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1.
Biochem Biophys Res Commun ; 732: 150406, 2024 Jul 16.
Article in English | MEDLINE | ID: mdl-39032412

ABSTRACT

Lignocellulose, the most abundant organic waste on Earth, is of economic value because it can be converted into biofuels like ethanol by enzymes such as ß-glucosidase. This study involved cloning a ß-glucosidase gene named JBG from the rumen fungus Neocallimastix patriciarum J11. When expressed recombinantly in Escherichia coli, the rJBG enzyme exhibited significant activity, hydrolyzing 4-nitrophenyl-ß-d-glucopyranoside and cellobiose to release glucose. Surprisingly, the rJBG enzyme also showed hydrolytic activity against ß-glucan, breaking it down into glucose, indicating that the rJBG enzyme possesses both ß-glucosidase and ß-glucanase activities, a characteristic rarely found in ß-glucosidases. When the JBG gene was expressed in Saccharomyces cerevisiae and the transformants were inoculated into a medium containing ß-glucan as the sole carbon source, the ethanol concentration in the culture medium increased from 0.17 g/L on the first day to 0.77 g/L on the third day, reaching 1.3 g/L on the fifth day, whereas no ethanol was detected in the yeast transformants containing the recombinant plasmid pYES-Sur under the same conditions. These results demonstrate that yeast transformants carrying the JBG gene can directly saccharify ß-glucan and ferment it to produce ethanol. This gene, with its dual ß-glucosidase and ß-glucanase activities, simplifies and reduces the cost of the typical process of converting lignocellulose into bioethanol using enzymes and yeast.

2.
Article in English | MEDLINE | ID: mdl-39031839

ABSTRACT

BACKGROUND AND AIM: Among low viral load (DNA of hepatitis B virus (HBV) was < 2000 IU/mL), the factor of the loss of hepatitis B surface antigen (HBsAg) remained elusive. METHODS: The retrospective study recruited patients with chronic hepatitis B (CHB) who were negative low for hepatitis B e-antigen (HBeAg), had a low viral load, and experienced HBsAg loss during follow-up. CHB patients with low-viral load but without consequent HBsAg loss were also enrolled at the ratio of 1:4. The factors contributing to HBsAg loss were analyzed. RESULTS: A total of 80 patients were recruited for the current study, with a mean age of 63.9 years and 61.3% being male. Among them, 62.5% patients (50/80) were treated with potent nucleoside/nucleotide analogues (NAs) during the follow-up period. Additionally, 12.5% patients (10/80) had a prior history of NAs treatment before enrolment. During the follow-up, HBsAg loss occurred in 17 patients (21.3%). Compared with patients without HBsAg loss, those with HBsAg loss were younger (57.9 years vs 65.5 years; P = 0.01), had lower HBV DNA levels (1.3 log10 IU/mL vs 2.3 log10 IU/mL; P = 0.003), and higher proportion of prior NAs-treated history. Logistic regression analysis revealed that the factors associated with factors associated with HBsAg loss were age < 60 years (OR/CI: 3.95/1.15-13.60, P = 0.03), prior NAs-treated history (OR/CI: 7.59/1.42-40.51, P = 0.01) and current NAs-treated (OR/CI: 0.19/0.05-0.71, P = 0.01). CONCLUSIONS: In the study, older age and prior NAs were positively associated with HBsAg loss, and current NAs was negatively associated with HBsAg loss. Additionally, some patients experienced HBsAg loss during the NAs therapy.

3.
Nutrients ; 15(7)2023 Mar 31.
Article in English | MEDLINE | ID: mdl-37049552

ABSTRACT

(1) Background: Non-alcoholic fatty liver disease (NAFLD) is a major global health concern. The increasing prevalence of NAFLD has been related to type 2 diabetes mellitus (T2D). However, the relationship between short-chain fatty acids (SCFAs) and NAFLD severity is ambiguous in T2D subjects. This study aimed to explore the association of SCFAs with the severity of NAFLD in T2D patients. (2) Methods: We employed echography to examine the severity of hepatic steatosis. The serum levels of nine SCFAs, namely, formate, acetate, propionate, butyrate, isobutyrate, methylbutyrate, valerate, isovalerate, and methylvalerate, were measured using gas chromatography mass spectrometry. (3) Results: A total of 259 T2D patients was enrolled in this cross-sectional study. Of these participants, 117 with moderate to severe NAFLD had lower levels of formate, isobutyrate, and methylbutyrate than the 142 without NAFLD or with mild NAFLD. Lower circulating levels of isobutyrate and methylbutyrate were associated with an increased severity of NAFLD. A relationship between NAFLD severity and circulating isobutyrate and methylbutyrate levels was found independently of a glycated hemoglobin (HbA1C) level of 7.0%. (4) Conclusion: Circulating levels of isobutyrate and methylbutyrate were significantly and negatively correlated with NAFLD severity in the enrolled T2D patients. SCFAs may be related to NAFLD severity in T2D patients.


Subject(s)
Diabetes Mellitus, Type 2 , Fatty Acids, Volatile , Fatty Liver , Non-alcoholic Fatty Liver Disease , Humans , Fatty Acids, Volatile/blood , Non-alcoholic Fatty Liver Disease/blood , Diabetes Mellitus, Type 2/blood , Ultrasonography , Fatty Liver/diagnostic imaging , Isobutyrates/blood , Cross-Sectional Studies , Male , Female , Adult , Middle Aged , Aged , Aged, 80 and over
4.
Biomedicines ; 10(11)2022 Nov 05.
Article in English | MEDLINE | ID: mdl-36359344

ABSTRACT

The regulatory role of microRNAs (miRNAs) in HBV-associated HCC pathogenesis has been reported previously. This study aimed to investigate the association between serum miR-125b and liver fibrosis progression in chronic hepatitis B (CHB) patients after nucleos(t)ide analog (NA) treatment. Baseline serum miR-125b levels and other relevant laboratory data were measured for 124 patients who underwent 12-month NA therapy. Post-12-month NA therapy, serum miR-125, platelet, AST, and ALT levels were measured again for post-treatment FIB-4 index calculation. Univariate and multivariate logistic regression analyses were performed to identify independent risk factors for a higher post-treatment FIB-4 index. Results showed that baseline miR-125b levels were inversely correlated with the post-treatment FIB-4 index (ρ = −0.2130, p = 0.0082). In logistic regression analyses, age (OR = 1.17, p < 0.0001), baseline platelet level (OR = 0.98, p = 0.0032), and ALT level (OR = 1.00, p = 0.0241) were independent predictors of FIB-index > 2.9 post-12-month treatment. The baseline miR-125b level was not significantly associated with a higher post-treatment FIB-4 index (p = 0.8992). In 59 patients receiving entecavir (ETV) monotherapy, the alternation of serum miR-125b in 12 months and age were substantially associated with a higher post-treatment FIB-4 index (>2.9), suggesting that miR-125b is a reliable biomarker for detecting early liver fibrosis under specific anti-HBV NA treatments (e.g., ETV).

5.
Int J Mol Sci ; 23(10)2022 May 13.
Article in English | MEDLINE | ID: mdl-35628265

ABSTRACT

As a conventional medical dressing, medical gauze does not adequately protect complex and hard-to-heal diabetic wounds and is likely to permit bacterial entry and infections. Therefore, it is necessary to develop novel dressings to promote wound healing in diabetic patients. Komagataeibacter intermedius was used to produce unmodified bacterial cellulose, which is rarely applied directly to diabetic wounds. The produced cellulose was evaluated for wound recovery rate, level of inflammation, epidermal histopathology, and antimicrobial activities in treated wounds. Diabetic mices' wounds treated with bacterial cellulose healed 1.63 times faster than those treated with gauze; the values for the skin indicators in bacterial cellulose treated wounds were more significant than those treated with gauze. Bacterial cellulose was more effective than gauze in promoting tissue proliferation with more complete epidermal layers and the formation of compact collagen in the histological examination. Moreover, wounds treated with bacterial cellulose alone had less water and glucose content than those treated with gauze; this led to an increase of 6.82 times in antimicrobial protection, lower levels of TNF-α and IL-6 (39.6% and 83.2%), and higher levels of IL-10 (2.07 times) than in mice wounds treated with gauze. The results show that bacterial cellulose produced using K. intermedius beneficially affects diabetic wound healing and creates a hygienic microenvironment by preventing inflammation. We suggest that bacterial cellulose can replace medical gauze as a wound dressing for diabetic patients.


Subject(s)
Cellulose , Diabetes Mellitus, Experimental , Acetobacteraceae , Animals , Cellulose/pharmacology , Humans , Inflammation , Mice , Wound Healing
6.
Int J Mol Sci ; 22(23)2021 Nov 23.
Article in English | MEDLINE | ID: mdl-34884465

ABSTRACT

Rhizopus oryzae is a fungus used to ferment tempeh in Indonesia and is generally recognized as safe (GRAS) for human consumption by the USA FDA. We previously assessed the effect of a tempeh extract on cortisol levels in zebrafish but did not include behavioral studies. Here, we measured the GABA content in three strains of Rhizopus oryzae, two isolated by us (MHU 001 and MHU 002) and one purchased. We then investigated the effect of tempeh on cortisol and the gut microbiota in a zebrafish experimental model. GABA concentration was the highest in MHU 002 (9.712 ± 0.404 g kg-1) followed by our MHU 001 strain and the purchased one. The fish were divided into one control group fed a normal diet and three experimental groups fed soybean tempeh fermented with one of the three strains of Rhizopus oryzae. After two weeks, individual fish were subjected to unpredicted chronic stress using the novel tank diving test and the tank light-dark test. Next-generation sequencing was used to analyze gut microbial communities and RT-PCR to analyze the expression of BDNF (brain-derived neurotrophic factor) gene and of other genes involved in serotonin signaling/metabolism in gut and brain. Tempeh-fed zebrafish exhibited increased exploratory behavior (less stress) in both tank tests. They also had significantly reduced gut Proteobacteria (include E. coli) (51.90% vs. 84.97%) and significantly increased gut Actinobacteria (include Bifidobacterium spp.) (1.80% vs. 0.79%). The content of Bifidobacteriumadolescentis, a "psychobiotic", increased ten-fold from 0.04% to 0.45%. Tempeh also increases BDNF levels in zebrafish brain. Rhizopus oryzae MHU 001 greatly improved the anti-stress effect of tempeh and microbiota composition in zebrafish gut.


Subject(s)
Bacteria/classification , DNA, Bacterial/genetics , Rhizopus oryzae/physiology , Soy Foods/microbiology , Zebrafish/physiology , Animal Feed/microbiology , Animals , Bacteria/genetics , Bacteria/isolation & purification , Brain-Derived Neurotrophic Factor/genetics , Fermentation , Gastrointestinal Microbiome , High-Throughput Nucleotide Sequencing , Hydrocortisone/analysis , Rhizopus oryzae/chemistry , Rhizopus oryzae/classification , Sequence Analysis, DNA , Stress, Physiological , Zebrafish Proteins/genetics , gamma-Aminobutyric Acid/analysis
7.
Microorganisms ; 8(9)2020 Aug 31.
Article in English | MEDLINE | ID: mdl-32878315

ABSTRACT

Tempeh is traditionally produced by fermenting soybean with the fungus Rhizopus oligosporus found in banana leafs. We wanted to investigate if Taiwan's flavorful red bean could be used as a healthy substitute for soybeans in tempeh. One bioactive component of tempeh is γ-Aminobutyric acid (GABA). We measured GABA content and shelf-life-related antimicrobial activity in red-bean tempeh made with four strains of Rhizopus, one purchased strain of Rhizopus, and an experimental co-cultured group (Rhizopus and Lactobacillus rhamnosus BCRC16000) as well as cortisol in red-bean-tempeh-treated zebrafish. GABA was highest in the co-culture group (19.028 ± 1.831 g kg-1), followed by screened Strain 1, the purchased strain, and screened Strain 4. All strains had antibacterial activity on S. aureus and B. cereus. The extract significantly reduced cortisol in zebrafish. However, Strain 1, with less GABA than some of the other strains, had the best effect on cortisol level, suggesting that other components in red-bean tempeh may also affect stress-related cortisol. We found the benefits of red-bean tempeh to be similar to those reported for soybean-produced tempeh, suggesting that it could be produced as an alternative product. Considering the Taiwanese appreciation of the red-bean flavor, it might find a welcoming market.

8.
Int J Biol Macromol ; 146: 202-211, 2020 Mar 01.
Article in English | MEDLINE | ID: mdl-31887379

ABSTRACT

Bacillus amyloliquefaciens is a probiotic for animals. A strain of B. amyloliquefaciens designated amy-1 was isolated from soil, and the exopolysaccharides (EPSs) of the strain were characterized in terms of their effect on glycemic control. The EPSs were composed of mannose, glucose, and galactose, with the major components being polymers larger than 1000 kDa as revealed by size-exclusion high-performance liquid chromatography. The EPSs reduced the elevation of blood glucose in mice on oral glucose tolerance tests. The hypoglycemic effect was still apparent when glucose was administered through intraperitoneal injection. Further investigation revealed that the EPSs stimulated glucagon-like peptide 1 (GLP-1) secretion from enteroendocrine cells in vitro and increased plasma GLP-1 level in vivo. Moreover, the EPSs promoted the glucose consumption of a liver cell line and an intestinal epithelial cell line. Therefore, the interaction between EPSs and intestinal tissues at least partially contributed to their hypoglycemic effect. The enhanced glucose uptake of cells was likely mediated by the activation of phosphatidylinositol-3-kinase and Akt and was independent of insulin receptor substrate and AMP-activated protein kinase. These findings suggest that EPSs likely involve in the hypoglycemic functions of probiotics and are potential new agents for glycemic control.


Subject(s)
Bacillus amyloliquefaciens/chemistry , Blood Glucose/metabolism , Hypoglycemic Agents/pharmacology , Polysaccharides, Bacterial/pharmacology , Proto-Oncogene Proteins c-akt/metabolism , Animals , Enzyme Activation/drug effects , Hypoglycemic Agents/chemistry , Male , Mice , Mice, Inbred ICR , Polysaccharides, Bacterial/chemistry
9.
Acta Crystallogr D Struct Biol ; 75(Pt 12): 1138-1147, 2019 Dec 01.
Article in English | MEDLINE | ID: mdl-31793907

ABSTRACT

The catalytic domain (residues 128-449) of the Orpinomyces sp. Y102 CelC7 enzyme (Orp CelC7) exhibits cellobiohydrolase and cellotriohydrolase activities. Crystal structures of Orp CelC7 and its cellobiose-bound complex have been solved at resolutions of 1.80 and 2.78 Å, respectively. Cellobiose occupies subsites +1 and +2 within the active site of Orp CelC7 and forms hydrogen bonds to two key residues: Asp248 and Asp409. Furthermore, its substrate-binding sites have both tunnel-like and open-cleft conformations, suggesting that the glycoside hydrolase family 6 (GH6) Orp CelC7 enzyme may perform enzymatic hydrolysis in the same way as endoglucanases and cellobiohydrolases. LC-MS/MS analysis revealed cellobiose (major) and cellotriose (minor) to be the respective products of endo and exo activity of the GH6 Orp CelC7.


Subject(s)
Bacterial Proteins/chemistry , Cellobiose/metabolism , Cellulase/chemistry , Cellulose 1,4-beta-Cellobiosidase/chemistry , Cellulose/metabolism , Neocallimastigales/enzymology , Trioses/metabolism , beta-Glucosidase/chemistry , Binding Sites , Crystallography, X-Ray/methods , Models, Molecular , Protein Conformation , Substrate Specificity
10.
Microbiol Resour Announc ; 8(32)2019 Aug 08.
Article in English | MEDLINE | ID: mdl-31395643

ABSTRACT

Massilia sp. strain Mn16-1_5 was isolated from serpentine soil. This strain is able to oxidize manganese and has the potential for bioremediation of chromium. Here, we present a 5.53-Mb draft genome sequence of this strain with a G+C content of 64.8% that might provide more information for species delineation and oxidase genes in this strain.

11.
Int J Biol Macromol ; 133: 575-582, 2019 Jul 15.
Article in English | MEDLINE | ID: mdl-31004639

ABSTRACT

Exopolysaccharides (EPSs) are metabolites of probiotics that have gained wide interest recently. A strain of Lactobacillus reuteri Mh-001 with high exopolysaccharide (EPS) production ability was isolated, identified, and were used to investigate the anti-inflammatory effects of the EPSs. Among the three unpurified EPSs, RAW246.7 murine macrophages treated with 5 ppm of EPS 1 revealed the lowest tumour necrosis factor α (TNF-α) secretion (325.32 ±â€¯51.10 pg/ug DNA). The second lowest TNF- α secretion occurred with EPS 2 (701.12 ±â€¯86.108 pg/ug DNA) from Mh-002. EPSs 4, 5, and 6 were further purified from EPS 1. Cells treated with 1 ppm of EPS 4 had the lowest TNF-α secretion of all (209.20 ±â€¯84.34 pg/ug DNA). The monosaccharide components, EPS 4 and EPS 1, had the highest galactose content (45 ±â€¯2.75% and 39 ±â€¯2.75%, respectively). The monosaccharide percentages (galactose > rhamnose > glucose) were related to the anti-inflammatory activity of the EPSs. The galactose content of EPSs enhanced their anti-inflammatory effects on the macrophages. These data indicate that EPS possesses beneficial physiological effects such as anti-inflammatory properties, and the monosaccharide content of the EPS was the factor influencing the anti-inflammatory properties.


Subject(s)
Immunologic Factors/chemistry , Immunologic Factors/pharmacology , Monosaccharides/analysis , Polysaccharides, Bacterial/chemistry , Polysaccharides, Bacterial/pharmacology , Probiotics/chemistry , Animals , Dose-Response Relationship, Drug , Hydrolysis , Mice , RAW 264.7 Cells , Structure-Activity Relationship , Tumor Necrosis Factor-alpha/biosynthesis
12.
Fish Shellfish Immunol ; 86: 410-419, 2019 Mar.
Article in English | MEDLINE | ID: mdl-30471337

ABSTRACT

Zebrafish (Danio rerio) are an excellent model for assessing the beneficial effects of probiotics before applying them in aquaculture. This study evaluated the effects on zebrafish of dietary supplementation with the probiotic Bacillus amyloliquefaciens R8, which heterologously expresses xylanase from rumen fungi. Nutrient metabolism, hepatic oxidative stress, and innate immunity against pathogen infections were investigated. Treated zebrafish received feed supplemented with B. amyloliquefaciens R8 for 30 days and then were compared to zebrafish that were fed a control diet. The treated fish showed significant increases in xylanase activity in the intestines. The livers of the treated fish showed increased mRNA expressions of glycolysis-related genes of hexokinase, glucokinase, glucose-6-phosphatase, and pyruvate kinase; and higher enzyme activities of 3-hydroxyacyl-coenzyme A dehydrogenase and citrate synthase which are associated with fatty acid ß-oxidation and mitochondrial integrity. The livers of treated fish also showed decreased mRNA expressions of oxidative stress-related genes (SOD, Gpx, NOS2, and Hsp70) and an apoptotic gene (tp53), as well as increased expression of an anti-apoptotic gene (bcl-2). The probiotics-treated fish had increased expression of innate immune-related genes (IL-1ß, IL-6, IL-21, TNF-α, and TLR-1, -3, and -4). Following challenge with Aeromonas hydrophila and Streptococcus agalactiae, treated fish showed increased a higher survival rate than control fish. Overall, results showed that the administration of xylanase-expressing B. amyloliquefaciens R8 can potentially improve nutrient metabolism and hepatic stress tolerance, and enhance immunity and disease resistance against A. hydrophila and S. agalactiae in zebrafish.


Subject(s)
Bacillus amyloliquefaciens/chemistry , Fish Diseases/immunology , Immunity, Innate/drug effects , Nutrients/metabolism , Oxidative Stress/drug effects , Probiotics/pharmacology , Zebrafish , Aeromonas hydrophila/physiology , Animal Feed/analysis , Animals , Diet/veterinary , Fish Diseases/microbiology , Gram-Negative Bacterial Infections/immunology , Gram-Negative Bacterial Infections/microbiology , Gram-Negative Bacterial Infections/veterinary , Liver/drug effects , Probiotics/administration & dosage , Streptococcal Infections/immunology , Streptococcal Infections/microbiology , Streptococcal Infections/veterinary , Streptococcus agalactiae/physiology
13.
3 Biotech ; 7(1): 3, 2017 May.
Article in English | MEDLINE | ID: mdl-28389897

ABSTRACT

Ribosome-inactivating proteins (RIPs) are a group of enzymes originally isolated from plants that possess the ability to damage ribosomes in an irreversible manner, leading to inhibition of protein synthesis in eukaryotic cells. In this study, we aimed to purify recombinant RIPs, investigate their function in the treatment of bacterial infection, and determine their toxicity in mice. We employed a pMAL protein fusion and purification system using E. coli transformed with a plasmid containing MBP-tagged MAP30 cDNA. MBP-tagged MAP30 was purified using a modified novel protocol to effectively produce highly active MAP30 of high purity. In an acute toxicity study in mice, no mortality occurred at doses lower than 1.25 mg/kg. MAP30 at both 0.42 and 0.14 mg/kg induced anti-MAP30 IgG, which reached a maximum titer at week 3. In conclusion, recombinant MAP30 prepared using our purification method possesses bioactivity, and has a synergistic bacteria-killing effect that can significantly reduce the required dosages of chloramphenicol and erythromycin. Therefore, when MAP30 is used in combination with chloramphenicol or erythromycin, it may of benefit in terms of reducing the side effects of the antibiotics, as lower concentrations of antibiotics are required.

14.
Fish Shellfish Immunol ; 58: 397-405, 2016 Nov.
Article in English | MEDLINE | ID: mdl-27670086

ABSTRACT

Bacillus amyloliquefaciens has attracted attention as a probiotic in aquaculture due to its immunostimulatory activity against pathogenic infection. Xylanases are extensively used in animal feed to degrade plant ingredients, enhancing nutrient utilization and increasing the growth rate of various animals. In the present study, the effects of dietary supplementation with B. amyloliquefaciens and xylanase-expressing B. amyloliquefaciens R8 on the growth of Nile tilapia (Oreochromis niloticus) and immunity against Aeromonas hydrophila were evaluated. The results showed that the xylanase activity in the intestine, weight gain (WG), feed efficiency (FE) and condition factor (CF) of Nile tilapia fed B. amyloliquefaciens R8 for 2 months were significantly increased compared with those of the fish fed the control diet and B. amyloliquefaciens. Moreover, the mRNA expression of growth- and metabolism-related genes, such as insulin-like growth factor-1 (igf-1), glucokinase (GK), glucose-6-phosphate 1-dehydrogenase (G6PD), and glucose-6-phosphatase (G6Pase), was significantly induced in Nile tilapia fed administered B. amyloliquefaciens R8, and this group also exhibited a higher survival rate than the control fish following a challenge with A. hydrophila. The phagocytic activity and respiratory burst activity of head kidney leukocytes as well as the serum lysozyme activity of B. amyloliquefaciens R8-fed Nile tilapia were significantly higher than those of fish fed the control diet for 2 months. Superoxide dismutase (SOD) levels in the head kidney leukocytes of Nile tilapia fed B. amyloliquefaciens R8 differed from those of fish fed the control diet, but this was not significant. These results indicate that dietary supplementation with xylanase-expressing B. amyloliquefaciens R8 improves growth performance and enhances immunity and disease resistance against A. hydrophila in Nile tilapia.


Subject(s)
Aeromonas hydrophila/physiology , Cichlids , Dietary Supplements , Fish Diseases/immunology , Gram-Negative Bacterial Infections/veterinary , Probiotics , Animal Feed/analysis , Animals , Cichlids/growth & development , Diet/veterinary , Disease Resistance , Endo-1,4-beta Xylanases/metabolism , Fish Diseases/microbiology , Gram-Negative Bacterial Infections/immunology , Gram-Negative Bacterial Infections/microbiology
15.
BMC Vet Res ; 11: 103, 2015 May 06.
Article in English | MEDLINE | ID: mdl-25944062

ABSTRACT

BACKGROUND: Autophagy is an essential process in the control of cellular homeostasis. It enables cells under certain stress conditions to survive by removing toxic cellular components, and may protect cells from apoptosis. In the present study, the signaling pathways involved in ARV S1133 regulated switch from autophagy to apoptosis were investigated. RESULTS: ARV S1133 infection caused autophagy in the early to middle infectious stages in Vero and DF1 cells, and apoptosis in the middle to late stages. Conversion of the autophagy marker LC3-I to LC3-II occurred earlier than cleavage of the apoptotic marker caspase-3. ARV S1133 also activated the Beclin-1 promoter in the early to middle stages of infection. Levels of RhoA-GTP and ROCK1 activity were elevated upon ARV S1133 infection, while inhibition of RhoA and ROCK1 reduced autophagy and subsequent apoptosis. Conversely, inhibition of caspase-3 did not affect the level of autophagy. Beclin-1 knockdown and treatment with autophagy inhibitors, 3-MA and Bafilomycin A1, suppressed ARV S1133-induced autophagy and apoptosis simultaneously, suggesting the shift from autophagy to apoptosis. A co-immunoprecipitation assay demonstrated that the formation of a RhoA, ROCK1 and Beclin-1 complex coincided with the induction of autophagy. CONCLUSION: Our results demonstrate that RhoA/ROCK1 signaling play critical roles in the transition of cell activity from autophagy to apoptosis in ARV S1133-infected cells.


Subject(s)
Apoptosis/physiology , Autophagy/physiology , Fibroblasts/metabolism , Orthoreovirus, Avian/physiology , rho-Associated Kinases/metabolism , rhoA GTP-Binding Protein/metabolism , Amides/pharmacology , Animals , Chickens , Chlorocebus aethiops , Fibroblasts/virology , Gene Expression Regulation/drug effects , Gene Expression Regulation/physiology , Orthoreovirus, Avian/classification , Pyridines/pharmacology , RNA Interference , RNA, Small Interfering , Signal Transduction , Vero Cells , rho-Associated Kinases/genetics , rhoA GTP-Binding Protein/genetics
16.
Apoptosis ; 20(4): 481-90, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25576194

ABSTRACT

In this study the mechanism of avian reovirus (ARV) S1133-induced pathogenesis was investigated, with a focus on the contribution of ER stress to apoptosis. Our results showed that upregulation of the ER stress response protein, as well as caspase-3 activation, occurred in ARV S1133-infected cultured cells and in SPF White Leghorn chicks organs. Upon infection, Bim was translocated specifically to the ER, but not mitochondria, in the middle to late infectious stages. In addition, ARV S1133 induced JNK phosphorylation and promoted JNK-Bim complex formation, which correlated with the Bim translocation and apoptosis induction that was observed at the same time point. Knockdown of BiP/GRP78 by siRNA and inhibition of BiP/GRP78 using EGCG both abolished the formation of the JNK-Bim complex, caspase-3 activation, and subsequent apoptosis induction by ARV S1133 efficiently. These results suggest that BiP/GRP78 played critical roles and works upstream of JNK-Bim in response to the ARV S1133-mediated apoptosis process.


Subject(s)
Apoptosis Regulatory Proteins/metabolism , Apoptosis , Endoplasmic Reticulum/metabolism , Heat-Shock Proteins/metabolism , Membrane Proteins/metabolism , Orthoreovirus, Avian/physiology , Poultry Diseases/metabolism , Proto-Oncogene Proteins/metabolism , Reoviridae Infections/metabolism , Reoviridae Infections/veterinary , Animals , Apoptosis Regulatory Proteins/genetics , Bcl-2-Like Protein 11 , Caspase 3/metabolism , Chickens , Endoplasmic Reticulum Chaperone BiP , Heat-Shock Proteins/genetics , Membrane Proteins/genetics , Orthoreovirus, Avian/genetics , Poultry Diseases/genetics , Poultry Diseases/physiopathology , Poultry Diseases/virology , Protein Transport , Proto-Oncogene Proteins/genetics , Reoviridae Infections/genetics , Reoviridae Infections/physiopathology , Reoviridae Infections/virology , Signal Transduction
17.
Enzyme Microb Technol ; 64-65: 6-10, 2014 Oct.
Article in English | MEDLINE | ID: mdl-25152410

ABSTRACT

The xylanase R8 gene (xynR8) from uncultured rumen fungi was cloned and successfully expressed in Lactobacillus reuteri. A xylanase activity of 132.1 U/mL was found in the broth of L. reuteri R8, the transformant containing pNZ3004 vector with xynR8 gene insertion. Two distinct forms of recombinant xylanase with different hydrophobicities and molecular weights were found in the broth after purification. According to the results of Western blotting, only the T7-tag, fused in the N-terminus of XynR8, could be bound to the expressed proteins, which indicated that the C-terminus of XynR8 had been truncated. These results, combined with tryptic digestion and mass spectrometry analyses, allow us to attribute the two xylanase forms to an optional cleavage of C-terminal sequences, and XynR8A, a 13 amino acid residues truncated form, and XynR8B, a 22 amino acid residues truncated form, were the main products in the extracellular fraction of L. reuteri R8. The specific activities of XynR8A and R8B were 1028 and 395 U/mg protein. Both forms of recombinant xylanase displayed a typical endoxylanase activity when they were reacted with xylan, but XynR8A demonstrated a better specific activity, catalytic efficiency and thermostability than XynR8B according to the results of enzyme characterization. These changes in enzyme properties were highly possibly caused by the present of the ß-sheet in the C-terminal undeleted fragment of XynR8A. This study demonstrates that modified forms with different enzyme properties could be produced when a gene was recombinantly expressed by a L. reuteri transformant.


Subject(s)
Fungal Proteins/metabolism , Limosilactobacillus reuteri/enzymology , Rumen/microbiology , Xylosidases/metabolism , Amino Acid Sequence , Animals , Enzyme Stability , Fungal Proteins/chemistry , Fungal Proteins/genetics , Genes, Fungal , Hydrolysis , Hydrophobic and Hydrophilic Interactions , Kinetics , Limosilactobacillus reuteri/genetics , Molecular Sequence Data , Molecular Weight , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Protein Structure, Secondary , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Xylosidases/chemistry , Xylosidases/genetics
18.
Bioresour Technol ; 170: 513-521, 2014 Oct.
Article in English | MEDLINE | ID: mdl-25164344

ABSTRACT

A new strain of rumen fungus was isolated from Bos taurus, identified and designated Orpinomyces sp.Y102. A clone, celC7, isolated from the cDNA library of Orpinomyces sp.Y102, was predicted to encode a protein containing a signal peptide (Residues 1-17), an N-terminal dockerin-containing domain, and a C-terminal cellobiohydrolase catalytic domain of glycoside hydrolase family 6. CelC7 was insoluble when expressed in Escherichia coli. Deletion of 17 or 105 residues from the N-terminus significantly improved its solubility. The resulting enzymes, CelC7(-17) and CelC7(-105), were highly active to ß-glucan substrates and were stable between pH 5.0 and 11.0. CelC7(-105) worked as an exocellulase releasing cellobiose and cellotriose from acid-swollen Avicel and cellooligosaccharides, and displayed a Vmax of 6321.64µmole/min/mg and a Km of 2.18mg/ml to barley ß-glucan. Further, the crude extract of CelC7(-105) facilitated ethanol fermentation from cellulose. Thus, CelC7(-105) is a good candidate for industrial applications such as biofuel production.


Subject(s)
Cattle/microbiology , Cellulases/metabolism , Cellulose 1,4-beta-Cellobiosidase/metabolism , Industrial Microbiology/methods , Neocallimastigales/enzymology , Rumen/microbiology , Animals , Base Sequence , Biofuels , Blotting, Western , Cellulases/genetics , Cellulose 1,4-beta-Cellobiosidase/genetics , Chromatography, Thin Layer , Cluster Analysis , DNA Primers/genetics , Escherichia coli , Gene Library , Molecular Sequence Data , Neocallimastigales/cytology , Neocallimastigales/genetics , Phylogeny , Sequence Analysis, DNA
19.
Biochem Biophys Res Commun ; 451(2): 190-5, 2014 Aug 22.
Article in English | MEDLINE | ID: mdl-25073115

ABSTRACT

Understanding the roles of the components of the multienzyme complex of the anaerobial cellulase system, acting on complex substrates, is crucial to the development of efficient cellulase systems for industrial applications such as converting lignocellulose to sugars for bioethanol production. In this study, we purified the multienzyme complex of Neocallimastix patriciarum J11 from a broth through cellulose affinity purification. The multienzyme complex is composed of at least 12 comprised proteins, based on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Eight of these constituents have demonstrated ß-glucanase activity on zymogram analysis. The multienzyme complex contained scaffoldings that respond to the gathering of the cellulolytic components. The levels and subunit ratio of the multienzyme complex from N. patriciarum J11 might have been affected by their utilized carbon sources, whereas the components of the complexes were consistent. The trypsin-digested peptides of six proteins were matched to the sequences of cellulases originating from rumen fungi, based on identification through liquid chromatography/mass spectrometry, revealing that at least three types of cellulase, including one endoglucanase and two exoglucanases, could be found in the multienzyme complex of N. patriciarum J11. The cellulolytic subunits could hydrolyze synergistically on both the internal bonds and the reducing and nonreducing ends of cellulose. Based on our research, our findings are the first to depict the composition of the multienzyme complex produced by N. patriciarum J11, and this complex is composed of scaffoldin and three types of cellulase.


Subject(s)
Cellulases/isolation & purification , Fungal Proteins/isolation & purification , Multienzyme Complexes/isolation & purification , Neocallimastix/enzymology , Animals , Blotting, Western , Buffaloes/microbiology , Cellulases/genetics , Cellulases/metabolism , Chromatography, Gel , Fungal Proteins/genetics , Fungal Proteins/metabolism , Multienzyme Complexes/genetics , Multienzyme Complexes/metabolism , Neocallimastix/isolation & purification , Protein Subunits , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Rumen/microbiology , Tandem Mass Spectrometry
20.
Protein Expr Purif ; 90(2): 153-9, 2013 Aug.
Article in English | MEDLINE | ID: mdl-23770555

ABSTRACT

An 1888-bp cDNA designated celA, isolated from a cDNA library of Neocallimastix patriciarum J11 was cloned. The celA had an open reading frame of 1530 bp encoding J11 CelA of 510 amino acids. The primary structure analysis of J11 CelA revealed a complete cellulose-binding domain at the N-terminal, followed by an Asn, Ala, Gly, Gln and Pro-rich linker and ending with a C-terminal glycosyl hydrolase family 6 catalytic domain. The mature J11 CelA was overexpressed in Escherichia coli and purified to homogeneity. This enzyme had high specific activities towards barley ß-glucan and lichenan, low toward carboxymethyl cellulose (CMC), Avicel, and H3PO4-swollen Avicel (PSA). The product of Avicel hydrolysis was cellobiose indicating that J11 CelA is a typical cellobiohydrolase. The recombinant J11 CelA had an optimal pH of 6.0 and was stable over a wide range of pH (5.2-11.3). The enzyme showed an optimal temperature of 50°C and was still maintained approximately 50% of the maximum activity in response to the treatment at 70°C for 1h. Cobalt and Fe(3+) at 1 mM greatly activated the enzyme activity. As a thermostable and pH stable enzyme with crystalline cellulose-degrading activity, J11 CelA is a potential candidate for the bioethanol industry.


Subject(s)
Cellulose 1,4-beta-Cellobiosidase/chemistry , Cellulose 1,4-beta-Cellobiosidase/genetics , Fungal Proteins/genetics , Neocallimastix/enzymology , Amino Acid Sequence , Carboxymethylcellulose Sodium/chemistry , Carboxymethylcellulose Sodium/metabolism , Cellulose 1,4-beta-Cellobiosidase/metabolism , Cloning, Molecular , DNA, Complementary/chemistry , DNA, Complementary/metabolism , Enzyme Stability , Escherichia coli/genetics , Fungal Proteins/chemistry , Fungal Proteins/metabolism , Hot Temperature , Hydrogen-Ion Concentration , Molecular Sequence Data , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism
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