Title: Crystal structure and biochemical analysis of acetylesterase (LgEstI) from Lactococcus garvieae Do H, Yoo W, Wang Y, Nam Y, Shin SC, Kim HW, Kim KK, Lee JH Ref: PLoS ONE, 18:e0280988, 2023 : PubMed
Esterase, a member of the serine hydrolase family, catalyzes the cleavage and formation of ester bonds with high regio- and stereospecificity, making them attractive biocatalysts for the synthesis of optically pure molecules. In this study, we performed an in-depth biochemical and structural characterization of a novel microbial acetylesterase, LgEstI, from the bacterial fish pathogen Lactococcus garvieae. The dimeric LgEstI displayed substrate preference for the short acyl chain of p-nitrophenyl esters and exhibited increased activity with F207A mutation. Comparative analysis with other esterases indicated that LgEstI has a narrow and shallow active site that may exhibit substrate specificity to short acyl chains. Unlike other esterases, LgEstI contains bulky residues such as Trp89, Phe194, and Trp217, which block the acyl chain channel. Furthermore, immobilized LgEstI retained approximately 90% of its initial activity, indicating its potential in industrial applications. This study expands our understanding of LgEstI and proposes novel ideas for improving its catalytic efficiency and substrate specificity for various applications.
Title: Structural and Biochemical Insights into Bis(2-hydroxyethyl) Terephthalate Degrading Carboxylesterase Isolated from Psychrotrophic Bacterium Exiguobacterium antarcticum Hwang J, Yoo W, Shin SC, Kim KK, Kim HW, Do H, Lee JH Ref: Int J Mol Sci, 24:12022, 2023 : PubMed
This study aimed to elucidate the crystal structure and biochemically characterize the carboxylesterase EaEst2, a thermotolerant biocatalyst derived from Exiguobacterium antarcticum, a psychrotrophic bacterium. Sequence and phylogenetic analyses showed that EaEst2 belongs to the Family XIII group of carboxylesterases. EaEst2 has a broad range of substrate specificities for short-chain p-nitrophenyl (pNP) esters, 1-naphthyl acetate (1-NA), and 1-naphthyl butyrate (1-NB). Its optimal pH is 7.0, losing its enzymatic activity at temperatures above 50 degreesC. EaEst2 showed degradation activity toward bis(2-hydroxyethyl) terephthalate (BHET), a polyethylene terephthalate degradation intermediate. We determined the crystal structure of EaEst2 at a 1.74 A resolution in the ligand-free form to investigate BHET degradation at a molecular level. Finally, the biochemical stability and immobilization of a crosslinked enzyme aggregate (CLEA) were assessed to examine its potential for industrial application. Overall, the structural and biochemical characterization of EaEst2 demonstrates its industrial potency as a biocatalyst.
Ferulic acid and related hydroxycinnamic acids, used as antioxidants and preservatives in the food, cosmetic, pharmaceutical and biotechnology industries, are among the most abundant phenolic compounds present in plant biomass. Identification of novel compounds that can produce ferulic acid and hydroxycinnamic acids, that are safe and can be mass-produced, is critical for the sustainability of these industries. In this study, we aimed to obtain and characterize a feruloyl esterase (LaFae) from Lactobacillus acidophilus. Our results demonstrated that LaFae reacts with ethyl ferulate and can be used to effectively produce ferulic acid from wheat bran, rice bran and corn stalks. In addition, xylanase supplementation was found to enhance LaFae enzymatic hydrolysis, thereby augmenting ferulic acid production. To further investigate the active site configuration of LaFae, crystal structures of unliganded and ethyl ferulate-bound LaFae were determined at 2.3 and 2.19 A resolutions, respectively. Structural analysis shows that a Phe34 residue, located at the active site entrance, acts as a gatekeeper residue and controls substrate binding. Mutating this Phe34 to Ala produced an approximately 1.6-fold increase in LaFae activity against p-nitrophenyl butyrate. Our results highlight the considerable application potential of LaFae to produce ferulic acid from plant biomass and agricultural by-products.
INTRODUCTION: Inflammation is a key pathogenic feature of abdominal aortic aneurysm (AAA). Soluble epoxide hydrolase (sEH) is a pro-inflammatory enzyme that converts cytochrome P450-derived epoxides of fatty acids to the corresponding diols, and pharmacological inhibition of sEH prevented AAA formation. Both cytochrome P450 enzymes and sEH are highly expressed in the liver. Here, we investigated the role of hepatic sEH in AAA using a selective pharmacological inhibitor of sEH and hepatocyte-specific Ephx2 (which encodes sEH gene) knockout (KO) mice in two models of AAA [angiotensin II (AngII) infusion and calcium chloride (CaCl (2) ) application]. METHODS AND RESULTS: sEH expression and activity were strikingly higher in mouse liver compared with aorta and further increased the context of AAA, in conjunction with elevated expression of the transcription factor Sp1 and the epigenetic regulator Jarid1b, which have been reported to positively regulate sEH expression. Pharmacological sEH inhibition, or liver-specific sEH disruption, achieved by crossing sEH floxed mice with albumin-cre mice, prevented AAA formation in both models, concomitant with reduced expression of hepatic sEH as well as complement factor 3 (C3) and serum amyloid A (SAA), liver-derived factors linked to AAA formation. Moreover, sEH antagonism markedly reduced C3 and SAA protein accumulation in the aortic wall. Co-incubation of liver ex vivo with aneurysm-prone aorta resulted in induction of sEH in the liver, concomitant with upregulation of Sp1, Jarid1b, C3 and SAA gene expression, suggesting that the aneurysm-prone aorta secretes factors that activate sEH and downstream inflammatory signaling in the liver. Using an unbiased proteomic approach, we identified a number of dysregulated proteins [ e.g., plastin-2, galectin-3 (gal-3), cathepsin S] released by aneurysm-prone aorta as potential candidate mediators of hepatic sEH induction. CONCLUSION: We provide the first direct evidence of the liver's role in orchestrating AAA via the enzyme sEH. These findings not only provide novel insight into AAA pathogenesis, but they have potentially important implications with regard to developing effective medical therapies for AAA.
Title: Structural and biochemical insights into PsEst3, a new GHSR-type esterase obtained from Paenibacillus sp. R4 Son J, Choi W, Kim H, Kim M, Lee JH, Shin SC, Kim HW Ref: IUCrJ, 10:220, 2023 : PubMed
PsEst3, a psychrophilic esterase obtained from Paenibacillus sp. R4, which was isolated from the permafrost of Alaska, exhibits relatively high activity at low temperatures. Here, crystal structures of PsEst3 complexed with various ligands were generated and studied at atomic resolution, and biochemical studies were performed to analyze the structure-function relationship of PsEst3. Certain unique characteristics of PsEst3 distinct from those of other classes of lipases/esterases were identified. Firstly, PsEst3 contains a conserved GHSRA/G pentapeptide sequence in the GxSxG motif around the nucleophilic serine. Additionally, it contains a conserved HGFR/K consensus sequence in the oxyanion hole, which is distinct from that in other lipase/esterase families, as well as a specific domain composition (for example a helix-turn-helix motif) and a degenerative lid domain that exposes the active site to the solvent. Secondly, the electrostatic potential of the active site in PsEst3 is positive, which may cause unintended binding of negatively charged chemicals in the active site. Thirdly, the last residue of the oxyanion hole-forming sequence, Arg44, separates the active site from the solvent by sealing the acyl-binding pocket, suggesting that PsEst3 is an enzyme that is customized to sense an unidentified substrate that is distinct from those of classical lipases/esterases. Collectively, this evidence strongly suggests that PsEst3 belongs to a distinct family of esterases.
A gene encoding LgEstI was cloned from a bacterial fish pathogen, Lactococcus garvieae. Sequence and bioinformatic analysis revealed that LgEstI is close to the acetyl esterase family and had maximum similarity to a hydrolase (UniProt: Q5UQ83) from Acanthamoeba polyphaga mimivirus (APMV). Here, we present the results of LgEstI overexpression and purification, and its preliminary X-ray crystallographic analysis. The wild-type LgEstI protein was overexpressed in Escherichia coli, and its enzymatic activity was tested using p-nitrophenyl of varying lengths. LgEstI protein exhibited higher esterase activity toward p-nitrophenyl acetate. To better understand the mechanism underlying LgEstI activity and subject it to protein engineering, we determined the high-resolution crystal structure of LgEstI. First, the wild-type LgEstI protein was crystallized in 0.1 M Tris-HCl buffer (pH 7.1), 0.2 M calcium acetate hydrate, and 19% (w/v) PEG 3000, and the native X-ray diffraction dataset was collected up to 2.0 A resolution. The crystal structure was successfully determined using a molecular replacement method, and structure refinement and model building are underway. The upcoming complete structural information of LgEstI may elucidate the substrate-binding mechanism and provide novel strategies for subjecting LgEstI to protein engineering.
Title: Dual functional roles of a novel bifunctional beta-lactamase/esterase from Lactococcus garvieae Le L, Yoo W, Wang Y, Jeon S, Kim KK, Kim HW, Kim TD Ref: Int J Biol Macromol, :, 2022 : PubMed
A novel bifunctional beta-lactamase/esterase (LgLacI), which is capable of hydrolyzing beta-lactam-containing antibiotics including ampicillin, oxacillin, and cefotaxime as well as synthesizing biodiesels, was cloned from Lactococcus garvieae. Unlike most bacterial esterases/lipases that have G-x-S-x-G motif, LgLacI, which contains S-x-x-K catalytic motif, has sequence similarities to bacterial family VIII esterase as well as beta-lactamases. The catalytic properties of LgLacI were explored using a wide range of biochemical methods including spectroscopy, assays, structural modeling, mutagenesis, and chromatography. We confirmed the bifunctional property of LgLacI hydrolyzing both esters and beta-lactam antibiotics. This study provides novel perspectives into a bifunctional enzyme from L. garvieae, which can degrade beta-lactam antibiotics with high esterase activity.
This report deals with the purification, characterization, and a preliminary crystallographic study of a novel cold-active esterase (HaEst1) from Halocynthiibacter arcticus. Primary sequence analysis reveals that HaEst1 has a catalytic serine in G-x-S-x-G motif. The recombinant HaEst1 was cloned, expressed, and purified. SDS-PAGE and zymographic analysis were carried out to characterize the properties of HaEst1. A single crystal of HaEst1 was obtained in a solution containing 10% (w/v) PEG 8000/8% ethylene glycol, 0.1 M Hepes-NaOH, pH 7.5. Diffraction data were collected to 2.10 A resolution with P21 space group. The final Rmerge and Rp.i.m values were 7.6% and 3.5% for 50-2.10 A resolution. The unit cell parameters were a = 35.69 A, b = 91.21 A, c = 79.15 A, and beta = 96.9deg
Title: Characterization and mutation anaylsis of a cold-active bacterial hormone-sensitive lipase from Salinisphaera sp. P7-4 Kim BY, Yoo W, Huong Luu Le LT, Kim KK, Kim HW, Lee JH, Kim YO, Kim TD Ref: Archives of Biochemistry & Biophysics, 663:132, 2019 : PubMed
In mammals, hormone sensitive lipase (EC 3.1.1.79, HSL) catalyzes the hydrolysis of triacylglycerols as well as the modifications of a broad range of hydrophobic substrates containing ester linkages. HSLs are composed of an N-terminal ligand-binding domain and a C-terminal catalytic domain. Bacterial hormone-sensitive lipases (bHSLs), which are homologous to the C-terminal domain of mammalian HSLs, have a catalytic triad composed of Ser, His, and Asp. Here, a novel cold-active hormone-sensitive lipase (SaHSL) from Salinisphaera sp. P7-4 was identified, functionally characterized, and subjected to site-directed mutations. The enzymatic properties of SaHSL were investigated using several biochemical and biophysical methods. Interestingly, SaHSL exhibited the ability to act on a broad range of substrates including glyceryl tributyrate and glucose pentaacetate. Homology modeling and site-directed mutagenesis indicated that hydrophobic residues (Leu(156), Phe(164), and Val(204)) around the substrate-binding pocket were involved in substrate recognition. In addition, highly conserved amino acids (Glu(201), Arg(207), Leu(208), and Asp(227)) in the regulatory regions were found to be responsible for substrate specificity, thermostability, and enantioselectivity. In summary, this work provides new insights into the understanding of the C-terminal domain of HSL family and evidence that SaHSL can be used in a wide range of industrial applications.
BACKGROUND: S-Formylglutathione is hydrolyzed to glutathione and formate by an S-formylglutathione hydrolase (SFGH) (3.1.2.12). This thiol esterase belongs to the esterase family and is also known as esterase D. SFGHs contain highly conserved active residues of Ser-Asp-His as a catalytic triad at the active site. Characterization and investigation of SFGH from Antarctic organisms at the molecular level is needed for industrial use through protein engineering. RESULTS: A novel cold-active S-formylglutathione hydrolase (SfSFGH) from Shewanella frigidimarina, composed of 279 amino acids with a molecular mass of ~ 31.0 kDa, was characterized. Sequence analysis of SfSFGH revealed a conserved pentapeptide of G-X-S-X-G found in various lipolytic enzymes along with a putative catalytic triad of Ser148-Asp224-His257. Activity analysis showed that SfSFGH was active towards short-chain esters, such as p-nitrophenyl acetate, butyrate, hexanoate, and octanoate. The optimum pH for enzymatic activity was slightly alkaline (pH 8.0). To investigate the active site configuration of SfSFGH, we determined the crystal structure of SfSFGH at 2.32 A resolution. Structural analysis shows that a Trp182 residue is located at the active site entrance, allowing it to act as a gatekeeper residue to control substrate binding to SfSFGH. Moreover, SfSFGH displayed more than 50% of its initial activity in the presence of various chemicals, including 30% EtOH, 1% Triton X-100, 1% SDS, and 5 M urea. CONCLUSIONS: Mutation of Trp182 to Ala allowed SfSFGH to accommodate a longer chain of substrates. It is thought that the W182A mutation increases the substrate-binding pocket and decreases the steric effect for larger substrates in SfSFGH. Consequently, the W182A mutant has a broader substrate specificity compared to wild-type SfSFGH. Taken together, this study provides useful structure-function data of a SFGH family member and may inform protein engineering strategies for industrial applications of SfSFGH.
Title: Effect of Placenta-Derived Mesenchymal Stem Cells in a Dementia Rat Model via Microglial Mediation: a Comparison between Stem Cell Transplant Methods Cho JS, Lee J, Jeong DU, Kim HW, Chang WS, Moon J, Chang JW Ref: Yonsei Med J, 59:406, 2018 : PubMed
PURPOSE: Loss of cholinergic neurons in the hippocampus is a hallmark of many dementias. Administration of stem cells as a therapeutic intervention for patients is under active investigation, but the optimal stem cell type and transplantation modality has not yet been established. In this study, we studied the therapeutic effects of human placenta-derived mesenchymal stem cells (pMSCs) in dementia rat model using either intracerebroventricular (ICV) or intravenous (IV) injections and analyzed their mechanisms of therapeutic action. MATERIALS AND METHODS: Dementia modeling was established by intraventricular injection of 192 IgG-saporin, which causes lesion of cholinergic neurons. Sixty-five male Sprague-Dawley rats were divided into five groups: control, lesion, lesion+ICV injection of pMSCs, lesion+IV injection of pMSCs, and lesion+donepezil. Rats were subjected to the Morris water maze and subsequent immunostaining analyses. RESULTS: Both ICV and IV pMSC administrations allowed significant cognitive recovery compared to the lesioned rats. Acetylcholinesterase activity was significantly rescued in the hippocampus of rats injected with pMSCs post-lesion. Choline acetyltransferase did not co-localize with pMSCs, showing that pMSCs did not directly differentiate into cholinergic cells. Number of microglial cells increased in lesioned rats and significantly decreased back to normal levels with pMSC injection. CONCLUSION: Our results suggest that ICV and IV injections of pMSCs facilitate the recovery of cholinergic neuronal populations and cognitive behavior. This recovery likely occurs through paracrine effects that resemble microglia function rather than direct differentiation of injected pMSCs into cholinergic neurons.
Title: Terminalia chebula extract prevents scopolamine-induced amnesia via cholinergic modulation and anti-oxidative effects in mice Kim MS, Lee DY, Lee J, Kim HW, Sung SH, Han JS, Jeon WK Ref: BMC Complement Altern Med, 18:136, 2018 : PubMed
BACKGROUND: Terminalia chebula Retz. (Combretaceae) is a traditional herbal medicine that is widely used in the treatment of diabetes, immunodeficiency diseases, and stomach ulcer in Asia. However, the anti-amnesic effect of T. chebula has not yet been investigated. The present study was designed to determine whether T. chebula extract (TCE) alleviates amnesia induced by scopolamine in mice. We also investigated possible mechanisms associated with cholinergic system and anti-oxidant effects. METHODS: TCE (100 or 200 mg/kg) was orally administered to mice for fourteen days (days 1-14), and scopolamine was intraperitoneally injected to induce memory impairment for seven days (days 8-14). Learning and memory status were evaluated using the Morris water maze. Hippocampal levels of acetylcholine (ACh), acetylcholinesterase (AChE) and choline acetyltransferase (ChAT) were measured ex vivo. Levels of reactive oxygen species (ROS), nitric oxide (NO), and malondialdehyde (MDA) in the hippocampus were also examined. RESULTS: In the Morris water maze task, TCE treatment reversed scopolamine-induced learning and memory deficits in acquisition and retention. TCE reduced hippocampal AChE activities and increased ChAT and ACh levels in the scopolamine-induced model. Moreover, TCE treatment suppressed scopolamine-induced oxidative damage by ameliorating the increased levels of ROS, NO, and MDA. CONCLUSION: These findings suggest that TCE exerts potent anti-amnesic effects via cholinergic modulation and anti-oxidant activity, thus providing evidence for its potential as a cognitive enhancer for amnesia.
Title: PsEst3, a new psychrophilic esterase from the Arctic bacterium Paenibacillus sp. R4: crystallization and X-ray crystallographic analysis Kim H, Park AK, Lee JH, Shin SC, Park H, Kim HW Ref: Acta Crystallographica F Struct Biol Commun, 74:367, 2018 : PubMed
Esterases are very useful biocatalysts in industry: they hydrolyze esters and split them into a carboxylic acid and an alcohol. The psychrophilic esterase PsEst3 was obtained from Paenibacillus sp. R4, which was isolated from the active layer of the permafrost in Council, Alaska. PsEst3 was successfully overexpressed using a psychrophilic chaperonin co-expression system and was purified by nickel-affinity and size-exclusion chromatography. Recombinant PsEst3 was crystallized at 290K using the hanging-drop vapour-diffusion method. X-ray diffraction data were collected to 2.1A resolution. The crystal was determined to belong to space group P4(1)32 or P4(3)32, with unit-cell parameters a = b = c = 145.33A. Further crystallographic analysis needs to be conducted to investigate the structure and function of this esterase.
Title: Crystal structure and functional characterization of a cold-active acetyl xylan esterase (PbAcE) from psychrophilic soil microbe Paenibacillus sp Park SH, Yoo W, Lee CW, Jeong CS, Shin SC, Kim HW, Park H, Kim KK, Kim TD, Lee JH Ref: PLoS ONE, 13:e0206260, 2018 : PubMed
Cold-active acetyl xylan esterases allow for reduced bioreactor heating costs in bioenergy production. Here, we isolated and characterized a cold-active acetyl xylan esterase (PbAcE) from the psychrophilic soil microbe Paenibacillus sp. R4. The enzyme hydrolyzes glucose penta-acetate and xylan acetate, reversibly producing acetyl xylan from xylan, and it shows higher activity at 4 degrees C than at 25 degrees C. We solved the crystal structure of PbAcE at 2.1-A resolution to investigate its active site and the reason for its low-temperature activity. Structural analysis showed that PbAcE forms a hexamer with a central substrate binding tunnel, and the inter-subunit interactions are relatively weak compared with those of its mesophilic and thermophilic homologs. PbAcE also has a shorter loop and different residue composition in the beta4-alpha3 and beta5-alpha4 regions near the substrate binding site. Flexible subunit movements and different active site loop conformations may enable the strong low-temperature activity and broad substrate specificity of PbAcE. In addition, PbAcE was found to have strong activity against antibiotic compound substrates, such as cefotaxime and 7-amino cephalosporanic acid (7-ACA). In conclusion, the PbAcE structure and our biochemical results provide the first example of a cold-active acetyl xylan esterase and a starting template for structure-based protein engineering.
A novel microbial esterase, EaEST, from a psychrophilic bacterium Exiguobacterium antarcticum B7, was identified and characterized. To our knowledge, this is the first report describing structural analysis and biochemical characterization of an esterase isolated from the genus Exiguobacterium. Crystal structure of EaEST, determined at a resolution of 1.9 A, showed that the enzyme has a canonical alpha/beta hydrolase fold with an alpha-helical cap domain and a catalytic triad consisting of Ser96, Asp220, and His248. Interestingly, the active site of the structure of EaEST is occupied by a peracetate molecule, which is the product of perhydrolysis of acetate. This result suggests that EaEST may have perhydrolase activity. The activity assay showed that EaEST has significant perhydrolase and esterase activity with respect to short-chain p-nitrophenyl esters (<=C8), naphthyl derivatives, phenyl acetate, and glyceryl tributyrate. However, the S96A single mutant had low esterase and perhydrolase activity. Moreover, the L27A mutant showed low levels of protein expression and solubility as well as preference for different substrates. On conducting an enantioselectivity analysis using R- and S-methyl-3-hydroxy-2-methylpropionate, a preference for R-enantiomers was observed. Surprisingly, immobilized EaEST was found to not only retain 200% of its initial activity after incubation for 1 h at 80 degrees C, but also retained more than 60% of its initial activity after 20 cycles of reutilization. This research will serve as basis for future engineering of this esterase for biotechnological and industrial applications.
Non-alcoholic fatty liver disease (NAFLD) is a prevalent obesity-related disease that affects large populations throughout the world due to excessive calorie intake and an increasingly sedentary lifestyle. Fibroblast growth factor 21 (FGF21) has recently emerged as a promising therapeutic candidate for the treatment of obesity and diabetes. FGF21 is a starvation-induced pleiotropic hormone with various beneficial metabolic effects, and pharmacological treatment in rodents has been shown to improve insulin sensitivity and decrease simple fatty liver disease. However, its effects on reversing the symptoms of advanced liver disease have yet to be validated. Here, we investigated the protective effects of the LY2405319 compound, an engineered FGF21 variant, in a non-alcoholic steatohepatitis (NASH) model using leptin-deficient ob/ob mice and a methionine- and choline-deficient (MCD) diet to induce steatohepatitis. LY2405319 treatment in ob/ob mice corroborated previous results showing that improvements in the metabolic parameters were due to increased mitochondrial oxygen consumption rate and fatty acid oxidation. LY2405319 treatment in ob/ob mice on an MCD diet significantly reduced the symptoms of steatohepatitis, as confirmed by Masson's trichrome staining intensity. Serum levels of AST and ALT were also reduced, suggesting an attenuation of liver injury, while detection of inflammatory markers showed decreased mRNA expression of TGF-beta1 and type-I collagen, and decreased phosphorylation of NF-kB p65, JNK1/2, and p38. Collectively, these data show that LY2405319 treatment attenuated MCD diet-induced NASH progression. We propose that the LY2405319 compound is a potential therapeutic candidate for the treatment of advanced liver disease.
Title: Enhancing extracellular lipolytic enzyme production in an arctic bacterium, Psychrobacter sp. ArcL13, by using statistical optimization and fed-batch fermentation Kim S, Wi AR, Park HJ, Kim D, Kim HW, Yim JH, Han SJ Ref: Preparative Biochemistry & Biotechnology, 45:348, 2015 : PubMed
A strain isolated from seawater samples in the Chuckchi Sea and exhibiting extracellular lipolytic activity was identified using 16S rRNA gene sequence analysis as Psychrobacter sp. ArcL13. The lipolytic enzyme exhibited cold-active properties and high hydrolytic activity toward p-nitrophenyl caprylate (C8), p-nitrophenyl decanoate (C10), and sunflower oil. Statistical optimization of the medium components was performed to enhance the production of cold-active extracellular lipolytic activity. Glucose, yeast extract (YE), and NaCl were selected as the main efficient nutrient sources. Fed-batch fermentation using optimized medium with concentrated YE as the main feeding material showed a maximum lipolytic activity of 10.7 U/mL, which was a 21-fold increase in production over unoptimized flask culture conditions. The information obtained in the present study could prove applicable to the production of cold-active lipase on a large scale.
Title: The effects of Betula platyphylla bark on amyloid beta-induced learning and memory impairment in mice Cho N, Lee HK, Jeon BJ, Kim HW, Kim HP, Lee JH, Kim YC, Sung SH Ref: Food & Chemical Toxicology, 74:156, 2014 : PubMed
Alzheimer's disease (AD) is closely associated with amyloid beta (Abeta)-induced neurotoxicity and oxidative stress in the brain. Betula platyphylla, which has been used to treat various oxidative-stressed related diseases, has recently received attention for its preventive activity on age-related neurodegenerative diseases. In this study, we attempted to investigate the effects of B. platyphylla bark (BPB-316) on Abeta1-42-induced neurotoxicity and memory impairment. Oral treatment using BPB-316 significantly attenuated Abeta-induced memory impairment which was evaluated by behavioral tests including the passive avoidance, Y-maze and Morris water maze test. BPB-316 also inhibited the elevation of beta-secretase activity accompanying the reduced Abeta1-42 levels in the hippocampus of the brain. Furthermore, BPB-316 significantly decreased the acetylcholinesterase activity and increased the glutathione content in the hippocampus. In addition, we confirmed that the expression of both cAMP responsive element-binding protein (CREB) and brain-derived neurotrophic factor (BDNF) in the hippocampus of Abeta1-42-injected mice were markedly upregulated by the treatment of BPB-316. Our data suggest that the extracts of B. platyphylla bark might be a potential therapeutic agent against AD.
Dipeptidyl peptidase IV (DPPIV) is an exopeptidase that modulates the function of several substrates, among which insulin-releasing incretin hormones are the most well known. DPPIV also modulate substrates involved in inflammation, cell migration, and cell differentiation. Although DPPIV is highly expressed in proximal renal tubular cells, the role of DPPIV inhibition in renal disease is not fully understood. For this reason, we investigated the effects of LC15-0444, a DPPIV inhibitor, on renal function in a mouse model of renal fibrosis. Eight-week-old C57/BL6 mice were subjected to unilateral ureteral obstruction (UUO) and were treated with LC15-0444 (a DPPIV inhibitor) at a dose of 150 mg/kg per day in food or vehicle for 14 days. DPPIV activity was significantly increased in obstructed kidneys, and reduced after treatment with LC15-0444. Administration of LC15-0444 resulted in a significant decrease in albuminuria, urinary excretion of 8-isoprostane, and renal fibrosis. DPPIV inhibition also substantially decreased the synthesis of several proinflammatory and profibrotic molecules, as well as the infiltration of macrophages. UUO significantly increased, and LC15-0444 markedly suppressed, levels of phosphorylated Smad2/3, TGFbeta1, toll-like receptor 4, high-mobility group box-1, NADPH oxidase 4, and NF-kappaB. These results suggest that activation of DPPIV in the kidney has a role in the progression of renal disease and that targeted therapy inhibiting DPPIV may prove to be a useful new approach in the management of progressive renal disease, independent of mechanisms mediated by glucagon-like peptide-1.
Title: Characterization and a point mutational approach of a psychrophilic lipase from an arctic bacterium, Bacillus pumilus Wi AR, Jeon SJ, Kim S, Park HJ, Kim D, Han SJ, Yim JH, Kim HW Ref: Biotechnol Lett, 36:1295, 2014 : PubMed
A bacterium with lipolytic activity was isolated from the Chukchi Sea within the Arctic Ocean. The lipase BpL5 from the isolate, Bacillus pumilus ArcL5, belongs to subfamily 4 of lipase family I. The optimum pH and temperature of the recombinant enzyme BpL5, as expressed in Escherichia coli, were 9.0 and 20 degrees C, respectively. The enzyme retained 85 % of its activity at 5 degrees C. There was a significant difference between temperatures for maximal activity (20 degrees C) and for protein denaturation (approx. 45 degrees C). The enzyme preferred middle-chain (C8) p-nitrophenyl substrates. Two mutants, S139A and S139Y, were rationally designed based on the 3D-structure model, and their activities were compared with that of the wild type. The both mutants showed significantly improved activity against tricaprylin.
Synaptotagmins are required for Ca(2+)-dependent membrane-trafficking in either neuronal synaptic vesicles or cellular membranes. Previous reports suggested that the synaptotagmin 11 (syt11) gene is involved in the development of schizophrenia based on the genomic analysis of patients. Parkin protein binds to the C2 domains of Syt11 which leads to the polyubiquitination of Syt11. However, where and how Syt11 performs its role in the brain is largely unknown. Here, we report that Syt11 is expressed mainly in the brain. In addition, exogenously expressed Syt11 in HEK293 cells can form higher molecular weight complex via its transmembrane domain. Also, Syt11 is targeted to both dendrite and axon compartments. Immunocytochemistry showed that Syt11 is juxtaposed to postsynaptic markers in both excitatory and inhibitory synapses. Both neuroligin 1 and 2, which are postsynaptic cell adhesion molecules and differentially induce excitatory and inhibitory presynapses, respectively, recruit Syt11 in neuron coculture. Immunogold electron microscopy analysis revealed that Syt11 exists mainly in presynaptic neurotransmitter vesicles and plasma membrane, and rarely in postsynaptic sites. These results suggest that Syt11 may contribute to the regulation of neurotransmitter release in the excitatory and inhibitory presynapses, and postsynapse-targeted membrane trafficking in dendrites.
Lactobacillus salivarius is a well-known lactic acid bacterium to which increasing attention has been paid recently for use as probiotics for humans and animals. L. salivarius NIAS840 was first isolated from broiler chicken feces, displaying antimicrobial activities against multidrug-resistant Staphylococcus aureus and Salmonella enterica serovar Typhimurium. Here, we report the genome sequence of L. salivarius NIAS840 (2,046,557 bp) including a small plasmid and two megaplasmids.
Title: Two juvenile hormone esterase-like carboxylesterase cDNAs from a Pandalus shrimp (Pandalopsis japonica): cloning, tissue expression, and effects of eyestalk ablation Lee SO, Jeon JM, Oh CW, Kim YM, Kang CK, Lee DS, Mykles DL, Kim HW Ref: Comparative Biochemistry & Physiology B Biochem Mol Biol, 159:148, 2011 : PubMed
Methyl farnesoate (MF), a crustacean juvenile hormone (JH) analog, plays important roles in the regulation of a number of physiological processes such as molting, metamorphosis, and reproduction. Understanding its metabolic pathway is a key for various potential applications in crustacean aquaculture, including artificial seed production and enhancement of growth. Although the synthetic pathway of MF is well established, little is known about its degradation and recycling in crustaceans. In insects, juvenile hormone esterase (JHE), a carboxylesterase, is responsible for JH inactivation. Two cDNAs, encoding JHE-like carboxylesterases (CXEs) from the hepatopancreas and ovary of Pandalopsis japonica, were isolated by using a combination of in-silico data mining from an expressed sequence tag (EST) database and traditional PCR-based cloning. The full length Pj-CXE1 (2084bp) and Pj-CXE2 (1985bp) cDNAs encoded proteins composed of 584 and 581 amino acids, respectively. The active site sequence and domain organization of the Pj-CXEs were highly conserved, including the catalytic triad and other motifs, which suggested that both Pj-CXEs are biologically active carboxylesterases. Phylogenetic analysis of the deduced sequences of Pj-CXEs showed that both were most closely related to the JHEs from non-lepidopteran insects. End-point RT-PCR showed that Pj-CXE1 was expressed primarily in the gonad, whereas Pj-CXE2 was expressed in both the hepatopancreas and hindgut. Quantitative PCR showed that Pj-CXE1 was upregulated in the gonads by eyestalk ablation (ESA). In contrast, ESA had no significant effect on Pj-CXE2 expression in hepatopancreas or gonad. This is the first report of the cloning of two JHE-like CXE cDNAs in decapods and the upregulation of Pj-CXE1 by acute withdrawal of eyestalk neuropeptides. Further study is needed to understand the function of CXEs in MF metabolism and its regulation by eyestalk neuropeptides.
Title: Pancreatic lipase-related protein (PY-PLRP) highly expressed in the vitellogenic ovary of the scallop, Patinopecten yessoensis Kim KS, Kim BK, Kim HJ, Yoo MS, Mykles DL, Kim HW Ref: Comparative Biochemistry & Physiology B Biochem Mol Biol, 151:52, 2008 : PubMed
A cDNA (1206 bp) encoding a pancreatic lipase-related protein (PY-PLRP) was obtained from the ovary of the scallop, Patinopecten yessoensis, using a differentially expressed gene system. The open reading frame specified a protein containing 353 amino acids (~38 kDa). The N-terminal catalytic domain, which contained the catalytic triad of serine, aspartate, and histidine residues, 10 cysteine residues involved in disulfide bridges, and the conserved lid domain, indicated that the protein would be catalytically active. However, PY-PLRP lacked the C-terminal colipase-binding domain present in mammalian PLRPs. Sequence analysis of the catalytic domains of PY-PLRP with members of the pancreatic triglyceride lipase (PTL) family suggested that PY-PLRP was related to mammalian PLRP1, PLRP2, and PTL. End-point reverse transcriptase-polymerase chain reaction (RT-PCR) after 25 cycles showed that PY-PLRP was expressed at a high level in the ovary and at low levels in testis and mantle; expression was not detected in gill, digestive gland, and adductor muscle. Quantitative PCR of RNA from ovaries at different stages of development showed that PY-PLRP was expressed at a significantly higher level in the late growth stage than in the ripe and spent stages. These data suggest that PY-PLRP plays a role in lipid metabolism associated with oocyte maturation and vitellogenesis that occurs during ovarian growth.
Title: Frequency Detection of Organophosphate Resistance Allele in Anopheles sinensis (Diptera: Culicidae) Populations by Real-time PCR Amplification of Specific Allele (rtPASA) Baek JH, Kim HW, Lee WJ, Lee SH Ref: Journal of Asia-Pacific Entomology, 9:375, 2006 : PubMed
A rapid, simple and accurate real-time PASA (PCR amplification of specific allele) (rtPASA) protocol was developed and optimized for the frequency estimation of the Glyl119Ser mutation in the type-I acetylcholinesterase locus, putatively associated with organophosphate resistance, in pooled DNA samples of Anopheles sinensis, a major vector mosquito of malaria in Korea. Performance of the rtPASA protocol was evaluated by comparing with the data generated from individual genotypings of a field population. The resistance allele frequency of the population (74.4%) predicted from the linear regression line of the rtPASA agreed well with that estimated from the individual genotyping (74.1%), demonstrating its reliability and accuracy. Using this rtPASA protocol, the resistance allele frequency in 10 local populations of An. sinensis was determined to range from 74.4% to 97.2%, suggestive of the widespread organophosphate resistance in An. sinensis in Korea.
Intrathecal (IT) injection of neostigmine (a cholinesterase inhibitor) has been reported to produce a significant anti-nociceptive effect in a number of inflammatory pain models. However, a potential anti-inflammatory effect of IT neostigmine in these models has not been investigated. In the present study, we have examined the 'anti-inflammatory effect of IT injection of neostigmine' (AI-NEO) using a standard mouse air pouch model by evaluating the effect of AI-NEO on zymosan-induced leukocyte migration and myeloperoxidase (MPO) release. IT neostigmine was found to suppress both leukocyte migration and MPO degranulation in a dose dependent manner. We then established which subtypes of cholinergic receptors were involved in this AI-NEO. IT pretreatment with atropine (a muscarinic receptor antagonist) but not hexamethonium (a nicotinic receptor antagonist) completely blocked the IT neostigmine anti-inflammatory effect. Subsequent experiments showed that IT pretreatment with methoctramine (a muscarinic type 2 (M2) receptor antagonist), but not pirenzepine (M1 receptor antagonist) or 4-DAMP (M3 receptor antagonist), suppressed the AI-NEO. We then evaluated whether adrenal glandular activity was important in the AI-NEO. Adrenalectomy significantly blocked the AI-NEO, while intraperitoneal pretreatment with the beta-adrenoceptor antagonist (propranolol), but not the corticosteroid antagonist (RU486) reversed AI-NEO. In conclusion, these results indicate that IT neostigmine facilitates the activation of spinal M2 receptors and this activation ultimately leads to release of adrenal catecholamines which contribute to the anti-inflammatory effect observed at the site of tissue inflammation.
