Gene expression analysis of inflammation-related genes in macrophages treated with α-(1 → 3, 1 → 6)-D-glucan extracted from Streptococcus mutans.

Streptococcus mutans is a gram-positive bacterium that causes tooth decay. The exopolyssacharides, mostly glucans synthesized by the bacterium are responsible for establishing pathogenic bio-films associated with dental caries disease. The regulatory immune and inflammatory reactions implicated by the synthesized glucans are still not clearly understood. In this study, a water-soluble exopolyssacharide (WSP) was extracted from culture of Str. mutans. The structural properties of WSP, [α-(1 â†’ 3, 1 â†’ 6)-D-glucan] were confirmed using Fourier-transform infrared spectroscopy and 13C-nuclear magnetic resonance spectroscopy. Furthermore, the effects of WSP on the global gene expression of the macrophage-like RAW 264.7 cells were analyzed using mRNA-seq analysis. Using Gene Ontology analysis, we compiled a total of 24,421 genes that were upregulated or downregulated by more than 5.0-fold and 0.3-fold, respectively. Most of the transcripts were grouped under immune response and inflammation-related gene categories. Among the 802 immunity-related genes analyzed, chemokine ligand 7 (Ccl7), interleukin-1ß (IL-1ß), interleukin-1α (IL-1α) and interleukin-6 (IL-6) were upregulated after WSP exposure. In addition, among a total of 344 genes related to inflammation, Ccl7, IL-1α and IL-6 were upregulated. These results suggest that [α-(1 â†’ 3, 1 â†’ 6)-D-glucan] from Str. mutans produces activates macrophages and may contribute to the immune and inflammatory response to periodontal disease.

Juglone Suppresses LPS-induced Inflammatory Responses and NLRP3 Activation in Macrophages.

The NLRP3 (NACHT, LRR and PYD domains-containing protein 3) inflammasome has been implicated in a variety of diseases, including atherosclerosis, neurodegenerative diseases, and infectious diseases. Thus, inhibitors of NLRP3 inflammasome have emerged as promising approaches to treat inflammation-related diseases. The aim of this study was to explore the effects of juglone (5-hydroxyl-1,4-naphthoquinone) on NLRP3 inflammasome activation. The inhibitory effects of juglone on nitric oxide (NO) production were assessed in lipopolysaccharide (LPS)-stimulated J774.1 cells by Griess assay, while its effects on reactive oxygen species (ROS) and NLRP3 ATPase activity were assessed. The expression levels of NLRP3, caspase-1, and pro-inflammatory cytokines (IL-1ß, IL-18) and cytotoxicity of juglone in J774.1 cells were also determined. Juglone was non-toxic in J774.1 cells when used at 10 µM (p < 0.01). Juglone treatment inhibited the production of ROS and NO. The levels of NLRP3 and cleaved caspase-1, as well as the secretion of IL-1ß and IL-18, were decreased by treatment with juglone in a concentration-dependent manner. Juglone also inhibited the ATPase activities of NLRP3 in LPS/ATP-stimulated J774.1 macrophages. Our results suggested that juglone could inhibit inflammatory cytokine production and NLRP3 inflammasome activation in macrophages, and should be considered as a therapeutic strategy for inflammation-related diseases.

Pine ( Pinus densiflora ) needle extract could promote the expression of PCNA and Ki-67 after partial hepatectomy in rat.

PURPOSE: To investigate the effects of pine needle extract (PNE) on the expression of proliferating cell nuclear antigen (PCNA) and Ki-67 during liver regeneration induced by 70% partial hepatectomy (PH) in rat. METHODS: Forty-eight male rats (SD, 7 weeks) had surgery (70% PH). They were randomly divided into two groups. PH + PNE group was only provided PNE diluted in water (10%) for drinking and PH group was provided water from 5 days before surgery to the time of sacrifice. PNE was made by pressing and filtering. Animals were sacrificed at 12h, 24h, 36h, 60h, 84h, 168h after PH, respectively. The expressions of PCNA and Ki-67 were determined as proliferation indices. RESULTS: Immunohistochemistry turned out to increase the expression of PCNA and Ki-67. PCNA expression of PH+PNE group increased up to twice of that of PH group. Western blot also seemed to increase the PCNA expression. These results indicated the promotion of cell proliferation in liver tissue and hepatic regeneration. CONCLUSIONS: Pine needle extract stimulates the expression of some mitotic proteins during liver regeneration induced by 70% PH in rats. It suggests that administration of pine needle extract could accelerate the liver regeneration after partial hepatectomy.

Pine ( Pinus densiflora ) needle extract could promote the expression of PCNA and Ki-67 after partial hepatectomy in rat

Abstract Purpose To investigate the effects of pine needle extract (PNE) on the expression of proliferating cell nuclear antigen (PCNA) and Ki-67 during liver regeneration induced by 70% partial hepatectomy (PH) in rat. Methods Forty-eight male rats (SD, 7 weeks) had surgery (70% PH). They were randomly divided into two groups. PH + PNE group was only provided PNE diluted in water (10%) for drinking and PH group was provided water from 5 days before surgery to the time of sacrifice. PNE was made by pressing and filtering. Animals were sacrificed at 12h, 24h, 36h, 60h, 84h, 168h after PH, respectively. The expressions of PCNA and Ki-67 were determined as proliferation indices. Results Immunohistochemistry turned out to increase the expression of PCNA and Ki-67. PCNA expression of PH+PNE group increased up to twice of that of PH group. Western blot also seemed to increase the PCNA expression. These results indicated the promotion of cell proliferation in liver tissue and hepatic regeneration. Conclusions Pine needle extract stimulates the expression of some mitotic proteins during liver regeneration induced by 70% PH in rats. It suggests that administration of pine needle extract could accelerate the liver regeneration after partial hepatectomy.

Immunomodulatory Effects of Extracellular ß-Glucan Isolated from the King Oyster Mushroom Pleurotus eryngii (Agaricomycetes) and Its Sulfated Form on Signaling Molecules Involved in Innate Immunity.

The aim of this study was to determine, using murine RAW 264.7 macrophages, the immunomodulatory effect of extracellular ß-glucan isolated from Pleurotus eryngii (PEBG) and its sulfated derivative (PEBG-S) on signaling molecules implicated in host innate immunity. ß-Glucan was extracted and purified from the mycelial culture using optimal medium concentrations. It was then chemically converted to its sulfated form. Monosaccharide composition of ß-glucan was characterized with p-aminobenzoic acid ethyl ester-derivatized sugars through highperformance liquid chromatography analysis. Fourier transform infrared structural analysis showed an S=O bond at 1250 cm-1 and C-S-O binding at 815 cm-1 in PEBG-S. 13C nuclear magnetic resonance analysis showed 1,3-linked α-D-mannopyranosyl and 1,3-ß-D-glucopyranosyl in PEBG-S. A concentration-dependent increase of nitric oxide production was noticed in RAW 264.7 cells treated with PEBG-S or PEBG; those treated with PEBG-S showed less cytotoxicity than those treated with PEBG. Cellular levels of tumor necrosis factor-α, interleukin-1ß, and interleukin-6 were increased by PEBG and PEBG-S treatment, suggesting that they have immunomodulatory activity. Real-time polymerase chain reaction array revealed that the expression levels of nuclear factor-κB and Toll-like receptor signaling genes in cells were upregulated by PEBG and PEBG-S. Moreover, the expression of the ß-glucan receptor dectin-2 was significantly upregulated by PEBG and PEBG-S treatment, reflecting immune activation through the dectin-2-Syk-(CARD9/Bcl-10/MALT1) pathway. Our results suggest that PEBG-S could be used as an effective adjuvant or immune enhancer that can be sustainably produced by recycling the by-product of mycelial culture.

Mutan: A mixed linkage α-[(1,3)- and (1,6)]-d-glucan from Streptococcus mutans, that induces osteoclast differentiation and promotes alveolar bone loss.

Mutan is an extracellular polysaccharide of Streptococcus mutans (S. mutans) that consists of α-(1,3)-linked glucose residues in main chains and α-(1,6) bonds in side chains. In the present study, mutan was isolated from S. mutans, and its structural characteristics were determined using Fourier-transform infrared spectroscopy (FT-IR) and (13)C nuclear magnetic resonance (NMR) spectroscopy. The effects of mutan on RANKL-induced osteoclast differentiation in RAW 264.7 cells were examined. Furthermore, microCT and morphometric analyses were used to determine the contribution of mutan to alveolar bone loss in the maxilla of a rat periodontitis model. Mutan increased (more than 2-fold) RANKL-induced osteoclast differentiation in a dose-dependent manner. Mutan also enhanced the alveolar bone loss in the rat maxilla 2.3-fold. In mutan-treated rats, the bone mineral density, bone volume, trabecular number, and trabecular thickness decreased, whereas trabecular separation significantly increased. In addition, mutan and lipopolysaccharide (LPS) induced similar microarray profiles in RAW 264.7 cells. A total of 43 genes related to osteoclastogenesis were differentially expressed after either mutan or LPS treatment. Five-fold increases in the expression of several genes, including IL-1ß, IL-1α, IL-6, and chemokine ligands, were observed in mutan-treated RAW 264.7 cells. These results suggest a molecular mechanism for the inflammation induced by S. mutans during the establishment of periodontal disease.

Brazilin Limits Inflammatory Responses through Induction of Prosurvival Autophagy in Rheumatoid Fibroblast-Like Synoviocytes.

Brazilin is an active compound of Caesalpinia sappan L. (Leguminosae), which possesses pro-apoptotic and anti-inflammation potentials depending on the specific cell type. However, it is largely unknown whether autophagy is implicated in the mechanism underlying its chemotherapeutic and anti-inflammatory effects in rheumatoid arthritis (RA). Here, we show that treatment of RA fibroblast-like synoviocytes (FLS) with brazilin results in enhanced level of autophagic flux, evidenced by accumulation of autophagosome and increased level of lipidated LC3 (LC3-II), which is mainly mediated by enhanced production of reactive oxygen species (ROS). Interestingly, long-term exposure of brazilin was able to restore cell survival against the cytotoxity, exclusively in RA FLS, but not in normal fibroblast. Importantly, such a restoration from brazilin-induced cytotoxity in RA FLS was completely abrogated after co-treatment with autophagy inhibitors including NH4Cl or chloroquine. Furthermore, we found that the pretreatment of RA FLS with brazilin reduced LPS- or TNF-induced NF-κB activation and the secretion of inflammatory cytokines in parallel with the enhanced autophagic flux. Such anti-NF-κB potentials of brazilin were drastically masked in RA FLS when autophagy was suppressed. These results suggest that brazilin is capable of activating autophagy exclusively in RA FLS, and such inducible autophagy promotes cell survival and limits inflammatory response.

Brazilin isolated from Caesalpinia sappan L. inhibits rheumatoid arthritis activity in a type-II collagen induced arthritis mouse model.

BACKGROUND: Caesalpinia sappan L. extracts exhibit great therapeutic potential, and have been shown to have analgesic and anti-inflammatory properties. This study aimed to understand the anti-rheumatoid activity of brazilin that was isolated from ethyl acetate extract of C. sappan L. The evaluations were conducted in mice with type-II collagen-induced arthritis (CIA). METHODS: Brazilin was purified via preparative HPLC and identified by mass spectrometry and 1H/13C NMR analysis. DBA/1J mice were divided into four groups (n=10). Three groups of mice received intradermal injections of inducer bovine type-II collagen (BTIIC; 2 mg/ml in 0.05 ml acetic acid) and 0.1 ml of booster complete Freund's adjuvant (CFA). A second injection of BTIIC with booster incomplete Freund's adjuvant (ICFA) was given subsequently after 21 days. On 22nd day, purified brazilin (10 mg/kg body weight) or the disease-modifying anti-rheumatic drug methotrexate (3 mg/kg body weight) was administered intraperitoneally daily or every three days for 21 days, respectively to two groups of mice. At the 42nd day, mice sera were collected, and the levels of pro-inflammatory cytokines and stress enzyme markers in serum were measured using standard immunoassay methods. The microstructure and morphometric analyses of the bones were assessed using high-resolution microfocal computed tomography. RESULTS: Brazilin isolated from C. sappan reduced the arthritis index score and the extent of acute inflammatory paw edema in CIA-mice. The bone mineral density was significantly (p<0.05) lower in only-CIA mice, and appeared to increase commensurate with methotrexate and brazilin administration. Brazilin prevented joint destruction, surface erosion, and enhanced bone formation as revealed by microstructural examinations. Brazilin markedly attenuated mouse CIA and reduced the serum levels of inflammatory cytokines including TNF-α, IL-1ß, and IL-6. CONCLUSIONS: Brazilin purified from C. sappan L. shows protective efficacy in CIA mouse, and may be useful to treat chronic inflammatory disorders including rheumatoid arthritis.

Anti-inflammatory activity of sappanchalcone isolated from Caesalpinia sappan L. in a collagen-induced arthritis mouse model.

Sappanchalcone, a bioactive flavonoid isolated from the heartwood of Caesalpinia sappan L. possesses anti-inflammatory effects. We studied the efficacy of sappanchalcone in attenuating collagen-induced arthritis (CIA) in a mouse model of rheumatoid arthritis. Sappanchalcone was purified to homogeneity from the chloroform fraction of the methanolic extract of C. sappan, and identified using mass spectrometry and (1)H-nuclear magnetic resonance spectroscopy. CIA-induced male DBA/1J mice were divided into control, sappanchalcone-treated, and methotrexate-treated groups (n = 10 per group). Paw swelling, arthritis severity, radiographic and histomorphometric changes were assessed to measure the protective role of sappanchalcone against chronic disease progression. Sappanchalcone administration significantly reduced clinical arthritis and inflammatory edema in paws. Bone mineral density and trabecular structure were maintained in CIA mice administered sappanchalcone. The levels of pro-inflammatory cytokines (TNF-α, IL-6, and 1L-1ß) were significantly lower in the serum of sappanchalcone-treated mice as compared with the control group. Our results suggest that sappanchalcone could be used as an anti-inflammatory and bone-protective agent during the treatment of rheumatoid arthritis.

Bacterial community dynamics of salted and fermented shrimp based on denaturing gradient gel electrophoresis.

The Korean traditional seafood jeotgal is consumed directly or as an additive in other foods to improve flavor or fermentation efficiency. Saeujot, made from salted and fermented tiny shrimp (SFS; Acetes japonicus), is the best-selling jeotgal in Korea. In this study, we reveal the microbial diversity and dynamics in naturally fermented shrimp by denaturing gradient gel electrophoresis (DGGE). The population fingerprints of the predominant microbiota and its succession were generated by DGGE analysis of universal V3 16S rDNA polymerase chain reaction (PCR) amplicons. Overall, 17 strains were identified from sequencing of 30 DGGE bands. The DGGE profiles showed diverse bacterial populations in the sample, throughout the fermentation of SFS. Staphylococcus equorum, Halanaerobium saccharolyticum, Salimicrobium luteum, and Halomonas jeotgali were the dominant bacteria, and their levels steadily increased during the fermentation process. Certain other bacteria, such as Psychrobacter jeotgali and Halomonas alimentaria appeared during the early-fermentation process, while Alkalibacterium putridalgicola, Tetragenococcus muriaticus, and Salinicoccus jeotgali appeared during the late-fermentation process. The members of the order Bacillales were found to be predominant during the fermentation of SFS. Furthermore, S. equorum was identified as the dominant bacterial isolate by the traditional method of culturing under aerobic and facultative anaerobic conditions. We expect that this information will facilitate the design of autochthonous starter cultures for the production of SFS with desired characteristic sensory profiles and shorter ripening times.

The role of wogonin in controlling SOCS3 expression in neuronal cells.

The mechanism underlying the wogonin-mediated increase in the expression of suppressor of cytokine signaling 3 (SOCS3) is unclear. Promoter deletion assay results revealed that wogonin-induced SOCS3 expression is dependent on the AP-1 consensus sequences and two STAT responsive elements (TTACAAGAA and TTCCAGGAA) in the 5'-flanking region of the SOCS3 gene in SH-SY5Y cells. Wogonin-induced SOCS3 expression was blocked by inhibitors of PI3K, Akt, Raf, p38, JNK, MEK, and STAT3, respectively. However, JAK2 inhibitors did not inhibit wogonin-induced SOCS3 expression. These results indicate that SOCS3-inducing effect of wogonin is caused by the activation of PI3K-mediated MAPK signaling pathways (Akt, ERK1/2, p38, and JNK), and the subsequent activation of AP-1 consensus sequences and STAT responsive elements in SH-SY5Y cells.

Brazilin selectively disrupts proximal IL-1 receptor signaling complex formation by targeting an IKK-upstream signaling components.

The ligation of interleukin-1 receptor (IL-1R) or tumor necrosis factor receptor 1 (TNFR1) induces the recruitment of adaptor proteins and their concomitant ubiquitination to the proximal receptor signaling complex, respectively. Such are upstream signaling events of IKK that play essential roles in NF-κB activation. Thus, the discovery of a substance that would modulate the recruitment of key proximal signaling elements at the upstream level of IKK has been impending in this field of study. Here, we propose that brazilin, an active compound of Caesalpinia sappan L. (Leguminosae), is a potent NF-κB inhibitor that selectively disrupts the formation of the upstream IL-1R signaling complex. Analysis of upstream signaling events revealed that brazilin markedly abolished the IL-1ß-induced polyubiquitination of IRAK1 and its interaction with IKK-γ counterpart. Notably, pretreatment of brazilin drastically interfered the recruitment of the receptor-proximal signaling components including IRAK1/4 and TRAF6 onto MyD88 in IL-1R-triggerd NF-κB activation. Interestingly, brazilin did not affect the TNF-induced RIP1 ubiquitination and the recruitment of RIP1 and TRAF2 to TNFR1, suggesting that brazilin is effective in selectively suppressing the proximal signaling complex formation of IL-1R, but not that of TNFR1. Moreover, our findings suggest that such a disruption of IL-1R-proximal complex formation by brazilin is not mediated by affecting the heterodimerization of IL-1R and IL-1RAcP. Taken together, the results suggest that the anti-IKK activity of brazilin is induced by targeting IKK upstream signaling components and subsequently disrupting proximal IL-1 receptor signaling complex formation.

Isolation and characterization of chitinase-producing Bacillus and Paenibacillus strains from salted and fermented shrimp, Acetes japonicus.

Chitinases catalyze the conversion of chitin and are produced by a wide range of bacteria. The biological applications of these enzymes have been exploited in food and pharmaceutical industries. We isolated 2 halophilic chitinase-producing novel strains of bacteria-SCH-1 and SCH-2 from Saeu-jeot, a traditional Korean salted and fermented food made with shrimp (Acetes japonicus). The isolated strains- SCH-1 and SCH-2 were Gram-positive, rod-shaped, endospore-forming facultative anaerobes, with strain SCH-2 showing peritrichous flagella. Molecular characterization of the 16S rRNA gene identified the strains SCH-1 and SCH-2 as Bacillus sp. and Paenibacillus sp. respectively. Basic Local Alignment Search Tool and subsequent phylogenetic analysis of strain SCH-1 showed an identity of 97.83% with Bacillus cereus ATCC 14579 (NR_074540), whereas strain SCH-2 showed an identity of 99.16% with Paenibacillus lautus JCM 9073 (NR_040882). Furthermore, the SCH-1 strain could use glucose, N-acetyl glucosamine, esculin, and maltose as carbon source substrates. Cellular fatty acid analysis showed that iso-C15:0 and anteiso-C15:0 are the major acids in strain SCH-1 and SCH-2, respectively. The SCH-1 strain showed a higher chitinase activity at 15.71 unit/mg protein compared with SCH-2 strain. Chitinase isozymes of Bacillus sp. SCH-1was expressed as 2 bands having sizes of 41 and 50 kDa, and as 4 bands with sizes of 30, 37, 45.7, and 50 kDa in Paenibacillus sp. SCH-2. The rich chitinase activity with the isozyme profiles of the isolated Bacillus and Paenibacillus strains provide advancement in the study of fermentation and may play putative functions in the chitin bioconversion of sea crustacean foods.

Effects of different selenium levels on growth and regulation of laccase and versatile peroxidase in white-rot fungus, Pleurotus eryngii.

This study was conducted to evaluate the physiological effects of different selenium (Se) levels on the growth of white-rot fungus, Pleurotus eryngii, with special reference to the regulation of ligninolytic enzymes such as laccase and versatile peroxidase. The fungus was grown in medium supplemented with 1, 10, 100, 1,000 and 10,000 µM of sodium selenite. Mycelial growth was stronger at lower Se levels, but declined significantly at higher concentrations of 1,000 and 10,000 µM, highlighting its association in mediating toxic responses. Inhibition of fungal growth was accompanied with dense and entangled hyphae taking the shape of irregular short strips. Additionally, hyphal swellings and septation were noticed which lead to a reduction in the advancement of the mycelium. Along with the inhibition of fungal biomass, the reducing sugar and protein concentrations increased to about 30.2 and 3.5 mg/ml respectively in the growth medium. Additionally, the laccase gene expression showed a twofold upregulation at higher levels of Se, although the activity of the enzyme was compromised with an inverse relationship with increased gene transcripts. The versatile peroxidase transcript showed a complete downregulation at 10,000 µM after an upregulation at lower levels of Se. We also confirmed the direct relationship of different Se levels on laccase activity of Rhus vernicifera that showed similar behavior to the fungal laccase. The results of the present study suggest that Se supplementation regulates mRNA levels of laccase and versatile peroxidase depending on exposure and may play a role in the toxicity associated with Se.

Tert-butylhydroquinone reduces lipid accumulation in C57BL/6 mice with lower body weight gain.

tert-Butylhydroquinone (tBHQ) is a commonly used antioxidant additive that is approved for human use by both the Food and Agriculture Organization and the World Health Organization (FAO/WHO). In this study, we examined the effect of tBHQ on body weight gain and found that food supplementation with 0.001 % (w/w) tBHQ inhibited 61.4 % (P < 0.01) of body weight gain in high-fat diet (HFD)-induced C57BL/6 mice, and the oral administration of tBHQ (1.5 mg/kg) reduced 47.5 % (P < 0.05) of body weight gain in normal diet fed db/db mice. The HFD increased lipid deposit in adipocytes, but these were reduced significantly by tBHQ treatment in C57BL/6 mice. tBHQ supplementation significantly lowered the plasma triglyceride and total cholesterol, with reduced size of accumulated fat mass. The rate limiting enzyme of beta-oxidation (ACOX1) was significantly over-expressed in the liver with tBHQ treatment. These results indicate that tBHQ suppresses body weight gain in mice, possibly at least related to the up-regulation of ACOX1 gene expression.

Single dose toxicity studies of sulfated water soluble beta-D-glucan in Sprague-Dawley rats.

The fungal beta-D-glucan is a biological response modifier (BRM), but a major obstacle to the clinical utilization of these BRMs is their relative insolubility in aqueous media. We made soluble sulfated-beta-glucan (SGL) from insoluble beta-glucan (IGL) by sulfation method. In single dose toxicity study of SGL for 7 days, no negative effects on body weight or food consumption of rats were evident below a dose rate of 2,000 mg kg(-1) SGL. No clinical pathology, functional/behavioral, or gross observations indicating toxicity were detected. In hematology and biochemistry, statistically significant increases of WBC and neutrophils (P < 0.01) in male and increase of MCV (P < 0.05) in females was observed. However, since the changes were not dose-responsive, the effects were considered to be of no toxicological significance. These results suggest that chemically modified sulfated-beta-D-glucan was lesstoxic than the insoluble b-glucan and not considered acutely toxic following peritoneal exposure to 2,000 mg kg(-1) day(-1) in Sprague-Dawley rats.

Resveratrol, purified from the stem of Vitis coignetiae Pulliat, inhibits food intake in C57BL/6J Mice.

Neuropeptide Y (NPY) and agouti-related protein (AgRP) have powerful stimulatory effects on food intake, which suggests that the downregulation of brain NPY or AgRP may help reduce obesity and diabetes by inhibiting food intake. To search for active compounds that inhibit NPY and AgRP expression, we made two luciferase reporter assay systems consisting of NPY and AgRP promoter-driven luciferase genes, together with the puromycin resistance gene, in a plasmid vector. Each plasmid was permanently transfected into N29-4 neuronal cells. Using the systems, resveratrol was purified from the stem of Vitis coignetiae Pulliat by activityguided fractionation. Resveratrol downregulated NPY and AgRP promoter-driven luciferase activity in a dose-dependent manner. The inhibitory concentrations (IC(50), 50% inhibition) of resveratrol against pNPY-luc and pAgRP-luc activities were 8.9 microM and 8.0 microM, respectively. Furthermore, one-time intraperitoneal injection of resveratrol (100 mg/kg) suppressed 20.0% and 17.2% of food intake during 24 and 48 h, respectively. These results indicated that resveratrol inhibited food intake, which may be related to the downregulation of NPY and AgRP gene expression.

Expression of recombinant hybrid peptide hinnavin II/alpha-melanocyte-stimulating hormone in Escherichia coli: purification and characterization.

The increasing problem of antibiotic resistance among pathogenic bacteria requires novel strategies for the construction of multiple, joined genes of antimicrobial agents. The strategy used in this study involved synthesis of a cDNA-encoding hinnavin II/alpha-melanocyte-stimulating hormone (hin/MSH) hybrid peptide, which was cloned into the pET32a (+) vector to allow expression of the hybrid peptide as a fusion protein in Escherichia coli BL21 (DE3). The resulting expression of fusion protein Trx-hin/MSH could reach up to 20% of the total cell proteins. More than 50% of the target protein was in a soluble form. The target fusion protein from the soluble fraction, Trx-hin/MSH, was easily purified by Ni(2+)-chelating chromatography. Then, enterokinase cleavage effectively cleaved the Trx-hin/MSH to release the recombinant hin/MSH (rhin/MSH) hybrid peptide. After removing the contaminants, we purified the recombinant hybrid peptide to homogeneity by reversed-phase FPLC and obtained 210 mg of pure, active rhin/MSH from 800 ml of culture medium. Antimicrobial activity assay demonstrated that rhin/MSH had a broader spectrum of activity than did the parental hinnavin II or MSH against fungi and Gram-positive and Gram-negative bacteria. These results suggest an efficient method for producing high-level expression of various kinds of antimicrobial peptides that are toxic to the host, a reliable and simple method for producing different hybrid peptides for biological studies.

Rutecarpine ameliorates bodyweight gain through the inhibition of orexigenic neuropeptides NPY and AgRP in mice.

Orexigenic neuropeptides NPY and AgRP play major roles in feeding and are closely related to obesity and diabetic metabolic syndrome. This study explored the inhibitory effect of rutecarpine on feeding and obesity in high-fat-diet-induced (C57BL/6) and leptin-deficient (ob/ob) obese mice. Both mice strains developed obesity, but the obesity was inhibited by the reduced food intake resulting from rutecarpine treatment (0.01%, p<0.01). Blood cholesterol, non-fasting glucose, insulin, and leptin levels were reduced, compared with the control group. Rutecarpine inhibited the expression of NPY and AgRP in the arcuate nucleus (ARC) of the hypothalamus and suppressed the expression of both neuropeptides in N29-4 neuronal cells. These results indicate that rutecarpine ameliorates obesity by inhibiting food intake, which involves inhibited expression of the orexigenic neuropeptides NPY and AgRP.

Solubilization of water-insoluble beta-glucan isolated from Ganoderma lucidum.

The fungal beta-D-glucan is a biological response modifier (BRM), but a major obstacle to the clinical utilization of beta-glucan BRMs is thei relative lack of solubility in aqueous media. Water insoluble fungal glucans extracted by alkali from the mycelia of Ganoderma lucidum were sulfated to yield their corresponding water-soluble derivatives. Insoluble glucan is dissolved in methyl sulfoxide and urea, and is partially sulfated with sulfuric acid. The sulfated glucan (SGL) yield prepared from insoluble glucan (IGL) was 85%, the sulfation degree of SGL was about 14.9%, and the solubility of SGL was above 95% in water. The monosugar SGL content was 34.9% alpha-glucose and 35.9% beta-glucose. The mean molecular weight (MW) of SGL was shown as a single peak on Sepharose CL-4B column chromatography, and their MW was approximately 9.3 kDa. The 13C NMR spectrum analysis shows that SGL has a high similarity with the beta-(1-->3)-linked triple-helical control.