ABSTRACT
Background: Hydrophobins are small proteins secreted by filamentous fungi, which show a highly surface activity. Because of the signally self-assembling abilities and surface activities, hydrophobins were considered as candidates in many aspects, for example, stabilizing foams and emulsions in food products. Lentinus tuber-regium, known as tiger milk mushroom, is both an edible and medicinal sclerotium-producing mushroom. Up to now, the hydrophobins of L. tuber-regium have not been identified. Results: In this paper, a Class I hydrophobin gene, Ltr.hyd, was cloned from L. tuber-regium and expressed in the yeast-like cells of Tremella fuciformis mediated by Agrobacterium tumefaciens. The expression vector pGEH-GH was under the control of T. fuciformis glyceraldehyde-3-phosphate dehydrogenase gene (gpd) promoter. The integration of Ltr.hyd into the genome of T. fuciformis was confirmed by PCR, Southern blot, fluorescence observation and quantitative real-time PCR (qRT-PCR). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that recombinant hydrophobin rLtr.HYD with an expected molecular mass of 13 kDa was extracted. The yield of rLtr.HYD was 0.66 mg/g dry weight. The emulsifying activity of rLtr.HYD was better than the typical food emulsifiers sodium caseinate and Tween 20. Conclusions: We evaluated the emulsifying property of hydrophobin Ltr.HYD, which can be potentially used as a food emulsifier.
Subject(s)
Basidiomycota/metabolism , Fungal Proteins/genetics , Lentinula/genetics , Lentinula/metabolism , Transformation, Genetic , Basidiomycota/enzymology , Yeasts , Fungal Proteins/metabolism , Blotting, Southern , Cloning, Molecular , Agrobacterium tumefaciens/metabolism , Sequence Analysis , Emulsifying Agents , Electrophoresis, Polyacrylamide Gel , Real-Time Polymerase Chain Reaction , Glyceraldehyde-3-Phosphate Dehydrogenases/metabolism , Microscopy, FluorescenceABSTRACT
High plasma levels of homocysteine (Hcy) promote the progression of neurodegenerative diseases. However, the mechanism by which Hcy mediates neurotoxicity has not been elucidated. We observed that upon incubation with Hcy, the viability of a neuroblastoma cell line Neuro2a declined in a dose-dependent manner, and apoptosis was induced within 48 h. The median effective concentration (EC50) of Hcy was approximately 5 mM. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) nuclear translocation and acylation has been implicated in the regulation of apoptosis. We found that nuclear translocation and acetylation of GAPDH increased in the presence of 5 mM Hcy and that higher levels of acetyltransferase p300/CBP were detected in Neuro2a cells. These findings implicate the involvement of GAPDH in the mechanism whereby Hcy induces apoptosis in neurons. This study highlights a potentially important pathway in neurodegenerative disorders, and a novel target pathway for neuroprotective therapy.
Subject(s)
Animals , Rabbits , Apoptosis/drug effects , Neuroprotective Agents/pharmacology , Glyceraldehyde-3-Phosphate Dehydrogenases/metabolism , Homocysteine/pharmacology , Acetylation , Acetyltransferases/analysis , Time Factors , Cell Count , Cell Extracts/chemistry , Cell Nucleus/metabolism , Cell Survival/physiology , Enzyme Induction , Blotting, Western , Fluorescent Antibody Technique , Apoptosis/physiology , Neuroprotective Agents/administration & dosage , Cell Line, Tumor , p300-CBP Transcription Factors/metabolism , Homocysteine/administration & dosageABSTRACT
OBJECTIVE@#To explore the relationship between degradation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA in the mouse liver and postmortem interval (PMI).@*METHODS@#Sixty NIH mice were sacrificed by cervical dislocation and suffocation, and then placed into 10 degrees C and 25 degrees C temperature-controlling systems. The changes of GAPDH mRNA in the liver were detected by two-step fluorimetric reverse transcriptase polymerase chain reaction (RT-PCR) technique and nucleic acids protein cryoscope from 0 to 48 h postmortem.@*RESULTS@#In the mouse liver, the amplification products of GAPDH mRNA could be examined within 48 h postmortem in 10 degrees C temperature-controlling system and within 36 h postmortem in 25 degrees C temperature-controlling system. The amplification products showed a decreasing tendency.@*CONCLUSION@#Degradation of GAPDH mRNA in the mouse liver is negative correlation with PMI. GAPDH mRNA could be a new marker for estimation of PMI.
Subject(s)
Animals , Female , Male , Mice , Forensic Pathology , Glyceraldehyde-3-Phosphate Dehydrogenases/metabolism , Liver/metabolism , Mice, Inbred Strains , Postmortem Changes , RNA Stability , RNA, Messenger/metabolism , Regression Analysis , Reverse Transcriptase Polymerase Chain Reaction , Temperature , Time FactorsABSTRACT
Rate studies using phosphoglycerate kinase (PGK)--glyceraldehyde-3-phosphate dehydrogenase (GPDH) enzyme pair have been carried out to distinguish between the two mechanisms of intermediate metabolite transfer, namely diffusion through the solvent versus "substrate channelling" within an enzyme-enzyme complex. A procedure has been described for the assay of the rates of PGK-catalysed and the PGK-GPDH coupled reactions at high (saturating) GPDH concentration. With PGKs of rabbit muscle and yeast, the coupled reaction proceeded faster than the PGK-catalysed reaction. At a high salt concentration (0.5 M KCl), where a PGK-GPDH complex is known to dissociate, the two reactions proceeded at almost equal rates. At fixed PGK concentration, the rate of the coupled reaction at high (saturating) GPDH concentration varied with the nature (biological origin) of the latter enzyme. In the presence of 0.5 M KCl, the saturating rate values with different GPDHs were almost equal. The PGK-catalysed reaction exhibited typical Michaelian behaviour on varying the substrate concentrations (linear double reciprocal plots). The Km values for 3-PGA (0.51 mM) and ATP (0.40 mM) were independent of the concentration of the second substrate. The double reciprocal plots for the coupled reaction showed downward curvature, i.e. activation at higher substrate concentrations. The ratio of the rate of the coupled reaction: the rate of the PGK catalysed reaction was found to be a function of the nature of PGK, nature of GPDH, nature of buffer, pH, salt concentration and substrate concentrations. The ratio varied between close to unity at low substrate concentrations, to three when the Vmax values of the two reactions were compared. At low substrate concentrations, the rate of the coupled reaction became independent of the nature of GPDH. It has been suggested that in the PGK-GPDH pair, the intermediate metabolite (BPG) is transferred directly from one enzyme to the other within an enzyme-enzyme complex, except at high salt or low substrate concentrations. Under the latter conditions, data were consistent with metabolite transfer by diffusion. Implications of these results for coupled enzyme assays have been discussed.
Subject(s)
Animals , Glyceraldehyde-3-Phosphate Dehydrogenases/metabolism , Kinetics , Phosphoglycerate Kinase/metabolism , Rabbits , SwineABSTRACT
The hypoglycemic effect of Bordetella pertussis (Challenge strain No.18323) purified cell extract (protein with traces of carbohydrates, 2 mg%) administered (0.1 mg/100 g body wt. i.v.) into mice on the activities of the key regulatory enzymes, viz. glucokinase, phosphofructokinase, pyruvate kinase, glyceraldehyde phosphodehydrogenase, glucose-6-phosphate dehydrogenase (G-6-PD) and lactate dehydrogenase, of glycolytic pathway in liver has been studied at varying intervals after injection. The maximum hypoglycaemic effect was observed at the end of 12 hr, while activities of all the enzymes studied showed significant enhancement after 18 hr, thus suggesting increased glucose utilization towards the formation of pyruvate. Actinomycin D is found to inhibit stimulation of G-6-PD activity in B. pertussis treated animals, thereby indicating the role of B. pertussis in synthesis of this enzyme.