Both PepT1 and GLUT Intestinal Transporters Are Utilized by a Novel Glycopeptide Pro-Hyp-CONH-GlcN

Pro-Hyp (PO) accounts for many beneficial biological effects of collagen hydrolysates for skin and bone health. The objective of this study was to conjugate PO with glucosamine (GlcN) to create a novel glycopeptide Pro-Hyp-CONH-GlcN (POGlcN) and then to investigate the potential involvement of multiple transepithelial transport pathways for this glycopeptide. Nuclear magnetic resonance results revealed the amide nature of this glycopeptide with α and β configurations derived from GlcN. This glycopeptide was very resistant to simulated gastrointestinal digestion. Also, it showed a rate of transepithelial transport [permeability coefficient (<i>P</i>_app) of (2.82 ± 0.15) × 10^–6 cm/s] across the Caco-2 cell monolayer superior to those of parental dipeptide PO and GlcN [<i>P</i>_app values of (1.45 ± 0.17) × 10^–6 and (1.87 ± 0.15) × 10^–6 cm/s, respectively]. A transport mechanism experiment indicated that the improved transport efficiency of POGlcN is attributed to the introduction of glucose transporters

Identification and Evaluation of Cryoprotective Peptides from Chicken Collagen: Ice-Growth Inhibition Activity Compared to That of Type I Antifreeze Proteins in Sucrose Model Systems

The ability of chicken collagen peptides to inhibit the growth of ice crystals was evaluated and compared to that of fish antifreeze proteins (AFPs). This ice inhibition activity was assessed using a polarized microscope by measuring ice crystal dimensions in a sucrose model system with and without collagen peptides after seven thermal cycles. The system was stabilized at −25 °C and cycled between −16 and −12 °C. Five candidate peptides with ice inhibition activity were identified using liquid chromatography and tandem mass spectrometry and were then synthesized. Their ice inhibition capacity was compared to that of type I AFPs in a 23% sucrose model system. Specific collagen peptides with certain amino acid sequences reduced the extent of ice growth by approximately 70% at a relatively low concentration (1 mg/mL). These results suggest that specific collagen peptides may act in a noncolligative manner, inhibiting ice crystal growth like type I AFPs, but less efficiently

Studies on the Formation of Maillard and Caramelization Products from Glucosamine Incubated at 37 °C

This experiment compared the in vitro degradation of glucosamine (GlcN), <i>N</i>-acetylglucosamine, and glucose in the presence of NH₃ incubated at 37 °C in phosphate buffer from 0.5 to 12 days. The reactions were monitored with UV–vis absorption and fluorescence emission spectroscopies, and the main products of degradation, quinoxaline derivatives of α-dicarbonyl compounds and condensation products, were determined using UHPLC-UV and Orbitrap mass spectrometry. GlcN produced two major dicarbonyl compounds, glucosone and 3-deoxyglucosone, ranging from 709 to 3245 mg/kg GlcN and from 272 to 4535 mg/kg GlcN, respectively. 3,4-Dideoxyglucosone-3-ene, glyoxal, hydroxypyruvaldehyde, methylglyoxal, and diacetyl were also detected in lower amounts compared to glucosone and 3-deoxyglucosone. Several pyrazine condensation products resulting from the reaction between dicarbonyls and GlcN were also identified. This study determined that GlcN is a significantly unstable molecule producing a high level of degradation products at 37 °C

Transport of the Glucosamine-Derived Browning Product Fructosazine (Polyhydroxyalkylpyrazine) Across the Human Intestinal Caco‑2 Cell Monolayer: Role of the Hexose Transporters

The transport mechanism of fructosazine, a glucosamine self-condensation product, was investigated using a Caco-2 cell model. Fructosazine transport was assessed by measuring the bidirectional permeability coefficient across Caco-2 cells. The mechanism of transport was evaluated using phlorizin, an inhibitor of sodium-dependent glucose cotransporters (SGLT) 1 and 2, phloretin and quercetin, inhibitors of glucose transporters (GLUT) 1 and 2, transcytosis inhibitor wortmannin, and gap junction disruptor cytochalasin D. The role of hexose transporters was further studied using downregulated or overexpressed cell lines. The apparent permeability (<i>P</i>_a,b) of fructosazine was 1.30 ± 0.02 × 10^–6 cm/s. No significant (<i>p</i> > 0.05) effect was observed in fructosazine transport by adding wortmannin and cytochalasin D. The presence of phlorizin, phloretin, and quercetin decreased fructosazine transport. The downregulated GLUT cells line was unable to transport fructosazine. In human intestinal epithelial Caco-2 cells, GLUT1 or GLUT2 and SGLT are mainly responsible for fructosazine transport

A deconvoluted ESI-MS spectra of Mb incubated at 37°C for various times in the presence of GlcNAc, Glc and GlcN.

<p>The experimental conditions were the same as those used to obtain the spectra in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139022#pone.0139022.g001" target="_blank">Fig 1</a>. Inset spectrum (A) shows the spectrum of GlcN incubated for 12 days in the region of 7000–18000 Da.</p

Concentration of the major α-dicarbonyl compound produced during incubation of Mb in the presence of GlcN from 0 to 12 days.

<p>The values are represented as mean ± standard deviation (calculated from three independent trials). G, glucosone; 3-DG, 3-deoxyglucosone; GO, glyoxal; MGO, methylglyoxal; DA, diacetyl. Different letters within each α-dicarbonyl compound indicate statistical significant difference (<i>p</i> < 0.05).</p

Fructosazine, a Polyhydroxyalkylpyrazine with Antimicrobial Activity: Mechanism of Inhibition against Extremely Heat Resistant <i>Escherichia coli</i>

Fructosazine is a polyhydroxyalkylpyrazine recently reported to have antimicrobial activity against heat-resistant <i>Escherichia coli</i> AW 1.7. This study investigated fructosazine’s antimicrobial mechanism of action and compared it to that of riboflavin. Fructosazine–acetic acid was effective in permeabilizing the outer membrane based on an evaluation of bacterial membrane integrity using 1-<i>N-</i>phenyl-1-naphthylamine and propidium iodide. The uptake of fructosazine by <i>E. coli</i> was pH-dependent with a greater uptake at pH 5 compared to pH 7 for all times throughout 16 h, except 2, 3, and 10 h. Fructosazine generates ¹O₂, which is partially why it damages <i>E. coli</i>. DNA fragmentation was confirmed by fluorescence microscopy, and the fructosazine–acetic acid was the second most intense treatment after riboflavin–acetic acid. Electron microscopy revealed membrane structural damage by fructosazine at pH 5 and 7. This study provides evidence that fructosazine exerts antimicrobial action by permeabilizing the cell membrane, damaging membrane integrity, and fragmenting DNA

Rapid Myoglobin Aggregation through Glucosamine-Induced α-Dicarbonyl Formation

<div><p>The extent of glycation and conformational changes of horse myoglobin (Mb) upon glycation with <i>N</i>-acetyl-glucosamine (GlcNAc), glucose (Glc) and glucosamine (GlcN) were investigated. Among tested sugars, the rate of glycation with GlcN was the most rapid as shown by MALDI and ESI mass spectrometries. Protein oxidation, as evaluated by the amount of carbonyl groups present on Mb, was found to increase exponentially in Mb-Glc conjugates over time, whereas in Mb-GlcN mixtures the carbonyl groups decreased significantly after maximum at 3 days of the reaction. The reaction between GlcN and Mb resulted in a significantly higher amount of α-dicarbonyl compounds, mostly glucosone and 3-deoxyglucosone, ranging from and 27 to 332 mg/L and from 14 to 304 mg/L, respectively. Already at 0.5 days, tertiary structural changes of Mb-GlcN conjugate were observed by altered tryptophan fluorescence. A reduction of metmyoglobin to deoxy-and oxymyoglobin forms was observed on the first day of reaction, coinciding with the greatest amount of glucosone produced. In contrast to native α-helical myoglobin, 41% of the glycated protein sequence was transformed into a β-sheet conformation, as determined by circular dichroism spectropolarimetry. Transmission electron microscopy demonstrated that Mb glycation with GlcN causes the formation of amorphous or fibrous aggregates, started already at 3 reaction days. These aggregates bind to an amyloid-specific dye thioflavin T. With the aid of α-dicarbonyl compounds and advanced products of reaction, this study suggests that the Mb glycation with GlcN induces the unfolding of an initially globular protein structure into amyloid fibrils comprised of a β-sheet structure.</p></div

UHPLC analyses of quinoxaline derivatives of α-dicarbonyl compounds produced from Mb-GlcN conjugates over time.

<p>(A) Chromatograms of (I) a reference quinoxaline mixture of glucosone (G), 3-deoxyglucosone (3-DG), glyoxal (GO), methylglyoxal (MGO) and diacetyl (DA). (II) Representative chromatogram of Mb-GlcN conjugate incubated for 1 d, derivatized with <i>o</i>-OPD and acquired by UHPLC with UV detection at 314 nm. Numbers indicate the peaks of the quinoxalines of (1) G, (2) unidentified, (3) 3-DG, (4) GO, (5) HPA, (6) 3,4- DGE, (7) MGO, (8) DA and a, b, c peaks corresponding to non-OPD derived GlcN condensation products.</p

Retention time, MS and MS/MS data of the α-dicarbonyl compounds detected Mb-GlcN conjugates.

<p>Retention time, MS and MS/MS data of the α-dicarbonyl compounds detected Mb-GlcN conjugates.</p