Analysis of Acetylated Hyaluronic Acid in Moisturizing Lotion and Milk Lotion by HPLC with Fluorescence Detection.

We developed a simple and sensitive analytical HPLC method for the determination of acetylated hyaluronic acid (AcHA) in moisturizing and milk lotions. AcHA with different molecular weights was separated as a single peak using a C4 column and detected through post-column derivatization using 2-cyanoacetamide. The limits of detection and quantification were 60 and 200 ng, respectively. We found that AcHA in water was successfully extracted into a strong anion exchange (SAX) spin column with a recovery rate of AcHA was 63.8 ± 1.8%. Although the supernatant from acetone precipitation of lotions could pass through the spin column, the recovery rate (%) and accuracy of AcHA were affected by the viscous properties of cosmetics and acidic and acetone-soluble ingredients. Upon conducting analytical methods in this study, the concentration of AcHA in nine lotions was found to have ranged from 7.50 to 83.3 µg/mL. These values are comparable to the concentration range of AcHA in emulsions that have been previously evaluated for their superior effects. We believe that the analytical and extraction method is useful for the qualitative analysis of AcHA in moisturizing and milk lotions.

Ischemic stroke disrupts the endothelial glycocalyx through activation of proHPSE via acrolein exposure.

Infiltration of peripheral immune cells after blood-brain barrier dysfunction causes severe inflammation after a stroke. Although the endothelial glycocalyx, a network of membrane-bound glycoproteins and proteoglycans that covers the lumen of endothelial cells, functions as a barrier to circulating cells, the relationship between stroke severity and glycocalyx dysfunction remains unclear. In this study, glycosaminoglycans, a component of the endothelial glycocalyx, were studied in the context of ischemic stroke using a photochemically induced thrombosis mouse model. Decreased levels of heparan sulfate and chondroitin sulfate and increased activity of hyaluronidase 1 and heparanase (HPSE) were observed in ischemic brain tissues. HPSE expression in cerebral vessels increased after stroke onset and infarct volume greatly decreased after co-administration of N-acetylcysteine + glycosaminoglycan oligosaccharides as compared with N-acetylcysteine administration alone. These results suggest that the endothelial glycocalyx was injured after the onset of stroke. Interestingly, scission activity of proHPSE produced by immortalized endothelial cells and HEK293 cells transfected with hHPSE1 cDNA were activated by acrolein (ACR) exposure. We identified the ACR-modified amino acid residues of proHPSE using nano LC-MS/MS, suggesting that ACR modification of Lys139 (6-kDa linker), Lys107, and Lys161, located in the immediate vicinity of the 6-kDa linker, at least in part is attributed to the activation of proHPSE. Because proHPSE, but not HPSE, localizes outside cells by binding with heparan sulfate proteoglycans, ACR-modified proHPSE represents a promising target to protect the endothelial glycocalyx.

Structure and Immunomodulatory Activity of Glycogen Derived from Honeybee Larvae (Apis mellifera).

Honeybee larvae have been recognized as nutrient-rich food in many countries. Although glycogen, a storage form of glucose in animals, is synthesized in honeybee larvae, there is no information on the structure of glycan and its biological activity. In this study, we successfully extracted glycogen from honeybee larvae using hot water extraction and investigated the structure and biological activity of glycan. It was found that the molecular weight of glycogen from honeybee larvae is higher than that of glycogen from bovine liver and oysters. In addition, treatment of RAW264.7 cells with glycogen from honeybee larvae resulted in a much higher production of tumor necrosis factor (TNF)-α and interleukin (IL)-6 than treatment with glycogen from either bovine liver or oysters. These results suggest that the high molecular weight glycogen from honeybee larvae is a functional food ingredient with immunomodulatory activity.

Preparation of 99mTc-Labeled Mannan-S-Cysteine and Effect of Molecular Size of Mannan on Its Biodistribution.

Macrophage mannose receptor (MMR/CD206) is a promising target for the detection and identification of sentinel lymph node (SLN). MMR-targeting probes have been developed using mannosylated dextran, however, impairment of efficient targeting of SLN was often caused because of retention of injection site in which macrophages and dendritic cells exist. In this study, we prepared new MMR-targeting probes from yeast mannan (85 kDa), and its bioditribution was investigated. In-vivo evaluation showed that 11.9% of injected dose of 99mTc-labeled mannan-S-cysteines (99mTc-MSCs) was accumulated in popliteal lymph node (the SLN in this model), however, significant level of radioactivity (approximately 80%) was remained in injection site. Interestingly, 99mTc-labeled low molecular weight mannan-S-cysteine mannan (99mTc-LSC) prepared from 50 and 25 kDa mannan showed a decreased specific accumulation of 99mTc-LSC in the popliteal lymph node, while the radioactivity at the injection site remained unchanged. These results suggest that the molecular size, or nature/shape of the sugar chain is important for the specific accumulation of 99mTc-MSC in popliteal lymph node.

Polyamines stimulate the CHSY1 synthesis through the unfolding of the RNA G-quadruplex at the 5'-untraslated region.

Glycosaminoglycans (GAGs), a group of structurally related acidic polysaccharides, are primarily found as glycan moieties of proteoglycans (PGs). Among these, chondroitin sulfate (CS) and dermatan sulfate, side chains of PGs, are widely distributed in animal kingdom and show structural variations, such as sulfation patterns and degree of epimerization, which are responsible for their physiological functions through interactions with growth factors, chemokines and adhesion molecules. However, structural changes in CS, particularly the ratio of 4-O-sulfation to 6-O-sulfation (4S/6S) and CS chain length that occur during the aging process, are not fully understood. We found that 4S/6S ratio and molecular weight of CS were decreased in polyamine-depleted cells. In addition, decreased levels of chondroitin synthase 1 (CHSY1) and chondroitin 4-O-sulfotransferase 2 proteins were also observed on polyamine depletion. Interestingly, the translation initiation of CHSY1 was suppressed by a highly structured sequence (positions -202 to -117 relative to the initiation codon) containing RNA G-quadruplex (G4) structures in 5'-untranslated region. The formation of the G4s was influenced by the neighboring sequences to the G4s and polyamine stimulation of CHSY1 synthesis disappeared when the formation of the G4s was inhibited by site-directed mutagenesis. These results suggest that the destabilization of G4 structures by polyamines stimulates CHSY1 synthesis and, at least in part, contribute to the maturation of CS chains.

Detection of Selenocyanate in Biological Samples by HPLC with Fluorescence Detection Using König Reaction.

We developed a simple and sensitive HPLC method for the determination of selenocyanate (SeCN-). The König reaction, which is generally used for the determination of cyanide and thiocyanate, was applied for the post-column detection, and using barbituric acid as a fluorogenic reagent made it possible to detect SeCN- with high sensitivity. The limits of detection (LOD) and quantification (LOQ) were 73.5 fmol and 245.1 fmol, respectively. Subsequently, the amounts of SeCN- in human blood and in cultured cell samples were analyzed, and no SeCN- was detected in human whole blood. Interestingly, we have found that some of the spiked SeCN- decomposed to cyanide in human whole blood. Ascorbic acid suppressed the decomposition of SeCN- to cyanide by reducing the ferric ion, which is typically involved in SeCN- decomposition. Then, SeCN- was detected in cultured HEK293 cells exposed to selenite. The established HPLC method with fluorescence detection of SeCN- is useful for investigating small amounts of SeCN- in biological samples.

Protective Effects of Brain Infarction by N-Acetylcysteine Derivatives.

BACKGROUND AND PURPOSE: We recently found that acrolein (CH2=CH-CHO) is more strongly involved in brain infarction compared with reactive oxygen species. In this study, we looked for acrolein scavengers with less side effects. METHODS: Photochemically induced thrombosis model mice were prepared by injection of Rose Bengal. Effects of N-acetylcysteine (NAC) derivatives on brain infarction were evaluated using the public domain National Institutes of Health image program. RESULTS: NAC, NAC ethyl ester, and NAC benzyl ester (150 mg/kg) were administered intraperitoneally at the time of induction of ischemia, or these NAC derivatives (50 mg/kg) were administered 3× at 24-h intervals before induction of ischemia and 1 more administration at the time of induction of ischemia. The size of brain infarction decreased in the order NAC benzyl ester>NAC ethyl ester>NAC in both experimental conditions. Detoxification of acrolein occurred through conjugation of acrolein with glutathione, which was catalyzed by glutathione S-transferases, rather than direct conjugation between acrolein and NAC derivatives. The level of glutathione S-transferases at the locus of brain infarction was in the order of administration of NAC benzyl ester>NAC ethyl ester>NAC>no NAC derivatives, suggesting that NAC derivatives stabilize glutathione S-transferases. CONCLUSIONS: The results indicate that detoxification of acrolein by NAC derivatives is caused through glutathione conjugation with acrolein catalyzed by glutathione S-transferases, which can be stabilized by NAC derivatives. This is a new concept of acrolein detoxification by NAC derivatives.

Sequencing the Dermatan Sulfate Chain of Decorin.

Glycomics represents one of the last frontiers and most challenging in omic analysis. Glycosylation occurs in the endoplasmic reticulum and the Golgi organelle and its control is neither well-understood nor predictable based on proteomic or genomic analysis. One of the most structurally complex classes of glycoconjugates is the proteoglycans (PGs) and their glycosaminoglycan (GAG) side chains. Previously, our laboratory solved the structure of the chondroitin sulfate chain of the bikunin PG. The current study examines the much more complex structure of the dermatan sulfate GAG chain of decorin PG. By utilizing sophisticated separation methods followed by compositional analysis, domain mapping, and tandem mass spectrometry coupled with analysis by a modified genetic algorithm approach, the structural motif for the decorin dermatan sulfate chain was determined. This represents the second example of a GAG with a prominent structural motif, suggesting that the structural variability of this class of glycoconjugates is somewhat simpler than had been expected.

Heparan sulfate alterations in extracellular matrix structures and fibroblast growth factor-2 signaling impairment in the aged neurogenic niche.

Adult neurogenesis in the subventricular zone of the lateral ventricle decreases with age. In the subventricular zone, the specialized extracellular matrix structures, known as fractones, contact neural stem cells and regulate neurogenesis. Fractones are composed of extracellular matrix components, such as heparan sulfate proteoglycans. We previously found that fractones capture and store fibroblast growth factor 2 (FGF-2) via heparan sulfate binding, and may deliver FGF-2 to neural stem cells in a timely manner. The heparan sulfate (HS) chains in the fractones of the aged subventricular zone are modified based on immunohistochemistry. However, how aging affects fractone composition and subsequent FGF-2 signaling and neurogenesis remains unknown. The formation of the FGF-fibroblast growth factor receptor-HS complex is necessary to activate FGF-2 signaling and induce the phosphorylation of extracellular signal-regulated kinase (Erk1/2). In this study, we observed a reduction in HS 6-O-sulfation, which is critical for FGF-2 signal transduction, and failure of the FGF-2-induced phosphorylation of Erk1/2 in the aged subventricular zone. In addition, we observed increased HS 6-O-endo-sulfatase, an enzyme that may be responsible for the HS modifications in aged fractones. In conclusion, the data revealed that heparan sulfate 6-O-sulfation is reduced and FGF-2-dependent Erk1/2 signaling is impaired in the aged subventricular zone. HS modifications in fractones might play a role in the reduced neurogenic activity in aging brains.

Identification of keratan sulfate disaccharide at C-3 position of glucuronate of chondroitin sulfate from Mactra chinensis.

Glycosaminoglycans (GAGs), including chondroitin sulfate (CS), dermatan sulfate, heparin, heparan sulfate and keratan sulfate (KS) are linear sulfated repeating disaccharide sequences containing hexosamine and uronic acid [or galactose (Gal) in the case of KS]. Among the GAGs, CS shows structural variations, such as sulfation patterns and fucosylation, which are responsible for their physiological functions through CS interaction with CS-binding proteins. Here, we solved the structure of KS-branched CS-E derived from a clam, Mactra chinensis KS disaccharide [d-GlcNAc6S-(1→3)-ß-d-Gal-(1→] was attached to the C-3 position of GlcA, and consecutive KS-branched disaccharide sequences were found in a CS chain. KS-branched polysaccharides clearly exhibited resistance to degradation by chondroitinase ABC or ACII (at low concentrations) compared with typical CS structures. Furthermore, KS-branched polysaccharides stimulated neurite outgrowth of hippocampal neurons. These results strongly suggest that M. chinensis is a rich source of KS-branched CS, and it has important biological activities.

Aggravation of brain infarction through an increase in acrolein production and a decrease in glutathione with aging.

We previously reported that tissue damage during brain infarction was mainly caused by inactivation of proteins by acrolein. This time, it was tested why brain infarction increases in parallel with aging. A mouse model of photochemically induced thrombosis (PIT) was studied using 2, 6, and 12 month-old female C57BL/6 mice. The size of brain infarction in the mouse PIT model increased with aging. The volume of brain infarction in 12 month-old mice was approximately 2-fold larger than that in 2 month-old mice. The larger brain infarction in 12 month-old mice was due to an increase in acrolein based on an increase in the activity of spermine oxidase, together with a decrease in glutathione (GSH), a major acrolein-detoxifying compound in cells, based on the decrease in one of the subunits of glutathione biosynthesizing enzymes, γ-glutamylcysteine ligase modifier subunit, with aging. The results indicate that aggravation of brain infarction with aging was mainly due to the increase in acrolein production and the decrease in GSH in brain.

Recent Progress in Analytical Methods for Determination of Urinary 3-Hydroxypropylmercapturic Acid, a Major Metabolite of Acrolein.

3-Hydroxypropylmercapturic acid (3-HPMA), a major metabolite of acrolein in urine, has been recognized as a noninvasive biomarker of exposure to cigarette smoke. Since acrolein is formed endogenously from polyamines and is also formed during oxidative stress and aggravates tissue damage by changing protein activity through its conjugation in pathological lesions, it is thought that the urinary 3-HPMA level is useful as a biomarker to monitor the severity of several diseases related to acrolein. To study the correlation between 3-HPMA and disease severity, it is important to understand the properties of analytical methods for determination of 3-HPMA. In this article, we summarize the analytical methods for determination of urinary 3-HPMA and discuss the utility of 3-HPMA as one of the biomarkers for the diagnosis of brain infarction.

Poly-ion Complex of Chondroitin Sulfate and Spermine and Its Effect on Oral Chondroitin Sulfate Bioavailability.

Chondroitin sulfate (CS) has been accepted as an ingredient in health foods for the treatment of symptoms related to arthritis and cartilage repair. However, CS is poorly absorbed through the gastrointestinal tract because of its high negative electric charges and molecular weight (MW). In this study, poly-ion complex (PIC) formation was found in aqueous solutions through electrostatic interaction between CS and polyamines-organic molecules having two or more primary amino groups ubiquitously distributed in natural products at high concentrations. Characteristic properties of various PICs generated by mixing CS and natural polyamines, including unusual polyamines, were studied based on the turbidity for PIC formation, the dynamic light scattering for the size of PIC particles, and ζ-potential measurements for the surface charges of PIC particles. The efficiency of PIC formation between CS and spermine increased in a CS MW-dependent manner, with 15 kDa CS being critical for the formation of PIC (particle size: 3.41 µm) having nearly neutral surface charge (ζ-potential: -0.80 mV). Comparatively, mixing tetrakis(3-aminopropyl)ammonium and 15 kDa of CS afforded significant levels of PIC (particle size: 0.42±0.16 µm) despite a strongly negative surface charge (-34.67±1.15 mV). Interestingly, the oral absorption efficiency of CS was greatly improved only when PIC possessing neutral surface charges was administered to mice. High formation efficiency and electrically neutral surface charge of PIC particles are important factors for oral CS bioavailability.

Spermidine and Ca(2+), but not Na(+), can permeate NMDA receptors consisting of GluN1 and GluN2A or GluN2B in the presence of Mg(2+).

N-Methyl-D-aspartate receptors (NMDA receptors) are known to be permeable to Na(+) and Ca(2+) ions. In this study, we tested whether polyamines (putrescine, spermidine, spermine), organic cations found in cells, can permeate NMDA receptors expressed in Xenopus laevis oocytes and HEK293 cells. It was found that polyamines, especially spermidine, can permeate NMDA channels expressed from GluN1/GluN2A or GluN1/GluN2B activated by glycine and glutamate. Furthermore, spermidine and Ca(2+) influx through NMDA receptors was observed in the presence of Mg(2+), although Na(+) influx was strongly inhibited by Mg(2+). The Km values for spermidine influx through GluN1/GluN2A and GluN1/GluN2B were 2.2 mM and 2.7 mM, respectively in the presence of isotonic extracellular ion solutions. Spermidine uptake by NMDA receptors was dependent on the presence of glycine and glutamate, and inhibited by Ca(2+) and by memantine, an NMDA receptor channel blocker. The Km values for Ca(2+) influx through GluN1/GluN2A and GluN1/GluN2B were 4.6 mM and 3.3 mM, respectively, under the same ionic conditions. The results indicate that spermidine and Ca(2+), but not Na(+), can permeate NMDA receptors in the presence of Mg(2+). Spermidine, if released locally from presynaptic terminals (where its concentration is high in synaptosomes and synaptic vesicles) could permeate NMDA receptors and play a role in synaptic plasticity mediated by NMDA receptors together with Ca(2+).

Sequence analysis and domain motifs in the porcine skin decorin glycosaminoglycan chain.

Decorin proteoglycan is comprised of a core protein containing a single O-linked dermatan sulfate/chondroitin sulfate glycosaminoglycan (GAG) chain. Although the sequence of the decorin core protein is determined by the gene encoding its structure, the structure of its GAG chain is determined in the Golgi. The recent application of modern MS to bikunin, a far simpler chondroitin sulfate proteoglycans, suggests that it has a single or small number of defined sequences. On this basis, a similar approach to sequence the decorin of porcine skin much larger and more structurally complex dermatan sulfate/chondroitin sulfate GAG chain was undertaken. This approach resulted in information on the consistency/variability of its linkage region at the reducing end of the GAG chain, its iduronic acid-rich domain, glucuronic acid-rich domain, and non-reducing end. A general motif for the porcine skin decorin GAG chain was established. A single small decorin GAG chain was sequenced using MS/MS analysis. The data obtained in the study suggest that the decorin GAG chain has a small or a limited number of sequences.

Method to detect contaminants in heparin using radical depolymerization and liquid chromatography-mass spectrometry.

Heparin is a critically important anticoagulant drug that was contaminated with a persulfonated polysaccharide in 2008, resulting in a number of severe adverse reactions, some leading to death. Controversy remains as to the precise composition of the 2008 contaminant, and new information suggests that heparin may now be subject to adulteration with a new, difficult to detect, contaminant, N-sulfo oversulfated chondroitin sulfate. This study synthesizes this new potential contaminant and describes the use of radical depolymerization followed by liquid chromatography-mass spectrometry to detect N-sulfo oversulfated chondroitin sulfate and to confirm the structure of the 2008 contaminant as oversulfated chondroitin sulfate and not oversulfated heparan sulfate.

Properties of putrescine uptake by PotFGHI and PuuP and their physiological significance in Escherichia coli.

Properties of putrescine uptake by PotFGHI and PuuP and their physiological significance were studied using a polyamine biosynthesis and uptake deficient Escherichia coli KK3131 transformed with pACYC184 containing potFGHI or puuP. Putrescine uptake activity of E. coli KK3131 transformed with pACYC184-PotFGHI was higher than that of E. coli 3131 transformed with pACYC-PuuP when cells were cultured in the absence of putrescine. Putrescine uptake by PotFGHI was both ATP and membrane potential dependent, while that by PuuP was membrane potential dependent. Feedback inhibition by polyamines occurred at the PotFGHI uptake system but not at the PuuP uptake system. Expression of PuuP was reduced in the presence of PuuR, a negative regulator for PuuP, and expression of PuuR was positively regulated by glucose, which reduces the level of cAMP. The complex of cAMP and CRP (cAMP receptor protein) inhibited the expression of PuuR in the absence of glucose. Thus, the growth rate of E. coli KK3131 in the presence of both 0.4% (22.2 mM) glucose and 10 mM putrescine was in the order of cells transformed with pACYC-PotFGHI > pACYC-PuuP > pACYC-PuuP + PuuR, which was parallel with the polyamine content in cells. The results indicate that PotFGHI is necessary for rapid cell growth in the presence of glucose as an energy source. When glucose in medium was depleted, however, PuuP was absolutely necessary for cell growth in the presence of putrescine, because accumulation of putrescine to a high level by PuuP was necessary for utilization of putrescine as an energy source.

Acrolein stimulates the synthesis of IL-6 and C-reactive protein (CRP) in thrombosis model mice and cultured cells.

Measurements of protein-conjugated acrolein (PC-Acro), IL-6, and C-reactive protein (CRP) in plasma were useful for identifying silent brain infarction with high sensitivity and specificity. The aim of this study was to determine whether acrolein causes increased production of IL-6 and CRP in thrombosis model mice and cultured cells. In mice with photochemically induced thrombosis, acrolein produced at the locus of infarction increased the level of IL-6 and then CRP in plasma. This was confirmed in cell culture systems - acrolein stimulated the production of IL-6 in mouse neuroblastoma Neuro-2a cells, mouse macrophage-like J774.1 cells, and human umbilical vein endothelial cells (HUVEC), and IL-6 in turn stimulated the production of CRP in human hepatocarcinoma cells. The level of IL-6 mRNA was increased by acrolein through an increase in phosphorylation of the transcription factors, c-Jun, and NF-κB p65. Furthermore, CRP stimulated IL-6 production in mouse macrophage-like J774.1 cells and HUVEC. IL-6 functioned as a protective factor against acrolein toxicity in Neuro-2a cells and HUVEC. These results show that acrolein stimulates the synthesis of IL-6 and CRP, which function as protecting factors against acrolein toxicity, and that the combined measurement of PC-Acro, IL-6, and CRP is effective for identification of silent brain infarction. The combined measurements of protein-conjugated acrolein (PC-Acro), IL-6, and C-reactive protein (CRP) in plasma were useful for identifying silent brain infarction. The aim of this study was to determine whether acrolein causes increased production of IL-6 and CRP, and indeed acrolein increased IL-6 synthesis and IL-6 in turn increased CRP synthesis. Furthermore, IL-6 decreased acrolein toxicity in several cell lines.

The proteoglycan bikunin has a defined sequence.

Proteoglycans are complex glycoconjugates that regulate critical biological pathways in all higher organisms. Bikunin, the simplest proteoglycan, with a single glycosaminoglycan chain, is a serine protease inhibitor used to treat acute pancreatitis. Unlike nucleic acids and proteins, whose synthesis is template driven, Golgi-synthesized glycosaminoglycans are not believed to have predictable or deterministic sequences. Bikunin peptidoglycosaminoglycans were prepared and fractionated to obtain a collection of size-similar and charge-similar chains. Fourier transform mass spectral analysis identified a small number of parent molecular ions corresponding to monocompositional peptidoglycosaminoglycans. Fragmentation using collision-induced dissociation unexpectedly afforded a single sequence for each monocompositional parent ion, unequivocally demonstrating the presence of a defined sequence. The biosynthetic pathway common to all proteoglycans suggests that even more structurally complex proteoglycans, such as heparan sulfate, may have defined sequences, requiring a readjustment in the understanding of information storage in complex glycans.

In vitro and in vivo evaluation of polymethylene tetraamine derivatives as NMDA receptor channel blockers.

The biological activities of six symmetrically substituted 2-methoxy-benzyl polymethylene tetraamines (1-4) and diphenylethyl polymethylene tetraamines (5 and 6) as N-methyl-D-aspartate (NMDA) receptor channel blockers, were evaluated in vitro and in vivo. Although all compounds exhibited stronger channel block activities in comparison to memantine in Xenopus oocytes voltage clamped at -70 mV, only compound 2 (0.4 mg/kg intravenous injection) decreased the size of brain infarction in a photochemically induced thrombosis model mice at the same extent of memantine (10mg/kg intravenous injection). Other compounds (1, 3, 4, 5 and 6) did not decrease the size of brain infarction significantly due to the limited injection doses. The present study suggests that compound 2 could represent a valuable lead compound to design low toxicity polyamines for clinical use against stroke.