucosal immunization.
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http://dx.doi.org/10.1007/s00253-018-9427-1DOI Listing
December 2018

Analysis of relationships between polymorphisms in the genes encoding the pentameric complex and neutralization of clinical cytomegalovirus isolates.

Vaccine 2018 09 30;36(40):5983-5989. Epub 2018 Aug 30.

Microbiology and Immunology, Gifu Pharmaceutical University, 1-25-4 Daigaku-nishi, Gifu-shi, Gifu 501-1196, Japan. Electronic address:

Introduction: As congenital cytomegalovirus (CMV) infection is one of the major causes of birth defects and developmental abnormalities, it is essential to develop vaccines and therapeutic antibodies against CMV. Clinical trials demonstrated that the subunit vaccine based on glycoprotein B, which had been believed to be the major target for neutralization, did not induce sufficient protective immunity. On the other hand, it has been reported that the immunization of animals with the Pentamer, the pentameric complex of gH/gL/UL128/UL130/UL131A, induced strong neutralizing antibodies. Here, we sought to clarify whether any polymorphic alterations present in the Pentamer of clinical isolates affect neutralization by anti-Pentamer antibodies.

Methods: Sequences of the genes encoding the Pentamer components of 25 Japanese clinical isolates were determined. Neutralization of infection by two seropositive sera and by anti-Pentamer serum was measured using a CMV reporter cell line based on ARPE-19.

Results: Polymorphisms of the amino acid sequence of UL128, UL130, and UL131A ORFs were limited and clustered into two major groups. The identified alterations, except UL128 I140T, were mapped outside of the reported regions recognized by neutralizing antibodies. Anti-Pentamer serum neutralized infection with all isolates to a similar degree and had no correlation with the polymorphic groups.

Conclusions: Our findings indicate that Pentamer antigens prepared from Merlin Fix strain induce antibodies that neutralize infection with all isolates to a similar level and that anti-Pentamer antibodies neutralize CMV infection better than do human sera, suggesting that vaccines and therapeutic antibodies based on Pentamer as an antigen have some promise.
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http://dx.doi.org/10.1016/j.vaccine.2018.08.054DOI Listing
September 2018

Differences in the effects of mutations in GP131, a guinea pig cytomegalovirus homologue of pentameric complex component UL130, on macrophage and epithelial cell infection.

J Gen Virol 2018 10 16;99(10):1425-1431. Epub 2018 Aug 16.

1​Laboratory of Microbiology and Immunology, Gifu Pharmaceutical University, Gifu, Japan.

As congenital cytomegalovirus (CMV) infection is the major cause of developmental abnormalities in children, the development of effective vaccines is critical to public health. Recent studies have demonstrated that the pentameric complex (Pentamer) of glycoproteins, which is required for human CMV infection of endothelial and epithelial cells, could be a potent vaccine antigen. As guinea pig CMV (GPCMV) infects congenitally and encodes homologues of all Pentamer components, GPCMV models are considered to be useful for the development of vaccine strategies. Here, to clarify the precise requirement of GP131, one of the GPCMV Pentamer components, for the infection of epithelial cells and macrophages, we prepared several mutants with a charged amino acid-to-alanine alteration in GP131 and found some differences in the effects of the mutations on the infection of the two cell types, suggesting the existence of cell type-dependent recognition or function of Pentamer in GPCMV infection.
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http://dx.doi.org/10.1099/jgv.0.001137DOI Listing
October 2018

Ingestion of bioactive collagen hydrolysates enhanced pressure ulcer healing in a randomized double-blind placebo-controlled clinical study.

Sci Rep 2018 07 30;8(1):11403. Epub 2018 Jul 30.

Aurous Health Care Research and Development Private Limited, No.180/109, Rangarajapuram Main Road, Kodambakkam, Chennai, 600 024, India.

We conducted a double blind, multi-centric, placebo-controlled, randomized trial to compare the Pressure Ulcer Scale for Healing (PUSH) and Pressure Sore Status Tool (PSST) scores and wound area measurements at 16 weeks of subjects with pressure ulcers who were given standard care plus one of two types of collagen hydrolysate (CH-a), which contained low levels of prolylhydroxyproline (Pro-Hyp) and hydroxyprolylglycine (Hyp-Gly), and CH-b, which contained high levels of Pro-Hyp and Hyp-Gly) with the placebo group. A total of 120 subjects with stage II or III pressure ulcers were entered into the trial and 112 subjects completed the study. The subjects were randomized to receive CH-a (n = 39), CH-b (n = 39), or a placebo (n = 42) twice daily (10 g per day) for 16 weeks. The PUSH score, PSST score, and wound area of the CH-b group were significantly lower than the placebo group at week 16 (PUSH score, P < 0.001; PSST score, P < 0.01; wound area, P < 0.05). The PUSH score of the CH-a group was significantly lower than the placebo group at week 16 (P < 0.05). This study demonstrated that CH-b ingestion helps healing of pressure ulcers as an add-on to the standard therapy.
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http://dx.doi.org/10.1038/s41598-018-29831-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6065362PMC
July 2018

Titration of cell-associated varicella-zoster virus with the MV9G reporter cell line for antiviral studies.

J Virol Methods 2018 10 30;260:14-20. Epub 2018 Jun 30.

Department of Microbiology and Immunology, Gifu Pharmaceutical University, Gifu 502-8585, Japan. Electronic address:

Titration of the cell-associated virus (CAV) of varicella-zoster virus (VZV) is essential for antiviral studies. A VZV reporter cell line, MV9G, generated in our previous study expresses firefly luciferase upon CAV infection in a dose-dependent manner, suggesting that use of the cell line for titration is feasible. In this study, MeWo cells infected with VZV vaccine Oka (vOka) strain or with clinical isolates obtained from patients with varicella or zoster were used as CAV. A co-culture of MV9G cells with the virus-infected MeWo cells were set up and optimized for titration of CAV. Luciferase activities of MV9G cells measured as relative light units (RLUs) of chemiluminescence correlated well (r > 0.9, p < 0.05) both with quantities of viral DNAs measured by TaqMan PCR and with numbers of viral foci detected by immunostaining with a monoclonal antibody against VZV IE62. In addition, the usefulness of MV9G for antiviral studies was exemplified by treatment of the VZV-infected cells with various concentrations of acyclovir. Thus, the reporter cell-based titration of CAV by measuring the induced RLUs may be a reliable way to estimate viral foci and viral DNAs.
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http://dx.doi.org/10.1016/j.jviromet.2018.06.019DOI Listing
October 2018

Vaccine Development for Cytomegalovirus.

Adv Exp Med Biol 2018 ;1045:271-296

Department of Virology I, National Institute of Infectious Diseases, Tokyo, Japan.

The development of a cytomegalovirus (CMV) vaccine has become a top priority due to its potential cost-effectiveness and associated public health benefits. However, there are a number of challenges facing vaccine development including the following: (1) CMV has many mechanisms for evading immune responses , and natural immunity is not perfect, (2) the immune correlates for protection are unclear, (3) a narrow range of CMV hosts limits the value of animal models, and (4) the placenta is a specialized organ formed transiently and its immunological status changes with time. In spite of these limitations, several types of CMV vaccine candidate, including live-attenuated, DISC , subunit, DNA, vectored, and peptide vaccines, have been developed or are currently under development. The recognition of the pentameric complex as the major neutralization target and identification of various strategies to block viral immune response evasion mechanisms have opened new avenues to CMV vaccine development. Here, we discuss the immune correlates for protection, the characteristics of the various vaccine candidates and their clinical trials, and the relevant animal models.
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http://dx.doi.org/10.1007/978-981-10-7230-7_13DOI Listing
November 2018

Effects of cationic liposomes with stearylamine against virus infection.

Int J Pharm 2018 May 3;543(1-2):311-317. Epub 2018 Apr 3.

Laboratory of Pharmaceutical Engineering, Gifu Pharmaceutical University, Gifu, Japan. Electronic address:

In this study, we demonstrated that cationic liposomes with incorporated stearylamine (SA) inhibit viral infectivity without preloaded active pharmaceutical ingredients. Specifically, we correlated physiochemical properties of liposomes, such as zeta potentials and particle sizes, with virus infectivity using the BacMam™ reagent, which is based on recombinant baculovirus (BV). Compared with neutral or negatively-charged liposomes, SA liposomes suppressed BV infectivity in several mammalian cell lines, including A549 cells. SA liposomes inhibited BV infection over 80% by optimizing the liposomal concentration and exposure time with cells. Moreover, these antiviral SA liposomes were not cytotoxic, and reducing the embedded cholesterol contents intensified the antiviral effects and simultaneously increased the binding of SA liposomes to the cell membranes. These data indicate that binding of SA liposomes to cell membranes may block virus entry. Finally, we also demonstrated the antiviral effects of SA liposomes on herpes simplex virus type 1 in A549 cells, and showed comparable efficacy to that of the antiviral drug acyclovir.
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http://dx.doi.org/10.1016/j.ijpharm.2018.04.001DOI Listing
May 2018

Characterization of phenyl pyrimidine derivatives that inhibit cytomegalovirus immediate-early gene expression.

Antivir Chem Chemother 2018 Jan-Dec;26:2040206618763193

1 Department of Microbiology and Immunology, Gifu Pharmaceutical University, Gifu, Japan.

Background Previously, we established a reporter cell line for human cytomegalovirus and screened anti-human cytomegalovirus compounds using the cell line. In this study, we characterized one of the identified compounds, 2,4-diamino-6-(4-methoxyphenyl)pyrimidine (coded as 35C10). Methods 50% Effective concentrations (EC50s) and 50% cytotoxic concentrations (CC50s) of 35C10 and its derivatives in human fibroblasts were determined by X-gal staining of the cells infected with human cytomegalovirus Towne strain expressing β-galactosidase. Results EC50 and CC50 of 35C10 were 4.3 µM and >200 µM, respectively. Among several 35C10 derivatives, only one lacking 4-amino group of pyrimidine showed a similar EC50. 35C10 weakly inhibited murine cytomegalovirus, herpes simplex virus type 1, and varicella-zoster virus. A "time of addition" experiment suggested that 35C10 inhibited an early phase of the infection. Although 35C10 did not inhibit viral attachment to the cells nor the delivery of viral DNA to the nuclei, it decreased the number of infected cells expressing immediate-early 1 and 2 (IE1/IE2) proteins. 35C10 also inhibited the activation of a promoter for TRL4 in the reporter cells upon human cytomegalovirus infection, but not in the same reporter cells transfected with a plasmid expressing IE2. Conclusion Our findings suggest that 35C10 is a novel compound that inhibits IE gene expression in human cytomegalovirus-infected cells.
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http://dx.doi.org/10.1177/2040206618763193DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5890547PMC
June 2019

Absorption and metabolism of orally administered collagen hydrolysates evaluated by the vascularly perfused rat intestine and liver in situ.

Biomed Res 2018 ;39(1):1-11

Faculty of Agriculture, Utsunomiya University.

A number of studies have shown that oral administration of collagen hydrolysate (CH) results in the absorption of di- and tri-peptides. In order to understand the dynamics of CH absorption and metabolism, molecular profiles of hydroxyproline (Hyp) and Hyp-containing peptides (HCPs) were analyzed by in situ perfusion of rat intestine and liver. The total amount of absorbed HCPs during 1 h of perfusion was 16.6 μmol, which was significantly higher than that of free Hyp (6.6 μmol). In addition, HCPs were also reliably detected in hepatic perfusate at the level higher than free Hyp. Thus, the results demonstrated that CH is absorbed predominantly as peptides, which subsequently enter systemic circulation. Size exclusion chromatography showed that perfusates include significant amount of HCPs larger than tripeptides, leading us to analyze these peptides in detail. Mass spectrometric analysis of intestinal perfusate finally identified three CH-derived peptides, which are surprisingly large as food-derived circulating peptides. Peptide quantitation by liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed that di- and tri-peptides, which are previously identified as major peptides in circulating blood, comprise only a part of HCPs in intestinal and liver perfusate. Finally, analysis of portal vein blood revealed that the larger peptides, such as pentadecapeptide identified in this study, could be absorbed in vivo. Taken all together, this study showed that peptides which are larger than tripeptide could reach to the circulation system after administration of CH, revealing previously unknown dynamics of absorption of CH.
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http://dx.doi.org/10.2220/biomedres.39.1DOI Listing
June 2018

A double-blind, placebo-controlled, randomised clinical study of the effect of pork collagen peptide supplementation on atherosclerosis in healthy older individuals.

Biosci Biotechnol Biochem 2018 May 15;82(5):893-895. Epub 2018 Feb 15.

Department of Geriatric Medicine and Neurology, Ehime University Graduate School of Medicine, Toon, Japan.

We examined whether baPWV could be affected by pork collagen peptide (CP) ingestion. Seventy subjects were randomized into two groups (2.5 g/day CP and 2.5 g/day placebo). A significant reduction in baPWV was observed in the CP group compared to the placebo group. This study demonstrated that pork CP may contribute to the prevention of atherosclerosis in elderly.
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http://dx.doi.org/10.1080/09168451.2018.1434406DOI Listing
May 2018

Peyer's Patches as a Portal for DNA Delivery by Lactococcus lactis in Vivo.

Biol Pharm Bull 2018 ;41(2):190-197

Microbiology and Immunology, Gifu Pharmaceutical University.

Application of food-grade Lactococcus lactis (L. lactis) as a safe delivery tool for DNA vaccines and therapeutic proteins has been well investigated. Although some studies showed that eukaryotic expression plasmids were transferred from L. lactis to enterocytes, the precise mechanism of the DNA transfer remains unknown. In this study, we generated an invasive L. lactis strain that expresses "murinized" Internalin A, an invasin of intracellular bacteria Listeria monocytogenes with two amino acid alterations for invasion into murine cells, and confirmed that this L. lactis strain delivered DNA in an invasin-dependent manner into a monolayer of epithelial cells polarized to mimic the gastrointestinal tract environment. Although invasive L. lactis inoculated orally can deliver DNA into enterocytes in the gastrointestinal tract of mice, the efficiency of DNA transfer was similar to that of non-invasive L. lactis strain, suggesting that the in vivo DNA transfer from L. lactis occurs invasin-independently. A ligated-intestinal loop assay, a method for a short-term culturing of the whole intestine filled with materials to evaluate the interaction of the materials with intestinal cells, demonstrated that both non-invasive and invasive L. lactis strains were present in the Peyer's patches of the small intestine. On the other hand, few L. lactis was detected in the non-Peyer's patch epithelial region. Thus, our observations lead us to speculate that DNA transfer from L. lactis occurs predominantly in the Peyer's patches in an invasin-independent manner.
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http://dx.doi.org/10.1248/bpb.b17-00657DOI Listing
August 2018

Knockdown of the mitochondria-localized protein p13 protects against experimental parkinsonism.

EMBO Rep 2018 03 25;19(3). Epub 2018 Jan 25.

Laboratory of Molecular Neuropharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka, Japan

Mitochondrial dysfunction in the nigrostriatal dopaminergic system is a critical hallmark of Parkinson's disease (PD). Mitochondrial toxins produce cellular and behavioural dysfunctions resembling those in patients with PD Causative gene products for familial PD play important roles in mitochondrial function. Therefore, targeting proteins that regulate mitochondrial integrity could provide convincing strategies for PD therapeutics. We have recently identified a novel 13-kDa protein (p13) that may be involved in mitochondrial oxidative phosphorylation. In the current study, we examine the mitochondrial function of p13 and its involvement in PD pathogenesis using mitochondrial toxin-induced PD models. We show that p13 overexpression induces mitochondrial dysfunction and apoptosis. p13 knockdown attenuates toxin-induced mitochondrial dysfunction and apoptosis in dopaminergic SH-SY5Y cells via the regulation of complex I. Importantly, we generate -deficient mice using the CRISPR/Cas9 system and observe that heterozygous knockout prevents toxin-induced motor deficits and the loss of dopaminergic neurons in the substantia nigra. Taken together, our results suggest that manipulating p13 expression may be a promising avenue for therapeutic intervention in PD.
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http://dx.doi.org/10.15252/embr.201744860DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5836091PMC
March 2018

Collagen-derived dipeptide prolyl-hydroxyproline promotes osteogenic differentiation through Foxg1.

Cell Mol Biol Lett 2017 1;22:27. Epub 2017 Dec 1.

Faculty of Pharmaceutical Sciences, Josai University, 1-1 Keyakidai, Sakado-shi, Saitama, 350-0295 Japan.

Prolyl-hydroxyproline (Pro-Hyp) is one of the major constituents of collagen-derived dipeptides. We previously reported that Pro-Hyp promotes the differentiation of osteoblasts by increasing Runx2, osterix and Col1α1 mRNA expression levels. Here, to elucidate the mechanism of Pro-Hyp promotion of osteoblast differentiation, we focus on the involvement of Foxo1 in osteoblast differentiation via Runx2 regulation and the role of Foxg1 in Foxo1 regulation. The addition of Pro-Hyp had no effect on MC3T3-E1 cell proliferation in Foxo1- or Foxg1-knockdown cells. In Foxo1-knockdown cells, the addition of Pro-Hyp increased ALP activity, but in Foxg1-knockdown cells, it had no effect on ALP activity. An enhancing effect of Pro-Hyp on the Runx2 and osterix expression levels was observed in Foxo1-knockdown cells. However, no enhancing effect of Pro-Hyp on osteoblastic gene expression was observed when Foxg1 was knocked down. These results demonstrate that Pro-Hyp promotes osteoblastic MC3T3-E1 cell differentiation and upregulation of osteogenic genes via Foxg1 expression.
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http://dx.doi.org/10.1186/s11658-017-0060-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5710072PMC
July 2018