The underlying causes of Alzheimer's disease (AD) remain a mystery, with multiple pathological components, including oxidative stress, acetylcholinesterase, amyloid-beta, and metal ions, all playing a role. Here we report a strategic approach to designing flavonoids that can effectively tackle multiple pathological elements involved in AD. Our systematic investigations revealed key structural features for flavonoids to simultaneously target and regulate pathogenic targets. Our findings led to the development of a highly promising flavonoid that exhibits a range of functions, based on a complete structure-activity relationship analysis. Furthermore, our mechanistic studies confirmed that this flavonoid's versatile reactivities are driven by its redox potential and direct interactions with pathogenic factors. This work highlights the potential of multi-target-directed flavonoids as a novel solution in the fight against AD.
Title: Affinity Purification of Glycosylphosphatidylinositol-anchored Proteins by Alpha-Toxin Huang K, Park S Ref: Methods Mol Biol, 2303:251, 2022 : PubMed
The glycosylphosphatidylinositol (GPI)-anchor modification attaches a lipid anchor to the C-terminus of a protein, tethering the protein to the cell surface membrane. From this membrane-bound state, GPI-anchored proteins (GPI-APs) can be released into the extracellular space by multiple mechanisms, including proteolytic shedding and GPI lipase activity. Since the core GPI structure is co-released with the protein by GPI lipase activity, while removed from the protein by proteolytic cleavage, affinity purification by alpha-toxin (alphaToxin), which binds to the core domain of the GPI-anchor, isolates GPI-containing proteins from the culture medium. The following method details a technique for affinity purification of GP-APs using His-tagged alphaToxin for identification of GPI-anchored proteins, analysis of the GPI-anchor status of a protein of interest, or purification for subsequent biochemical analysis.
Although current antiretroviral therapy can control HIV-1 replication and prevent disease progression, it is not curative. Identifying mechanisms that can lead to eradication of persistent viral reservoirs in people living with HIV-1 (PLWH) remains an outstanding challenge to achieving cure. Utilizing a phenotypic screen, we identified a novel chemical class capable of killing HIV-1 infected peripheral blood mononuclear cells. Tool compounds ICeD-1 and ICeD-2 ("inducer of cell death-1 and 2"), optimized for potency and selectivity from screening hits, were used to deconvolute the mechanism of action using a combination of chemoproteomic, biochemical, pharmacological, and genetic approaches. We determined that these compounds function by modulating dipeptidyl peptidase 9 (DPP9) and activating the caspase recruitment domain family member 8 (CARD8) inflammasome. Efficacy of ICeD-1 and ICeD-2 was dependent on HIV-1 protease activity and synergistic with efavirenz, which promotes premature activation of HIV-1 protease at high concentrations in infected cells. This in vitro synergy lowers the efficacious cell kill concentration of efavirenz to a clinically relevant dose at concentrations of ICeD-1 or ICeD-2 that do not result in complete DPP9 inhibition. These results suggest engagement of the pyroptotic pathway as a potential approach to eliminate HIV-1 infected cells.
No study has revealed the effect of porcine brain enzyme hydrolysate (PBEH) on memory impairment. We aimed to examine the hypothesis that PBEH intake modulates memory deficits and cognitive behavior in scopolamine (SC)-induced amnesia rats, and its mechanism, including gut microbiota changes, was determined. Sprague-Dawley male rats had intraperitoneal injections of SC (2 mg/kg body weight/day) at 30 min after daily feeding of casein (MD-control), PBEH (7 mg total nitrogen/mL) at 0.053 mL (Low-PBEH), 0.159 mL (Medium-PBEH), 0.478 mL (High-PBEH), or 10 mg donepezil (Positive-control) per kilogram body weight per day through a feeding needle for six weeks. The Normal-control rats had casein feeding without SC injection. PBEH dose-dependently protected against memory deficits determined by passive avoidance test, Y-maze, water-maze, and novel object recognition test in SC-induced rats compared to the MD-control. The High-PBEH group had a similar memory function to the Positive-control group. Systemic insulin resistance determined by HOMA-IR was lower in the PBEH groups than in the Normal-control but not the Positive-control. In parallel with systemic insulin resistance, decreased cholesterol and increased glycogen contents in the hippocampus in the Medium-PBEH and High-PBEH represented reduced brain insulin resistance. PBEH intake prevented the increment of serum TNF-alpha and IL-1beta concentrations in the SC-injected rats. Hippocampal lipid peroxide and TNF-alpha contents and mRNA TNF-alpha and IL-1beta expression were dose-dependently reduced in PBEH and Positive-control. PBEH decreased the hippocampal acetylcholinesterase activity compared to the MD-control, but not as much as the Positive-control. PBEH intake increased the alpha-diversity of the gut microbiota compared to the MD-control, and the gut microbiota community was separated from MD-control. In metagenome function analysis, PBEH increased the energy metabolism-related pathways of the gut microbiota, including citric acid cycle, oxidative phosphorylation, glycolysis, and amino acid metabolism, which were lower in the MD-control than the Normal-control. In conclusion, alleviated memory deficit by PBEH was associated potentially with not only reducing acetylcholinesterase activity but also improving brain insulin resistance and neuroinflammation potentially through modulating gut microbiota. PBEH intake (1.5-4.5 mL of 7 mg total nitrogen/mL for human equivalent) can be a potential therapeutic agent for improving memory impairment.
Title: Mitigation of Memory Impairment with Fermented Fucoidan and lambda-Carrageenan Supplementation through Modulating the Gut Microbiota and Their Metagenome Function in Hippocampal Amyloid-beta Infused Rats Zhang T, Wu X, Yuan H, Huang S, Park S Ref: Cells, 11:, 2022 : PubMed
Attenuating acetylcholinesterase and insulin/insulin-like growth factor-1 signaling in the hippocampus is associated with Alzheimer's disease (AD) development. Fucoidan and carrageenan are brown and red algae, respectively, with potent antibacterial, anti-inflammatory, antioxidant and antiviral activities. This study examined how low-molecular-weight (MW) and high-MW fucoidan and lambda-carrageenan would improve memory impairment in Alzheimer's disease-induced rats caused by an infusion of toxic amyloid-beta(Abeta). Fucoidan and lambda-carrageenan were dissected into low-MW by Luteolibacter algae and Pseudoalteromonas carrageenovora. Rats receiving an Abeta(25-35) infusion in the CA1 region of the hippocampus were fed dextrin (AD-Con), 1% high-MW fucoidan (AD-F-H), 1% low-MW fucoidan (AD-F-L), 1% high-MW lambda-carrageenan (AD-C-H), and 1% low-MW lambda-carrageenan (AD-C-L) for six weeks. Rats to receive saline infusion (Normal-Con) had an AD-Con diet. The AD-F-L group showed an improved memory function, which manifested as an enhanced Y-maze spontaneous alternation test, water maze, and passive avoidance tests, similar to the Normal-Con group. AD-F-L also potentiated hippocampal insulin signaling and increased the expression of ciliary neurotrophic factor (CNTF) and brain-derived neurotrophic factor (BDNF) in the hippocampus. AD-C-L improved the memory function mainly by increasing the BDNF content. AD-F-H and AD-C-H did not improve the memory function. Compared to AD-Con, the ascending order of AD-C-H, AD-F-H, AD-C-L, and AD-F-L increased insulin signaling by enhancing the pSTAT3(a)pAkt(a)pGSK-3beta pathway. AD-F-L improved glucose tolerance the most. Compared to AD-CON, the AD-F-L treatment increased the serum acetate concentrations and compensated for the defect of cerebral glucose metabolism. AD-Con increased Clostridium, Terrisporobacter and Sporofaciens compared to Normal-Con, and AD-F-L and AD-C-L increased Akkermentia. In conclusion, AD-F-L and AD-C-L alleviated the memory function in the rats with induced AD symptoms by modulating.
Title: Skin commensal fungus Malassezia and its lipases Park M, Park S, Jung WH Ref: J Microbiol Biotechnol, :, 2021 : PubMed
Malassezia is the most abundant genus in the fungal microflora found on human skin, and it is associated with various skin diseases. Among the 18 different species of Malassezia that have been identified to date, M. restricta and M. globosa are the most predominant fungal species found on human skin. Several studies have suggested a possible link between Malassezia and skin disorders. However, our knowledge on the physiology and pathogenesis of Malassezia in human body is still limited. Malassezia is unable to synthesize fatty acids; hence, it uptakes external fatty acids as a nutrient source for survival, a characteristic compensated by the secretion of lipases and degradation of sebum to produce and uptake external fatty acids. Although it has been reported that the activity of secreted lipases may contribute to pathogenesis of Malassezia, majority of the data were indirect evidences; therefore, enzymes' role in the pathogenesis of Malassezia infections is still largely unknown. This review focuses on the recent advances on Malassezia in the context of an emerging interest for lipases and summarizes the existing knowledge on Malassezia, diseases associated with the fungus, and the role of the reported lipases in its physiology and pathogenesis.
Title: Novel Hybrid Molecules Based on (-)-Epigallocatechin Gallate as Potent Anti-adipogenic Agents Jeong GH, Cho JH, Jo C, Park S, Kim SB, Kim TH Ref: Chem Pharm Bull (Tokyo), 68:1155, 2020 : PubMed
A series of novel (-)-epigallocatechin gallate (EGCG)-phloroglucinol hybrid compounds 1-4 has been successfully synthesized by employing a simple and efficient methodology using a dielectric barrier discharge (DBD) plasma irradiation. The new hybrid structures were determined by interpretation of spectroscopic data, with the absolute configurations being established by analysis of the circular dichroism (CD) spectra. The novel hybrids 1 and 2 showed highly improved anti-adipogenic potencies toward both pancreatic lipase and preadipocytes differentiation in 3T3-L1 compared to the original EGCG and phloroglucinol. A novel hybrid 1 represent an interesting subclass of anti-adipogenic candidates that need further research.
Title: Characterization of a novel carboxylesterase belonging to family VIII hydrolyzing beta-lactam antibiotics from a compost metagenomic library Park JM, Won SM, Kang CH, Park S, Yoon JH Ref: Int J Biol Macromol, 164:4650, 2020 : PubMed
A novel esterase, EstCS3, was isolated from a metagenomic library constructed from a compost. The EstCS3, which consists of 409 amino acids with an anticipated molecular mass of 44 kDa, showed high amino acid sequence identities to predicted esterases, serine hydrolases and beta-lactamases from uncultured and cultured bacteria. Phylogenetic analysis suggested that EstCS3 belongs to family VIII of lipolytic enzymes. EstCS3 had catalytic Ser78 residue in the consensus tetrapeptide motif SXXK, which is characteristic of family VIII esterases. Two conserved YXX and W(H or K)XG motifs in an oxyanion hole of family VIII esterases were also present in EstCS3. EstCS3 demonstrated the highest activity toward p-nitrophenyl butyrate (C4) and was stable up to 70 degreesC with optimal activity at 55 degreesC. EstCS3 had optimal activity at pH 8 and maintained its stability within pH range of 7-10. EstCS3 had over 70% activity in the presence of 20% (v/v) methanol and DMSO and hydrolyzed sterically hindered tertiary alcohol esters of t-butyl acetate and linalyl acetate. EstCS3 hydrolyzed ampicillin, cephalothin and cefepime. The properties of EstCS3, including moderate thermostability, stability against organic solvents and activity toward esters of tertiary alcohols, indicated that it has the potential to be used in industrial applications.
Upon invading target cells, multifunctional autoprocessing repeats-in-toxin (MARTX) toxins secreted by bacterial pathogens release their disease-related modularly structured effector domains. However, it is unclear how a diverse repertoire of effector domains within these toxins are processed and activated. Here, we report that Makes caterpillars floppy-like effector (MCF)-containing MARTX toxins require ubiquitous ADP-ribosylation factor (ARF) proteins for processing and activation of intermediate effector modules, which localize in different subcellular compartments following limited processing of holo effector modules by the internal cysteine protease. Effector domains structured tandemly with MCF in intermediate modules become disengaged and fully activated by MCF, which aggressively interacts with ARF proteins present at the same location as intermediate modules and is converted allosterically into a catalytically competent protease. MCF-mediated effector processing leads ultimately to severe virulence in mice via an MCF-mediated ARF switching mechanism across subcellular compartments. This work provides insight into how bacteria take advantage of host systems to induce systemic pathogenicity.
Neuronal morphology and circuitry established during early development must often be maintained over the entirety of animal lifespans. Compared with neuronal development, the mechanisms that maintain mature neuronal structures and architecture are little understood. The conserved disco-interacting protein 2 (DIP2) consists of a DMAP1-binding domain and two adenylate-forming domains (AFDs). We show that the Caenorhabditis elegans DIP-2 maintains morphology of mature neurons. dip-2 loss-of-function mutants display a progressive increase in ectopic neurite sprouting and branching during late larval and adult life. In adults, dip-2 also inhibits initial stages of axon regeneration cell autonomously and acts in parallel to DLK-1 MAP kinase and EFA-6 pathways. The function of DIP-2 in maintenance of neuron morphology and in axon regrowth requires its AFD domains and is independent of its DMAP1-binding domain. Our findings reveal a new conserved regulator of neuronal morphology maintenance and axon regrowth after injury.
The mechanisms underlying axon regeneration in mature neurons are relevant to the understanding of normal nervous system maintenance and for developing therapeutic strategies for injury. Here, we report novel pathways in axon regeneration, identified by extending our previous function-based screen using the C. elegans mechanosensory neuron axotomy model. We identify an unexpected role of the nicotinamide adenine dinucleotide (NAD(+)) synthesizing enzyme, NMAT-2/NMNAT, in axon regeneration. NMAT-2 inhibits axon regrowth via cell-autonomous and non-autonomous mechanisms. NMAT-2 enzymatic activity is required to repress regrowth. Further, we find differential requirements for proteins in membrane contact site, components and regulators of the extracellular matrix, membrane trafficking, microtubule and actin cytoskeleton, the conserved Kelch-domain protein IVNS-1, and the orphan transporter MFSD-6 in axon regrowth. Identification of these new pathways expands our understanding of the molecular basis of axonal injury response and regeneration.
Title: Ionic liquids as cosolvents for the lipase-catalyzed kinetic resolution of ketoprofen Park S, Doan TTN, Koo Y-M, Oh KK, Lee SH Ref: Molecular Catalysis, 459:113, 2018 : PubMed
This study examined the use of ionic liquids (ILs) as cosolvents for the Candida rugosa lipase-catalyzed enantioselective hydrolysis of racemic ketoprofen ethyl ester. To determine the effect of the IL anion structures on the lipase activity and enantioselectivity, six ILs containing the 1-butyl-3-methylimidazolium ([Bmim]) cation were used as cosolvents and their solvent properties were correlated with various reaction characteristics. The highest lipase activities were obtained using [Bmim][BF4] and [Bmim][MeSO4] as cosolvents. The enantioselectivity (E) of lipase was enhanced by a factor of 50 with 5% [Bmim][MeSO4]. Although the conversion, enantiomeric excess of product (eep), and E value in 5% [Bmim][MeSO4] were 47.3%, >99%, and -300, respectively, the corresponding lipase stability is inadequate for commercial application. Excluding [Bmim][MeSO4], the lipase enantioselectivity increased with decreasing hydrogen bond basicity (beta) of the IL when the log E values of lipase in 5% ILs were correlated with IL solvent parameters. With [Bmim][PF6] as the cosolvent, which has the lowest beta value among all ILs examined, the E value was enhanced by a factor of 32. The conversion and eep in 20% [Bmim][PF6] were 48.0% and 96.9%, respectively. Additionally, the lipase stability in aqueous [Bmim][PF6] solution was much higher than that in aqueous [Bmim][MeSO4] solution.
Colletotrichum species are major fungal pathogens that cause devastating anthracnose diseases in many economically important crops. In this study, we observed the hydrolyzing activity of a fungus-inducible pepper carboxylesterase (PepEST) on cell walls of C. gloeosporioides, causing growth retardation of the fungus by blocking appressorium formation. To determine the cellular basis for the growth inhibition, we observed the localization of PepEST on the fungus and found the attachment of the protein on surfaces of conidia and germination tubes. Moreover, we examined the decomposition of cell-wall materials from the fungal surface after reaction with PepEST, which led to the identification of 1,2-dithiane-4,5-diol (DTD) by gas chromatography mass spectrometry analysis. Exogenous DTD treatment did not elicit expression of defense-related genes in the host plant but did trigger the necrosis of C. gloeosporioides. Furthermore, the DTD compound displayed protective effects on pepper fruits and plants against C. gloeosporioides and C. coccodes, respectively. In addition, DTD was also effective in preventing other diseases, such as rice blast, tomato late blight, and wheat leaf rust. Therefore, our results provide evidence that PepEST is involved in hydrolysis of the outmost layer of the fungal cell walls and that DTD has antifungal activity, suggesting an alternative strategy to control agronomically important phytopathogens.
Tenofovir disoproxil fumarate (TDF), a prodrug of tenofovir, has oral bioavailability (25%) limited by intestinal transport (P-glycoprotein), and intestinal degradation (carboxylesterase). However, the influence of luminal pancreatic enzymes is not fully understood. Physiologically based pharmacokinetic (PBPK) modeling has utility for estimating drug exposure from in vitro data. This study aimed to develop a PBPK model that included luminal enzyme activity to inform dose reduction strategies. TDF and tenofovir stability in porcine pancrelipase concentrations was assessed (0, 0.48, 4.8, 48, and 480 U/ml of lipase; 1 mM TDF; 37 degrees C; 0 to 30 min). Samples were analyzed using mass spectrometry. TDF stability and permeation data allowed calculation of absorption rates within a human PBPK model to predict plasma exposure following 6 days of once-daily dosing with 300 mg of TDF. Regional absorption of drug was simulated across gut segments. TDF was degraded by pancrelipase (half-lives of 0.07 and 0.62 h using 480 and 48 U/ml, respectively). Previously reported maximum concentration (Cmax; 335 ng/ml), time to Cmax (Tmax; 2.4 h), area under the concentration-time curve from 0 to 24 h (AUC0-24; 3,045 ng . h/ml), and concentration at 24 h (C24; 48.3 ng/ml) were all within a 0.5-fold difference from the simulated Cmax (238 ng/ml), Tmax (3 h), AUC0-24 (3,036 ng . h/ml), and C24 (42.7 ng/ml). Simulated TDF absorption was higher in duodenum and jejunum than in ileum (p<0.05). These data support that TDF absorption is limited by the action of intestinal lipases. Our results suggest that bioavailability may be improved by protection of drug from intestinal transporters and enzymes, for example, by coadministration of enzyme-inhibiting agents or nanoformulation strategies.
Tenofovir disoproxil fumarate (TDF), the bisphosphonate ester prodrug of tenofovir (TFV), has poor bioavailability due to intestinal degradation and efflux transport. Reformulation using U.S. Food and Drug Administration-approved esterase and efflux inhibitors to increase oral bioavailability could provide lower dose alternatives and reduce costs for patients with HIV in resource-limited settings. Inhibition of mucosal and intracellular esterases was studied in human and rat intestinal extracts (S9), where TDF was protected by the carboxylesterase inhibitor bis-para-nitrophenylphosphate, the ester mix EM1, and the generally recognized-as-safe (GRAS) excipient propylparaben. Permeability studies using Madin-Darby canine kidney and Caco-2 cell monolayers demonstrated that TDF was a substrate for the permeability glycoprotein with permeability glycoprotein inhibitors reducing basolateral to apical transport of TDF. These studies also showed that transport was increased by esterase inhibitors. TDF, TFV, and tenofovir monophosphonate ester transport across Caco-2 monolayers with esterase and efflux inhibitors revealed a maximum 38.7-fold increase in apical to basolateral TDF transport with the potent non-GRAS combination of EM1 and GF120918. Transport was increased 22.8-fold by the GRAS excipients, propylparaben, and d-a-tocopheryl polyethylene glycol 1000 succinate (a vitamin E derivative). TFV pharmacokinetics in rats following oral administration of TDF and GRAS esterase and efflux inhibitors confirmed enhanced bioavailability. Area under the curve increased 1.5- to 2.1-fold with various combinations of parabens and d-a-tocopheryl polyethylene glycol 1000 succinate. This significant inhibition of TDF hydrolysis and efflux in vivo exhibits the potential to safely increase TDF bioavailability in humans.
The advent of super-resolution imaging (SRI) has created a need for optimized labelling strategies. We present a new method relying on fluorophore-conjugated monomeric streptavidin (mSA) to label membrane proteins carrying a short, enzymatically biotinylated tag, compatible with SRI techniques including uPAINT, STED and dSTORM. We demonstrate efficient and specific labelling of target proteins in confined intercellular and organotypic tissues, with reduced steric hindrance and no crosslinking compared with multivalent probes. We use mSA to decipher the dynamics and nanoscale organization of the synaptic adhesion molecules neurexin-1beta, neuroligin-1 (Nlg1) and leucine-rich-repeat transmembrane protein 2 (LRRTM2) in a dual-colour configuration with GFP nanobody, and show that these proteins are diffusionally trapped at synapses where they form apposed trans-synaptic adhesive structures. Furthermore, Nlg1 is dynamic, disperse and sensitive to synaptic stimulation, whereas LRRTM2 is organized in compact and stable nanodomains. Thus, mSA is a versatile tool to image membrane proteins at high resolution in complex live environments, providing novel information about the nano-organization of biological structures.
Candida antarctica lipase B (CAL-B) exhibits remarkable enantioselectivity for various chiral sec-alcohols, and the enantioselectivity is structurally well-understood. Two substituents at the chiral center of a sec-alcohol separately bind two pockets, namely, large and medium binding pockets. It has been believed that the medium pocket is too small to accommodate a large substituent (larger than an ethyl group), and thus, bulky sec-alcohols bearing two large substituents have been regarded as a poor substrate for CAL-B. However, we found that CAL-B can catalyze the transesterification of N-Boc-protected rac-2-amino-1-phenylethanol (1a) enantioselectively with a moderate reaction rate. X-ray crystallography and computer modeling revealed that the rotation of the Leu278 side chain creates a space to accept the N-Boc-aminomethylene group of 1a. Moreover, a sec-alcohol substrate with less than one hydrogen atom at the gamma-position from the hydroxyl group is required to achieve a moderate reaction rate. On the basis of this observation, we diversified bulky N-Boc-protected rac-2-amino-1-arylethanols for the transesterifications with high enantioselectivities (E > 200).
Most lipases resolve secondary alcohols in accordance with the "Kazlauskas rule" to give the R enantiomers. In a similar manner to other lipases, Candida rugosa lipase (CRL) exhibits R enantioselectivity towards heptan-2-ol, although the enantiomeric ratio (E) is low (E=1.6). However, unexpected enantioselectivity (i.e., S enantioselectivity, E=58) of CRL towards 4-(tert-butoxycarbonylamino)butan-2-ol, which has a similar chain length to heptan-2-ol, has been observed. To develop a deeper understanding of the molecular basis for this unusual enantioselectivity, we have conducted a series of molecular modeling and substrate engineering experiments. The results of these computational and experimental analyses indicated that a hydrogen bond between the Ser450 residue and the nitrogen atom of the carbamate group is critical to stabilize the transition state of the S enantiomer.
Comparative Gene Identification-58 (CGI-58) is an alpha/beta hydrolase-type protein that regulates lipid homeostasis and signaling in eukaryotes by interacting with and stimulating the activity of several different types of proteins, including a lipase in mammalian cells and a peroxisomal ABC transporter (PXA1) in plant cells. Here we show that plant CGI-58 also interacts with spermidine synthase 1 (SPDS1), an enzyme that plays a central role in polyamine metabolism by converting putrescine into spermidine. Analysis of polyamine contents in Arabidopsis thaliana plants revealed that spermidine levels were significantly reduced, and putrescine increased, in both cgi-58 and cgi-58/pxa1 mutant plants, relative to pxa1 mutant or wild-type plants. Evaluation of polyamine-related gene expression levels, however, revealed similar increases in transcript abundance in all mutants, including cgi-58, pxa1, and cgi-58/pxa1, in comparison to wild type. Taken together, the data support a model whereby CGI-58 and PXA1 contribute to the regulation of polyamine metabolism at the transcriptional level, perhaps through a shared lipid-signaling pathway, and that CGI-58 also acts independently of PXA1 to increase spermidine content at a post-transcriptional level, possibly through protein-protein interaction with SPDS1.
Title: Sphaerotilus natans encrusted with nanoball-shaped Fe(III) oxide minerals formed by nitrate-reducing mixotrophic Fe(II) oxidation Park S, Kim DH, Lee JH, Hur HG Ref: FEMS Microbiol Ecol, 90:68, 2014 : PubMed
Ferrous iron has been known to function as an electron source for iron-oxidizing microorganisms in both anoxic and oxic environments. A diversity of bacteria has been known to oxidize both soluble and solid-phase Fe(II) forms coupled to the reduction of nitrate. Here, we show for the first time Fe(II) oxidation by Sphaerotilus natans strain DSM 6575(T) under mixotrophic condition. Sphaerotilus natans has been known to form a sheath structure enclosing long chains of rod-shaped cells, resulting in a thick biofilm formation under oxic conditions. Here, we also demonstrate that strain DSM 6575(T) grows mixotrophically with pyruvate, Fe(II) as electron donors and nitrate as an electron acceptor and single cells of strain DSM 6575(T) are dominant under anoxic conditions. Furthermore, strain DSM 6575(T) forms nanoball-shaped amorphous Fe(III) oxide minerals encrusting on the cell surfaces through the mixotrophic iron oxidation reaction under anoxic conditions. We propose that cell encrustation results from the indirect Fe(II) oxidation by biogenic nitrite during nitrate reduction and that causes the bacterial morphological change to individual rod-shaped single cells from filamentous sheath structures. This study extends the group of existing microorganisms capable of mixotrophic Fe(II) oxidation by a new strain, S. natans strain DSM 6575(T) , and could contribute to biogeochemical cycles of Fe and N in the environment.
BACKGROUND: Although ginsenosides such as Rg1, Rb1 and Rg3 have shown promise as potential nutraceuticals for cognitive impairment, their use has been limited due to high production cost and low potency. In particular, the process of extracting pure Rg3 from ginseng is laborious and expensive. METHODS: We described the methods in preparing ginseol k-g3, an Rg3-enriched fraction, and evaluated its effects on scopolamine-induced memory impairment in mice. RESULTS: Ginseol k-g3 (25-200 mg/kg) significantly reversed scopolamine-induced cognitive impairment in the passive avoidance, but not in Y-maze testing. Ginseol k-g3 (50 and 200 mg/kg) improved escape latency in training trials and increased swimming times within the target zone of the Morris water maze. The effect of ginseol k-g3 on the water maze task was more potent than that of Rg3 or Red ginseng. Acute or subchronic (6 d) treatment of ginseol k-g3 did not alter normal locomotor activity of mice in an open field. Ginseol k-g3 did not inhibit acetylcholinesterase activity, unlike donezepil, an acetylcholinesterase inhibitor. Rg3 enrichment through the ginseol k-g3 fraction enhanced the efficacy of Rg3 in scopolamine-induced memory impairment in mice as demonstrated in the Morris water maze task. CONCLUSION: The effects of ginseol k-g3 in ameliorating scopolamine-induced memory impairment in the passive avoidance and Morris water maze tests indicate its specific influence on reference or long-term memory. The mechanism underlying the reversal of scopolamine-induced amnesia by ginseol k-g3 is not yet known, but is not related to anticholinesterase-like activity.
Title: Cloning and Characterization of a Novel Esterase from Rhodococcus sp. for Highly Enantioselective Synthesis of a Chiral Cilastatin Precursor Zhang Y, Pan J, Luan ZJ, Xu GC, Park S, Xu JH Ref: Applied Environmental Microbiology, 80:7348, 2014 : PubMed
A novel nonheme chloroperoxidase (RhEst1), with promiscuous esterase activity for enantioselective hydrolysis of ethyl (S)-2,2-dimethylcyclopropanecarboxylate, was identified from a shotgun library of Rhodococcus sp. strain ECU1013. RhEst1 was overexpressed in Escherichia coli BL21(DE3), purified to homogeneity, and functionally characterized. Fingerprinting analysis revealed that RhEst1 prefers para-nitrophenyl (pNP) esters of short-chain acyl groups. pNP esters with a cyclic acyl moiety, especially that with a cyclobutanyl group, were also substrates for RhEst1. The Km values for methyl 2,2-dimethylcyclopropanecarboxylate (DmCpCm) and ethyl 2,2-dimethylcyclopropane carboxylate (DmCpCe) were 0.25 and 0.43 mM, respectively. RhEst1 could serve as an efficient hydrolase for the bioproduction of optically pure (S)-2,2-dimethyl cyclopropane carboxylic acid (DmCpCa), which is an important chiral building block for cilastatin. As much as 0.5 M DmCpCe was enantioselectively hydrolyzed into (S)-DmCpCa, with a molar yield of 47.8% and an enantiomeric excess (ee) of 97.5%, indicating an extremely high enantioselectivity (E = 240) of this novel and unique biocatalyst for green manufacturing of highly valuable chiral chemicals.
Title: Glycerol assimilation and production of 1,3-propanediol by Citrobacter amalonaticus Y19 Ainala SK, Ashok S, Ko Y, Park S Ref: Applied Microbiology & Biotechnology, 97:5001, 2013 : PubMed
Citrobacter amalonaticus Y19 (Y19) was isolated because of its ability for carbon monoxide-dependent hydrogen production (water-gas shift reaction). This paper reports the assimilation of glycerol and the production of 1,3-propanediol (1,3-PDO) by Y19. Genome sequencing revealed that Y19 contained the genes for the utilization of glycerol and 1,2-propanediol (pdu operon) along with those for the synthesis of coenzyme B12 (cob operon). On the other hand, it did not possess the genes for the fermentative metabolism of glycerol of Klebsiella pneumoniae, which consists of both the oxidative (dhaD and dhaK) and reductive (dhaB and dhaT) pathways. In shake-flask cultivation under aerobic conditions, Y19 could grow well with glycerol as the sole carbon source and produced 1,3-PDO. The level of 1,3-PDO production was improved when vitamin B12 was added to the culture medium under aerobic conditions. Under anaerobic conditions, cell growth and 1,3-PDO production on glycerol was also possible, but only when an exogenous electron acceptor, such as nitrate or fumarate, was added. This is the first report of the glycerol metabolism and 1,3-PDO production by C. amalonaticus Y19.
Title: Complete Genome Sequence of Pseudomonas denitrificans ATCC 13867 Ainala SK, Somasundar A, Park S Ref: Genome Announc, 1:, 2013 : PubMed
Pseudomonas denitrificans ATCC 13867, a Gram-negative facultative anaerobic bacterium, is known to produce vitamin B12 under aerobic conditions. This paper reports the annotated whole-genome sequence of the circular chromosome of this organism.
COMPARATIVE GENE IDENTIFICATION-58 (CGI-58) is a key regulator of lipid metabolism and signaling in mammals, but its underlying mechanisms are unclear. Disruption of CGI-58 in either mammals or plants results in a significant increase in triacylglycerol (TAG), suggesting that CGI-58 activity is evolutionarily conserved. However, plants lack proteins that are important for CGI-58 activity in mammals. Here, we demonstrate that CGI-58 functions by interacting with the PEROXISOMAL ABC-TRANSPORTER1 (PXA1), a protein that transports a variety of substrates into peroxisomes for their subsequent metabolism by beta-oxidation, including fatty acids and lipophilic hormone precursors of the jasmonate and auxin biosynthetic pathways. We also show that mutant cgi-58 plants display changes in jasmonate biosynthesis, auxin signaling, and lipid metabolism consistent with reduced PXA1 activity in planta and that, based on the double mutant cgi-58 pxa1, PXA1 is epistatic to CGI-58 in all of these processes. However, CGI-58 was not required for the PXA1-dependent breakdown of TAG in germinated seeds. Collectively, the results reveal that CGI-58 positively regulates many aspects of PXA1 activity in plants and that these two proteins function to coregulate lipid metabolism and signaling, particularly in nonseed vegetative tissues. Similarities and differences of CGI-58 activity in plants versus animals are discussed.
Title: The toxicity of mixtures of specific organophosphate compounds is modulated by paraoxonase 1 status Cole TB, Jansen K, Park S, Li WF, Furlong CE, Costa LG Ref: Advances in Experimental Medicine & Biology, 660:47, 2010 : PubMed
Most chemical exposures involve complex mixtures. The role of paraoxonase 1 (PON1) and the Q192R polymorphism in the detoxication of individual organophosphorous (OP) compounds has been well-established. The extent to which PON1 protects against a given OP is determined by its catalytic efficiency. We used a humanized transgenic mouse model of the Q192R polymorphism to demonstrate that PON1 modulates the toxicity of OP mixtures by altering the activity of another detoxication enzyme, carboxylesterase (CaE). Chlorpyrifos oxon (CPO), diazoxon (DZO), and paraoxon (PO) are potent inhibitors of CaE, both in vitro and in vivo. We hypothesized that exposure of mice to these OPs would increase their sensitivity to the CaE substrate, malaoxon (MO), and that the degree of effect would vary among PON1 genotypes if the OP was a physiologically relevant PON1 substrate. When wild-type mice were exposed dermally to CPO, DZO, or PO and then, after 4 h, to different doses of MO, the toxicity of MO was increased compared to mice that received MO alone. The potentiation of MO toxicity by CPO and DZO was higher in PON1 knockout mice, which are less able to detoxify CPO or DZO. Potentiation by CPO was higher in Q192 mice than in R192 mice due to the decreased ability of PON1(Q192) to detoxify CPO. Potentiation by DZO was similar in the Q192 and R192 mice, due to their equivalent effectiveness at detoxifying DZO. PO exposure resulted in equivalent potentiation of MO toxicity among all four genotypes. These results indicate that PON1 status modulates the ability of CaE to detoxicate OP compounds from specific mixed insecticide exposures. PON1 status can also impact the capacity to metabolize drugs or other CaE substrates following insecticide exposure.
Expression and purification of recombinant human paraoxonase-1 (rHuPON1) from bacterial systems have proven elusive. Most systems for successful production of recombinant PON1 have relied on either eukaryotic expression in baculovirus or prokaryotic expression of synthetic, gene-shuffled rabbit-mouse-human PON1 hybrid molecules. We review here methods and protocols for the production of pure, native rHuPON1 using an E. coli expression system followed by conventional column chromatographic purification. The resulting rHuPON1 is stable, active, and capable of protecting PON1 knockout mice (PON1(-/-)) from exposure to high levels of the organophosphorus (OP) compound diazoxon. Bacterially-derived rHuPON1 can be produced in large quantities and lacks the glycosylation of eukaryotic systems that produces immunogenic complications when used as a therapeutic. The rHuPON1 should be useful for treating insecticide OP exposures and reducing risks of other diseases resulting from low PON1 status. The ease of mutagenesis in bacterial systems will also allow for the generation and screening of rHuPON1 variants with enhanced catalytic efficiencies against nerve agents and other OP compounds.
Title: Recyclable chaperone-conjugated magnetic beads for in vitro refolding of Burkholderia cepacia lipase Jung S, Park S Ref: Biotechnol Lett, 31:107, 2009 : PubMed
Polymer-coated magnetic beads have become widely used in biological applications because of their facile recovery and easily modifiable surface. Herein, we report the application of magnetic beads to in vitro refolding of B. cepacia lipase. Magnetic particles (Fe3O4) prepared by co-precipitation of Fe2+ and Fe3+ ions under basic conditions were subsequently coated with carboxylic acid-containing polystyrene by emulsion polymerization. The polymer-coated magnetic beads were then conjugated with molecular chaperone proteins to assist with refolding. The chaperone-conjugated magnetic beads efficiently refolded B. cepacia lipase and were easily reused. The beads showed comparable refolding activity to the soluble chaperone, and retained more than 95% of their refolding activity after five cycles of refolding B. cepacia lipase.
Title: Kdr allelic variation in a sodium channel gene from a population of South Carolina Heliothis virescens (Fabricius) Cho J, Park S, Lim C, Park YC, Hur JH, Hong S, Brown TM, Cho S Ref: Journal of Asia-Pacific Entomology, 11:117, 2008 : PubMed
Mutations at V421M and L1029H in the hscp sodium channel gene are known to contribute to knockdown resistance (kdr) in the Woodrow, Dalzell, and PTJ strains of H. virescens (tobacco budworm) from the cotton fields of South Carolina, USA. In the IS6 region of the sodium channel gene, the frequencies of the mutant allele methionine in the Woodrow and Dalzell strains were 0.07 and 0.1, respectively. For the IIS6 region, the frequencies of the mutant allele histidine in Woodrow and Dalzell strains were 0.175 and 0.263, respectively. In the PTJ strain, the frequencies of methionine and histidine alleles were 0 and 0.1, respectively. The Hpy3 allele, which is strongly linked to the histidine mutant allele, was also found in Woodrow and Dalzell strains. In addition, we found a new allele, which is one nucleotide different from Hpy3, called Hpy3-1, and found that it is also linked to histidine.
Title: Improving the expression yield of Candida antarctica lipase B in Escherichia coli by mutagenesis Jung S, Park S Ref: Biotechnol Lett, 30:717, 2008 : PubMed
Increasing the expression yield of active Candida antarctica lipase B (CAL-B) in Escherichia coli was achieved by using a codon-optimized synthetic gene and by mutagenesis to introduce hydrophilic residues on the surface of CAL-B. Five residues (four leucines and one isoleucine) on the surface of CAL-B were selected and changed with aspartate after codon optimization. While the codon-optimized synthetic gene of CAL-B did not increase the expression yield, the mutation increased the activity of the enzyme three-fold (3.3 mg/l of culture) compared to the wild type. The mutant enzyme had similar hydrolytic activity toward hydrolysis of p-nitrophenyl acetate or p-nitrophenyl butyrate and enantioselectivity toward hydrolysis of (R, S)-1-phenylethyl acetate compared to the wild-type enzyme.
Neuroligin-1 is a potent trigger for the de novo formation of synaptic connections, and it has recently been suggested that it is required for the maturation of functionally competent excitatory synapses. Despite evidence for the role of neuroligin-1 in specifying excitatory synapses, the underlying molecular mechanisms and physiological consequences that neuroligin-1 may have at mature synapses of normal adult animals remain unknown. By silencing endogenous neuroligin-1 acutely in the amygdala of live behaving animals, we have found that neuroligin-1 is required for the storage of associative fear memory. Subsequent cellular physiological studies showed that suppression of neuroligin-1 reduces NMDA receptor-mediated currents and prevents the expression of long-term potentiation without affecting basal synaptic connectivity at the thalamo-amygdala pathway. These results indicate that persistent expression of neuroligin-1 is required for the maintenance of NMDAR-mediated synaptic transmission, which enables normal development of synaptic plasticity and long-term memory in the amygdala of adult animals.
Title: Enantioselective resolution of racemic styrene oxide at high concentration using recombinant Pichia pastoris expressing epoxide hydrolase of Rhodotorula glutinis in the presence of surfactant and glycerol Yoo SS, Park S, Lee EY Ref: Biotechnol Lett, 30:1807, 2008 : PubMed
The reaction medium was optimized to accomplish epoxide hydrolase-catalyzed, batch enantioselective hydrolysis of racemic styrene oxide at high initial substrate concentrations. The recombinant Pichia pastoris containing the epoxide hydrolase gene of Rhodotorula glutinis was used as the biocatalyst. Enantiopure (S)-styrene oxide with 98% ee was obtained with 41% yield (maximum yield = 50%) from 1.8 M racemic styrene oxide at pH 8.0, 4 degrees C in the presence of 40% (v/v) Tween 20 and 5% (v/v) glycerol.
Genome sequences for most metazoans and plants are incomplete because of the presence of repeated DNA in the heterochromatin. The heterochromatic regions of Drosophila melanogaster contain 20 million bases (Mb) of sequence amenable to mapping, sequence assembly, and finishing. We describe the generation of 15 Mb of finished or improved heterochromatic sequence with the use of available clone resources and assembly methods. We also constructed a bacterial artificial chromosome-based physical map that spans 13 Mb of the pericentromeric heterochromatin and a cytogenetic map that positions 11 Mb in specific chromosomal locations. We have approached a complete assembly and mapping of the nonsatellite component of Drosophila heterochromatin. The strategy we describe is also applicable to generating substantially more information about heterochromatin in other species, including humans.
Title: Cloning, expression and enantioselective hydrolytic catalysis of a microsomal epoxide hydrolase from a marine fish, Mugil cephalus Lee SJ, Kim HS, Kim SJ, Park S, Kim BJ, Shuler ML, Lee EY Ref: Biotechnol Lett, 29:237, 2007 : PubMed
The cDNA of a marine fish microsomal epoxide hydrolase (mEH) gene from Mugil cephalus was cloned by rapid amplification of cDNA ends (RACE) techniques. The homology model for the mEH of M. cephalus showed a characteristic structure of alpha/beta-hydrolase-fold main domain with a lid domain over the active site. The characteristic catalytic triad, consisting of Asp(238), His(444), and Glu(417), was highly conserved. The cloned mEH gene was expressed in Escherichia coli and the recombinant mEH exhibited (R)-preferred hydrolysis activity toward racemic styrene oxide. We obtained enantiopure (S)-styrene oxide with a high enantiopurity of more than 99% enantiomeric excess and yield of 15.4% by batch kinetic resolution of 20 mM racemic styrene oxide.
Title: Molecular basis for the enhanced lipase-catalyzed N-acylation of 1-phenylethanamine with methoxyacetate Cammenberg M, Hult K, Park S Ref: Chembiochem, 7:1745, 2006 : PubMed
One of the commercial methods for preparing enantiopure amines is lipase-catalyzed kinetic resolution, although lipases catalyze aminolysis with only low activity. Interestingly, in 1997 Balkenhohl et al. used ethyl methoxyacetate instead of ethyl butyrate as an acylation reagent for the aminolysis of 1-phenylethanamine and increased the reaction rate more than a 100-fold. This method has been applied to other aminolysis reactions, but the molecular basis for the enhanced rate is not understood. A molecular-modeling study of the transition-state analogue for the aminolysis showed that an interaction between the beta-oxygen atom in methoxyacetate and the amine nitrogen atom might be a key factor in the rate enhancement. Other acylation reagents, such as methyl 3-methoxypropionate and methyl 4-methoxybutyrate, were chosen to test the influence of this interaction because these molecules can be spatially arranged to have similar interactions. The results were similar to that in the acylation with methoxyacetate. The initial aminolysis rates were improved (11-fold and sixfold, respectively) compared to that with butyrate. In contrast, alcoholysis with 1-phenylethanol afforded the same rate with all acyl donors.
Title: Detection of maternal uniparental disomy at the two imprinted genes on chromosome 7, GRB10 and PEG1/MEST, in a Silver-Russell syndrome patient using methylation-specific PCR assays Kim Y, Kim SS, Kim G, Park S, Park IS, Yoo HW Ref: Clin Genet, 67:267, 2005 : PubMed
Title: Focusing mutations into the P. fluorescens esterase binding site increases enantioselectivity more effectively than distant mutations Park S, Morley KL, Horsman GP, Holmquist M, Hult K, Kazlauskas RJ Ref: Chemical Biology, 12:45, 2005 : PubMed
Rational design of enzymes with improved properties, such as enantioselectivity, usually focuses mutations within the substrate binding site. On the other hand, directed evolution of enzymes usually targets the entire protein and discovers beneficial mutations far from the substrate binding site. In this paper, we propose an explanation for this discrepancy and show that a combined approach--random mutagenesis within the substrate binding site--is better. To increase the enantioselectivity (E) of a Pseudomonas fluorescens esterase (PFE) toward methyl 3-bromo-2-methylpropionate, we focused mutagenesis into the substrate binding site at Trp28, Val121, Phe198, and Val225. Five of the catalytically active mutants (13%) showed better enantioselectivity than wild-type PFE. The increases in enantioselectivity were higher (up to 5-fold, reaching E = 61) than with mutants identified by random mutagenesis of the entire enzyme.
The structure of PFE, an aryl esterase from Pseudomonas fluorescens, has been solved to a resolution of 1.8 A by X-ray diffraction and shows a characteristic alpha/beta-hydrolase fold. In addition to catalyzing the hydrolysis of esters in vitro, PFE also shows low bromoperoxidase activity. PFE shows highest structural similarity, including the active-site environment, to a family of non-heme bacterial haloperoxidases, with an r.m.s. deviation in 271 C(alpha) atoms between PFE and its five closest structural neighbors averaging 0.8 A. PFE has far less similarity (r.m.s. deviation in 218 C(alpha) atoms of 5.0 A) to P. fluorescens carboxyl esterase. PFE favors activated esters with small acyl groups, such as phenyl acetate. The X-ray structure of PFE reveals a significantly occluded active site. In addition, several residues, including Trp28 and Met95, limit the size of the acyl-binding pocket, explaining its preference for small acyl groups.
Title: Effects of methanol extract of Uncariae Ramulus et Uncus on ibotenic acid-induced amnesia in the rat Kim JH, Chung JY, Lee YJ, Park S, Hahm DH, Lee HJ, Shim I Ref: J Pharmacol Sci, 96:314, 2004 : PubMed
In the present study, we investigated the effects of Uncariae Ramulus et Uncus (UR) on learning and memory in the Morris water maze task and the central cholinergic system of rats with excitotoxic medial septum (MS) lesion. In the water maze test, the animals were trained to find a platform in a fixed position during 6 days and then received a 60-s probe trial in which the platform was removed from the pool on the 7th day. Ibotenic lesion of the MS showed impaired performance of the maze test and severe cell losses in the septohippocampal cholinergic system (SHC), as indicated by decreased choline acetyltransferase-immunoreactivity and acetylcholinesterase-reactivity in the hippocampus. Daily administrations of UR (100 mg/kg, i.p.) for 21 consecutive days produced significant reversals of ibotenic acid-induced deficit in learning and memory. These treatments also reduced the loss of cholinergic immunoreactivity in the hippocampus induced by ibotenic acid. These results demonstrated that impairments of spatial learning and memory may be attributable to degeneration of SHC neurons and that UR ameliorated learning and memory deficits partly through neuroprotective effects on the central acetylcholine system. Our studies suggest that UR may be useful in the treatment of Alzheimer's disease.
Title: Production of (S)-styrene oxide by recombinant Pichia pastoris containing epoxide hydrolase from Rhodotorula glutinis Lee EY, Yoo SS, Kim HS, Lee SJ, Oh YK, Park S Ref: Enzyme Microb Technol, 35:624, 2004 : PubMed
A recombinant yeast Pichia pastoris carrying the gene encoding epoxide hydrolase (EH) of Rhodotorula glutinis was constructed and used for producing (S)-styrene oxide by enantioselective hydrolysis of racemic mixtures of styrene oxides. The EH gene was obtained by PCR amplification of cDNA of R. glutinis and integrated into the chromosomal DNA of P. pastoris to express EH under the control of AOX promoter. The recombinant yeast has a high hydrolytic activity toward (R)-styrene oxide as 358 nmol min-1 (mg cell)-1, which is about 10-fold higher than that of wild type R. glutinis. When kinetic resolution was conducted by the recombinant yeast at a high initial epoxides concentration of 526 mM that constitutes an epoxidewater two-liquid phase, chiral (S)-styrene oxide with an enantiomeric excess (e.e.) higher than 98% was obtained as 36% yield (theoretical, 50%) at 16 h
We used a systematic approach to build a network of genes associated with developmental and stress responses in rice by identifying interaction domains for 200 proteins from stressed and developing tissues, by measuring the associated gene expression changes in different tissues exposed to a variety of environmental, biological, and chemical stress treatments, and by localizing the cognate genes to regions of stress-tolerance trait genetic loci. The integrated data set suggests that similar genes respond to environmental cues and stresses, and some may also regulate development. We demonstrate that the data can be used to correctly predict gene function in monocots and dicots. As a result, we have identified five genes that contribute to disease resistance in Arabidopsis.
BACKGROUND: It is widely accepted that comparative sequence data can aid the functional annotation of genome sequences; however, the most informative species and features of genome evolution for comparison remain to be determined. RESULTS: We analyzed conservation in eight genomic regions (apterous, even-skipped, fushi tarazu, twist, and Rhodopsins 1, 2, 3 and 4) from four Drosophila species (D. erecta, D. pseudoobscura, D. willistoni, and D. littoralis) covering more than 500 kb of the D. melanogaster genome. All D. melanogaster genes (and 78-82% of coding exons) identified in divergent species such as D. pseudoobscura show evidence of functional constraint. Addition of a third species can reveal functional constraint in otherwise non-significant pairwise exon comparisons. Microsynteny is largely conserved, with rearrangement breakpoints, novel transposable element insertions, and gene transpositions occurring in similar numbers. Rates of amino-acid substitution are higher in uncharacterized genes relative to genes that have previously been studied. Conserved non-coding sequences (CNCSs) tend to be spatially clustered with conserved spacing between CNCSs, and clusters of CNCSs can be used to predict enhancer sequences. CONCLUSIONS: Our results provide the basis for choosing species whose genome sequences would be most useful in aiding the functional annotation of coding and cis-regulatory sequences in Drosophila. Furthermore, this work shows how decoding the spatial organization of conserved sequences, such as the clustering of CNCSs, can complement efforts to annotate eukaryotic genomes on the basis of sequence conservation alone.
BACKGROUND: The Drosophila melanogaster genome was the first metazoan genome to have been sequenced by the whole-genome shotgun (WGS) method. Two issues relating to this achievement were widely debated in the genomics community: how correct is the sequence with respect to base-pair (bp) accuracy and frequency of assembly errors? And, how difficult is it to bring a WGS sequence to the accepted standard for finished sequence? We are now in a position to answer these questions. RESULTS: Our finishing process was designed to close gaps, improve sequence quality and validate the assembly. Sequence traces derived from the WGS and draft sequencing of individual bacterial artificial chromosomes (BACs) were assembled into BAC-sized segments. These segments were brought to high quality, and then joined to constitute the sequence of each chromosome arm. Overall assembly was verified by comparison to a physical map of fingerprinted BAC clones. In the current version of the 116.9 Mb euchromatic genome, called Release 3, the six euchromatic chromosome arms are represented by 13 scaffolds with a total of 37 sequence gaps. We compared Release 3 to Release 2; in autosomal regions of unique sequence, the error rate of Release 2 was one in 20,000 bp. CONCLUSIONS: The WGS strategy can efficiently produce a high-quality sequence of a metazoan genome while generating the reagents required for sequence finishing. However, the initial method of repeat assembly was flawed. The sequence we report here, Release 3, is a reliable resource for molecular genetic experimentation and computational analysis.
BACKGROUND: A collection of sequenced full-length cDNAs is an important resource both for functional genomics studies and for the determination of the intron-exon structure of genes. Providing this resource to the Drosophila melanogaster research community has been a long-term goal of the Berkeley Drosophila Genome Project. We have previously described the Drosophila Gene Collection (DGC), a set of putative full-length cDNAs that was produced by generating and analyzing over 250,000 expressed sequence tags (ESTs) derived from a variety of tissues and developmental stages. RESULTS: We have generated high-quality full-insert sequence for 8,921 clones in the DGC. We compared the sequence of these clones to the annotated Release 3 genomic sequence, and identified more than 5,300 cDNAs that contain a complete and accurate protein-coding sequence. This corresponds to at least one splice form for 40% of the predicted D. melanogaster genes. We also identified potential new cases of RNA editing. CONCLUSIONS: We show that comparison of cDNA sequences to a high-quality annotated genomic sequence is an effective approach to identifying and eliminating defective clones from a cDNA collection and ensure its utility for experimentation. Clones were eliminated either because they carry single nucleotide discrepancies, which most probably result from reverse transcriptase errors, or because they are truncated and contain only part of the protein-coding sequence.
A yellow-eyed mutant was discovered in a strain of Heliothis virescens, the tobacco budworm, that already exhibited a mutation for yellow scale, y. We investigated the inheritance of these visible mutations as candidate markers for transgenesis. Yellow eye was controlled by a single, recessive, autosomal factor, the same type of inheritance previously known for y. Presence of the recombinant mutants with yellow scales and wild type eyes in test crosses indicated independent segregation of genes for these traits. The recombinant class with wild type scales and yellow eyes was completely absent and there was a corresponding increase of the double mutant parental class having yellow scales and yellow eyes. These results indicated that a single factor for yellow eye also controlled yellow scales independently of y. This gene was named yes, for yellow eye and scale. We hypothesize that yes controls both eye and scale color through a deficiency in transport of pigment precursors in both the ommochrome and melanin pathways. The unlinked gene y likely controls an enzyme affecting the melanin pathway only. Both y and yes segregated independently of AceIn, acetylcholinesterase insensitivity, and sodium channel hscp, which are genes related to insecticide resistance.
Title: Improved preparation and use of room-temperature ionic liquids in lipase-catalyzed enantio- and regioselective acylations Park S, Kazlauskas RJ Ref: J Org Chem, 66:8395, 2001 : PubMed
Polar organic solvents such as methanol or N-methylformamide inactivate lipases. Although ionic liquids such as 3-alkyl-1-methylimidazolium tetrafluoroborates have polarities similar to these polar organic solvents, they do not inactivate lipases. To get reliable lipase-catalyzed reactions in ionic liquids, we modified their preparation by adding a wash with aqueous sodium carbonate. Lipase-catalyzed reactions that previously did not occur in untreated ionic liquids now occur at rates comparable to those in nonpolar organic solvents such as toluene. Acetylation of 1-phenylethanol catalyzed by lipase from Pseudomonas cepacia (PCL) was as fast and as enantioselective in ionic liquids as in toluene. Ionic liquids permit reactions in a more polar solvent than previously possible. Acetylation of glucose catalyzed by lipase B from Candida antarctica (CAL-B) was more regioselective in ionic liquids because glucose is up to one hundred times more soluble in ionic liquids. Acetylation of insoluble glucose in organic solvents yielded the more soluble 6-O-acetyl glucose, which underwent further acetylation to give 3,6-O-diacetyl glucose (2-3:1 mixture). However, acetylation of glucose in ionic liquids yielded only 6-O-acetyl glucose (>13:1 and up to >50:1).
Title: Identification of myelinated motor and sensory axons in a regenerating mixed nerve Kawasaki Y, Yoshimura K, Harii K, Park S Ref: J Hand Surg [Am], 25:104, 2000 : PubMed
The common peroneal nerves of Wistar rats were transected and repaired to compare the sequential changes in the numbers of regenerating motor and sensory myelinated axons in a single mixed nerve. At sequential intervals (2, 4, and 12 weeks) after nerve repair, 3 kinds of staining were performed: cholinesterase staining (Karnovsky's staining) for motor axons, carbonic anhydrase staining for sensory axons, and antineurofilament immunohistochemical staining for all axons. At 2 weeks there was a large number of carbonic anhydrase-positive axons (600 +/- 98; mean +/- SD) and cholinesterase-positive axons were occasionally seen. Subsequently, there was a striking increase of cholinesterase-positive myelinated axons, reaching to 302 +/- 50 at 12 weeks. The results suggest that the myelinated sensory axons regenerate faster in the early stage of nerve regeneration and that regeneration of the myelinated motor axons is prominent in the subsequent stage. (J Hand Surg 2000; 25A:104-111.)
