Assessment of Oxidative Stress-Induced Oral Epithelial Toxicity.

Reactive oxygen species (ROS) are highly reactive molecules generated in living organisms and an excessive production of ROS culminates in oxidative stress and cellular damage. Notably, oxidative stress plays a critical role in the pathogenesis of a number of oral mucosal diseases, including oral mucositis, which remains one of cancer treatments' most common side effects. We have shown previously that oral keratinocytes are remarkably sensitive to oxidative stress, and this may hinder the development and reproducibility of epithelial cell-based models of oral disease. Here, we examined the oxidative stress signatures that parallel oral toxicity by reproducing the initial events taking place during cancer treatment-induced oral mucositis. We used three oral epithelial cell lines (an immortalized normal human oral keratinocyte cell line, OKF6, and malignant oral keratinocytes, H357 and H400), as well as a mouse model of mucositis. The cells were subjected to increasing oxidative stress by incubation with hydrogen peroxide (H2O2) at concentrations of 100 µM up to 1200 µM, for up to 24 h, and ROS production and real-time kinetics of oxidative stress were investigated using fluorescent dye-based probes. Cell viability was assessed using a trypan blue exclusion assay, a fluorescence-based live-dead assay, and a fluorometric cytotoxicity assay (FCA), while morphological changes were analyzed by means of a phase-contrast inverted microscope. Static and dynamic real-time detection of the redox changes in keratinocytes showed a time-dependent increase of ROS production during oxidative stress-induced epithelial injury. The survival rates of oral epithelial cells were significantly affected after exposure to oxidative stress in a dose- and cell line-dependent manner. Values of TC50 of 800 µM, 800 µM, and 400 µM were reported for H400 cells (54.21 ± 9.04, p < 0.01), H357 cells (53.48 ± 4.01, p < 0.01), and OKF6 cells (48.64 ± 3.09, p < 0.01), respectively. Oxidative stress markers (MPO and MDA) were also significantly increased in oral tissues in our dual mouse model of chemotherapy-induced mucositis. In summary, we characterized and validated an oxidative stress model in human oral keratinocytes and identified optimal experimental conditions for the study of oxidative stress-induced oral epithelial toxicity.

Novel, acidic, and cold-adapted glycoside hydrolase family 8 endo-ß-1,4-glucanase from an Antarctic lichen-associated bacterium, Lichenicola cladoniae PAMC 26568.

Endo-ß-1,4-glucanase is a crucial glycoside hydrolase (GH) involved in the decomposition of cellulosic materials. In this study, to discover a novel cold-adapted ß-1,4-D-glucan-degrading enzyme, the gene coding for an extracellular endo-ß-1,4-glucanase (GluL) from Lichenicola cladoniae PAMC 26568, an Antarctic lichen (Cladonia borealis)-associated bacterium, was identified and recombinantly expressed in Escherichia coli BL21. The GluL gene (1044-bp) encoded a non-modular polypeptide consisting of a single catalytic GH8 domain, which shared the highest sequence identity of 55% with that of an uncharacterized protein from Gluconacetobacter takamatsuzukensis (WP_182950054). The recombinant endo-ß-1,4-glucanase (rGluL: 38.0 kDa) most efficiently degraded sodium carboxymethylcellulose (CMC) at pH 4.0 and 45°C, and showed approximately 23% of its maximum degradation activity even at 3°C. The biocatalytic activity of rGluL was noticeably enhanced by >1.3-fold in the presence of 1 mM Mn2+ or NaCl at concentrations between 0.1 and 0.5 M, whereas the enzyme was considerably downregulated by 1 mM Hg2+ and Fe2+ together with 5 mM N-bromosuccinimide and 0.5% sodium dodecyl sulfate. rGluL is a true endo-ß-1,4-glucanase, which could preferentially decompose D-cellooligosaccharides consisting of 3 to 6 D-glucose, CMC, and barley ß-glucan, without other additional glycoside hydrolase activities. The specific activity (15.1 U mg-1) and k cat/K m value (6.35 mg-1 s-1mL) of rGluL toward barley ß-glucan were approximately 1.8- and 2.2-fold higher, respectively, compared to its specific activity (8.3 U mg-1) and k cat/K m value (2.83 mg-1 s-1mL) toward CMC. The enzymatic hydrolysis of CMC, D-cellotetraose, and D-cellohexaose yielded primarily D-cellobiose, accompanied by D-glucose, D-cellotriose, and D-cellotetraose. However, the cleavage of D-cellopentaose by rGluL resulted in the production of only D-cellobiose and D-cellotriose. The findings of the present study imply that rGluL is a novel, acidic, and cold-adapted GH8 endo-ß-1,4-glucanase with high specific activity, which can be exploited as a promising candidate in low-temperature processes including textile and food processes.

Oxidative Stress and Chemoradiation-Induced Oral Mucositis: A Scoping Review of In Vitro, In Vivo and Clinical Studies.

Chemoradiation-induced mucositis is a debilitating condition of the gastrointestinal tract eventuating from antineoplastic treatment. It is believed to occur primarily due to oxidative stress mechanisms, which generate Reactive Oxygen Species (ROS). The aim of this scoping review was to assess the role of oxidative stress in the development of Oral Mucositis (OM). Studies from the literature, published in MEDLINE and SCOPUS, that evaluated the oxidative stress pathways or antioxidant interventions for OM, were retrieved to elucidate the current understanding of their relationship. Studies failing inclusion criteria were excluded, and those suitable underwent data extraction, using a predefined data extraction table. Eighty-nine articles fulfilled criteria, and these were sub-stratified into models of study (in vitro, in vivo, or clinical) for evaluation. Thirty-five clinical studies evaluated antioxidant interventions on OM's severity, duration, and pain, amongst other attributes. A number of clinical studies sought to elucidate the protective or therapeutic effects of compounds that had been pre-determined to have antioxidant properties, without directly assessing oxidative stress parameters (these were deemed "indirect evidence"). Forty-seven in vivo studies assessed the capacity of various compounds to prevent OM. Findings were mostly consistent, reporting reduced OM severity associated with a reduction in ROS, malondialdehyde (MDA), myeloperoxidase (MPO), but higher glutathione (GSH) and superoxide dismutase (SOD) activity or expression. Twenty-one in vitro studies assessed potential OM therapeutic interventions. The majority demonstrated successful a reduction in ROS, and in select studies, secondary molecules were assessed to identify the mechanism. In summary, this review highlighted numerous oxidative stress pathways involved in OM pathogenesis, which may inform the development of novel therapeutic targets.

Novel Bi-Modular GH19 Chitinase with Broad pH Stability from a Fibrolytic Intestinal Symbiont of Eisenia fetida, Cellulosimicrobium funkei HY-13.

Endo-type chitinase is the principal enzyme involved in the breakdown of N-acetyl-d-glucosamine-based oligomeric and polymeric materials through hydrolysis. The gene (966-bp) encoding a novel endo-type chitinase (ChiJ), which is comprised of an N-terminal chitin-binding domain type 3 and a C-terminal catalytic glycoside hydrolase family 19 domain, was identified from a fibrolytic intestinal symbiont of the earthworm Eisenia fetida, Cellulosimicrobium funkei HY-13. The highest endochitinase activity of the recombinant enzyme (rChiJ: 30.0 kDa) toward colloidal shrimp shell chitin was found at pH 5.5 and 55 °C and was considerably stable in a wide pH range (3.5-11.0). The enzyme exhibited the highest biocatalytic activity (338.8 U/mg) toward ethylene glycol chitin, preferentially degrading chitin polymers in the following order: ethylene glycol chitin > colloidal shrimp shell chitin > colloidal crab shell chitin. The enzymatic hydrolysis of N-acetyl-ß-d-chitooligosaccharides with a degree of polymerization from two to six and colloidal shrimp shell chitin yielded primarily N,N'-diacetyl-ß-d-chitobiose together with a small amount of N-acetyl-d-glucosamine. The high chitin-degrading ability of inverting rChiJ with broad pH stability suggests that it can be exploited as a suitable biocatalyst for the preparation of N,N'-diacetyl-ß-d-chitobiose, which has been shown to alleviate metabolic dysfunction associated with type 2 diabetes.

Short-term storage of semen samples in acidic extender increases the proportion of females in pigs.

BACKGROUND: Sex preselection is a desired goal of the animal industry to improve production efficiency, depending on industry demand. In the porcine industry, there is a general preference for pork from female and surgically castrated male pigs. Therefore, the birth of more females than males in a litter leads to economic benefits and improved animal welfare in the pig production industry. Our previous study suggested that the porcine semen extender (BTS) adjusted to pH 6.2 maximises the differences in viability between X-chromosome-bearing (X) spermatozoa and Y-chromosome-bearing (Y) spermatozoa without affecting sperm's functional parameters. In this study we aimed to evaluate whether the pH 6.2 extender is applicable at the farm level for increasing the number of female piglets without a decline in spermatozoa fertility. Artificial insemination (AI) was carried out with spermatozoa stored at pH 6.2 and pH 7.2 (original BTS) at day 1 and day 2 of storage. Next, the functional parameters of the spermatozoa, litter size, farrowing rate, and female-to-male ratio of offspring were determined. RESULTS: Although sperm motility decreased significantly after 2 d of storage, the viability of spermatozoa was preserved at pH 6.2 for 3 d. There was no significant difference in the farrowing rate and average litter size between the group inseminated with the spermatozoa stored in (pH 7.2) and that inseminated with spermatozoa stored in acidic BTS. The percentage of female piglets was approximately 1.5-fold higher in sows inseminated on day 1 in the pH 6.2 than in the pH 7.2 group. Furthermore, although there was no significant difference in the female-to-male ratio, the percentage of female piglets born was slightly higher in the pH 6.2 group than in the pH 7.2 group on day 2. CONCLUSIONS: The method optimised in our study is simple, economical, and may enhance the number of female births without any decline in spermatozoa fertility.

Novel Anti-Fungal d-Laminaripentaose-Releasing Endo-ß-1,3-glucanase with a RICIN-like Domain from Cellulosimicrobium funkei HY-13.

Endo-ß-1,3-glucanase plays an essential role in the deconstruction of ß-1,3-d-glucan polysaccharides through hydrolysis. The gene (1650-bp) encoding a novel, bi-modular glycoside hydrolase family 64 (GH64) endo-ß-1,3-glucanase (GluY) with a ricin-type ß-trefoil lectin domain (RICIN)-like domain from Cellulosimicrobium funkei HY-13 was identified and biocatalytically characterized. The recombinant enzyme (rGluY: 57.5 kDa) displayed the highest degradation activity for laminarin at pH 4.5 and 40 °C, while the polysaccharide was maximally decomposed by its C-terminal truncated mutant enzyme (rGluYΔRICIN: 42.0 kDa) at pH 5.5 and 45 °C. The specific activity (26.0 U/mg) of rGluY for laminarin was 2.6-fold higher than that (9.8 U/mg) of rGluYΔRICIN for the same polysaccharide. Moreover, deleting the C-terminal RICIN domain in the intact enzyme caused a significant decrease (>60%) of its ability to degrade ß-1,3-d-glucans such as pachyman and curdlan. Biocatalytic degradation of ß-1,3-d-glucans by inverting rGluY yielded predominantly d-laminaripentaose. rGluY exhibited stronger growth inhibition against Candida albicans in a dose-dependent manner than rGluYΔRICIN. The degree of growth inhibition of C. albicans by rGluY (approximately 1.8 µM) was approximately 80% of the fungal growth. The superior anti-fungal activity of rGluY suggests that it can potentially be exploited as a supplementary agent in the food and pharmaceutical industries.

Combination Treatment of Arazyme and Soy Leaf Extract Attenuates Hyperglycemia and Hepatic Steatosis in High-Fat Diet-Fed C57BL/6J Mice.

Arazyme and extracts of soy leaves (ESLs) are used as ingredients for functional foods; however, their combined administration has not been studied. This study assessed the combined effect of Arazyme and ESLs in high-fat-diet (HFD)-induced obese C57BL/6J mice fed 2 mg/kg Arazyme, 50 mg/kg ESLs, or a combination of 2 mg/kg Arazyme and 50 mg/kg ESLs by oral gavage for 13 weeks. Individually, Arazyme and ESLs had no effect on the HFD-induced phenotypes. The combination of Arazyme and ESLs significantly suppressed body weight gain, improved glucose and insulin tolerance, and suppressed hepatic steatosis by reducing lipid synthesis and enhancing lipid utilization gene expression. Furthermore, the combination significantly reduced HFD-induced plasma bile acid reabsorption by suppressing bile acid transporter expression, including the ATP biding cassette subfamily B member 11 (Abcb11), solute carrier family 10 member 1 (Slc10a1), Slc10a2, Slc51a, and Slc51b in the liver and gut. Moreover, the combination of Arazyme and ESLs significantly prevented HFD-induced islet compensation in the pancreas. These results suggest that the incorporation of Arazyme combined with ESLs reduces HFD-induced body weight, hyperglycemia, and hepatic steatosis by regulating liver-gut bile acid circulation in HFD-fed mice. This combination can markedly reduce treatment doses and enhance their therapeutic effects, thereby reducing therapeutic healthcare costs.

Development of a novel denture care agent with highly active enzyme, arazyme.

BACKGROUND: The importance of efficient denture deposit removal and oral hygiene has been further underscored by the continuous increase of denture wearers. Denture hygiene management has also become an important aspect associated with denture-induced stomatitis. This study aims to evaluate the denture cleaning effect of arazyme, the metalloprotease produced from the Serratia proteamaculans HY-3. We performed growth inhibition tests against oral opportunistic pathogens to be used as a potential oral health care agent. METHODS: The proteolytic activities of arazyme was evaluated over broad ranges of temperature, pH, and denture components compared to those of subtilisin in commercially available denture cleansers. The washing effects of arazyme were also measured by using homogeneously soiled EMPA 105 cottons. To investigate the denture cleaning capability of arazyme, artificially contaminated dentures were treated with arazyme, subtilisin (Everlase 6.0T), and Polident®, respectively. The growth kinetics of Candida albicans, Enterococcus faecalis, Staphylococcus epidermis, and Streptococcus mutans were evaluated in the presence of different concentrations of arazyme to estimate the prevention effects of arazyme against major oral opportunistic pathogens. RESULTS: Arazyme showed strong proteolytic activities over wide temperature and pH ranges compared with the serine protease of the subtilisin family. Arazyme demonstrated efficient removal and decomposition of artificially contaminated dentures and showed explicit washing effects against soiled cottons. Moreover arazyme inhibited the growth of oral opportunistic pathogens, including C. albicans, E. faecalis, S. epidermis, and S. mutans, with more than 80% inhibition against C. albicans, the major cause of denture stomatitis, with 250 mg/mL arazyme. CONCLUSIONS: Arazyme shows promise as a biological oral health care agent with effective cleaning and antimicrobial activities and is a potential source for developing novel denture care agents.

Identification and Characterization of a Novel, Cold-Adapted d-Xylobiose- and d-Xylose-Releasing Endo-ß-1,4-xylanase from an Antarctic Soil Bacterium, Duganella sp. PAMC 27433.

Endo-ß-1,4-xylanase is a key enzyme in the degradation of ß-1,4-d-xylan polysaccharides through hydrolysis. A glycoside hydrolase family 10 (GH10) endo-ß-1,4-xylanase (XylR) from Duganella sp. PAMC 27433, an Antarctic soil bacterium, was identified and functionally characterized. The XylR gene (1122-bp) encoded an acidic protein containing a single catalytic GH10 domain that was 86% identical to that of an uncultured bacterium BLR13 endo-ß-1,4-xylanase (ACN58881). The recombinant enzyme (rXylR: 42.0 kDa) showed the highest beechwood xylan-degrading activity at pH 5.5 and 40 °C, and displayed 12% of its maximum activity even at 4 °C. rXylR was not only almost completely inhibited by 5 mM N-bromosuccinimide or metal ions (each 1 mM) including Hg2+, Ca2+, or Cu2+ but also significantly suppressed by 1 mM Ni2+, Zn2+, or Fe2+. However, its enzyme activity was upregulated (>1.4-fold) in the presence of 0.5% Triton X-100 or Tween 80. The specific activities of rXylR toward beechwood xylan, birchwood xylan, oat spelts xylan, and p-nitrophenyl-ß-d-cellobioside were 274.7, 103.2, 35.6, and 365.1 U/mg, respectively. Enzymatic hydrolysis of birchwood xylan and d-xylooligosaccharides yielded d-xylose and d-xylobiose as the end products. The results of the present study suggest that rXylR is a novel cold-adapted d-xylobiose- and d-xylose-releasing endo-ß-1,4-xylanase.

Arazyme Suppresses Hepatic Steatosis and Steatohepatitis in Diet-Induced Non-Alcoholic Fatty Liver Disease-Like Mouse Model.

Arazyme, a metalloprotease from the spider Nephila clavata, exerts hepatoprotective activity in CCL4-induced acute hepatic injury. This study investigated the hepatoprotective effects in high-fat diet (HFD)-induced non-alcoholic fatty liver disease-like C57BL/6J mice. The mice were randomly divided into four groups (n = 10/group): the normal diet group, the HFD group, the arazyme group (HFD with 0.025% arazyme), and the milk thistle (MT) group (HFD with 0.1% MT). Dietary supplementation of arazyme for 13 weeks significantly lowered plasma triglyceride (TG) and non-esterified fatty acid levels. Suppression of HFD-induced hepatic steatosis in the arazyme group was caused by the reduced hepatic TG and total cholesterol (TC) contents. Arazyme supplementation decreased hepatic lipogenesis-related gene expression, sterol regulatory element-binding transcription protein 1 (Srebf1), fatty acid synthase (Fas), acetyl-CoA carboxylase 1 (Acc1), stearoyl-CoA desaturase-1 (Scd1), Scd2, glycerol-3-phosphate acyltransferase (Gpam), diacylglycerol O-acyltransferase 1 (Dgat1), and Dgat2. Arazyme directly reduced palmitic acid (PA)-induced TG accumulation in HepG2 cells. Arazyme suppressed macrophage infiltration and tumor necrosis factor α (Tnfa), interleukin-1ß (Il1b), and chemokine-ligand-2 (Ccl2) expression in the liver, and inhibited secretion of TNFα and expression of inflammatory mediators, Tnfa, Il1b, Ccl2, Ccl3, Ccl4, and Ccl5, in PA-induced RAW264.7 cells. Arazyme effectively protected hepatic steatosis and steatohepatitis by inhibiting SREBP-1-mediated lipid accumulation and macrophage-mediated inflammation.

Genetic and functional characterization of a novel GH10 endo-ß- 1,4-xylanase with a ricin-type ß-trefoil domain-like domain from Luteimicrobium xylanilyticum HY-24.

The gene (1488-bp) encoding a novel GH10 endo-ß-1,4-xylanase (XylM) consisting of an N-terminal catalytic GH10 domain and a C-terminal ricin-type ß-trefoil lectin domain-like (RICIN) domain was identified from Luteimicrobium xylanilyticum HY-24. The GH10 domain of XylM was 72% identical to that of Micromonospora lupini endo-ß-1,4-xylanase and the RICIN domain was 67% identical to that of Actinospica robiniae hypothetical protein. The recombinant enzyme (rXylM: 49kDa) exhibited maximum activity toward beechwood xylan at 65°C and pH 6.0, while the optimum temperature and pH of its C-terminal truncated mutant (rXylM△RICIN: 35kDa) were 45°C and 5.0, respectively. After pre-incubation of 1h at 60°C, rXylM retained over 80% of its initial activity, but the thermostability of rXylM△RICIN was sharply decreased at temperatures exceeding 40°C. The specific activity (254.1Umg-1) of rXylM toward oat spelts xylan was 3.4-fold higher than that (74.8Umg-1) of rXylM△RICIN when the same substrate was used. rXylM displayed superior binding capacities to lignin and insoluble polysaccharides compared to rXylM△RICIN. Enzymatic hydrolysis of ß-1,4-d-xylooligosaccharides (X3-X6) and birchwood xylan yielded X3 as the major product. The results suggest that the RICIN domain in XylM might play an important role in substrate-binding and biocatalysis.

Applications of capacitation status for litter size enhancement in various pig breeds.

OBJECTIVE: Several studies have reported the development of new molecular methods for the prognosis and diagnosis of male fertility based on biomarkers aimed at overcoming the limitations of conventional male fertility analysis tools. However, further studies are needed for the field application of these methods. Therefore, alternative methods based on existing semen analysis methods are required to improve production efficiency in the animal industry. METHODS: we examined the possibility of improving litter size in various pig breeds using combined Hoechst 33258/chlortetracycline fluorescence (H33258/CTC) staining. The correlation between field fertility and capacitation status by combined H33258/CTC staining in different ejaculates spermatozoa (n = 3) from an individual boar (20 Landrace, 20 Yorkshire, and 20 Duroc) was evaluated as well as overall accuracy. RESULTS: The acrosome reacted (AR) pattern after capacitation (%) was positively correlated with the litter size of Landrace, Yorkshire, and Duroc pigs and the overall accuracy was 75%, 75%, and 70% in Landrace, Yorkshire, and Duroc pigs, respectively. The difference (Δ) in AR pattern before and after capacitation was positively correlated with the litter size of Landrace, Yorkshire, and Duroc pigs and the overall accuracy was 80%, 65%, and 55% in Landrace, Yorkshire, and Duroc pigs, respectively. However, the difference (Δ) in capacitated (B) pattern before and after capacitation was negatively correlated with the litter size of Landrace pigs and the overall accuracy was 75%. Moreover, average litter size was significantly altered according to different combined H33258/CTC staining parameters. CONCLUSION: These results show that combined H33258/CTC staining may be used to predict male fertility in various breeds. However, the selection of specific efficiency combined H33258/CTC staining parameters requires further consideration. Taken together, these findings suggest that combined H33258/CTC staining may constitute an alternative method for predicting male fertility until such time as fertility-related biomarkers are further validated.

Biocatalytic characterization of an endo-ß-1,4-mannanase produced by Paenibacillus sp. strain HY-8.

OBJECTIVES: To evaluate the biocatalytic characteristics of a new endo-ß-1,4-D-mannan-degrading enzyme (ManP) from Paenibacillus sp. strain HY-8, a gut bacterium of the longicorn beetle Moechotypa diphysis. RESULTS: Purified ManP (32 kDa) with an N-terminal amino acid sequence of APSFAVGADFSYVPG displayed the greatest degree of biocatalytic activity toward locust bean gum (LBG) at 55 °C and pH 7.0. The enzyme degraded LBG, guar gum, ivory nut mannan, and mannooligosaccharides (M2-M5), but did not exhibit any hydrolytic activity against structurally unrelated substrates. The biocatalytic activity of ManP against LBG and guar gum was 695 and 450 U mg-1, respectively. Especially, enzymatic hydrolysis of mannobiose yielded a mixture of mannose (16.6 %) and mannobiose (83.4 %), although the degree of mannobiose degradation by ManP with was relatively limited. CONCLUSION: The present results suggest that ManP is an endo-ß-1,4-mannanase and is distinct from various other characterized endo-ß-1,4-mannanases.

Beneficial Effects of Pterocarpan-High Soybean Leaf Extract on Metabolic Syndrome in Overweight and Obese Korean Subjects: Randomized Controlled Trial.

Pterocarpans are known to have antifungal and anti-inflammatory properties. However, little is known about the changes in transcriptional profiles in response to a pterocarpan-high soybean leaf extract (PT). Therefore, this study investigated the effects of PT on blood glucose and lipid levels, as well as on the inflammation-related gene expression based on a peripheral blood mononuclear cells (PBMCs) mRNA sequencing analysis in Korean overweight and obese subjects with mild metabolic syndrome. The participants were randomly assigned to two groups and were administered either placebo (starch, 3 g/day) or PT (2 g/day) for 12 weeks. The PT intervention did not change body weight, body fat percentage and body mass index (BMI). However, PT significantly decreased the glycosylated hemoglobin (HbA1c), plasma glucose, free fatty acid, total cholesterol, and non-HDL cholesterol levels after 12 weeks. Furthermore, PT supplementation significantly lowered the homeostatic index of insulin resistance, as well as the plasma levels of inflammatory markers. Finally, the mRNA sequencing analysis revealed that PT downregulated genes related to immune responses. PT supplementation is beneficial for the improvement of metabolic syndrome by altering the fasting blood and plasma glucose, HbA1c, plasma lipid levels and inflammation-related gene expression in PBMCs.

Genetic and functional characterization of an extracellular modular GH6 endo-ß-1,4-glucanase from an earthworm symbiont, Cellulosimicrobium funkei HY-13.

The gene (1608-bp) encoding a GH6 endo-ß-1,4-glucanase (CelL) from the earthworm-symbiotic bacterium Cellulosimicrobium funkei HY-13 was cloned from its whole genome sequence, expressed recombinantly, and biochemically characterized. CelL (56.0 kDa) is a modular enzyme consisting of an N-terminal catalytic GH6 domain (from Val57 to Pro396), which is 71 % identical to a GH6 protein (accession no.: WP_034662937) from Cellulomonas sp. KRMCY2, together with a C-terminal CBM 2 domain (from Cys429 to Cys532). The highest catalytic activity of CelL toward carboxymethylcellulose (CMC) was observed at 50 °C and pH 5.0, and was relatively stable at a broad pH range of 4.0-10.0. The enzyme was capable of efficiently hydrolyzing the cellulosic polymers in the order of barley ß-1,3-1,4-D-glucan > CMC > lichenan > Avicel > konjac glucomannan. However, cellobiose, cellotriose, p-nitrophenyl derivatives of mono- and disaccharides, or structurally unrelated carbohydrate polymers including ß-1,3-D-glucan, ß-1,4-D-galactomannan, and ß-1,4-D-xylan were not susceptible to CelL. The enzymatic hydrolysis of cellopentaose resulted in the production of a mixture of 68.6 % cellobiose and 31.4 % cellotriose but barley ß-1,3-1,4-D-glucan was 100 % degraded to cellotriose by CelL. The enzyme strongly bound to Avicel, ivory nut mannan, and chitin but showed relatively weak binding affinity to lichenan, lignin, or poly(3-hydroxybutyrate) granules.

Soy Leaf Extract Containing Kaempferol Glycosides and Pheophorbides Improves Glucose Homeostasis by Enhancing Pancreatic ß-Cell Function and Suppressing Hepatic Lipid Accumulation in db/db Mice.

This study investigated the molecular mechanisms underlying the antidiabetic effect of an ethanol extract of soy leaves (ESL) in db/db mice. Control groups (db/+ and db/db) were fed a normal diet (ND), whereas the db/db-ESL group was fed ND with 1% ESL for 8 weeks. Dietary ESL improved glucose tolerance and lowered plasma glucose, glycated hemoglobin, HOMA-IR, and triglyceride levels. The pancreatic insulin content of the db/db-ESL group was significantly greater than that of the db/db group. ESL supplementation altered pancreatic IRS1, IRS2, Pdx1, Ngn3, Pax4, Ins1, Ins2, and FoxO1 expression. Furthermore, ESL suppressed lipid accumulation and increased glucokinase activity in the liver. ESL primarily contained kaempferol glycosides and pheophorbides. Kaempferol, an aglycone of kaempferol glycosides, improved ß-cell proliferation through IRS2-related FoxO1 signaling, whereas pheophorbide a, a product of chlorophyll breakdown, improved insulin secretion and ß-cell proliferation through IRS1-related signaling with protein kinase A in MIN6 cells. ESL effectively regulates glucose homeostasis by enhancing IRS-mediated ß-cell insulin signaling and suppressing SREBP-1-mediated hepatic lipid accumulation in db/db mice.

Anti-Obesity Effects of Soy Leaf via Regulation of Adipogenic Transcription Factors and Fat Oxidation in Diet-Induced Obese Mice and 3T3-L1 Adipocytes.

The anti-obesity effects of extracts from soy leaves (SLE) cultivated for 8 weeks (8W) or 16 weeks (16W) were investigated in diet-induced obese mice. The effects of kaempferol, an aglycone of the kaempferol glycosides that are the major component of 8W-SLE, and coumestrol, the major component of 16W-SLE, were also investigated in 3T3-L1 adipocytes. Eight-week-old male C57BL/6J mice were randomly divided into normal diet, high-fat diet (HFD), 8W-SLE (HFD+8W-SLE 50 mg kg(-1) day(-1)), 16W-SLE (HFD+16W-SLE 50 mg kg(-1) day(-1)), and Garcinia cambogia extracts (GE) (HFD+GE 50 mg kg(-1) day(-1)) groups. Body weight gain and fat accumulation of white adipose tissue (WAT) were highly suppressed by daily oral administration of 8W-SLE and 16W-SLE for 10 weeks. Supplementing a HFD with 8W-SLE and 16W-SLE regulated the mRNA expression of peroxisome proliferator-activated receptor gamma (PPARγ), CCAAT/enhancer-binding protein alpha (c/EBPα), sterol regulatory element-binding protein-1 (SREBP-1), adipocyte protein 2, and fatty acid synthase (FAS), which are related to adipogenesis, in addition to hormone-sensitive lipase (HSL), carnitine palmitoyl transferase 1 (CPT-1), and uncoupling protein 2 (UCP2), which are related to fat oxidation in WAT. In 3T3-L1 adipocytes, kaempferol and coumestrol exhibited anti-adipogenic effects via downregulation of PPARγ, c/EBPα, SREBP-1, and FAS. Kaempferol and coumestrol increased the expression of HSL, CPT-1, and UCP2.

Inhibitory effect of arazyme on the development of atopic dermatitis-like lesions in BALB/c and Nc/Nga mice.

Arazyme is a metalloprotease released by Aranicola proteolyticus that was shown to inhibit cytokine release in HaCaT and endothelial cells. However, the regulatory effects of arazyme in atopic dermatitis remain to be fully understood. In the present study, the anti­inflammatory effects of arazyme in BALB/c and Nc/Nga mice induced with 2,4­dinitrochlrobenzene (DNCB) were investigated. BALB/c mice were sensitized with DNCB and were subsequently administered arazyme for 4 weeks either orally, dorsally or orally/dorsally. Arazyme administration significantly reduced epidermal thickening and infiltration of inflammatory cells into the dermis compared with the DNCB group. However, serum immunoglobulin E (IgE) levels were not altered by arazyme treatment. Additionally, the level of secretion of interleukins (IL)­4, ­5 and ­13 in the splenocytes of BALB/c mice was elevated following stimulation with concanavalin A, while the increase of IL­4 and IL­13 was inhibited by arazyme. Administration of arazyme (25 mg/kg in phosphate­buffered saline) to Nc/Nga mice that had been sensitized with DNCB for 6 weeks reduced the skin severity score compared with that in the DNCB group and inhibited the histological manifestations of atopic dermatitis­like skin lesions. In addition, the serum IgE levels were reduced in the arazyme­treated NC/Nga mice relative to the DNCB group. Collectively, these results indicated that arazyme attenuates the development of atopic dermatitis­like lesions via lowering the levels of IgE and inflammatory cytokines. The results of the present study will aid in the development of effective therapeutic strategies for the treatment of allergic diseases, including atopic dermatitis.

Involvement of Nrf2-GSH signaling in TGFß1-stimulated epithelial-to-mesenchymal transition changes in rat renal tubular cells.

Transforming growth factor-ß1 (TGFß1) induces epithelial-to-mesenchymal transition (EMT) in cultured renal tubular epithelial cells. This phenotypic transition has been known to be involved in the development of chronic kidney diseases by activating profibrotic gene expression. Since oxidative stress has been recognized as one of the contributors to this TGFß1-mediated pathology, we investigated the potential involvement of nuclear factor (erythroid-derived 2)-like 2 (Nrf2), which is a key transcription factor for the regulation of multiple antioxidant genes, in TGFß1-stimulated EMT gene changes using the rat proximal tubular epithelial cell line NRK52E. The treatment of NRK52E with TGFß1 led to changes in EMT gene expression, including increased α-Sma and decreased E-cadherin expression. In these cells, the TGFß1 treatment decreased the transcript level of the catalytic subunit of γ-glutamate cysteine ligase (Gclc), a glutathione (GSH) biosynthetic enzyme, and reduced the total GSH content with a concomitant decrease in Nrf2 transcription activity. Accordantly, pre-incubation with the GSH precursor N-acetylcysteine attenuated TGFß1-stimulated EMT gene changes. The involvement of Nrf2 in EMT gene changes has been demonstrated using NRK52E cells with nrf2 knockdown or pharmacological activation. When the expression of Nrf2 was stably silenced in NRK52E cells using interfering RNA administration, Gclc expression was significantly reduced and the increase in the levels of α-Sma and fibronectin-1 by TGFß1 was greater than those in the nonspecific RNA control group. Conversely, Nrf2 activation and subsequent Gclc increase by Nrf2-activating sulforaphane alleviated the TGFß1-stimulated α-Sma increase and E-cadherin decrease. Collectively, these results indicate that Nrf2-GSH signaling can modulate TGFß1-stimulated EMT gene changes and further suggest a beneficial role of Nrf2 inducers in renal pathogenesis.

Pterocarpan-enriched soy leaf extract ameliorates insulin sensitivity and pancreatic ß-cell proliferation in type 2 diabetic mice.

In Korea, soy (Glycine max (L.) Merr.) leaves are eaten as a seasonal vegetable or pickled in soy sauce. Ethyl acetate extracts of soy leaves (EASL) are enriched in pterocarpans and have potent α-glucosidase inhibitory activity. This study investigated the molecular mechanisms underlying the anti-diabetic effect of EASL in C57BL/6J mice with high-fat diet (HFD)-induced type 2 diabetes. Mice were randomly divided into normal diet (ND), HFD (60 kcal% fat diet), EASL (HFD with 0.56% (wt/wt) EASL), and Pinitol (HFD with 0.15% (wt/wt) pinitol) groups. Weight gain and abdominal fat accumulation were significantly suppressed by EASL. Levels of plasma glucose, HbA1c, and insulin in the EASL group were significantly lower than those of the HFD group, and the pancreatic islet of the EASL group had greater size than those of the HFD group. EASL group up-regulated neurogenin 3 (Ngn3), paired box 4 (Pax4), and v-maf musculoaponeurotic fibrosarcoma oncogene homolog A (MafA), which are markers of pancreatic cell development, as well as insulin receptor substrate 1 (IRS1), IRS2, and glucose transporter 4 (GLUT4), which are related to insulin sensitivity. Furthermore, EASL suppressed genes involved in hepatic gluconeogenesis and steatosis. These results suggest that EASL improves plasma glucose and insulin levels in mice with HDF-induced type 2 diabetes by regulating ß-cell proliferation and insulin sensitivity.