Collagen-Derived Dipeptide Pro-Hyp Enhanced ATDC5 Chondrocyte Differentiation under Hypoxic Conditions.

Chondrocytes are surrounded by a lower oxygen environment than other well-vascularized tissues with higher oxygenation levels. Prolyl-hydroxyproline (Pro-Hyp), one of the final collagen-derived peptides, has been previously reported to be involved in the early stages of chondrocyte differentiation. However, whether Pro-Hyp can alter chondrocyte differentiation under physiological hypoxic conditions is still unclear. This study aimed to investigate whether Pro-Hyp affects the differentiation of ATDC5 chondrogenic cells under hypoxic conditions. The addition of Pro-Hyp resulted in an approximately 18-fold increase in the glycosaminoglycan staining area compared to the control group under hypoxic conditions. Moreover, Pro-Hyp treatment significantly upregulated the expression of SOX9, Col2a1, Aggrecan, and MMP13 in chondrocytes cultured under hypoxic conditions. These results demonstrate that Pro-Hyp strongly promotes the early differentiation of chondrocytes under physiological hypoxic conditions. Therefore, Pro-Hyp, a bioactive peptide produced during collagen metabolism, may function as a remodeling factor or extracellular matrix remodeling signal that regulates chondrocyte differentiation in hypoxic cartilage.

High Incidence of Hippocampal Abnormalities in Pediatric Patients with Congenital Cytomegalovirus Infection.

BACKGROUND: Congenital cytomegalovirus (CMV) infection exhibits polymicrogyria, intracranial calcification, white matter lesions, and several types of intracranial lesions on magnetic resonance imaging (MRI), in addition to various developmental disorders and epilepsies. However, little is known on the presence of hippocampal abnormality in this affliction. The aim of this study is to clarify the incidence of hippocampal abnormality in congenital CMV infection. METHODS: Seventeen children diagnosed as having congenital CMV infection along with 17 age-matched pediatric controls were retrospectively evaluated by brain MRI and clinical review. The measurement data were obtained from conventional coronal sections in this retrospective study. Hippocampal malrotation (HIMAL) was defined as a hippocampal diameter ratio (i.e., the ratio of the height and width of the hippocampus) of >0.92. RESULTS: Hippocampal diameter ratios were significantly higher in the congenital CMV infection group (0.99 [range: 0.70-1.58] on the right side and 0.85 [range: 0.66-1.39] on the left side) than in controls (0.71 [range: 0.58-0.91] and 0.70 [range: 0.50-1.00], respectively). HIMAL was present in 17 of 34 hippocampi (50%) in the congenital CMV infection group and 1 of 34 hippocampi (2.9%) in controls. No correlations were detected between HIMAL and intelligence quotient/developmental quotient or the occurrences of autism spectrum disorder or epilepsy. CONCLUSION: This study is the first to demonstrate the incidence of hippocampal abnormality to be significantly higher in congenital CMV infection patients than in age-matched controls. Further study is necessary to clarify the associations of HIMAL with other clinical and developmental features.

Collagen-derived dipeptide prolyl hydroxyproline directly binds to Foxg1 to change its conformation and inhibit the interaction with Runx2.

Collagen-derived dipeptide prolyl hydroxyproline (Pro-Hyp) is involved in the proliferation and differentiation of various types of cultured cells. To elucidate the mechanism underlying Pro-Hyp actions during osteoblast differentiation, we hypothesized that proteins binding to Pro-Hyp serve to mediate cellular signaling, affecting Runx2 expression. Recently, we performed the characterization of Foxg1, that it enhances Runx2 expression in the presence of Pro-Hyp. Our findings indicate that Pro-Hyp directly binds to the Foxg1 recombinant protein, which leads to the structural alteration of the Foxg1 protein. In addition, Foxg1 appears to interact with Runx2 in the absence of Pro-Hyp, with Pro-Hyp disrupting the interaction between Foxg1 and Runx2. Collectively, our results indicate that the Pro-Hyp bound Foxg1 alters the structured conformation of Foxg1, resulting in conformational changes that lead to dissociation from Runx2. These novel findings suggest that during osteoblast differentiation, Pro-Hyp mediates Runx2 activity though directly binding to Foxg1 and increases Runx2 expression. Abbreviations: CPT: collagen peptide; GST: Glutathione S-transferase; PAGE: Polyacrylamide gel electrophoresis; PCR: Polymerase chain reaction; prolyl hydroxyproline: Pro-Hyp.

Collagen-derived dipeptide prolyl-hydroxyproline cooperates with Foxg1 to activate the PGC-1α promoter and induce brown adipocyte-like phenotype in rosiglitazone-treated C3H10T1/2 cells.

Background: The global obesity epidemic is a significant public health issue, often leading to metabolic disorders such as diabetes and cardiovascular diseases. Collagen peptides (CP) and their bioactive component, Prolyl-hydroxyproline (Pro-Hyp), have shown potential in reducing adipocyte size, with unclear mechanisms concerning brown adipocyte differentiation. Methods: We investigated the effects of Pro-Hyp on the differentiation of brown adipocytes in C3H10T1/2 mesenchymal stem cells, focusing on its impact on adipocyte size, gene expression related to brown fat function, and mitochondrial activity. Results: Pro-Hyp treatment decreased adipocyte size and upregulated brown fat-specific genes, including C/EBPα, PGC-1α, and UCP-1. Remarkably, it did not alter PPARγ expression. Pro-Hyp also elevated mitochondrial activity, suggesting enhanced brown adipocyte functionality. A Pro-Hyp responsive element was identified in the PGC-1α gene promoter, which facilitated the binding of the Foxg1 transcription factor, indicating a novel regulatory mechanism. Conclusion: Pro-Hyp promotes brown adipocyte differentiation, potentially offering a therapeutic strategy for obesity management. This study provides a molecular basis for the anti-obesity effects of CP, although further in vivo studies are needed to confirm these findings and to investigate the potential impact on beige adipocyte differentiation.

Preventive Effects of Collagen-Derived Dipeptide Prolyl-Hydroxyproline against Dexamethasone-Induced Muscle Atrophy in Mouse C2C12 Skeletal Myotubes.

Glucocorticoids, commonly used to manage inflammatory diseases, can induce muscle atrophy by accelerating the breakdown of muscle proteins. This research delves into the influence of Prolyl-hydroxyproline (Pro-Hyp), a collagen-derived peptide, on muscle atrophy induced with dexamethasone (DEX), a synthetic glucocorticoid, in mouse C2C12 skeletal myotubes. Exposure to DEX (10 µM) for 6 days resulted in a decrease in myotube diameter, along with elevated mRNA and protein levels of two muscle-atrophy-related ubiquitin ligases, muscle atrophy F-box (MAFbx, also known as atrogin-1) and muscle ring finger 1 (MuRF-1). Remarkably, treatment with 0.1 mM of Pro-Hyp mitigated the reduction in myotube thickness caused by DEX, while promoting the phosphorylation of Akt, mammalian target of rapamycin (mTOR), and forkhead box O3a (Foxo3a). This led to the inhibition of the upregulation of the ubiquitin ligases atrogin-1 and MuRF-1. These findings indicate the potential significance of Pro-Hyp as a promising therapeutic target for countering DEX-induced muscle atrophy.

Successful treatment of congenital myasthenic syndrome caused by a novel compound heterozygous variant in RAPSN.

BACKGROUND: Congenital myasthenic syndrome (CMS) is a clinically and genetically heterogeneous neuromuscular disorder characterized by muscle weakness and caused by mutations in more than 35 different genes. This condition should not be overlooked as a subset of patients with CMS are treatable. However, the diagnosis of CMS is often difficult due to the broad variability in disease severity and course. CASE REPORT: A five-year-old boy without remarkable family history was born with marked general muscle hypotonia and weakness, respiratory insufficiency, anomalies, and multiple joint contractures. Congenital myopathy was suspected based upon type 1 fiber predominance on muscle biopsy. However, he was diagnosed with CMS at age 4 years when his ptosis and ophthalmoplegia were found to be improved by edrophonium chloride and repetitive nerve stimulation showed attenuation of compound muscle action potentials. An exome sequencing identified a compound heterozygous missense variant of c.737C > T (p.A246V) and a novel intronic insertion c.1166 + 4_1166 + 5insAAGCCCACCAC in RAPSN. RT-PCR analysis which showed the skipping of exon 7 in a skeletal muscle sample confirmed that the intronic insertion was pathogenic. His myasthenic symptoms were remarkably improved by pyridostigmine. CONCLUSION: The patient's diagnosis of CMS was confirmed by exome sequencing, and RT-PCR revealed that the skipping of exon 7 in RAPSN was caused by a novel intronic insertion. The genetic information uncovered in this case should therefore be added to the collection of tools for diagnosing and treating CMS.

Stimulation of the Runx2 P1 promoter by collagen-derived dipeptide prolyl-hydroxyproline bound to Foxg1 and Foxo1 in osteoblasts.

Collagen-derived dipeptide prolyl-hydroxyproline (Pro-Hyp) directly binds to the forkhead box g1 (Foxg1) protein and causes it to undergo structural alteration. Pro-Hyp also promotes the production of a regulator of osteoblast differentiation, Runt-related transcription factor 2 (Runx2), through Foxg1, inducing osteoblast differentiation. In addition, Pro-Hyp disrupts the interaction between Foxg1 and Runx2, and Foxg1 appears to interact with Runx2 in the absence of Pro-Hyp. To elucidate the mechanism of Pro-Hyp that promotes osteoblast differentiation, we investigated whether Pro-Hyp regulates the Runx2 P1 promoter together with Foxg1. The present study revealed that Pro-Hyp is taken up by osteoblastic cells via the solute carrier family 15 member (Slc15a) 4. In the presence of Pro-Hyp, Runx2 is translocated from the nucleus to the cytoplasm and Foxg1 is translocated from the cytoplasm to the nucleus. We also found that Pro-Hyp promoted the interaction between Forkhead box o1 (Foxo1) and Runx2 and the dissociation of Foxg1 from Runx2. Moreover, we identified the Pro-Hyp response element in the Runx2 distal P1 promoter at nt -375 to -316, including the Runx2 binding sites and Fox core sequence. In the presence of Pro-Hyp, Runx2 is dissociated from the Pro-Hyp response element in the Runx2 distal P1 promoter. Subsequently, Foxg1 and Foxo1 activated the Runx2 promoter by binding to the Pro-Hyp response element. In summary, we delineated the mechanism by which Pro-Hyp stimulates the bone-related Runx2 distal P1 promoter activity in osteoblastic cells through Foxg1, Foxo1, and Runx2.

Monitoring urinary collagen metabolite changes following collagen peptide ingestion and physical activity using ELISA with anti active collagen oligopeptide antibody.

Active collagen oligopeptides (ACOP) are bioactive collagen-derived peptides detected by a recently-established ELISA. To facilitate studies of the function and metabolism of these products, this study aims to determine which of these peptides is recognized by a novel anti-ACOP antibody used in this ELISA. We then investigate the effect of collagen peptide (CP) ingestion and exercise on urinary ACOP concentrations in a cohort of university student athletes using colorimetric, LC-MS/MS, and ELISA. We observed that the antibody showed strong cross-reactivity to Pro-Hyp and Gly-Pro-Hyp and weak cross-reactivity to commercial CP. CP ingestion increased the urinary level of ACOP over time, which correlated highly with urinary levels of peptide forms of Hyp and Pro-Hyp. Physical activity significantly decreased the urinary ACOP level. This study demonstrates changes in urinary ACOP following oral CP intake and physical activity using ELISA with the novel anti-ACOP antibody. Thus, ACOP may be useful as a new biomarker for collagen metabolism.

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

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.

The enhancement of fluorescence quantum yields of anilino naphthalene sulfonic acids by inclusion of various cyclodextrins and cucurbit[7]uril.

The association constants (K) for the inclusion complexation of four kinds of cyclodextrins (CDs (ß- and γ-), 2,6-di-O-methylated ß-CD, and 2,3,6-tri-O-methylated ß-CD) and cucurbit[7]uril (CB[7]) with 1,8- and 2,6-anilinonaphthalene sulfonic acids (ANSs) were determined from fluorescence spectra enhanced by inclusion. Various CDs and CB[7] form stable 1:1 inclusion complexes with 1,8- and 2,6-ANSs: K=80-11700 M(-1) for 2,6-ANS and 50-195 M(-1) for 1,8-ANS. The high stability of the inclusion complexes of 2,6-ANS with CB[7] and 2,6-di-O-methylated ß-CD is shown. Further, we determined the fluorescence quantum yields (Φ values) for the inclusion complexes of ANSs by using a fluorescence spectrophotometer equipped with a half-moon unit. The Φ values of 1,8- and 2,6-ANSs were largely enhanced by the inclusion of methylated ß-CDs and did not correlate with the degree of stability (K) of the inclusion complexes. We characterized the structures of the inclusion complexes by 2D ROESY-NMR measurements. In addition, the microenvironmental polarity inside the hydrophobic CD and CB[7] cavities was evaluated using the fluorescence probe 2,6-ANS. Based on the emission mechanism and the aspect of inclusion in a hydrophobic cavity, we have suggested that the microenvironmental polarity and viscosity for the excited state of ANS plays an important role for the Φ values of inclusion complexes.