Title: Characterization of a novel thermostable alkaline lipase derived from a compost metagenomic library and its potential application in the detergent industry Li QQ, Zhu ZR, Liu QG, An YT, Wang YX, Zhang SB, Li G Ref: Front Microbiol, 13:1088581, 2022 : PubMed
Using composted soil samples, a metagenomic library consisting of 36,000 clones was constructed. Then, a novel lipase, Lip54q, which belongs to the VIII family of lipolytic enzymes, was identified from the metagenomic library by functional screening. To explore the enzymatic properties of Lip54q, lip54q was heterologous expressed in Escherichia coli with a high expression level of recombinant protein up to 720mg/L. The recombinant enzyme showed the highest activity (28,160U/mg) against a C10 substrate at pH 9.0 and 47 degreesC, and was stable at temperatures >=50 degreesC and pH 8.0-11.0. Of particular interest, the surfactants, Tween-20, Tween-80 and Tritonx-100, exhibited strong promoting effects on Lip54q activities regardless of whether low concentrations (0.1%) or high concentrations (10%) were used. Application studies of Lip54q using six commercial detergents indicated that the enzyme had strong tolerance and immersion resistance to all six detergents. The results of oil-stain removal experiments suggested that addition of the enzyme to various commercial detergents could significantly improve the abilities of these detergents to remove oil-stains. Furthermore, the results of a molecular docking analysis of Lip54q showed that both the C10 substrate and linoleic acid molecules could form hydrogen bond interactions with the catalytic amino acids, Ser-268, Glu-168, and Asp-192, in the catalytic center of the enzyme, and the hydrogen bond distances were shorter. The electrostatic attraction between the enzyme and the substrate formed by the hydrogen bond with a shorter distance is stronger, which is conducive to the formation of a more stable complex between the enzyme and the substrate, thus increasing the activity of the enzyme to such substrate. These results 1ay a good foundation for application of this enzyme in the detergent industry in the future.
We have previously reported that the single transmembrane protein Dipeptidyl Peptidase Like 6 (DPP6) impacts neuronal and synaptic development. DPP6-KO mice are impaired in hippocampal-dependent learning and memory and exhibit smaller brain size. Recently, we have described novel structures in hippocampal area CA1 in aging mice, apparently derived from degenerating presynaptic terminals, that are significantly more prevalent in DPP6-KO mice compared to WT mice of the same age and that these structures were observed earlier in development in DPP6-KO mice. These novel structures appear as clusters of large puncta that colocalize NeuN, synaptophysin, and chromogranin A, and also partially label for MAP2, amyloid beta, APP, alpha-synuclein, and phosphorylated tau, with synapsin-1 and VGluT1 labeling on their periphery. In this current study, using immunofluorescence and electron microscopy, we confirm that both APP and amyloid beta are prevalent in these structures; and we show with immunofluorescence the presence of similar structures in humans with Alzheimer's disease. Here we also found evidence that aging DPP6-KO mutants show additional changes related to Alzheimer's disease. We used in vivo MRI to show reduced size of the DPP6-KO brain and hippocampus. Aging DPP6-KO hippocampi contained fewer total neurons and greater neuron death and had diagnostic biomarkers of Alzheimer's disease present including accumulation of amyloid beta and APP and increase in expression of hyper-phosphorylated tau. The amyloid beta and phosphorylated tau pathologies were associated with neuroinflammation characterized by increases in microglia and astrocytes. And levels of proinflammatory or anti-inflammatory cytokines increased in aging DPP6-KO mice. We finally show that aging DPP6-KO mice display circadian dysfunction, a common symptom of Alzheimer's disease. Together these results indicate that aging DPP6-KO mice show symptoms of enhanced neurodegeneration reminiscent of dementia associated with a novel structure resulting from synapse loss and neuronal death. This study continues our laboratory's work in discerning the function of DPP6 and here provides compelling evidence of a direct role of DPP6 in Alzheimer's disease.
The roots of Piper nigrum L., a seasoning for cooking various types of broths, are renowned for their high nutritional content and potential medicinal benefits. In this study, nine pairs of novel cyclohexene-type bisamide alkaloids (1a/1b-9a/9b) were isolated from the pepper roots using molecular network analysis strategies. Their structures were determined by extensive spectroscopic data, electronic circular dichroism (ECD) calculations, and X-ray diffraction analyses. Using an intermolecular Diels-Alder reaction, a strategy for the synthesis of bisamide alkaloids from different monomeric amide alkaloids was developed. Furthermore, these compounds were chirally separated for the first time, and compounds 3a and 5a/5b showed significant anti-neuroinflammation effects in the models of lipopolysaccharide(LPS)-induced BV2 microglial cells. Meanwhile, compounds 6b and 7a displayed concentration-dependent inhibitory activities against acetylcholinesterase with IC(50) values of 6.05 +/- 1.10 and 3.81 +/- 0.10 microM, respectively. These findings confirmed that these bisamide alkaloids could be applied in functional food formulations and pharmaceutical products as well as facilitate the further development and usage of pepper roots.
Title: Notum palmitoleoyl-protein carboxylesterase regulates Fas cell surface death receptor-mediated apoptosis via the Wnt signaling pathway in colon adenocarcinoma Gong H, Niu Q, Zhou Y, Wang YX, Xu XF, Hou KZ Ref: Bioengineered, 12:5241, 2021 : PubMed
Colon adenocarcinoma (COAD) is one of the most common types of malignancy and accounts for >3 million deaths worldwide each year. The present study aimed to evaluate the role of notum palmitoleoyl-protein carboxylesterase (NOTUM) in in vivo and in vitro, and to identify the relationship between NOTUM and the apoptosis of COAD. Moreover, the present study aimed to investigate whether NOTUM regulated Fas cell surface death receptor (FAS)-mediated apoptosis was affected by the Wnt signaling pathway. Gene expression profiling interactive analysis (GEPIA) was used to predict the potential function of NOTUM. Western blotting and reverse transcription-quantitative PCR were conducted to detect the protein and mRNA expression levels of NOTUM in different tissues or cell lines. The occurrence and development of COAD was detected after NOTUM knockdown lentivirus administration. The apoptosis of COAD was also observed. SKL2001 was applied to examine whether the role of NOTUM was regulated by Wnt. GEPIA analysis demonstrated that NOTUM expression in COAD tumor tissue was higher compared with in normal tissues. Pair-wise gene correlation analysis identified a potential relationship between NOTUM and Wnt. NOTUM protein and mRNA expression levels in colon carcinoma tissues and RKO cells were increased. NOTUM knockdown lentivirus serves a role in inhibiting COAD development by reducing tumor proliferation, reducing tumor size, and increasing the level of apoptosis in vitro and in vivo. Moreover, NOTUM could increase apoptosis in COAD, which was regulated by FAS, and SKL2001 blocked the progress of apoptosis after NOTUM regulation by NOTUM knockdown lentivirus in vitro and in vivo. Collectively, the present results suggested that NOTUM may be able to regulate the apoptosis of COAD, and that Wnt may be the down-stream target signaling of NOTUM in apoptosis.
Title: A novel structure associated with aging is augmented in the DPP6-KO mouse brain Lin L, Petralia RS, Lake R, Wang YX, Hoffman DA Ref: Acta Neuropathologica Commun, 8:197, 2020 : PubMed
In addition to its role as an auxiliary subunit of A-type voltage-gated K(+) channels, we have previously reported that the single transmembrane protein Dipeptidyl Peptidase Like 6 (DPP6) impacts neuronal and synaptic development. DPP6-KO mice are impaired in hippocampal-dependent learning and memory and exhibit smaller brain size. Using immunofluorescence and electron microscopy, we report here a novel structure in hippocampal area CA1 that was significantly more prevalent in aging DPP6-KO mice compared to WT mice of the same age and that these structures were observed earlier in development in DPP6-KO mice. These novel structures appeared as clusters of large puncta that colocalized NeuN, synaptophysin, and chromogranin A. They also partially labeled for MAP2, and with synapsin-1 and VGluT1 labeling on their periphery. Electron microscopy revealed that these structures are abnormal, enlarged presynaptic swellings filled with mainly fibrous material with occasional peripheral, presynaptic active zones forming synapses. Immunofluorescence imaging then showed that a number of markers for aging and especially Alzheimer's disease were found as higher levels in these novel structures in aging DPP6-KO mice compared to WT. Together these results indicate that aging DPP6-KO mice have increased numbers of novel, abnormal presynaptic structures associated with several markers of Alzheimer's disease.
GDSL-type esterase/lipase proteins (GELPs) belong to the SGNH hydrolase superfamily and contain a conserved GDSL motif at their N-terminus. GELPs are widely distributed in nature, from microbes to plants, and play crucial roles in growth and development, stress responses and pathogen defense. However, the identification and functional analysis of GELP genes are hardly explored in soybean. This study describes the identification of 194 GELP genes in the soybean genome and their phylogenetic classification into 11 subfamilies (A-K). GmGELP genes are disproportionally distributed on 20 soybean chromosomes. Large-scale WGD/segmental duplication events contribute greatly to the expansion of the soybean GDSL gene family. The Ka/Ks ratios of more than 70% of duplicated gene pairs ranged from 0.1-0.3, indicating that most GmGELP genes were under purifying selection pressure. Gene structure analysis indicate that more than 74% of GmGELP genes are interrupted by 4 introns and composed of 5 exons in their coding regions, and closer homologous genes in the phylogenetic tree often have similar exon-intron organization. Further statistics revealed that approximately 56% of subfamily K members contain more than 4 introns, and about 28% of subfamily I members consist of less than 4 introns. For this reason, the two subfamilies were used to simulate intron gain and loss events, respectively. Furthermore, a new model of intron position distribution was established in current study to explore whether the evolution of multi-gene families resulted from the diversity of gene structure. Finally, RNA-seq data were used to investigate the expression profiles of GmGELP gene under different tissues and multiple abiotic stress treatments. Subsequently, 7 stress-responsive GmGELP genes were selected to verify their expression levels by RT-qPCR, the results were consistent with RNA-seq data. Among 7 GmGELP genes, GmGELP28 was selected for further study owing to clear responses to drought, salt and ABA treatments. Transgenic Arabidopsis thaliana and soybean plants showed drought and salt tolerant phenotype. Overexpression of GmGELP28 resulted in the changes of several physiological indicators, which allowed plants to adapt adverse conditions. In all, GmGELP28 is a potential candidate gene for improving the salinity and drought tolerance of soybean.
In the brain, many types of interneurons make functionally diverse inhibitory synapses onto principal neurons. Although numerous molecules have been identified to function in inhibitory synapse development, it remains unknown whether there is a unifying mechanism for development of diverse inhibitory synapses. Here we report a general molecular mechanism underlying hippocampal inhibitory synapse development. In developing neurons, the establishment of GABAergic transmission depends on Neuroligin 2 (NL2), a synaptic cell adhesion molecule (CAM). During maturation, inhibitory synapse development requires both NL2 and Slitrk3 (ST3), another CAM. Importantly, NL2 and ST3 interact with nanomolar affinity through their extracellular domains to synergistically promote synapse development. Selective perturbation of the NL2-ST3 interaction impairs inhibitory synapse development with consequent disruptions in hippocampal network activity and increased seizure susceptibility. Our findings reveal how unique postsynaptic CAMs work in concert to control synaptogenesis and establish a general framework for GABAergic synapse development.
Title: Enzyme-responsive supramolecular polymers by complexation of bis(p-sulfonatocalixarenes) with suberyl dicholine-based pseudorotaxane Guo DS, Zhang TX, Wang YX, Liu Y Ref: Chem Commun (Camb), 49:6779, 2013 : PubMed
A linear supramolecular ternary polymer was fabricated by iteratively threading cyclodextrin with suberyl dicholine and end-capping with bis-calixarenes, showing desired cholinesterase response.
Title: Cholinesterase-responsive supramolecular vesicle Guo DS, Wang K, Wang YX, Liu Y Ref: Journal of the American Chemical Society, 134:10244, 2012 : PubMed
Enzyme-responsive, amphiphilic self-assembly represents one of the increasingly significant topics in biomaterials research and finds feasible applications to the controlled release of therapeutic agents at specific sites where the target enzyme is located. The supramolecular approach, using "superamphiphiles", provides a smart way to fabricate drug delivery systems responsive to enzymatic catalysis. In this work based on the concept of supramolecular chemistry, we report an enzyme-responsive vesicle using p-sulfonatocalix[4]arene as the macrocyclic host and natural enzyme-cleavable myristoylcholine as the guest molecule. The complexation of p-sulfonatocalix[4]arene with myristoylcholine directs the formation of a supramolecular binary vesicle, which is dissipated by cholinesterase with high specificity and efficiency. Cholinesterase is a key protein overexpressed in Alzheimer's disease, and therefore, the present system may have potential for the delivery of Alzheimer's disease drugs.
1-(1-Acetyl-piperidin-4-yl)-3-adamantan-1-yl-urea 14a (AR9281), a potent and selective soluble epoxide hydrolase inhibitor, was recently tested in a phase 2a clinical setting for its effectiveness in reducing blood pressure and improving insulin resistance in pre-diabetic patients. In a mouse model of diet induced obesity, AR9281 attenuated the enhanced glucose excursion following an intraperitoneal glucose tolerance test. AR9281 also attenuated the increase in blood pressure in angiotensin-II-induced hypertension in rats. These effects were dose-dependent and well correlated with inhibition of the sEH activity in whole blood, consistent with a role of sEH in the observed pharmacology in rodents.
Endothelial dysfunction is a hallmark of, and plays a pivotal role in the pathogenesis of cardiometabolic diseases, including type II diabetes, obesity, and hypertension. It has been well established that epoxyeicosatrienoic acids (EETs) act as an endothelial derived hyperpolarization factor (EDHF). Soluble epoxide hydrolase (s-EH) rapidly hydrolyses certain epoxylipids (e.g. EETs) to less bioactive diols (DHETs), thereby attenuating the evoked vasodilator effects. The aim of the present study was to examine if inhibition of s-EH can restore impaired endothelial function in three animal models of cardiometabolic diseases. Isolated vessel rings of the aorta and/or mesenteric artery from mice or rats were pre-contracted using phenylephrine or U46619. Endothelium-dependent and independent vasorelaxation to acetylcholine and sodium nitroprusside (SNP) were measured using wire myography in vessels isolated from db/db or diet-induced obesity (DIO) mice, and angiotensin II-induced hypertensive rats treated chronically with s-EH inhibitors AR9281 or AR9276 or with vehicle. Vasorelaxation to acetylcholine, but not to SNP was severely impaired in all three animal models. Oral administration of AR9281 or AR9276 abolished whole blood s-EH activity, elevated epoxy/diol lipid ratio, and abrogated endothelial dysfunction in all three models. Incubating the mesenteric artery of db/db mice with L-NAME and indomethacin to block nitric oxide (NO) and prostacyclin formation did not affect AR9821-induced improvement of endothelial function. These data indicate that inhibition of s-EH ameliorates endothelial dysfunction and that effects in the db/db model are independent of the presence of NO and cyclooxygenase derived prostanoids. Thus, preserving vasodilator EETs by inhibition of s-EH may be of therapeutic benefit by improving endothelial function in cardiometabolic diseases.
BACKGROUND: Excessive accumulation of lipids in the adipose tissue is a major problem in the present-day broiler industry. However, few studies have analyzed the expression of adipose tissue genes that are involved in pathways and mechanisms leading to adiposity in chickens. Gene expression profiling of chicken adipose tissue could provide key information about the ontogenesis of fatness and clarify the molecular mechanisms underlying obesity. In this study, Chicken Genome Arrays were used to construct an adipose tissue gene expression profile of 7-week-old broilers, and to screen adipose tissue genes that are differentially expressed in lean and fat lines divergently selected over eight generations for high and low abdominal fat weight. RESULTS: The gene expression profiles detected 13,234-16,858 probe sets in chicken adipose tissue at 7 weeks, and genes involved in lipid metabolism and immunity such as fatty acid binding protein (FABP), thyroid hormone-responsive protein (Spot14), lipoprotein lipase(LPL), insulin-like growth factor binding protein 7(IGFBP7) and major histocompatibility complex (MHC), were highly expressed. In contrast, some genes related to lipogenesis, such as leptin receptor, sterol regulatory element binding proteins1 (SREBP1), apolipoprotein B(ApoB) and insulin-like growth factor 2(IGF2), were not detected. Moreover, 230 genes that were differentially expressed between the two lines were screened out; these were mainly involved in lipid metabolism, signal transduction, energy metabolism, tumorigenesis and immunity. Subsequently, real-time RT-PCR was performed to validate fifteen differentially expressed genes screened out by the microarray approach and high consistency was observed between the two methods. CONCLUSION: Our results establish the groundwork for further studies of the basic genetic control of growth and development of chicken adipose tissue, and will be beneficial in clarifying the molecular mechanism of obesity in chickens.
Title: Ontogeny of postsynaptic density proteins at glutamatergic synapses Petralia RS, Sans N, Wang YX, Wenthold RJ Ref: Molecular & Cellular Neurosciences, 29:436, 2005 : PubMed
In glutamatergic synapses, glutamate receptors (GluRs) associate with many other proteins involved in scaffolding and signal transduction. The ontogeny of these postsynaptic density (PSD) proteins involves changes in their composition during development, paralleling changes in GluR type and function. In the CA1 region of the hippocampus, at postnatal day 2 (P2), many synapses already have a distinct PSD. We used immunoblot analysis, subcellular fractionation, and quantitative immunogold electron microscopy to examine the distribution of PSD proteins during development of the hippocampus. Synapses at P2 contained substantial levels of NR1 and NR2B and most GluR-associated proteins, including SAP102, SynGAP, the chain of proteins from GluRs/SAP102 through GKAP/Shank/Homer and metabotropic glutamate receptors, and the adhesion factors, cadherin, catenin, neuroligin, and Nr-CAM. Development was marked by substantial decreases in NR2B and SAP102 and increases in NR2A, PSD-95, AMPA receptors, and CaMKII. Other components showed more moderate changes.
Title: Molecular simulation of a single-chain antibody against AChE to explore molecular basis of inhibitory effect of 3F3 McAb on enzyme activity Guo CZ, Wu JH, Wang YX, Hu YD, Li S, Sun MJ Ref: Acta Pharmacol Sin, 24:460, 2003 : PubMed
AIM: To explore the molecular basis of the inhibitory effect of 3F3, a monoclonal antibody against acetylcholinesterase (AChE), by computer-aided molecular simulation. METHODS: The single-chain 3F3 antibody (Sc3F3) was designed by joining VH and VL via a flexible linker (Gly4Ser)3. The amino acid sequence of the recombinant Sc3F3 was then subjected to computer-aided molecular modeling, and docking with the antigen molecule AChE to mimic the immunoactive interaction in a three-dimensional fashion. RESULTS: The modeled structure of Sc3F3 manifested the common features of a classical antibody. Both VH and VL were composed of two beta-sheets and connecting loops. The docking profile of the action between Sc3F3 with AChE demonstrated the formation of a stable structure. The van der Waals force played an important role suggesting that the complex was formed mainly via hydrophobic interactions between Sc3F3 and AChE molecules. CONCLUSION: The spatial structure of the complex of Sc3F3 and AChE showed that Sc3F3 overlaid the entrance of the active center gorge of AChE blocking the access of substrate.
Staphylococcus epidermidis strains are diverse in their pathogenicity; some are invasive and cause serious nosocomial infections, whereas others are non-pathogenic commensal organisms. To analyse the implications of different virulence factors in Staphylococcus epidermidis infections, the complete genome of Staphylococcus epidermidis strain ATCC 12228, a non-biofilm forming, non-infection associated strain used for detection of residual antibiotics in food products, was sequenced. This strain showed low virulence by mouse and rat experimental infections. The genome consists of a single 2499 279 bp chromosome and six plasmids. The chromosomal G + C content is 32.1% and 2419 protein coding sequences (CDS) are predicted, among which 230 are putative novel genes. Compared to the virulence factors in Staphylococcus aureus, aside from delta-haemolysin and beta-haemolysin, other toxin genes were not found. In contrast, the majority of adhesin genes are intact in ATCC 12228. Most strikingly, the ica operon coding for the enzymes synthesizing interbacterial cellular polysaccharide is missing in ATCC 12228 and rearrangements of adjacent genes are shown. No mec genes, IS256, IS257, were found in ATCC 12228. It is suggested that the absence of the ica operon is a genetic marker in commensal Staphylococcus epidermidis strains which are less likely to become invasive.
M2 receptor stimulation results in the gating of nonselective cation channels in several smooth muscle cell types. However the requirement for current activation includes a rise in cytosolic calcium mediated by M3 receptor induced calcium release. This complex signaling system confers substantial complexity on the interpretation of pharmacological experiments. M2 and M3 receptor stimulation has also been linked to the inhibition of potassium channels in smooth muscle. These signaling events are likely to play important roles in excitation/contraction coupling.
Title: Monoclonal antibody 3F3 against conformational epitope of Torpedo acetylcholinesterase Fu FH, Wang YX, Li FZ, Xin YB, Sun MJ Ref: Acta Pharmacol Sin, 18:284, 1997 : PubMed
AIM To study the type of epitope of native Torpedo acetylcholinesterase (AChE) directed by its monoclonal antibody (McAb) 3F3.
METHODS:
Enzyme-linked immunosorbent assay (ELISA) was used for the assay of the reaction between antigen and antibody.
RESULTS:
McAb 3F3 immunoreacted well with the native AChE, but not with the reduced- and alkylated-AChE (RA-AChE) at all. Soman did not interfere the binding of 3F3 with AChE molecule. The synthesized 24-peptide containing the active serine residue of the AChE active center did not react with McAb 3F3.
CONCLUSION:
3F3 is a monoclonal antibody against the conformational epitope of Torpedo AChE active center, but dose not occupy the active serine residue of the enzyme.
