Targeted delivery of immunomodulators to the lymphatic system has the potential to enhance therapeutic efficacy by increasing colocalization of drugs with immune targets such as lymphocytes. A triglyceride (TG)-mimetic prodrug strategy has been recently shown to enhance the lymphatic delivery of a model immunomodulator, mycophenolic acid (MPA), via incorporation into the intestinal TG deacylation-reacylation and lymph lipoprotein transport pathways. In the current study, a series of structurally related TG prodrugs of MPA were examined to optimize structure-lymphatic transport relationships for lymph-directing lipid-mimetic prodrugs. MPA was conjugated to the sn-2 position of the glyceride backbone of the prodrugs using linkers of different chain length (5-21 carbons) and the effect of methyl substitutions at the alpha and/or beta carbons to the glyceride end of the linker was examined. Lymphatic transport was assessed in mesenteric lymph duct cannulated rats, and drug exposure in lymph nodes was examined following oral administration to mice. Prodrug stability in simulated intestinal digestive fluid was also evaluated. Prodrugs with straight chain linkers were relatively unstable in simulated intestinal fluid; however, co-administration of lipase inhibitors (JZL184 and orlistat) was able to reduce instability and increase lymphatic transport (2-fold for a prodrug with a 6-carbon spacer, i.e., MPA-C6-TG). Methyl substitutions to the chain resulted in similar trends in improving intestinal stability and lymphatic transport. Medium- to long-chain spacers (C12, C15) between MPA and the glyceride backbone were most effective in promoting lymphatic transport, consistent with increases in lipophilicity. In contrast, short-chain (C6-C10) linkers appeared to be too unstable in the intestine and insufficiently lipophilic to associate with lymph lipid transport pathways, while very long-chain (C18, C21) linkers were also not preferred, likely as a result of increases in molecular weight reducing solubility or permeability. In addition to more effectively promoting drug transport into mesenteric lymph, TG-mimetic prodrugs based on a C12 linker resulted in marked increases (>40 fold) in the exposure of MPA in the mesenteric lymph nodes in mice when compared to administration of MPA alone, suggesting that optimizing prodrug design has the potential to provide benefit in targeting and modulating immune cells.
Title: Upregulation of Spinal MDGA1 in Rats After Nerve Injury Alters Interactions Between Neuroligin-2 and Postsynaptic Scaffolding Proteins and Increases GluR1 Subunit Surface Delivery in the Spinal Cord Dorsal Horn Li HL, Guo RJ, Ai ZR, Han S, Guan Y, Li JF, Wang Y Ref: Neurochem Res, :, 2023 : PubMed
Previous studies suggested that postsynaptic neuroligin-2 may shift from inhibitory toward excitatory function under pathological pain conditions. We hypothesize that nerve injury may increase the expression of spinal MAM-domain GPI-anchored molecule 1 (MDGA1), which can bind to neuroligin-2 and thereby, alter its interactions with postsynaptic scaffolding proteins and increase spinal excitatory synaptic transmission, leading to neuropathic pain. Western blot, immunofluorescence staining, and co-immunoprecipitation studies were conducted to examine the critical role of MDGA1 in the lumbar spinal cord dorsal horn in rats after spinal nerve ligation (SNL). Small interfering ribonucleic acids (siRNAs) targeting MDGA1 were used to examine the functional roles of MDGA1 in neuropathic pain. Protein levels of MDGA1 in the ipsilateral dorsal horn were significantly upregulated at day 7 post-SNL, as compared to that in naive or sham rats. The increased levels of GluR1 in the synaptosomal membrane fraction of the ipsilateral dorsal horn tissues at day 7 post-SNL was normalized to near sham level by pretreatment with intrathecal MDGA1 siRNA(2308), but not scrambled siRNA or vehicle. Notably, knocking down MDGA1 with siRNAs reduced the mechanical and thermal pain hypersensitivities, and inhibited the increased excitatory synaptic interaction between neuroligin-2 with PSD-95, and prevented the decreased inhibitory postsynaptic interactions between neuroligin-2 and Gephyrin. Our findings suggest that SNL upregulated MDGA1 expression in the dorsal horn, which contributes to the pain hypersensitivity through increasing the net excitatory interaction mediated by neuroligin-2 and surface delivery of GluR1 subunit in dorsal horn neurons.
Triple-negative breast cancer (TNBC) cells reprogram their metabolism to provide metabolic flexibility for tumor cell growth and survival in the tumor microenvironment. While our previous findings indicated that endothelial lipase (EL/LIPG) is a hallmark of TNBC, the precise mechanism through which LIPG instigates TNBC metabolism remains undefined. Here, we report that the expression of LIPG is associated with long non-coding RNA DANCR and positively correlates with gene signatures of mitochondrial metabolism-oxidative phosphorylation (OXPHOS). DANCR binds to LIPG, enabling tumor cells to maintain LIPG protein stability and OXPHOS. As one mechanism of LIPG in the regulation of tumor cell oxidative metabolism, LIPG mediates histone deacetylase 6 (HDAC6) and histone acetylation, which contribute to changes in IL-6 and fatty acid synthesis gene expression. Finally, aided by a relaxed docking approach, we discovered a new LIPG inhibitor, cynaroside, that effectively suppressed the enzyme activity and DANCR in TNBC cells. Treatment with cynaroside inhibited the OXPHOS phenotype of TNBC cells, which severely impaired tumor formation. Taken together, our study provides mechanistic insights into the LIPG modulation of mitochondrial metabolism in TNBC and a proof-of-concept that targeting LIPG is a promising new therapeutic strategy for the treatment of TNBC.
Title: Efficient Improvement of Surface Displayed Lipase from Rhizomucor miehei in PichiaPink(TM) Protease-deficient System Li Z, Miao Y, Yang J, Zhao F, Lin Y, Han S Ref: Protein Expr Purif, :105804, 2020 : PubMed
Lipase from Rhizomucor miehei (RML) is a versatile biocatalyst used in food industry, fine chemicals, and biodiesel production. Yeast surface display allows direct application of lipase in form of whole-cell biocatalyst, avoiding purification and immobilization process, but the protease of the host cell may affect the activity of displayed lipase. Herein, we used the protease-deficient Pichia pastoris, PichiaPink(TM) to efficiently display RML. RML gene, GCW21 gene and alpha-factor gene were co-cloned into plasmid pPink LC/HC and transformed into protease-deficient P. pastoris. After inducible expression for 96 h, the lipase activity of displayed RML reached 121.72 U/g in proteinase-A-deficient P. pastoris harboring high-copy plasmid, which exhibited 46.7% higher than recombinant P. pastoris without protease defect. Displayed RML occurred the maximum activity at pH 8.0 and 45 degC and the optimal substrate was p-nitrophenyl octanoate. Metal ions Li(+), Na(+), K(+), and Mg(2+) of 1-10 mM had activation towards displayed RML. Displayed RML was effectively improved in PichiaPink(TM) protease-deficient system, which may promote the further research and development of the industrial application of RML.
Lysosomal acid lipase (LAL) is a serine hydrolase which hydrolyzes cholesteryl ester and triglycerides delivered to the lysosomes into free cholesterol and fatty acids. LAL deficiency due to mutations in the LAL gene (LIPA) results in accumulation of triglycerides and cholesterol esters in various tissues of the body leading to pathological conditions such as Wolman's disease (WD) and Cholesteryl ester storage disease (CESD). Here we present the first crystal structure of recombinant human LAL to 2.6 A resolution in its closed form. The crystal structure was enabled by mutating three of the six potential glycosylation sites. The overall structure of human LAL (HLAL) closely resembles that of the evolutionarily related human gastric lipase (HGL). It consists of a core domain belonging to the classical alpha/beta hydrolase-fold family with a classical catalytic triad (Ser-153, His-353, Asp-324), an oxyanion hole and a "cap" domain, which regulates substrate entry to the catalytic site. Most significant structural differences between HLAL and HGL exist at the lid region. Deletion of the short helix, 238NLCFLLC244, at the lid region implied a possible role in regulating the highly hydrophobic substrate binding site from self-oligomerization during interfacial activation. We also performed molecular dynamic simulations of dog gastric lipase (DGL), lid open form and HLAL to gain insights and speculated a possible role of the human mutant, H274Y, leading to CESD.
Title: Expression of ADAM29 and FAM135B in the pathological evolution from normal esophageal epithelium to esophageal cancer: Their differences and clinical significance Wang T, Lv X, Jiang S, Han S, Wang Y Ref: Oncol Lett, 19:1727, 2020 : PubMed
A Disintegrin And Metalloprotease Domain 29 (ADAM29) and Family with sequence similarity 135 member B (FAM135B) genes have been reported to be associated with a carcinogenic risk of esophageal squamous cell carcinoma (ESCC). However, to the best of our knowledge, the expression of ADAM29 and FAM135B in the pathological evolution from normal esophageal epithelial cells to ESCC has not yet been investigated. The present study aimed to investigate the expression of ADAM29 and FAM135B in normal esophageal mucosal epithelium, low-grade and high-grade esophageal intraepithelial neoplasia, and ESCC. Furthermore, the present study aimed to investigate the role of ADAM29 and FAM135B in the development of esophageal lesions. Immunohistochemistry was performed in order to detect the expression levels of ADAM29 and FAM135B proteins in normal esophageal mucosa samples (40 cases), low-grade intraepithelial neoplasia samples (20 cases), high-grade intraepithelial neoplasia samples (20 cases) and ESCC samples (40 cases). The results of the present study demonstrated that the positive rates of ADAM29 and FAM135B proteins increased gradually from normal esophageal mucosal epithelium and esophageal intraepithelial neoplasia, to ESCC (P<0.05). Furthermore, the expression levels of ADAM29 and FAM135B proteins in ESCC were not associated with age and the tumor size (P>0.05); however, the protein levels were associated with the pathological stage, clinical stage and lymph node metastasis of ESCC (P<0.05). In addition, there was a significant association between the expression levels of ADAM29 protein and FAM135B protein ((2)=60.071; P<0.001). The results of the present study demonstrated that the expression levels of ADAM29 and FAM135B were associated with the tumor behavior characteristics and the progression of esophageal cancer, the expression of which could be used for the diagnosis of early esophageal cancer, and provide the basis for guiding individualized treatment.
Title: Cell Surface Display of Thermomyces lanuginosus Lipase in Pichia pastoris Yang J, Huang K, Xu X, Miao Y, Lin Y, Han S Ref: Front Bioeng Biotechnol, 8:544058, 2020 : PubMed
A cell surface displayed system in Pichia pastoris GS115 was developed by using GCW61, a glycosylphosphatidylinositol-modified cell wall protein from P. pastoris, as the anchor protein. Thermomyces lanuginosus lipase (TLL) was successfully displayed on the P. pastoris cell wall by fusing GCW61 gene with TLL2 gene (NCBI Accession: O59952) that was optimized with codon bias and synthesized. Cell surface displayed TLL2 was confirmed by the immunofluorescence microscopy. Flask fermentation was performed for 144 h with lipase activity up to 1964.76 U/g. Enzymatic properties of cell surface displayed TLL2 were also investigated. Displayed TLL2 occurred the maximum activity at pH 9 and 55 degC and demonstrated characteristics of wide thermal adaptability and alkaline pH resistance. The optimum substrate was p-nitrophenyl hexanoate. Bivalent metal ions Ca(2+), Mn(2+), and Zn(2+) had the activation effect on displayed TLL2, while Cu(2+), Fe(2+), Fe(3+), K(+), Li(+), Na(+), and Co(2+) ions had the inhibitory effect on it. Since cell surface displayed TLL2 required less purification steps compared with free enzyme and showed high enzyme activities, it would be able to be further applied in various potential applications.
Title: Elevated Human Dipeptidyl Peptidase 4 Expression Reduces the Susceptibility of hDPP4 Transgenic Mice to Middle East Respiratory Syndrome Coronavirus Infection and Disease Algaissi A, Agrawal AS, Han S, Peng BH, Luo C, Li F, Chan TS, Couch RB, Tseng CK Ref: J Infect Dis, 219:829, 2019 : PubMed
BACKGROUND: The ongoing Middle East respiratory syndrome coronavirus (MERS-CoV) infections pose threats to public health worldwide, making an understanding of MERS pathogenesis and development of effective medical countermeasures (MCMs) urgent. METHODS: We used homozygous (+/+) and heterozygous (+/-) human dipeptidyl peptidase 4 (hDPP4) transgenic mice to study the effect of hDPP4 on MERS-CoV infection. Specifically, we determined values of 50% lethal dose (LD50) of MERS-CoV for the 2 strains of mice, compared and correlated their levels of soluble (s)hDPP4 expression to susceptibility, and explored recombinant (r)shDPP4 as an effective MCM for MERS infection. RESULTS: hDPP4+/+ mice were unexpectedly more resistant than hDPP4+/- mice to MERS-CoV infection, as judged by increased LD50, reduced lung viral infection, attenuated morbidity and mortality, and reduced histopathology. Additionally, the resistance to MERS-CoV infection directly correlated with increased serum shDPP4 and serum virus neutralizing activity. Finally, administration of rshDPP4 led to reduced lung virus titer and histopathology. CONCLUSIONS: Our studies suggest that the serum shDPP4 levels play a role in MERS pathogenesis and demonstrate a potential of rshDPP4 as a treatment option for MERS. Additionally, it offers a validated pair of Tg mice strains for characterizing the effect of shDPP4 on MERS pathogenesis.
Title: Kinetic resolution of sec-alcohols catalysed by Candida antarctica lipase B displaying Pichia pastoris whole-cell biocatalyst Zhang K, Pan Z, Diao Z, Liang S, Han S, Zheng S, Lin Y Ref: Enzyme Microb Technol, 110:8, 2018 : PubMed
Kinetic resolution of sec-alcohols is a green process with biocatalyst. Candida antarctica lipase B (CALB) displayed on Pichia pastoris cell-surface (Pp-CALB) was characterized in kinetic resolution of sec-alcohols with different structures. The reaction parameters including acyl donors, molar ratio of substrates, solvents and temperatures were examined with 2-octanol as model substrate. 47.4% molar conversion of 2-octanol and 99.7% eep were obtained after a 5h reaction with Pp-CALB, and 90% of its original activity still remained after being reused for 10 cycles. Pp-CALB was then used to several sec-alcohols and it showed great enzymatic activity and enantioselectivity to all tested sec-alcohols, more than 93.1% of eep. The enantioselective characteristics of Pp-CALB catalysed sec-alcohols with different structures were compared with Novozyme 435 which was almost the same. Solvent free system as one way of green chemistry was applied to Pp-CALB and Pp-CALB showed great catalytic activity and enantioselectivity. Pp-CALB was potential biocatalyst of high enzymatic activity and enantioselectivity using in resolution of sec-alcohols.
Title: The bacterial quorum-sensing molecule, N-3-oxo-dodecanoyl-L-homoserine lactone, inhibits mediator release and chemotaxis of murine mast cells Khambati I, Han S, Pijnenburg D, Jang H, Forsythe P Ref: Inflamm Res, 66:259, 2017 : PubMed
OBJECTIVE: Bacterial colonization relies on communication between bacteria via so-called "quorum-sensing molecules", which include the acyl-homoserine lactone group. Certain acyl-homoserine lactones can modulate mammalian cell function and are thought to contribute to bacterial pathogenicity. Given the role of mast cells in host defense, we investigated the ability of acyl-homoserine lactones to modulate mast cell function. METHODS: We utilized murine primary mast cell cultures to assess the effect of acyl-homoserine lactones on degranulation and cytokine release in response to different stimuli. We also assessed cell migration in response to chemoattractants. The effect of acyl-homoserine lactones in vivo was tested using a passive cutaneous anaphylaxis model. RESULTS: Two of the tested quorum-sensing molecules, N-3-oxo-dodecanoyl-L-homoserine lactone and N-Dodecanoyl-L-homoserine lactone, inhibited IgE dependent and independent degranulation and mediator release from primary mast cells. Further testing of N-3-oxo-dodecanoyl-L-homoserine lactone, the most potent inhibitor and a product of Pseudomonas aeruginosa, revealed that it also attenuated chemotaxis and LPS induced cytokine production. In vivo, N-3-oxo-dodecanoyl-L-homoserine lactone inhibited the passive cutaneous anaphylaxis response in mice. CONCLUSION: The ability of N-3-oxo-dodecanoyl-L-homoserine lactone to stabilize mast cells may contribute to the pathogenicity of P. aeruginosa but could potentially be exploited therapeutically in allergic disease.
BACKGROUND: The 'Asia-Pacific Expert Panel (APEX) for donepezil 23 mg' met in November 2015 to review evidence for the recently approved high dose of donepezil and to provide recommendations to help physicians in Asia make informed clinical decisions about using donepezil 23 mg in patients with moderate-to-severe Alzheimer's disease (AD). SUMMARY: In a global phase III study (study 326) in patients with moderate-to-severe AD, donepezil 23 mg/day demonstrated significantly greater cognitive benefits versus donepezil 10 mg/day, with a between-treatment difference in mean change in the Severe Impairment Battery score of 2.2 points (p < 0.001) in the overall population and 3.1 points (p < 0.001) in patients with advanced AD. A subanalysis of study 326 demonstrated that the benefits and risks associated with donepezil 23 mg/day versus donepezil 10 mg/day in Asian patients with moderate-to-severe AD were comparable to those in the global study population. KEY MESSAGE: Donepezil 23 mg is a valuable treatment for patients with AD, particularly those with advanced disease. The APEX emphasized the importance of patient selection (AD severity, tolerability of lower doses of donepezil, and absence of contraindications), a stepwise titration strategy for dose escalation, and appropriate monitoring and counseling of patients and caregivers in the management of patients with AD.
Title: Display of fungal hydrophobin on the Pichia pastoris cell surface and its influence on Candida antarctica lipase B Wang P, He J, Sun Y, Reynolds M, Zhang L, Han S, Liang S, Sui H, Lin Y Ref: Applied Microbiology & Biotechnology, 100:5883, 2016 : PubMed
To modify the Pichia pastoris cell surface, two classes of hydrophobins, SC3 from Schizophyllum commune and HFBI from Trichoderma reesei, were separately displayed on the cell wall. There was an observable increase in the hydrophobicity of recombinant strains. Candida antarctica lipase B (CALB) was then co-displayed on the modified cells, generating strains GS115/SC3-61/CALB-51 and GS115/HFBI-61/CALB-51. Interestingly, the hydrolytic and synthetic activities of strain GS115/HFBI-61/CALB-51 increased by 37 and 109 %, respectively, but decreased by 26 and 43 %, respectively, in strain GS115/SC3-61/CALB-51 compared with the hydrophobin-minus recombinant strain GS115/CALB-GCW51. The amount of glycerol by-product from the transesterification reaction adsorbed on the cell surface was significantly decreased following hydrophobin modification, removing the glycerol barrier and allowing substrates to access the active sites of lipases. Electron micrographs indicated that the cell wall structures of both recombinant strains appeared altered, including changes to the inner glucan layer and outer mannan layer. These results suggest that the display of hydrophobins can change the surface structure and hydrophobic properties of P. pastoris and affect the catalytic activities of CALB displayed on the surface of P. pastoris cells.
ABT-089, an alpha4beta2 neuronal nicotinic receptor partial agonist, was evaluated for efficacy and safety in mild to moderate Alzheimer disease patients receiving stable doses of acetylcholinesterase inhibitors. This phase 2 double-blind, placebo-controlled, proof-of-concept, and dose-finding study adaptively randomized patients to receive ABT-089 (5, 10, 15, 20, 30, or 35 mg once daily) or placebo for 12 weeks. The primary efficacy endpoint was the Alzheimer's Disease Assessment Scale, cognition subscale (ADAS-Cog) total score. A Bayesian response-adaptive randomization algorithm dynamically assigned allocation probabilities based on interim ADAS-Cog total scores. A normal dynamic linear model for dose-response relationships and a longitudinal model for predicting final ADAS-cog score were employed in the algorithm. Stopping criteria for futility or success were defined. The futility stopping criterion was met, terminating the study with 337 patients randomized. No dose-response relationship was observed and no dose demonstrated statistically significant improvement over placebo on ADAS-Cog or any secondary endpoint. ABT-089 was well tolerated at all dose levels. When administered as adjunctive therapy to acetylcholinesterase inhibitors, ABT-089 was not efficacious in mild to moderate Alzheimer disease. The adaptive study design enabled the examination of a broad dose range, enabled rapid determination of futility, and reduced patient exposure to nonefficacious doses of the investigational compound.
Lysosomal acid lipase (LAL) is a serine hydrolase which hydrolyzes cholesteryl ester and triglycerides delivered to the lysosomes into free cholesterol and free fatty acids. Mutations in the LAL gene (LIPA) result in accumulation of triglycerides and cholesterol esters in various tissues of the body, leading to pathological conditions such as Wolman's disease (WD) and cholesteryl ester storage disease (CESD). CESD patients homozygous for His295Tyr (H295Y) mutation have less than 5% of normal LAL activity. To shed light on the molecular basis for this loss-of-function phenotype, we have generated the recombinant H295Y enzyme and studied its biophysical and biochemical properties. No significant differences were observed in the expression levels or glycosylation patterns between the mutant and the wild type LAL. However, the H295Y mutant displayed only residual enzymatic activity (<5%) compared to the wild type. While wild type LAL is mostly a monomer at pH 5.0, the vast majority H295Y exists as a high molecular soluble aggregate. Besides, the H295Y mutant has a 20 degrees C lower melting temperature compared to the wild type. Transient expression studies in WD fibroblasts showed that mutation of His295 to other amino acids resulted in a significant loss of enzymatic activity. A homology model of LAL revealed that His295 is located on an alpha-helix of the cap domain and could be important for tethering it to its core domain. The observed loss-of-function phenotype in CESD patients might arise from a combination of protein destabilization and the shift to a non-functional soluble aggregate.
Protein phosphatase methylesterase 1 (PPME1) is a protein phosphatase 2A (PP2A)-specific methyl esterase that negatively regulates PP2A through demethylation at its carboxy terminal leucine 309 residue. Emerging evidence shows that the upregulation of PPME1 is associated with poor prognosis in glioblastoma patients. By performing an array comparative genomic hybridization analysis to detect copy number changes, we have been the first to identify PPME1 gene amplification in 3.8% (5/131) of Chinese gastric cancer (GC) samples and 3.1% (4/124) of Chinese lung cancer (LC) samples. This PPME1 gene amplification was confirmed by fluorescence in situ hybridization analysis and is correlated with elevated protein expression, as determined by immunohistochemistry analysis. To further investigate the role of PPME1 amplification in tumor growth, short-hairpin RNA-mediated gene silencing was employed. A knockdown of PPME1 expression resulted in a significant inhibition of cell proliferation and induction of cell apoptosis in PPME1-amplified human cancer cell lines SNU668 (GC) and Oka-C1 (LC), but not in nonamplified MKN1 (GC) and HCC95 (LC) cells. The PPME1 gene knockdown also led to a consistent decrease in PP2A demethylation at leucine 309, which was correlated with the downregulation of cellular Erk and AKT phosphorylation. Our data indicate that PPME1 could be an attractive therapeutic target for a subset of GCs and LCs.
Neuronal nicotinic acetylcholine receptor (nAChR) genes (CHRNA5/CHRNA3/CHRNB4) have been reproducibly associated with nicotine dependence, smoking behaviors, and lung cancer risk. Of the few reports that have focused on early smoking behaviors, association results have been mixed. This meta-analysis examines early smoking phenotypes and SNPs in the gene cluster to determine: (1) whether the most robust association signal in this region (rs16969968) for other smoking behaviors is also associated with early behaviors, and/or (2) if additional statistically independent signals are important in early smoking. We focused on two phenotypes: age of tobacco initiation (AOI) and age of first regular tobacco use (AOS). This study included 56,034 subjects (41 groups) spanning nine countries and evaluated five SNPs including rs1948, rs16969968, rs578776, rs588765, and rs684513. Each dataset was analyzed using a centrally generated script. Meta-analyses were conducted from summary statistics. AOS yielded significant associations with SNPs rs578776 (beta = 0.02, P = 0.004), rs1948 (beta = 0.023, P = 0.018), and rs684513 (beta = 0.032, P = 0.017), indicating protective effects. There were no significant associations for the AOI phenotype. Importantly, rs16969968, the most replicated signal in this region for nicotine dependence, cigarettes per day, and cotinine levels, was not associated with AOI (P = 0.59) or AOS (P = 0.92). These results provide important insight into the complexity of smoking behavior phenotypes, and suggest that association signals in the CHRNA5/A3/B4 gene cluster affecting early smoking behaviors may be different from those affecting the mature nicotine dependence phenotype.
Title: Effects of Di-n-butyl Phthalate and Diethyl Phthalate on Acetylcholinesterase Activity and Neurotoxicity Related Gene Expression in Embryonic Zebrafish Xu H, Shao X, Zhang Z, Zou Y, Chen Y, Han S, Wang S, Wu X, Yang L, Chen Z Ref: Bulletin of Environmental Contamination & Toxicology, 91:635, 2013 : PubMed
In the present study, zebrafish embryos were used to assess the neurotoxicity of di-n-butyl phthalate (DBP), diethyl phthalate (DEP) and their mixture. Four-hour post-fertilization (hpf) zebrafish embryos were exposed to various concentrations of DBP, DEP and their mixture (DBP-DEP) until 96 hpf. The transcriptions levels of selected neuron-related genes reported as neurotoxicity biomarkers were analyzed. The results showed that transcripts of growth associated protein 43 (gap43), embryonic lethal abnormal vision-like 3 (elavl3), glial fibrillary acidic protein (gfap), myelin basic protein (mbp), alpha1-tubulin and neurogenin1 (ngn1) were significantly up-regulated after DBP, DEP and DBP-DEP mixture exposure. In addition, acetylcholinesterase activity was significantly inhibited in the embryos. These results indicate that DBP and DEP have the potential neurotoxicity in zebrafish embryos.
Title: Mixed-effect circadian rhythm model for human erythrocyte acetylcholinesterase activity--application to the proof of concept of cholinesterase inhibition by acorn extract in healthy subjects with galantamine as positive control Han S, Lee J, Jeon S, Hong T, Yim DS Ref: European Journal of Clinical Pharmacology, 68:599, 2012 : PubMed
PURPOSE: The aim of this study was to develop a non-linear mixed effect circadian rhythm model of acetylcholinesterase (AChE) activity variation and to evaluate the inhibitory effect of acorn extract (2 g) and galantamine (16 mg), used as positive control, on human AChE in red blood cells (RBC). METHODS: This was an open-label, randomized, three-way crossover study involving 12 healthy subjects who received one of the treatments in each study period: no treatment, acorn extract, and galantamine. RBC AChE activity was measured in peripheral blood samples collected at 0 (pre-dose), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16 and 24 h post-dose administration. Non-linear mixed effect modeling was performed using NONMEM (ver. 7.0). RESULTS: The circadian variation of AChE activity was best described using two mixed effect cosine functions, with periods of 24 and 12 h, respectively. When the inhibitory effect terms were added, the model was significantly improved for both acorn extract and galantamine. In terms of the effect, a 2-g single dose of acorn extract showed AChE inhibition (about 5%) similar to that of a 16-mg single dose of galantamine, in the first 24 h after administration. CONCLUSIONS: Based on the very pronounced inter- and intra-day variation in AChE activity in RBC, we conclude that the model-based approach is essential for the proof of concept and quantitation of AChE inhibition in human subjects.
CONTEXT: Recent studies have shown an association between cigarettes per day (CPD) and a nonsynonymous single-nucleotide polymorphism in CHRNA5, rs16969968. OBJECTIVE: To determine whether the association between rs16969968 and smoking is modified by age at onset of regular smoking. DATA SOURCES: Primary data. STUDY SELECTION: Available genetic studies containing measures of CPD and the genotype of rs16969968 or its proxy. DATA EXTRACTION: Uniform statistical analysis scripts were run locally. Starting with 94,050 ever-smokers from 43 studies, we extracted the heavy smokers (CPD >20) and light smokers (CPD </=10) with age-at-onset information, reducing the sample size to 33,348. Each study was stratified into early-onset smokers (age at onset </=16 years) and late-onset smokers (age at onset >16 years), and a logistic regression of heavy vs light smoking with the rs16969968 genotype was computed for each stratum. Meta-analysis was performed within each age-at-onset stratum. DATA SYNTHESIS: Individuals with 1 risk allele at rs16969968 who were early-onset smokers were significantly more likely to be heavy smokers in adulthood (odds ratio [OR] = 1.45; 95% CI, 1.36-1.55; n = 13,843) than were carriers of the risk allele who were late-onset smokers (OR = 1.27; 95% CI, 1.21-1.33, n = 19,505) (P = .01). CONCLUSION: These results highlight an increased genetic vulnerability to smoking in early-onset smokers.
Marinomonas posidonica IVIA-Po-181(T) Lucas-Elio et al. 2011 belongs to the family Oceanospirillaceae within the phylum Proteobacteria. Different species of the genus Marinomonas can be readily isolated from the seagrass Posidonia oceanica. M. posidonica is among the most abundant species of the genus detected in the cultured microbiota of P. oceanica, suggesting a close relationship with this plant, which has a great ecological value in the Mediterranean Sea, covering an estimated surface of 38,000 Km(2). Here we describe the genomic features of M. posidonica. The 3,899,940 bp long genome harbors 3,544 protein-coding genes and 107 RNA genes and is a part of the GenomicEncyclopedia ofBacteriaandArchaea project.
Title: Genome sequence of Corynebacterium glutamicum ATCC 14067, which provides insight into amino acid biosynthesis in coryneform bacteria Lv Y, Liao J, Wu Z, Han S, Lin Y, Zheng S Ref: Journal of Bacteriology, 194:742, 2012 : PubMed
We report the genome sequence of Corynebacterium glutamicum ATCC 14067 (once named Brevibacterium flavum), which is useful for taxonomy research and further molecular breeding in amino acid production. Preliminary comparison with those of the reported coryneform strains revealed some notable differences that might be related to the difficulties in molecular manipulation.
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.
Here we describe the genome of Mesotoga prima MesG1.Ag4.2, the first genome of a mesophilic Thermotogales bacterium. Mesotoga prima was isolated from a polychlorinated biphenyl (PCB)-dechlorinating enrichment culture from Baltimore Harbor sediments. Its 2.97 Mb genome is considerably larger than any previously sequenced Thermotogales genomes, which range between 1.86 and 2.30 Mb. This larger size is due to both higher numbers of protein-coding genes and larger intergenic regions. In particular, the M. prima genome contains more genes for proteins involved in regulatory functions, for instance those involved in regulation of transcription. Together with its closest relative, Kosmotoga olearia, it also encodes different types of proteins involved in environmental and cell-cell interactions as compared with other Thermotogales bacteria. Amino acid composition analysis of M. prima proteins implies that this lineage has inhabited low-temperature environments for a long time. A large fraction of the M. prima genome has been acquired by lateral gene transfer (LGT): a DarkHorse analysis suggests that 766 (32%) of predicted protein-coding genes have been involved in LGT after Mesotoga diverged from the other Thermotogales lineages. A notable example of a lineage-specific LGT event is a reductive dehalogenase gene-a key enzyme in dehalorespiration, indicating M. prima may have a more active role in PCB dechlorination than was previously assumed.
The genus Caldicellulosiruptor contains the most thermophilic, plant biomass-degrading bacteria isolated to date. Previously, genome sequences from three cellulolytic members of this genus were reported (C. saccharolyticus, C. bescii, and C. obsidiansis). To further explore the physiological and biochemical basis for polysaccharide degradation within this genus, five additional genomes were sequenced: C. hydrothermalis, C. kristjanssonii, C. kronotskyensis, C. lactoaceticus, and C. owensensis. Taken together, the seven completed and one draft-phase Caldicellulosiruptor genomes suggest that, while central metabolism is highly conserved, significant differences in glycoside hydrolase inventories and numbers of carbohydrate transporters exist, a finding which likely relates to variability observed in plant biomass degradation capacity.
Nocardioides sp. strain JS614 grows on ethene and vinyl chloride (VC) as sole carbon and energy sources and is of interest for bioremediation and biocatalysis. Sequencing of the complete genome of JS614 provides insight into the genetic basis of alkene oxidation, supports ongoing research into the physiology and biochemistry of growth on ethene and VC, and provides biomarkers to facilitate detection of VC/ethene oxidizers in the environment. This is the first genome sequence from the genus Nocardioides and the first genome of a VC/ethene-oxidizing bacterium.
"Pedosphaera parvula" Ellin514 is an aerobically grown verrucomicrobial isolate from pasture soil. It is one of the few cultured representatives of subdivision 3 of the phylum Verrucomicrobia. Members of this group are widespread in terrestrial environments.
Title: Pharmacogenetic Regulation of Acetylcholinesterase Activity in Drosophila Reveals the Regulatory Mechanisms of AChE Inhibitors in Synaptic Plasticity Kim W, Lee D, Choi J, Kim A, Han S, Park K, Kim J, Choi Y, Lee SH, Koh YH Ref: Neurochem Res, 36:879, 2011 : PubMed
We conducted experiments in Drosophila to investigate the consequences of altered acetylcholinesterase (AChE) activity in the nervous system. In ace hypomorphic mutant larvae, the amount of ace mRNA and the activity of AChE both in vivo and in vitro were significantly reduced compared with those of controls. Reduced Ace in Drosophila larvae resulted in significant down-regulation of branch length and the number of boutons in Type 1 glutamatergic neuromuscular junctions (NMJs). These defects in ace hypomorphic mutant larvae were suppressed when Musca domestica AChE was transgenically expressed. Because AChE inhibitors are utilized for medications for Alzheimer's disease, we investigated whether pharmacological inhibition of AChE activity induced any synaptic defects. We found that controls exposed to a sublethal dose of DDVP phenocopied the synaptic structural defects of the ace hypomorphic mutant. These results suggest that down-regulation of AChE activity, regardless of whether it is due to genetic or pharmacological manipulations, results in altered synaptic architecture. Our study suggests that exposure to AChE inhibitors for 6-12 months may induce altered synaptic architectures in human brains with Alzheimer's diseases, similar to those reported here. These changes may underlie or contribute to the loss of efficacy of AChE inhibitors after prolonged treatment.
Title: Biochemical characterization of digestive enzymes in the black soldier fly, Hermetia illucens (Diptera: Stratiomyidae) Kim W, Bae S, Park K, Lee S, Choi Y, Han S, Koh Youngho Ref: Journal of Asia-Pacific Entomology, 14:11, 2011 : PubMed
The black soldier fly, Hermetia illucens, is beneficial because its larvae feed on organic materials derived from plants, animals and humans and promote the recycling of food waste and organic materials. We investigated the biochemical properties of digestive enzymes released from the salivary gland and gut of the black soldier fly. Because the gut extracts of the black soldier fly larvae had high amylase, lipase and protease activities, we suggested that the black soldier fly might belong to the polyphagous insect group. In addition, a strong trypsin-like protease activity was observed in the gut extracts of the black soldier fly larvae. Higher activities of leucine arylamidase, +/--galactosidase, beta-galactosidase, +/--mannosidase and +/--fucosidase were observed from the gut extracts of the black soldier fly larvae compared with those of house fly larvae. These findings may explain previous reports that the black soldier fly larvae can digest food wastes and organic materials more efficiently than any other known species of fly.
Glaciecola sp. strain 4H-3-7+YE-5 was isolated from subseafloor sediments at Suruga Bay in Japan and is capable of efficiently hydrolyzing cellulose and xylan. The complete genome sequence of Glaciecola sp. 4H-3-7+YE-5 revealed several genes encoding putatively novel glycoside hydrolases, offering a high potential for plant biomass degradation.
Two members of the family Flavobacteriaceae were isolated from subseafloor sediments using artificial seawater with cellulose, xylan, and chitin as the sole carbon and energy sources. Here, we present the complete genome sequences of Krokinobacter sp. strain 4H-3-7-5 and Lacinutrix sp. strain 5H-3-7-4, which both encode putatively novel enzymes involved in cellulose, hemicellulose, and chitin metabolism.
Title: Genome sequence of Corynebacterium glutamicum S9114, a strain for industrial production of glutamate Lv Y, Wu Z, Han S, Lin Y, Zheng S Ref: Journal of Bacteriology, 193:6096, 2011 : PubMed
Here we report the genome sequence of Corynebacterium glutamicum S9114, an industrial producer widely used in production of glutamate in China. Preliminary comparison with the sequences of the Corynebacterium glutamicum strains ATCC 13032 and R revealed some notable mutagenesis that might be related to the high yield of glutamate.
Parvibaculum lavamentivorans DS-1(T) is the type species of the novel genus Parvibaculum in the novel family Rhodobiaceae (formerly Phyllobacteriaceae) of the order Rhizobiales of Alphaproteobacteria. Strain DS-1(T) is a non-pigmented, aerobic, heterotrophic bacterium and represents the first tier member of environmentally important bacterial communities that catalyze the complete degradation of synthetic laundry surfactants. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 3,914,745 bp long genome with its predicted 3,654 protein coding genes is the first completed genome sequence of the genus Parvibaculum, and the first genome sequence of a representative of the family Rhodobiaceae.
Methylocystis sp. strain Rockwell (ATCC 49242) is an aerobic methane-oxidizing alphaproteobacterium isolated from an aquifer in southern California. Unlike most methanotrophs in the Methylocystaceae family, this strain has a single pmo operon encoding particulate methane monooxygenase but no evidence of the genes encoding soluble methane monooxygenase. This is the first reported genome sequence of a member of the Methylocystis species of the Methylocystaceae family in the order Rhizobiales.
Ketogulonicigenium vulgare is characterized by the efficient production of 2KGA from L-sorbose. Ketogulonicigenium vulgare Y25 is known as a 2-keto-L-gulonic acid-producing strain in the vitamin C industry. Here we report the finished, annotated genome sequence of Ketogulonicigenium vulgare Y25.
Victivallis vadensis ATCC BAA-548 represents the first cultured representative from the novel phylum Lentisphaerae, a deep-branching bacterial lineage. Few cultured bacteria from this phylum are known, and V. vadensis therefore represents an important organism for evolutionary studies. V. vadensis is a strictly anaerobic sugar-fermenting isolate from the human gastrointestinal tract.
Title: Whole genome sequences of two Xylella fastidiosa strains (M12 and M23) causing almond leaf scorch disease in California Chen J, Xie G, Han S, Chertkov O, Sims D, Civerolo EL Ref: Journal of Bacteriology, 192:4534, 2010 : PubMed
Xylella fastidiosa is a Gram-negative plant-pathogenic bacterium causing many economically important diseases, including almond leaf scorch disease (ALSD) in California. Genome information greatly facilitates research on this nutritionally fastidious organism. Here we report the complete genome sequences of two ALSD strains of this bacterium, M12 and M23.
Methylosinus trichosporium OB3b (for "oddball" strain 3b) is an obligate aerobic methane-oxidizing alphaproteobacterium that was originally isolated in 1970 by Roger Whittenbury and colleagues. This strain has since been used extensively to elucidate the structure and function of several key enzymes of methane oxidation, including both particulate and soluble methane monooxygenase (sMMO) and the extracellular copper chelator methanobactin. In particular, the catalytic properties of soluble methane monooxygenase from M. trichosporium OB3b have been well characterized in context with biodegradation of recalcitrant hydrocarbons, such as trichloroethylene. The sequence of the M. trichosporium OB3b genome is the first reported from a member of the Methylocystaceae family in the order Rhizobiales.
Title: [Expression of Candida antarctica lipase B on yeast surface and synthesis of ethyl hexanoate catalyzed by CALB] Pan Z, Han S, Lin Y, Zheng S Ref: Sheng Wu Gong Cheng Xue Bao, 24:673, 2008 : PubMed
Short-chain esters play a significant role in the food industry as flavor and aroma constituents. Candida antarctica lipase B (CALB) is one of the most effective catalysts for organic synthesis. We constructed a CALB-displaying yeast whole-cell biocatalyst and applied it to esterification from caproic acid and ethanol. CALB was fused with the alpha-agglutinin C-terminal and the signal peptide of Glucoamylase in pICAS, a yeast surface display vector, to construct plasmid pICAS-CALB. An extremely Asn-rich linker, named celAL was inserted in the Xho I of pICAS-CALB to construct plasmid pICAS-celAL-CALB. The fused gene was under the control of GAPDH promoter. After incubated at 30 degrees C for 96 h the lipase hydrolytic activity of the yeast whole cells reached a plateau, 26.26 u/(g x dry cell). In nonaqeous media, the yield of 98.0% ethyl hexanoate was obtained after 24 h esterification from caproic acid and ethanol (the molar ratio of caproic acid : ethanol = 1 : 1.25) using lyophilized CALB displaying yeast whole cells.
Title: Differentiation of human neuroblastoma by phenylacetate is mediated by peroxisome proliferator-activated receptor gamma Han S, Wada RK, Sidell N Ref: Cancer Research, 61:3998, 2001 : PubMed
Phenylacetate (PA) is a member of a class of aromatic fatty acids that has demonstrated antitumor activity in experimental models and in humans. Previous reports have shown that PA and its analogues can act as ligands for the peroxisome proliferator-activated receptor (PPAR) and thereby regulate certain gene expression through peroxisome proliferator response elements. The role of this activity in the antitumor activity of PA has not been determined. To address this question, we have used the human neuroblastoma cell line LA-N-5, which expresses PPARgamma and can be induced to differentiate with PA and with classical PPARgamma ligands. Our results indicated that the PPARgamma ligands 15-deoxy- prostaglandin J2 and GW1929 as well as PA induced LA-N-5 cells to differentiate to a similar phenotype as evidenced by inhibition of cell proliferation, neurite outgrowth, increased acetylcholinesterase activity, and decreased N-myc gene expression. Furthermore, induction with all of the compounds was accompanied by up-regulation of mRNA levels of the nuclear retinoic acid receptor beta (RARbeta) and specific activation of a reporter gene construct (SVbetaRE-CAT) that contains the canonical RA response element located in the RARbeta promoter. All of the assessed functional and molecular effects of PA on LA-N-5 cells, as well as those of the classical PPARgamma ligands, were inhibited by cotreatment with specific PPARgamma antagonists (GW9662 and/or GW0072). Taken together, these studies have confirmed a role for PPARgamma in neuroblastoma cell biology and indicated that the PPARgamma signaling pathway plays a direct role in the PA-induced differentiation response of this cell type.
Title: Age Variation in Insecticide Susceptibility and Biochemical Changes of Beet Armyworm, Spodoptera exigua (Hubner) Kim Y, Lee J, Kang S, Han S Ref: Journal of Asia-Pacific Entomology, 1:109, 1998 : PubMed
The susceptibility of Spodoptera exigua (Hubner) to bifenthrin and chlorpyrifosmethyl in relation to larval development was investigated. Increased tolerances to these insecticides were associated with increasing larval instars. The insecticide tolerance increased linealy with larval body weights in bifenthrin but did exponentially in chlorpyrifos-methyl. Esterase (EST), acetylcholinesterase (AChE), and glutathione S-transferase(GST) of different larval ages were analyzed to elucidate variation of insecticide susceptibilities according to larval development within a population. Total EST activity increased linearly with body weights though the specific activities were not varied among ages. Both total and specific activity changes of GST, however, surpassed the rates of body weight gains as the larvae developed. AChE activities decreased significantly with larval development. This change of AChE activities was due to the developmental change of its catalytic function. The fifth instar larvae had the lowest catalytic capacity: the highest Km(187.39muM) and the lowest Vmax (0.58 nM/min/mug). Therefore, different insecticide tolerance of S. exigua according to larval ages can be explained by both enhanced detoxification enzymes and altered AchE.
