Evaluation of Monomer-containing Isocyanate Groups on Stabilizing Demineralized Dentin Matrix Against Bond Interface Degradation.

PURPOSE: To evaluate the effect of urethane methacrylate precursor (UMP) on the enzymatic resistance of demineralized dentin (DD) matrices. MATERIALS AND METHODS: Experimental treatments containing 0 (control), 1, and 5 mmol/L UMP dissolved in an acetone (Ace) solution were formulated. Dentin matrix specimens were demineralized in vitro and immersed in the experimental treatments for 1 h. The treated specimens were then stored in 0.1 mg/mL collagenase solution for 24 h, after which their dry mass loss and hydroxyproline (HYP) release were assessed. The swelling ratios of specimens in each group were also evaluated. The interaction between UMP and the dentin matrix was observed using field-emission scanning electron microscopy (FE-SEM). Endogenous enzyme activity in dentin was evaluated using confocal laser scanning microscopy (CLSM). RESULTS: Compared with the other treatment groups, treatment with 1 mM and 5 mM UMP-Ace significantly decreased the dry mass loss, HYP release and swelling ratio of the DD matrix (p < 0.05). FE-SEM and CLSM observations showed that treatment with UMP-Ace protected the structure of the dentin matrix and decreased porosity within the dentin-collagen network. CONCLUSION: Treatment with 1 mM and 5 mM UMP-Ace protects DD matrix against collagenase degradation and may be clinically useful for improving the durability of the hybrid layer.

Upcycled canola meal extract mitigates UVB-induced skin wrinkling by regulating photoaging-related biomarkers in hairless mice.

Canola meal, a by-product of processing canola into oil, reportedly contains high amounts of phenolic compounds and proteins. However, as canola meal is primarily used as feed for livestock, advances in multiple research fields are required to broaden its potential applications. Photoaging is caused by continuous exposure to ultraviolet (UV) radiation from sunlight. UV radiation generates reactive oxygen species and destroys collagen in the skin, thickening the epidermis, reducing elasticity, and causing wrinkles. We hypothesized that canola meal extract (CME) can mitigate the damage to skin associated with wrinkles induced by exposure to UVB radiation. To evaluate the anti-wrinkle effect, we administered CME orally to 40 female Hos:HR-1 hairless mice divided into 5 groups: (1) control mice, (2) a UVB group, and (3-5) CME-treated groups (CME-250, 500, and 1000 mg/kg body weight/day, respectively). All groups except the controls were irradiated with UVB 3 times a week to create wrinkles due to photoaging. CME administration inhibited the increase of the number, mean length, and mean depth of wrinkles induced by UVB radiation as assessed using a skin replica. Histopathological image analysis revealed that CME administration resulted in a decrease in epidermal thickness and an increase in collagen content, while increasing catalase activity and hydroxyproline content in skin tissues. CME administration inhibited the phosphorylation of mitogen-activated protein kinase and decreased the production of collagenase and gelatinase. These results suggest that CME, an upcycled material, has the potential to develop into a healthful and functional food ingredient with anti-wrinkling effects.

Effects of hydroxyproline supplementation in low fish meal diets on collagen synthesis, myofiber development and muscular texture of juvenile Pacific white shrimp (Litopenaeus vannamei).

This experiment aimed to evaluate the impact of dietary hydroxyproline (Hyp) supplementation on the muscle quality of juvenile Pacific white shrimp (Litopenaeus vannamei) fed a low fishmeal diet. Six formulated diets included one high fishmeal (HF; 25% fishmeal content) and five low fishmeal diets (10% fishmeal content) with 0%, 0.2%, 0.4%, 0.6% and 0.8% Hyp (LF0, LF2, LF4, LF6 and LF8, respectively). Each diet was assigned to four replicates, and 40 shrimp (0.32 ± 0.00 g) per replicate were fed four times a day for 8 weeks. Dietary Hyp supplementation had little effects on growth performance, but increased the contents of Hyp, prolyl 4-hydroxylases (P4Hs), and collagen. The meat yield, springiness, hardness, chewiness, and cohesiveness of muscle were the highest in the LF4 group among the low fishmeal groups (P < 0.05). Cooking loss and freezing loss of muscle were the lowest in the LF4 group (P < 0.05). Dietary supplementation with 0.4% Hyp increased the myofiber density and decreased the myofiber diameter of muscle (P < 0.05). Supplementation of Hyp in the diet up-regulated the mRNA expression of smyhc5, smyhc15, col1a1, col1a2, igf-1f, tgf-ß and tor and down-regulated the mRNA expression of smyhc 1, smyhc 2, smyhc 6a (P < 0.05). Supplementation of Hyp in the diet up-regulated the protein expression of P-4E-BP1, P-AKT, AKT and P-AKT/AKT (P < 0.05). These results suggested that the addition of 0.4% Hyp to low fishmeal diets improved the muscle quality of L. vannamei.

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.

Elucidating the modulatory role of dietary hydroxyproline on the integrity and functional performance of the intestinal barrier in early-weaned piglets: A comprehensive analysis of its interplay with the gut microbiota and metabolites.

Piglets receive far less hydroxyproline (Hyp) from a diet after weaning than they obtained from sow's milk prior to weaning, suggesting that Hyp may play a protective role in preserving intestinal mucosal homeostasis. This study aimed to evaluate the effect of Hyp on intestinal barrier function and its associated gut microbiota and metabolites in early-weaned piglets. Eighty weaned piglets were divided into four groups and fed diets containing different Hyp levels (0 %, 0.5 %, 1 %, or 2 %) for 21 days. Samples, including intestinal contents, tissues, and blood, were collected on day 7 for analysis of microbial composition, intestinal barrier function, and metabolites. We demonstrated that dietary supplementation with 2 % Hyp improved the feed conversion ratio and reduced the incidence of diarrhea in early-weaned piglets compared to the control group. Concurrently, Hyp enhanced intestinal barrier function by facilitating tight junction protein (zonula occludens (ZO)-1 and occludin) expression and mucin production in the jejunal, ileal, and colonic mucosas. It also improved mucosal immunity (by increasing the amount of secretory IgA (sIgA) and the ratio of CD4+/CD8+ T lymphocytes and decreasing NF-κB phosphorylation) and increased antioxidant capacity (by raising total antioxidant capacity (T-AOC) and glutathione levels) in the intestinal mucosa. In addition, Hyp supplementation resulted in an increase in the levels of glycine, glutathione, and glycine-conjugated bile acids, while decreasing the concentrations of cortisol and methionine sulfoxide in plasma. Intriguingly, piglets fed diet containing Hyp exhibited a remarkable increase in the abundance of probiotic Enterococcus faecium within their colonic contents. This elevation occurred alongside an attenuation of pro-inflammatory responses and an enhancement in intestinal barrier integrity. Further, these changes were accompanied by a rise in anti-inflammatory metabolites, specifically glycochenodeoxycholic acid and guanosine, along with a suppression of pro-inflammatory lipid peroxidation products, including (12Z)-9,10-dihydroxyoctadec-12-enoic acid (9,10-DHOME) and 13-L-hydroperoxylinoleic acid (13(S)-HPODE). In summary, Hyp holds the capacity to enhance the intestinal barrier function in weaned piglets; this effect is correlated with changes in the gut microbiota and metabolites. Our findings provide novel insights into the role of Hyp in maintaining gut homeostasis, highlighting its potential as a dietary supplement for promoting intestinal health in early-weaned piglets.

Development of a two-dimensional LC-MS/MS system for the determination of proline and 4-hydroxyproline enantiomers in biological and food samples.

A two-dimensional LC-MS/MS system has been developed for the enantioselective determination of proline (Pro), cis-4-hydroxyproline (cis-4-Hyp) and trans-4-hydroxyproline (trans-4-Hyp) in a variety of biological samples. The amino acids were pre-column derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), and the NBD-derivatives were separated by a reversed-phase column (Singularity RP18) as their D plus L mixtures in the first dimension. The collected target fractions were then introduced into the second dimension where the enantiomers were separated by a Pirkle-type enantioselective column (Singularity CSP-001S) and determined by a tandem mass spectrometer (Triple Quad™ 5500). The method was validated by the standard amino acids and also by human plasma, and sufficient results were obtained for the calibration, precision and accuracy. The method was applied to human plasma and urine, bivalve tissues and fermented food/beverages. D-Pro was widely found in the human physiological fluids, bivalves and several fermented products. Although trans-4-D-Hyp was not found in all the tested samples, cis-4-D-Hyp was present in human urine and tissues of the ark shell, and further studies focusing on the origin and physiological significance of these D-enantiomers are expected.

Investigation of the effects of white tea on liver fibrosis: An experimental animal model.

Liver fibrosis is a common, progressive disease that affects millions of patients worldwide. In this study, it was aimed at investigating the effect of white tea on liver fibrosis in an in-vivo environment by creating an experimental liver fibrosis model on rats. In this study, an experimental liver fibrosis model was created with carbon tetrachloride (CCl4) in Sprague-Dawley rats to investigate the effect of white tea on liver fibrosis. Rats are treated with CCl4 (1 mL/kg) to constitute the liver fibrosis model. White tea was given ad libitum with drinking water. As a result of the study, liver tissue hydroxyproline levels were found to be significantly lower (p = .001) in the white tea group. Histopathologically, it was found that the liver tissue histopathological damage score (LHDS) and fibrosis scoring were significantly lower (p < .001) in the white tea group. However, although it was not statistically significant in the group given white tea, compared with the fibrosis group, it was found that the malondialdehyde (MDA) level in the liver tissues was lower, the glutathione (GSH) level was higher, and the serum alanine aminotransferase (ALT) levels were lower. The study explained the effect of white tea on liver fibrosis and suggested that white tea might be beneficial in reducing the progression of liver fibrosis.

Effect of collagen cross-linking agents on the depth of penetration of bioceramic sealer and release of hydroxyproline: An in vitro study.

Context: During endodontic treatment, sealers seal off dentinal tubules and prevent microbial attack. Bioceramic sealers have excellent bioactivity, but its high alkalinity is found to have detrimental effects on radicular collagen. Collagen cross linkers have the ability to chemically modify collagen and can prevent the detrimental effects of the sealer. Aim: This research was aimed to assess the effect of collagen cross-linking agents on the integrity of radicular collagen matrix and depth of penetration of sealer. Materials and Methods: Mandibular premolars (n = 48) were taken. Teeth were decoronated; canals were prepared till ProTaper size F2 and were irrigated with 5 mL of 2.5% NaOCl, followed by 3 mL of 17% ethylenediaminetetraacetic acid between instrumentation and finally rinsed with saline following which teeth were divided into three groups based on the surface treatments: Group 1: 6.5% proanthocyanin (PA), Group 2: chlorhexidine (CHX), and Group 3: saline. Teeth were obturated using gutta-percha and bioceramic sealer and stored in artificial saliva. Hydroxyproline (HYP) release was assessed after 14 and 21 days using spectrophotometer. Sealer penetration was assessed using the scanning electron microscope. Statistical Analysis: Wilcoxon signed-rank test and Kruskal-Wallis test for release of HYP and paired t-test and ANOVA for sealer penetration were performed. Results: Significantly lower release of HYP was seen in proanthocyanin-treated group. Sealer penetration was better for both the proanthocyanin- and CHX-treated groups when compared to saline. Conclusion: Surface treatment with collagen cross-linkers caused a decrease in the amount of HYP released, indicating lesser degradation of collagen. Sealer penetration was better due to the removal of smear layer following the surface treatments.

GC-MS analysis of 4-hydroxyproline: elevated proline hydroxylation in metformin-associated lactic acidosis and metformin-treated Becker muscular dystrophy patients.

Metformin (N,N-dimethylbiguanide), an inhibitor of gluconeogenesis and insulin sensitizer, is widely used for the treatment of type 2 diabetes. In some patients with renal insufficiency, metformin can accumulate and cause lactic acidosis, known as metformin-associated lactic acidosis (MALA, defined as lactate ≥ 5 mM, pH < 7.35, and metformin concentration > 38.7 µM). Here, we report on the post-translational modification (PTM) of proline (Pro) to 4-hydroxyproline (OH-Pro) in metformin-associated lactic acidosis and in metformin-treated patients with Becker muscular dystrophy (BMD). Pro and OH-Pro were measured simultaneously by gas chromatography-mass spectrometry before, during, and after renal replacement therapy in a patient admitted to the intensive care unit (ICU) because of MALA. At admission to the ICU, plasma metformin concentration was 175 µM, with a corresponding lactate concentration of 20 mM and a blood pH of 7.1. Throughout ICU admission, the Pro concentration was lower compared to healthy controls. Renal excretion of OH-Pro was initially high and decreased over time. Moreover, during the first 12 h of ICU admission, OH-Pro seems to be renally secreted while thereafter, it was reabsorbed. Our results suggest that MALA is associated with hyper-hydroxyprolinuria due to elevated PTM of Pro to OH-Pro by prolyl-hydroxylase and/or inhibition of OH-Pro metabolism in the kidneys. In BMD patients, metformin, at the therapeutic dose of 3 × 500 mg per day for 6 weeks, increased the urinary excretion of OH-Pro suggesting elevation of Pro hydroxylation to OH-Pro. Our study suggests that metformin induces specifically the expression/activity of prolyl-hydroxylase in metformin intoxication and BMD.

Structure-guided discovery of protein and glycan components in native mastigonemes.

Mastigonemes, the hair-like lateral appendages lining cilia or flagella, participate in mechanosensation and cellular motion, but their constituents and structure have remained unclear. Here, we report the cryo-EM structure of native mastigonemes isolated from Chlamydomonas at 3.0 Å resolution. The long stem assembles as a super spiral, with each helical turn comprising four pairs of anti-parallel mastigoneme-like protein 1 (Mst1). A large array of arabinoglycans, which represents a common class of glycosylation in plants and algae, is resolved surrounding the type II poly-hydroxyproline (Hyp) helix in Mst1. The EM map unveils a mastigoneme axial protein (Mstax) that is rich in heavily glycosylated Hyp and contains a PKD2-like transmembrane domain (TMD). Mstax, with nearly 8,000 residues spanning from the intracellular region to the distal end of the mastigoneme, provides the framework for Mst1 assembly. Our study provides insights into the complexity of protein and glycan interactions in native bio-architectures.

Chromatographic separation by RPLC-ESI-MS of all hydroxyproline isomers for the characterization of collagens from different sources.

During collagen biosynthesis, proline is post-translationally converted to hydroxyproline by specific enzymes. This amino acid, unique to collagen, plays a crucial role in stabilizing the collagen triple helix structure and could serve as an important biomarker for collagen content and quality analysis. Hydroxyproline has four isomers, depending on whether proline is hydroxylated at position 4 or 3 and on whether the cis- or trans- conformation is formed. Moreover, as extensive hydrolysis of collagen is required for its amino acid analysis, epimerization may also occur, although to a lesser extent, giving a total of eight possible isomers. The aim of the present study was to develop a reversed-phase high-performance liquid chromatography-UV-mass spectrometry (RPLC-UV-MS) method for the separation and quantification of all eight hydroxyproline isomers. After the chiral derivatization of the hydroxyproline isomers with Nα-(2,4-dinitro-5-fluorophenyl)-L-valinamide (L-FDVA), to enable their UV detection, the derivatized diastereoisomers were separated by testing different C18 column technologies and morphologies and optimizing operative conditions such as the mobile phase composition (solvent, additives), elution mode, flow rate and temperature. Baseline resolution of all eight isomers was achieved on a HALO® ES-C18 reversed-phase column (150×1.5 mm, 2.7 µm, 160 Å) using isocratic elution and MS-compatible mobile phase. The optimized method was validated for the quantification of hydroxyproline isomers and then applied to different collagen hydrolysates to gain insight and a deeper understanding of hydroxyproline abundances in different species (human, chicken) and sources (native, recombinant).

Mastigoneme structure reveals insights into the O-linked glycosylation code of native hydroxyproline-rich helices.

Hydroxyproline-rich glycoproteins (HRGPs) are a ubiquitous class of protein in the extracellular matrices and cell walls of plants and algae, yet little is known of their native structures or interactions. Here, we used electron cryomicroscopy (cryo-EM) to determine the structure of the hydroxyproline-rich mastigoneme, an extracellular filament isolated from the cilia of the alga Chlamydomonas reinhardtii. The structure demonstrates that mastigonemes are formed from two HRGPs (a filament of MST1 wrapped around a single copy of MST3) that both have hyperglycosylated poly(hydroxyproline) helices. Within the helices, O-linked glycosylation of the hydroxyproline residues and O-galactosylation of interspersed serine residues create a carbohydrate casing. Analysis of the associated glycans reveals how the pattern of hydroxyproline repetition determines the type and extent of glycosylation. MST3 possesses a PKD2-like transmembrane domain that forms a heteromeric polycystin-like cation channel with PKD2 and SIP, explaining how mastigonemes are tethered to ciliary membranes.

Gum Arabic-Derived Hydroxyproline-Rich Peptides Stimulate Intestinal Nonheme Iron Absorption via HIF2α-Dependent Upregulation of Iron Transport Proteins.

The stimulation of host iron absorption is a promising antianemia strategy adjunctive/alternative to iron intervention. Here, gum arabic (GA) containing 3.14 ± 0.56% hydroxyproline-rich protein with repetitive X-(Pro/Hyp)n motifs was found to increase iron reduction, uptake, and transport to upregulate duodenal cytochrome b (Dcytb), divalent metal transporter 1 (DMT1), ferroportin, and hephaestin to inhibit hypoxia-inducible factor (HIF) prolyl hydroxylase (PHD) and to stabilize HIF2α in polarized Caco-2 cell monolayers in a dose-dependent manner, and this was dependent on its protein fraction, rather than the polysaccharide fraction. Three abundant GA-derived hydroxyproline-containing dipeptides of Hyp-Hyp, Pro-Hyp, and Ser-Hyp were detected by liquid chromatography-mass spectrometry in the lysates of polarized Caco-2 cell monolayers at the maximum levels of  0.167 ± 0.021, 0.134 ± 0.017, and 0.089 ± 0.015 µg/mg of protein, respectively, and showed desirable docking affinity energy values of -7.53, - 7.91, and -7.39 kcal/mol, respectively, against human PHD3. GA-derived peptides also acutely increased duodenal HIF2α stability and Dcytb, DMT1, ferroportin, and hephaestin transcription in rats (P < 0.05). Overall, GA-derived hydroxyproline-rich peptides stimulated intestinal iron absorption via PHD inhibition, HIF2α stabilization, and subsequent upregulation of iron transport proteins.

Polyphenolic characterization and biological assessment of Acacia nilotica (L.) wild. Ex delilie: An In vitro and In vivo appraisal of wound healing potential.

ETHNOPHARMACOLOGICAL RELEVANCE: Acacia nilotica (L.) Wild. Ex Delilie is a shrub with significant ethnomedicinal stature. Therefore, in the undertaken study, its wound healing attributes are determined. AIM OF THE STUDY: The current study provided evidence of the traditional use of A. nilotica species and conferred A. nilotica bark extract as a potent candidate for wound healing agents. MATERIALS & METHODS: A. nilotica leaves extract (ANL-E); A. nilotica bark extract (ANB-E), and A. nilotica stem extract (ANS-E) were prepared using methanol-chloroform (1:1). Phytochemical analysis was performed using gallic acid equivalent (GAE) total phenolic content (TPC), quercetin equivalent (QE) total flavonoid content (TFC) assays and High-performance liquid chromatography (HPLC). In vitro antioxidant potential (free radical scavenging activity (FRSA), total antioxidant capacity (TAC), and ferric reducing antioxidant power (FRAP) assay), antibacterial activity (broth microdilution method) and hemolytic analysis was carried out. Wound healing proficiency of ANB-E was determined by wound excision model followed by estimating hydroxyproline content and endogenous antioxidant markers. RESULTS: Maximum phenolic and flavonoid content were depicted by ANB-E i.e., 50.9 ± 0.34 µg gallic acid equivalent/mg extract and 28.7 ± 0.13 µg quercetin equivalent/mg extract, respectively. HPLC analysis unraveled the presence of a significant amount of catechin in ANL-E, ANB-E and ANS-E (54.66 ± 0.02, 44.9 ± 0.004 and 31.36 ± 0.02 µg/mg extract) respectively. Highest percent free radical scavenging activity, total antioxidant capacity, and ferric reducing action power (i.e., 93.3 ± 0.42 %, 222.10 ± 0.76, and 222.86 ± 0.54 µg ascorbic acid equivalent/mg extract) were exhibited by ANB-E. Maximum antibacterial potential against Staphylococcus aureus was exhibited by ANB-E (MIC 12.5 µg/ml). Two of the extracts i.e., ANL-E and ANB-E were found biocompatible with less than 5 % hemolytic potential. Based upon findings of in vitro analysis, ANB-E (10, 5, and 2.5 % w/w, C1, C2, and C3, respectively) was selected for evaluating its in vivo wound healing potential. Maximum contraction of wound area and fastest epithelization i.e., 98 ± 0.05 % and 11.2 ± 1.00 (day) was exhibited by C1. Maximum hydroxyproline content, glutathione, catalase, and peroxidase were demonstrated by C1 i.e., 15.9 ± 0.52 µg/mg, 9.3 ± 0.17 mmol/mg, 7.2 ± 0.17 and 6.2 ± 0.14 U/mg, respectively. Maximal curbed lipid peroxidation i.e., 0.7 ± 0.15 mmol/mg was also depicted by C1. CONCLUSIONS: In a nutshell, the current investigation endorsed the wound healing potential of ANB-E suggesting it to be an excellent candidate for future studies.

An improved Simon reaction method to discriminate between methamphetamine and false-positive substances.

The conventional methamphetamine (MA) detection method using the Simon reaction can be affected by false positives owing to compounds similar to aliphatic secondary amines. In this study, we examined the new Simon reaction to improve the qualitative accuracy of MA detection to discriminate substances that give false positives in a conventional Simon reaction. After the conventional Simon reaction for MA and false positives (N-isopropylbenzylamine (NIP-BA), N-methylbenzylamine (NMe-BA), L-proline (Pro), and L-hydroxyproline (HYP)), which are colored blue, di-tert-butyl dicarbonate (t-Boc) reagent was added, and color tone changes were observed. When t-Boc was added to the false positives (NIP-BA, NMe-BA, Pro, and HYP), the colors of MA, Pro, and HYP changed to purple; NIP-BA changed to blue; and NMe-BA changed to light pink after 3 min. These results suggested that MA can be differentiated from NIP-BA and NMe-BA. Furthermore, the solid-phase chromogenic method was examined, and it was confirmed that MA could be differentiated from Pro and HYP. The method developed in this study should increase the accuracy of MA appraisal at crime scenes and contribute to the reduction of misclassifications arising from false-positive substances.

Dimethyl α-Ketoglutarate Promotes the Synthesis of Collagen and Inhibits Metalloproteinases in HaCaT Cells.

We observed that treatment with dimethyl α-ketoglutarate (DMK) increased the amount of intracellular α-ketoglutarate significantly more than that of α-ketoglutarate in HaCaT cells. DMK also increased the level of intracellular 4-hydroxyproline and promoted the production of collagen in HaCaT cells. In addition, DMK decreased the production of collagenase and elastase and down-regulated the expression of selected matrix metalloproteinases (MMPs), such as MMP-1, MMP-9, MMP-10, and MMP-12, via transcriptional inhibition. The inhibition of MMPs by DMK was mediated by the suppression of the IL-1 signaling cascade, leading to the attenuation of ERK1/2 phosphorylation and AP-1 transactivation. Our study results illustrate that DMK, an alkylated derivative of α-ketoglutarate, increased the level of 4-hydroxyproline, promoted the production of collagen, and inhibited the expression of selected MMPs by affecting the IL-1 cascade and AP-1 transactivation in HaCaT cells. The results suggest that DMK might be useful as an anti-wrinkle ingredient.

Glyoxylate reductase: Definitive identification in human liver mitochondria, its importance for the compartment-specific detoxification of glyoxylate.

Glyoxylate is a key metabolite generated from various precursor substrates in different subcellular compartments including mitochondria, peroxisomes, and the cytosol. The fact that glyoxylate is a good substrate for the ubiquitously expressed enzyme lactate dehydrogenase (LDH) requires the presence of efficient glyoxylate detoxification systems to avoid the formation of oxalate. Furthermore, this detoxification needs to be compartment-specific since LDH is actively present in multiple subcellular compartments including peroxisomes, mitochondria, and the cytosol. Whereas the identity of these protection systems has been established for both peroxisomes and the cytosol as concluded from the deficiency of alanine glyoxylate aminotransferase (AGT) in primary hyperoxaluria type 1 (PH1) and glyoxylate reductase (GR) in PH2, the glyoxylate protection system in mitochondria has remained less well defined. In this manuscript, we show that the enzyme glyoxylate reductase has a bimodal distribution in human embryonic kidney (HEK293), hepatocellular carcinoma (HepG2), and cervical carcinoma (HeLa) cells and more importantly, in human liver, and is actively present in both the mitochondrial and cytosolic compartments. We conclude that the metabolism of glyoxylate in humans requires the complicated interaction between different subcellular compartments within the cell and discuss the implications for the different primary hyperoxalurias.

Collagen prolyl 4-hydroxylase isoenzymes I and II have sequence specificity towards different X-Pro-Gly triplets.

Collagen biosynthesis requires several co- and post-translational modifications of lysine and proline residues to form structurally and functionally competent collagen molecules. Formation of 4-hydroxyproline (4Hyp) in Y-position prolines of the repetitive -X-Y-Gly- sequences provides thermal stability for the triple-helical collagen molecules. 4Hyp formation is catalyzed by a collagen prolyl 4-hydroxylase (C-P4H) family consisting of three isoenzymes. Here we identify specific roles for the two main C-P4H isoenzymes in collagen hydroxylation by a detailed 4Hyp analysis of type I and IV collagens derived from cell and tissue samples. Loss of C-P4H-I results in underhydroxylation of collagen where the affected prolines are not uniformly distributed, but mainly present in sites where the adjacent X-position amino acid has a positively charged or a polar uncharged side chain. In contrast, loss of C-P4H-II results in underhydroxylation of triplets where the X-position is occupied by a negatively charged amino acid glutamate or aspartate. Hydroxylation of these triplets was found to be important as loss of C-P4H-II alone resulted in reduced collagen melting temperature and altered assembly of collagen fibrils and basement membrane. The observed C-P4H isoenzyme differences in substrate specificity were explained by selective binding of the substrate to the active site resulting in distinct differences in Km and Vmax values. Furthermore, our results clearly show that the substrate proline selection is not dependent on the collagen type, but the main determinant is the X-position amino acid of the -X-Pro-Gly- triplet. Although our data clearly shows the necessity of both C-P4H-I and II for normal prolyl 4-hydroxylation and function of collagens, the mRNA expression of the isoenzymes with various procollagens was, surprisingly, not tightly coordinated, suggesting additional levels of control. In conclusion, this study provides a molecular level explanation for the need of multiple C-P4H isoenzymes to generate collagen molecules capable to assemble into intact extracellular matrix structures.

N-acetyl-L-hydroxyproline - A potent skin anti-ageing active preventing advanced glycation end-product formation in vitro and ex vivo.

OBJECTIVE: Advanced glycation end-products (AGEs) represent a large group of compounds generated by a non-enzymatic reaction between reducing sugars and amino groups. The formation and accumulation of AGEs in the skin lead to protein crosslinking, dermal stiffening and yellowing, which ultimately contribute to cutaneous ageing. Amino acids have been described to exhibit anti-glycation effects. The objective of this study was to understand the inhibitory role of the amino acid derivative N-acetyl-L-hydroxyproline (NAHP) as an anti-glycation active for human skin. METHODS: A cell-free assay investigating the inhibition of glycation of serum albumin by NAHP was used to determine the capability of NAHP to decrease AGE formation. Also, by assessing the amount of the AGE N-(carboxymethyl)lysine (CML) the anti-glycation abilities of NAHP were investigated utilizing dot blot analysis. The improvement of cell-matrix interaction by NAHP was determined in vitro using a glycated fibroblast-populated collagen lattice (FPCL) dermis model. In skin biopsies, AGE autofluorescence was determined after treatment with NAHP and/or glucose ex vivo. RESULTS: NAHP significantly and dose-dependently inhibited levels of AGEs, which were induced by the glycation of a protein solution. This decrease could be visualized by showing that the brownish appearance as well as the AGE-specific fluorescence of glucose-treated samples were reduced after the application of increasing amounts of NAHP. Also, CML formation was dose-dependently inhibited by NAHP. In FPCLs, the contractile capacity of fibroblasts was significantly disturbed after glycation. This could be prevented by the addition of NAHP. Compared to glyoxal-treated samples, the co-application of NAHP significantly decreased the diameter as well as the weight of glycated FPCLs. Ex vivo application of glucose to skin explants showed a higher AGE fluorescence signal compared to control explants. Co-treatment with NAHP and glucose decreased the level of AGE fluorescence in comparison to glucose-treated explants. CONCLUSION: These data provide clear evidence that under glycation stress conditions treatment with NAHP inhibited AGE formation in vitro and ex vivo and prevented the loss of cellular contractile forces in a glycated dermis model. Thus, NAHP obviously provides a beneficial treatment option to counteract AGE-related changes in human skin such as dermal stiffening and yellowish skin appearance.

OBJECTIF: Les produits finis de glycation avancée (AGE) représentent un grand groupe de composés générés par une réaction non enzymatique entre des sucres réduits et des groupes amino. La formation et l'accumulation d'AGE dans la peau entraînent une réticulation protéique, un raidissement de la peau et un jaunissement, qui finissent par contribuer au vieillissement cutané. Les acides aminés ont été décrits comme ayant des effets d'anti­glycation. L'objectif de cette étude était de comprendre le rôle inhibiteur du dérivé d'acide aminé N­acétyl­L­hydroxyproline (NAHP) en tant qu'actif anti­glycation pour la peau humaine. MÉTHODES: Un test acellulaire étudiant l'inhibition de la glycation de l'albumine sérique par la NAHP a été utilisé pour déterminer la capacité de la NAHP à diminuer la formation d'AGE. En évaluant la quantité de l'AGE N­(carboxyméthyl)lysine (CML), les capacités d'anti­glycation de la NAHP ont également été étudiées à l'aide d'une analyse par dot blot. L'amélioration de l'interaction cellule­matrice par la NAHP a été déterminée in vitro à l'aide d'un modèle de derme de lattices de collagène composées de fibroblastes glyqués. Dans des biopsies cutanées, l'autofluorescence des AGE a été déterminée après un traitement par NAHP et/ou glucose ex vivo. RÉSULTATS: La NAHP a inhibé de manière significative et dose­dépendante les taux d'AGE induits par la glycation d'une solution protéique. Cette diminution a pu être visualisée en montrant que l'aspect brunâtre ainsi que la fluorescence spécifique aux AGE des échantillons traités par glucose ont été réduits après l'application de quantités croissantes de NAHP. En outre, la formation de CML était inhibée de manière dose­dépendante par la NAHP. Dans des lattices de collagène composées de fibroblastes, la capacité contractile des fibroblastes était significativement perturbée après la glycation. Cela a pu être évité par l'ajout de NAHP. Par rapport aux échantillons traités au glyoxal, la co­application de NAHP a significativement réduit le diamètre ainsi que le poids des lattices de collagène composées de fibroblastes glyquées. L'application ex vivo de glucose sur les explants de peau a montré un signal de fluorescence des AGE plus élevé que les explants témoins. Le traitement concomitant par NAHP et glucose a réduit le niveau de fluorescence des AGE par rapport aux explants traités par glucose. CONCLUSION: Ces données fournissent des preuves évidentes que, dans des conditions de stress par glycation, le traitement par NAHP a inhibé la formation d'AGE in vitro et ex vivo, et a prévenu la perte des forces contractiles cellulaires dans un modèle de derme glyqué. Ainsi, la NAHP constitue manifestement une option de traitement bénéfique pour contrer les changements liés aux AGE dans la peau humaine, tels que le raidissement du derme et l'aspect jaunâtre de la peau.

Therapeutic targeting of HYPDH/PRODH2 with N-propargylglycine offers a Hyperoxaluria treatment opportunity.

N-propargylglycine prevents 4-hydroxyproline catabolism in mouse liver and kidney. N-propargylglycine is a novel suicide inhibitor of PRODH2 and induces mitochondrial degradation of PRODH2. PRODH2 is selectively expressed in liver and kidney and contributes to primary hyperoxaluria (PH). Preclinical evaluation of N-propargylglycine efficacy as a new PH therapeutic is warranted.