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1.  Infection of Pericytes In Vitro by Japanese Encephalitis Virus Disrupts the Integrity of the Endothelial Barrier 
Journal of Virology  2014;88(2):1150-1161.
Though the compromised blood-brain barrier (BBB) is a pathological hallmark of Japanese encephalitis-associated neurological sequelae, the underlying mechanisms and the specific cell types involved are not understood. BBB characteristics are induced and maintained by cross talk between brain microvascular endothelial cells and neighboring elements of the neurovascular unit. In this study, we show a potential mechanism of disruption of endothelial barrier integrity during the course of Japanese encephalitis virus (JEV) infection through the activation of neighboring pericytes. We found that cultured brain pericytes were susceptible to JEV infection but were without signs of remarkable cytotoxicity. JEV-infected pericytes were found to release biologically active molecules which activated ubiquitin proteasome, degraded zonula occludens-1 (ZO-1), and disrupted endothelial barrier integrity in cultured brain microvascular endothelial cells. Infection of pericytes with JEV was found to elicit elevated production of interleukin-6 (IL-6), which contributed to the aforementioned endothelial changes. We further demonstrated that ubiquitin-protein ligase E3 component n-recognin-1 (Ubr 1) was a key upstream regulator which caused proteasomal degradation of ZO-1 downstream of IL-6 signaling. During JEV central nervous system trafficking, endothelial cells rather than pericytes are directly exposed to cell-free viruses in the peripheral bloodstream. Therefore, the results of this study suggest that subsequent to primary infection of endothelial cells, JEV infection of pericytes might contribute to the initiation and/or augmentation of Japanese encephalitis-associated BBB breakdown in concerted action with other unidentified barrier disrupting factors.
PMCID: PMC3911661  PMID: 24198423
2.  Crystal Structures of Complexes of the Branched-Chain Aminotransferase from Deinococcus radiodurans with α-Ketoisocaproate and l-Glutamate Suggest the Radiation Resistance of This Enzyme for Catalysis 
Journal of Bacteriology  2012;194(22):6206-6216.
Branched-chain aminotransferases (BCAT), which utilize pyridoxal 5′-phosphate (PLP) as a cofactor, reversibly catalyze the transfer of the α-amino groups of three of the most hydrophobic branched-chain amino acids (BCAA), leucine, isoleucine, and valine, to α-ketoglutarate to form the respective branched-chain α-keto acids and glutamate. The BCAT from Deinococcus radiodurans (DrBCAT), an extremophile, was cloned and expressed in Escherichia coli for structure and functional studies. The crystal structures of the native DrBCAT with PLP and its complexes with l-glutamate and α-ketoisocaproate (KIC), respectively, have been determined. The DrBCAT monomer, comprising 358 amino acids, contains large and small domains connected with an interdomain loop. The cofactor PLP is located at the bottom of the active site pocket between two domains and near the dimer interface. The substrate (l-glutamate or KIC) is bound with key residues through interactions of the hydrogen bond and the salt bridge near PLP inside the active site pocket. Mutations of some interaction residues, such as Tyr71, Arg145, and Lys202, result in loss of the specific activity of the enzymes. In the interdomain loop, a dynamic loop (Gly173 to Gly179) clearly exhibits open and close conformations in structures of DrBCAT without and with substrates, respectively. DrBCAT shows the highest specific activity both in nature and under ionizing radiation, but with lower thermal stability above 60°C, than either BCAT from Escherichia coli (eBCAT) or from Thermus thermophilus (HB8BCAT). The dimeric molecular packing and the distribution of cysteine residues at the active site and the molecular surface might explain the resistance to radiation but small thermal stability of DrBCAT.
PMCID: PMC3486342  PMID: 22984263
3.  Induction of Apoptosis by Luteolin Involving Akt Inactivation in Human 786-O Renal Cell Carcinoma Cells 
There is a growing interest in the health-promoting effects of natural substances obtained from plants. Although luteolin has been identified as a potential therapeutic and preventive agent for cancer because of its potent cancer cell-killing activity, the molecular mechanisms have not been well elucidated. This study provides evidence of an alternative target for luteolin and sheds light on the mechanism of its physiological benefits. Treatment of 786-O renal cell carcinoma (RCC) cells (as well as A498 and ACHN) with luteolin caused cell apoptosis and death. This cytotoxicity was caused by the downregulation of Akt and resultant upregulation of apoptosis signal-regulating kinase-1 (Ask1), p38, and c-Jun N-terminal kinase (JNK) activities, probably via protein phosphatase 2A (PP2A) activation. In addition to being a concurrent substrate of caspases and event of cell death, heat shock protein-90 (HSP90) cleavage might also play a role in driving further cellular alterations and cell death, at least in part, involving an Akt-related mechanism. Due to the high expression of HSP90 and Akt-related molecules in RCC and other cancer cells, our findings suggest that PP2A activation might work in concert with HSP90 cleavage to inactivate Akt and lead to a vicious caspase-dependent apoptotic cycle in luteolin-treated 786-O cells.
PMCID: PMC3576787  PMID: 23476679
4.  Stereoselective Esterase from Pseudomonas putida IFO12996 Reveals α/β Hydrolase Folds for d-β-Acetylthioisobutyric Acid Synthesis 
Journal of Bacteriology  2005;187(24):8470-8476.
Esterase (EST) from Pseudomonas putida IFO12996 catalyzes the stereoselective hydrolysis of methyl dl-β-acetylthioisobutyrate (dl-MATI) to produce d-β-acetylthioisobutyric acid (DAT), serving as a key intermediate for the synthesis of angiotensin-converting enzyme inhibitors. The EST gene was cloned and expressed in Escherichia coli; the recombinant protein is a non-disulfide-linked homotrimer with a monomer molecular weight of 33,000 in both solution and crystalline states, indicating that these ESTs function as trimers. EST hydrolyzed dl-MATI to produce DAT with a degree of conversion of 49.5% and an enantiomeric excess value of 97.2% at an optimum pH of about 8 to 10 and an optimum temperature of about 57 to 67°C. The crystal structure of EST has been determined by X-ray diffraction to a resolution of 1.6 Å, confirming that EST is a member of the α/β hydrolase fold superfamily of enzymes and includes a catalytic triad of Ser97, Asp227, and His256. The active site is located approximately in the middle of the molecule at the end of a pocket ∼12 Å deep. EST can hydrolyze the methyl ester group without affecting the acetylthiol ester moiety in dl-MATI. The examination of substrate specificity of EST toward other linear esters revealed that the enzyme showed specific activity toward methyl esters and that it recognized the configuration at C-2.
PMCID: PMC1317000  PMID: 16321951
5.  Susceptibility of Human Embryonic Stem Cell-Derived Neural Cells to Japanese Encephalitis Virus Infection 
PLoS ONE  2014;9(12):e114990.
Pluripotent human embryonic stem cells (hESCs) can be efficiently directed to become immature neuroepithelial precursor cells (NPCs) and functional mature neural cells, including neurotransmitter-secreting neurons and glial cells. Investigating the susceptibility of these hESCs-derived neural cells to neurotrophic viruses, such as Japanese encephalitis virus (JEV), provides insight into the viral cell tropism in the infected human brain. We demonstrate that hESC-derived NPCs are highly vulnerable to JEV infection at a low multiplicity of infection (MOI). In addition, glial fibrillary acid protein (GFAP)-expressing glial cells are also susceptible to JEV infection. In contrast, only a few mature neurons were infected at MOI 10 or higher on the third day post-infection. In addition, functional neurotransmitter-secreting neurons are also resistant to JEV infection at high MOI. Moreover, we discover that vimentin intermediate filament, reported as a putative neurovirulent JEV receptor, is highly expressed in NPCs and glial cells, but not mature neurons. These results indicate that the expression of vimentin in neural cells correlates to the cell tropism of JEV. Finally, we further demonstrate that membranous vimentin is necessary for the susceptibility of hESC-derived NPCs to JEV infection.
PMCID: PMC4269419  PMID: 25517725
6.  Structures of Plasmodium vivax serine hydroxymethyltransferase: implications for ligand-binding specificity and functional control 
Crystal structures of P. vivax serine hydroxymethyltransferase (PvSHMT) in complex with l-serine and with d-serine and 5-formyltetrahydrofolate provide better understanding of ligand binding and the catalytic mechanism. Features that are important for controlling the activity and specificity of PvSHMT such as stereoselectivity and redox status are addressed.
Plasmodium parasites, the causative agent of malaria, rely heavily on de novo folate biosynthesis, and the enzymes in this pathway have therefore been explored extensively for antimalarial development. Serine hydroxymethyltransferase (SHMT) from Plasmodium spp., an enzyme involved in folate recycling and dTMP synthesis, has been shown to catalyze the conversion of l- and d-serine to glycine (Gly) in a THF-dependent reaction, the mechanism of which is not yet fully understood. Here, the crystal structures of P. vivax SHMT (PvSHMT) in a binary complex with l-serine and in a ternary complex with d-serine (d-Ser) and (6R)-5-formyl­tetra­hydro­folate (5FTHF) provide clues to the mechanism underlying the control of enzyme activity. 5FTHF in the ternary-complex structure was found in the 6R form, thus differing from the previously reported structures of SHMT–Gly–(6S)-5FTHF from other organisms. This suggested that the presence of d-Ser in the active site can alter the folate-binding specificity. Investigation of binding in the presence of d-Ser and the (6R)- or (6S)-5FTHF enantiomers indicated that both forms of 5FTHF can bind to the enzyme but that only (6S)-5FTHF gives rise to a quinonoid intermediate. Likewise, a large surface area with a highly positively charged electrostatic potential surrounding the PvSHMT folate pocket suggested a preference for a polyglutamated folate substrate similar to the mammalian SHMTs. Furthermore, as in P. falciparum SHMT, a redox switch created from a cysteine pair (Cys125–Cys364) was observed. Overall, these results assert the importance of features such as stereoselectivity and redox status for control of the activity and specificity of PvSHMT.
PMCID: PMC4257618  PMID: 25478836
Plasmodium vivax; serine hydroxymethyltransferase; antimalarial targets; d-serine; (6R)-5-formyltetrahydrofolate; redox switch
7.  Direct phase selection of initial phases from single-wavelength anomalous dispersion (SAD) for the improvement of electron density and ab initio structure determination 
A novel direct phase-selection method to select optimized phases from the ambiguous phases of a subset of reflections to replace the corresponding initial SAD phases has been developed. With the improved phases, the completeness of built residues of protein molecules is enhanced for efficient structure determination.
Optimization of the initial phasing has been a decisive factor in the success of the subsequent electron-density modification, model building and structure determination of biological macromolecules using the single-wavelength anomalous dispersion (SAD) method. Two possible phase solutions (ϕ1 and ϕ2) generated from two symmetric phase triangles in the Harker construction for the SAD method cause the well known phase ambiguity. A novel direct phase-selection method utilizing the θDS list as a criterion to select optimized phases ϕam from ϕ1 or ϕ2 of a subset of reflections with a high percentage of correct phases to replace the corresponding initial SAD phases ϕSAD has been developed. Based on this work, reflections with an angle θDS in the range 35–145° are selected for an optimized improvement, where θDS is the angle between the initial phase ϕSAD and a preliminary density-modification (DM) phase ϕDM NHL. The results show that utilizing the additional direct phase-selection step prior to simple solvent flattening without phase combination using existing DM programs, such as RESOLVE or DM from CCP4, significantly improves the final phases in terms of increased correlation coefficients of electron-density maps and diminished mean phase errors. With the improved phases and density maps from the direct phase-selection method, the completeness of residues of protein molecules built with main chains and side chains is enhanced for efficient structure determination.
PMCID: PMC4157445  PMID: 25195747
direct phase selection; ab initio structure determination; electron-density improvement
8.  Detection of subtle neurological alterations by the Catwalk XT gait analysis system 
A new version of the CatWalk XT system was evaluated as a tool for detecting very subtle alteration in gait based on higher speed sample rate; the system could also demonstrate minor changes in neurological function. In this study, we evaluated the neurological outcome of sciatic nerve injury intervened by local injection of hyaluronic acid. Using the CatWalk XT system, we looked for differences between treated and untreated groups and differences within the same group as a function of time so as to assess the power of the Catwalk XT system for detecting subtle neurological change.
Peripheral nerve injury was induced in 36 Sprague–Dawley rats by crushing the left sciatic nerve using a vessel clamp. The animals were randomized into one of two groups: Group I: crush injury as the control; Group II: crush injury and local application with hyaluronic acid. These animals were subjected to neurobehavior assessment, histomorphology evaluation, and electrophysiology study periodically. These data were retrieved for statistical analysis.
The density of neurofilament and S-100 over the distal end of crushed nerve showed significant differences either in inter-group comparison at various time points or intra-group comparison from 7 to 28 days. Neuronal structure architecture, axon counts, intensity of myelination, electrophysiology, and collagen deposition demonstrate significant differences between the two groups. There was significant difference of SFI and angle of ankle in inter- group analysis from 7 to 28 days, but there were no significant differences in SFI and angle of ankle at time points of 7 and 14 days. In the Cat Walk XT analysis, the intensity, print area, stance duration, and swing duration all showed detectable differences at 7, 14, 21, and 28 days, whereas there were no significant difference at 7 and 14 days with CatWalk 7 testing. In addition, there were no significant differences of step sequence or regularity index between the two versions.
Hyaluronic acid augmented nerve regeneration as early as 7 days after crush injury. This subtle neurological alteration could be detected through the CatWalk XT gait analysis but not the SFI, angle of ankle, or CatWalk 7 methods.
PMCID: PMC3997750  PMID: 24739213
9.  Divergent MicroRNA Targetomes of Closely Related Circulating Strains of a Polyomavirus 
Journal of Virology  2013;87(20):11135-11147.
Hundreds of virus-encoded microRNAs (miRNAs) have been uncovered, but an in-depth functional understanding is lacking for most. A major challenge for the field is separating those miRNA targets that are biologically relevant from those that are not advantageous to the virus. Here, we show that miRNAs from related variants of the polyomavirus simian vacuolating virus 40 (SV40) have differing host target repertoires (targetomes) while their direct autoregulatory activity on virus-encoded early gene products is completely preserved. These results underscore the importance of miRNA-mediated viral gene autoregulation in some polyomavirus life cycles. More broadly, these findings imply that some host targets of virus-encoded miRNAs are likely to be of little selective advantage to the virus, and our approach provides a strategy for prioritizing relevant targets.
PMCID: PMC3807300  PMID: 23926342
10.  FeoC from Klebsiella pneumoniae Contains a [4Fe-4S] Cluster 
Journal of Bacteriology  2013;195(20):4726-4734.
Iron is essential for pathogen survival, virulence, and colonization. Feo is suggested to function as the ferrous iron (Fe2+) transporter. The enterobacterial Feo system is composed of 3 proteins: FeoB is the indispensable component and is a large membrane protein likely to function as a permease; FeoA is a small Src homology 3 (SH3) domain protein that interacts with FeoB; FeoC is a winged-helix protein containing 4 conserved Cys residues in a sequence suitable for harboring a putative iron-sulfur (Fe-S) cluster. The presence of an iron-sulfur cluster on FeoC has never been shown experimentally. We report that under anaerobic conditions, the recombinant Klebsiella pneumoniae FeoC (KpFeoC) exhibited hyperfine-shifted nuclear magnetic resonance (NMR) and a UV-visible (UV-Vis) absorbance spectrum characteristic of a paramagnetic center. The electron paramagnetic resonance (EPR) and extended X-ray absorption fine structure (EXAFS) results were consistent only with the [4Fe-4S] clusters. Substituting the cysteinyl sulfur with oxygen resulted in significantly reduced cluster stability, establishing the roles of these cysteines as the ligands for the Fe-S cluster. When exposed to oxygen, the [4Fe-4S] cluster degraded to [3Fe-4S] and eventually disappeared. We propose that KpFeoC may regulate the function of the Feo transporter through the oxygen- or iron-sensitive coordination of the Fe-S cluster.
PMCID: PMC3807426  PMID: 23955005
11.  Viral MicroRNA Effects on Pathogenesis of Polyomavirus SV40 Infections in Syrian Golden Hamsters 
PLoS Pathogens  2014;10(2):e1003912.
Effects of polyomavirus SV40 microRNA on pathogenesis of viral infections in vivo are not known. Syrian golden hamsters are the small animal model for studies of SV40. We report here effects of SV40 microRNA and influence of the structure of the regulatory region on dynamics of SV40 DNA levels in vivo. Outbred young adult hamsters were inoculated by the intracardiac route with 1×107 plaque-forming units of four different variants of SV40. Infected animals were sacrificed from 3 to 270 days postinfection and viral DNA loads in different tissues determined by quantitative real-time polymerase chain reaction assays. All SV40 strains displayed frequent establishment of persistent infections and slow viral clearance. SV40 had a broad tissue tropism, with infected tissues including liver, kidney, spleen, lung, and brain. Liver and kidney contained higher viral DNA loads than other tissues; kidneys were the preferred site for long-term persistent infection although detectable virus was also retained in livers. Expression of SV40 microRNA was demonstrated in wild-type SV40-infected tissues. MicroRNA-negative mutant viruses consistently produced higher viral DNA loads than wild-type SV40 in both liver and kidney. Viruses with complex regulatory regions displayed modestly higher viral DNA loads in the kidney than those with simple regulatory regions. Early viral transcripts were detected at higher levels than late transcripts in liver and kidney. Infectious virus was detected infrequently. There was limited evidence of increased clearance of microRNA-deficient viruses. Wild-type and microRNA-negative mutants of SV40 showed similar rates of transformation of mouse cells in vitro and tumor induction in weanling hamsters in vivo. This report identified broad tissue tropism for SV40 in vivo in hamsters and provides the first evidence of expression and function of SV40 microRNA in vivo. Viral microRNA dampened viral DNA levels in tissues infected by SV40 strains with simple or complex regulatory regions.
Author Summary
The recent discovery of virally encoded microRNAs (miRNAs) raises the possibility of additional regulatory processes being involved in viral replication, immune recognition, and host cell survival. In this study, we sought to characterize the effect of SV40-encoded miRNAs and the structure of the viral regulatory region on infections in outbred Syrian golden hamsters. Results revealed that SV40 has a wide tissue tropism, including liver, kidney, spleen, lung, and brain, with kidney the preferred site for long-term persistent infection. Significant increases in tissue-associated viral DNA loads were observed with miRNA-negative mutant strains, whereas the presence of SV40 miRNAs had no effect on tumor induction and little effect on viral clearance. Our results provide the first evidence for SV40 miRNA expression and function in an in vivo animal model and highlight the complexity of regulation of SV40 viral replication and persistent infections.
PMCID: PMC3916418  PMID: 24516384
12.  Degradation of high affinity HuD targets releases Kv1.1 mRNA from miR-129 repression by mTORC1 
The Journal of Cell Biology  2013;202(1):53-69.
Inhibition of mTORC1 leads to the degradation of high affinity HuD target mRNAs, freeing HuD to bind Kv1.1 mRNA and promote its translation by overcoming miR-129–mediated repression.
Little is known about how a neuron undergoes site-specific changes in intrinsic excitability during neuronal activity. We provide evidence for a novel mechanism for mTORC1 kinase–dependent translational regulation of the voltage-gated potassium channel Kv1.1 messenger RNA (mRNA). We identified a microRNA, miR-129, that repressed Kv1.1 mRNA translation when mTORC1 was active. When mTORC1 was inactive, we found that the RNA-binding protein, HuD, bound to Kv1.1 mRNA and promoted its translation. Unexpectedly, inhibition of mTORC1 activity did not alter levels of miR-129 and HuD to favor binding to Kv1.1 mRNA. However, reduced mTORC1 signaling caused the degradation of high affinity HuD target mRNAs, freeing HuD to bind Kv1.1 mRNA. Hence, mTORC1 activity regulation of mRNA stability and high affinity HuD-target mRNA degradation mediates the bidirectional expression of dendritic Kv1.1 ion channels.
PMCID: PMC3704988  PMID: 23836929
13.  Depression-Like Effect of Prenatal Buprenorphine Exposure in Rats 
PLoS ONE  2013;8(12):e82262.
Studies indicate that perinatal opioid exposure produces a variety of short- and long-term neurobehavioral consequences. However, the precise modes of action are incompletely understood. Buprenorphine, a mixed agonist/antagonist at the opioid receptors, is currently being used in clinical trials for managing pregnant opioid addicts. This study provides evidence of depression-like consequence following prenatal exposure to supra-therapeutic dose of buprenorphine and sheds light on potential mechanisms of action in a rat model involving administration of intraperitoneal injection to pregnant Sprague-Dawley rats starting from gestation day 7 and lasting for 14 days. Results showed that pups at postnatal day 21 but not the dams had worse parameters of depression-like neurobehaviors using a forced swimming test and tail suspension test, independent of gender. Neurobehavioral changes were accompanied by elevation of oxidative stress, reduction of plasma levels of brain-derived neurotrophic factor (BDNF) and serotonin, and attenuation of tropomyosin-related kinase receptor type B (TrkB) phosphorylation, extracellular signal-regulated kinase (ERK) phosphorylation, protein kinase A activity, cAMP response element-binding protein (CREB) phosphorylation, and CREB DNA-binding activity. Since BDNF/serotonin and CREB signaling could orchestrate a positive feedback loop, our findings suggest that the induction of oxidative stress, reduction of BDNF and serotonin expression, and attenuation of CREB signaling induced by prenatal exposure to supra-therapeutic dose of buprenorphine provide evidence of potential mechanism for the development of depression-like neurobehavior.
PMCID: PMC3867331  PMID: 24367510
14.  Purification, crystallization and preliminary X-ray crystallographic analysis of the receiver and stalk domains (PA3346RS) of the response regulator PA3346 from Pseudomonas aeruginosa PAO1 
The crystallization of PA3346RS from P. aeruginosa is reported.
The regulatory domain (PA3346RS), comprising the receiver and stalk domains, of the response regulator PA3346 requires phosphorylation for activation with magnesium ions as cofactors in order to modulate the downstream protein phosphatase activity for the regulation of swarming motility in Pseudomonas aeruginosa PAO1. Fusion-tagged recombinant PA3346RS of total molecular mass 25.3 kDa has been overexpressed in Escherichia coli, purified using Ni2+–NTA and Q-­Sepharose ion-exchange columns and crystallized using the hanging-drop vapour-diffusion method. X-ray diffraction data were collected from PA3346RS crystals to 2.0 Å resolution. The crystal belonged to space group P41 or P43, with unit-cell parameters a = 82.38, c = 73.34 Å. Preliminary analysis indicated the presence of a dimer of PA3346RS in the asymmetric unit, with a solvent content of 48.6%.
PMCID: PMC3151133  PMID: 21821900
receiver domain; stalk domain; response regulator; Pseudomonas aeruginosa
15.  Oligomerization of Peptides LVEALYL and RGFFYT and Their Binding Affinity to Insulin 
PLoS ONE  2013;8(6):e65358.
Recently it has been proposed a model for fibrils of human insulin in which the fibril growth proceeds via stacking LVEALYL (fragment 11–17 from chain B of insulin) into pairs of tightly interdigitated -sheets. The experiments have also shown that LVEALYL has high propensity to self-assembly and binding to insulin. This necessitates study of oligomerization of LVEALYL and its binding affinity to full-length insulin. Using the all-atom simulations with Gromos96 43a1 force field and explicit water it is shown that LVEALYL can aggregate. Theoretical estimation of the binding free energy of LVEALYL to insulin by the molecular mechanic Poisson-Boltzmann surface area method reveals its strong binding affinity to chain B, implying that, in agreement with the experiments, LVEALYL can affect insulin aggregation via binding mechanism. We predict that, similar to LVEALYL, peptide RGFFYT (fragment B22-27) can self-assemble and bind to insulin modulating its fibril growth process. The binding affinity of RGFFYT is shown to be comparable with that of LVEALYL.
PMCID: PMC3689759  PMID: 23805182
16.  Crystal Structure of Dimeric Flavodoxin from Desulfovibrio gigas Suggests a Potential Binding Region for the Electron-Transferring Partner 
Flavodoxins, which exist widely in microorganisms, have been found in various pathways with multiple physiological functions. The flavodoxin (Fld) containing the cofactor flavin mononucleotide (FMN) from sulfur-reducing bacteria Desulfovibrio gigas (D. gigas) is a short-chain enzyme that comprises 146 residues with a molecular mass of 15 kDa and plays important roles in the electron-transfer chain. To investigate its structure, we purified this Fld directly from anaerobically grown D. gigas cells. The crystal structure of Fld, determined at resolution 1.3 Å, is a dimer with two FMN packing in an orientation head to head at a distance of 17 Å, which generates a long and connected negatively charged region. Two loops, Thr59–Asp63 and Asp95–Tyr100, are located in the negatively charged region and between two FMN, and are structurally dynamic. An analysis of each monomer shows that the structure of Fld is in a semiquinone state; the positions of FMN and the surrounding residues in the active site deviate. The crystal structure of Fld from D. gigas agrees with a dimeric form in the solution state. The dimerization area, dynamic characteristics and structure variations between monomers enable us to identify a possible binding area for its functional partners.
PMCID: PMC3565340  PMID: 23322018
flavodoxin (Fld); flavin mononucleotide (FMN); crystal structure; dimer; binding region
17.  Crystallization and preliminary X-ray crystallographic analysis of a full-length active form of the Cry4Ba toxin from Bacillus thuringiensis  
The crystallization of the Cry4Ba toxin from B. thuringiensis is described.
To obtain a complete structure of the Bacillus thuringiensis Cry4Ba mosquito-larvicidal protein, a 65 kDa functional form of the Cry4Ba-R203Q mutant toxin was generated for crystallization by eliminating the tryptic cleavage site at Arg203. The 65 kDa trypsin-resistant fragment was purified and crystallized using the sitting-drop vapour-diffusion method. The crystals belonged to the rhombohedral space group R32, with unit-cell parameters a = b = 184.62, c = 187.36 Å. Diffraction data were collected to at least 2.07 Å resolution using synchrotron radiation and gave a data set with an overall R merge of 9.1% and a completeness of 99.9%. Preliminary analysis indicated that the asymmetric unit contained one molecule of the active full-length mutant, with a V M coefficient and solvent content of 4.33 Å3 Da−1 and 71%, respectively.
PMCID: PMC2882780  PMID: 20516610
Cry4Ba mosquito-larvicidal protein; Bacillus thuringiensis
18.  Structure of Bacillus amyloliquefaciens α-amylase at high resolution: implications for thermal stability 
The crystal structure of B. amyloliquefaciens α-amylase (BAA) at 1.4 Å resolution revealed ambiguities in the thermal adaptation of homologous proteins in this family.
The crystal structure of Bacillus amyloliquefaciens α-amylase (BAA) at 1.4 Å resolution revealed ambiguities in the thermal adaptation of homologous proteins in this family. The final model of BAA is composed of two molecules in a back-to-back orientation, which is likely to be a consequence of crystal packing. Despite a high degree of identity, comparison of the structure of BAA with those of other liquefying-type α-amylases indicated moderate discrepancies at the secondary-structural level. Moreover, a domain-displacement survey using anisotropic B-factor and domain-motion analyses implied a significant con­tribution of domain B to the total flexibility of BAA, while visual inspection of the structure superimposed with that of B. licheniformis α-amylase (BLA) indicated higher flexibility of the latter in the central domain A. Therefore, it is suggested that domain B may play an important role in liquefying α-­amylases, as its rigidity offers a substantial improvement in thermostability in BLA compared with BAA.
PMCID: PMC2815676  PMID: 20124706
α-amylases; thermostability; flexibility; alignment
19.  Insights into Polyomaviridae MicroRNA Function Derived from Study of the Bandicoot Papillomatosis Carcinomatosis Viruses▿ 
Journal of Virology  2011;85(9):4487-4500.
Several different members of the Polyomaviridae, including some human pathogens, encode microRNAs (miRNAs) that lie antisense with respect to the early gene products, the tumor (T) antigens. These miRNAs negatively regulate T antigen expression by directing small interfering RNA (siRNA)-like cleavage of the early transcripts. miRNA mutant viruses of some members of the Polyomaviridae express increased levels of early proteins during lytic infection. However, the importance of miRNA-mediated negative regulation of the T antigens remains uncertain. Bandicoot papillomatosis carcinomatosis virus type 1 (BPCV1) is associated with papillomas and carcinomas in the endangered marsupial the western barred bandicoot (Perameles bougainville). BPCV1 is the founding member of a new group of viruses that remarkably share distinct properties in common with both the polyomavirus and papillomavirus families. Here, we show that BPCV1 encodes, in the same orientation as the papillomavirus-like transcripts, a miRNA located within a long noncoding region (NCR) of the genome. Furthermore, this NCR serves the function of both promoter and template for the primary transcript that gives rise to the miRNA. Unlike the polyomavirus miRNAs, the BPCV1 miRNA is not encoded antisense to the T antigen transcripts but rather lies in a separate, proximal region of the genome. We have mapped the 3′ untranslated region (UTR) of the BPCV1 large T antigen early transcript and identified a functional miRNA target site that is imperfectly complementary to the BPCV1 miRNA. Chimeric reporters containing the entire BPCV1 T antigen 3′ UTR undergo negative regulation when coexpressed with the BPCV1 miRNA. Notably, the degree of negative regulation observed is equivalent to that of an identical reporter that is engineered to bind to the BPCV1 miRNA with perfect complementarity. We also show that this miRNA and this novel mode of early gene regulation are conserved with the related BPCV2. Finally, papillomatous lesions from a western barred bandicoot express readily detectable levels of this miRNA, stressing its likely importance in vivo. Combined, the alternative mechanisms of negative regulation of T antigen expression between the BPCVs and the polyomaviruses support the importance of miRNA-mediated autoregulation in the life cycles of some divergent polyomaviruses and polyomavirus-like viruses.
PMCID: PMC3126244  PMID: 21345962
20.  Purification, crystallization and preliminary X-ray crystallographic analysis of xylose reductase from Candida tropicalis  
The crystallization of xylose reductase from C. tropicalis is reported.
Xylose reductase (XR), which requires NADPH as a co-substrate, catalyzes the reduction of d-xylose to xylitol, which is the first step in the metabolism of d-­xylose. The detailed three-dimensional structure of XR will provide a better understanding of the biological significance of XR in the efficient production of xylitol from biomass. XR of molecular mass 36.6 kDa from Candida tropicalis was crystallized using the hanging-drop vapour-diffusion method. According to X-ray diffraction data from C. tropicalis XR crystals at 2.91 Å resolution, the unit cell belongs to space group P31 or P32. Preliminary analysis indicated the presence of four XR molecules in the asymmetric unit, with 68.0% solvent content.
PMCID: PMC2664776  PMID: 19342796
xylose reductase; Candida tropicalis
21.  Purification, crystallization and preliminary X-ray analysis of an aminoacylhistidine dipeptidase (PepD) from Vibrio alginolyticus  
The aminoacylhistidine dipeptidase encoded by V. alginolyticus pepD has been overexpressed and crystallized.
The aminoacylhistidine dipeptidase (PepD) protein encoded by Vibrio algino­lyticus pepD was successfully overexpressed and characterized and the putative active-site residues responsible for metal binding and catalysis were identified. The purified enzyme contained two zinc ions per monomer. The recombinant dipeptidase enzyme, which was identified as a homodimer in solution, exhibited broad substrate specificity for Xaa-His dipeptides, with highest activity towards the His-His dipeptide. The purified protein was crystallized using the hanging-drop vapour-diffusion method. Preliminary crystallographic analysis showed that the crystal belonged to space group P61 or P65, with unit-cell parameters a = b = 80.42, c = 303.11 Å. The crystal contained two molecules per asymmetric unit and the predicted solvent content was 53.4%.
PMCID: PMC2650446  PMID: 19255468
aminoacylhistidine dipeptidases; Vibrio alginolyticus; metallopeptidases
22.  Comparison of the intervertebral disc spaces between axial and anterior lean cervical traction 
European Spine Journal  2009;18(11):1669-1676.
The insufficient investigations on the changes of spinal structures during traction prevent further exploring the possible therapeutic mechanism of cervical traction. A blind randomized crossover-design study was conducted to quantitatively compare the intervertebral disc spaces between axial and anterior lean cervical traction in sitting position. A total of 96 radiographic images from the baseline measurements, axial and anterior lean tractions in 32 asymptomatic subjects were digitized for further analysis. The intra- and inter-examiner reliabilities for measuring the intervertebral disc spaces were in good ranges (ICCs = 0.928–0.942). With the application of anterior lean traction, the statistical increases were detected both in anterior and in posterior disc spaces compared to the baseline (0.29 mm and 0.24 mm; both P < 0.01) and axial traction (0.16 mm and 0.35 mm; both P < 0.01). The greater intervertebral disc spaces obtained during anterior lean traction might be associated with the more even distribution of traction forces over the anterior and posterior neck structures. The neck extension moment through mandible that generally occurred in the axial traction could be counteracted by the downward force of head weight during anterior lean traction. This study quantitatively demonstrated that anterior lean traction in sitting position provided more intervertebral disc space enlargements in both anterior and posterior aspects than axial traction did. These findings may serve as a therapeutic reference when cervical traction is suggested.
PMCID: PMC2899394  PMID: 19533177
Axial traction; Anterior lean traction; Intervertebral disc space; Image analysis
23.  Protective effects of luteolin against lipopolysaccharide-induced acute lung injury involves inhibition of MEK/ERK and PI3K/Akt pathways in neutrophils 
Acta Pharmacologica Sinica  2010;31(7):831-838.
To investigate whether luteolin, the major polyphenolic components of Lonicera japonica, has beneficial effects against lipopolysaccharide (LPS)-induced acute lung injury (ALI) and to determine whether the protective mechanism involves anti-inflammatory effects on neutrophils.
ALI was induced with intratracheal instillation of LPS in mice. The level of ALI was determined by measuring the cell count and protein content in bronchoalveolar lavage (BAL) fluid. Neutrophils were stimulated with formyl-Met-Leu-Phe (fMLP) or LPS in vitro. Chemotaxis and superoxide anion generation were measured to evaluate neutrophil activation. The potential involvement of intracellular signaling molecules in regulating neutrophil activation was analyzed by using Western blot.
LPS induced ALI in mice, as evidenced with leukocyte infiltration and protein leakage into the lungs. Luteolin attenuated LPS-induced leukocyte infiltration and protein extravasation. In cell studies, luteolin attenuated the fMLP-induced neutrophil chemotaxis and respiratory burst (IC50 0.2±0.1 μmol/L and 2.2±0.8 μmol/L, respectively), but had a negligible effect on superoxide anion generation during phorbol myristate acetate stimulation. Furthermore luteolin effectively blocked MAPK/ERK kinase 1/2 (MEK), extracellular signal-regulated kinase (ERK), and Akt phosphorylation in fMLP- and LPS-stimulated neutrophils.
These results indicate that luteolin has beneficial effects against LPS-induced ALI in mice, and the attenuation of neutrophil chemotaxis and respiratory burst by luteolin involves the blockade of MEK-, ERK-, and Akt-related signaling cascades.
PMCID: PMC4007725  PMID: 20562902
acute lung injury; chemotaxis; luteolin; mitogen activated protein kinase; neutrophils; respiratory burst; PI3K/Akt
24.  Crystal Structure of Adenylylsulfate Reductase from Desulfovibrio gigas Suggests a Potential Self-Regulation Mechanism Involving the C Terminus of the β-Subunit ▿ †  
Journal of Bacteriology  2009;191(24):7597-7608.
Adenylylsulfate reductase (adenosine 5′-phosphosulfate [APS] reductase [APSR]) plays a key role in catalyzing APS to sulfite in dissimilatory sulfate reduction. Here, we report the crystal structure of APSR from Desulfovibrio gigas at 3.1-Å resolution. Different from the α2β2-heterotetramer of the Archaeoglobus fulgidus, the overall structure of APSR from D. gigas comprises six αβ-heterodimers that form a hexameric structure. The flavin adenine dinucleotide is noncovalently attached to the α-subunit, and two [4Fe-4S] clusters are enveloped by cluster-binding motifs. The substrate-binding channel in D. gigas is wider than that in A. fulgidus because of shifts in the loop (amino acid 326 to 332) and the α-helix (amino acid 289 to 299) in the α-subunit. The positively charged residue Arg160 in the structure of D. gigas likely replaces the role of Arg83 in that of A. fulgidus for the recognition of substrates. The C-terminal segment of the β-subunit wraps around the α-subunit to form a functional unit, with the C-terminal loop inserted into the active-site channel of the α-subunit from another αβ-heterodimer. Electrostatic interactions between the substrate-binding residue Arg282 in the α-subunit and Asp159 in the C terminus of the β-subunit affect the binding of the substrate. Alignment of APSR sequences from D. gigas and A. fulgidus shows the largest differences toward the C termini of the β-subunits, and structural comparison reveals notable differences at the C termini, activity sites, and other regions. The disulfide comprising Cys156 to Cys162 stabilizes the C-terminal loop of the β-subunit and is crucial for oligomerization. Dynamic light scattering and ultracentrifugation measurements reveal multiple forms of APSR upon the addition of AMP, indicating that AMP binding dissociates the inactive hexamer into functional dimers, presumably by switching the C terminus of the β-subunit away from the active site. The crystal structure of APSR, together with its oligomerization properties, suggests that APSR from sulfate-reducing bacteria might self-regulate its activity through the C terminus of the β-subunit.
PMCID: PMC2786610  PMID: 19820092
25.  Rapid SYBR Green I and Modified Probe Real-Time Reverse Transcription-PCR Assays Identify Influenza H1N1 Viruses and Distinguish between Pandemic and Seasonal Strains ▿  
Journal of Clinical Microbiology  2009;47(11):3714-3716.
A rapid SYBR green I real-time reverse transcription-PCR (RT-PCR) assay was developed to identify pandemic influenza H1N1 virus from clinical specimens in less than 1 h. Probe real-time RT-PCR influenza A/B, H1/H3, and swNP/swHA assays were modified into the same PCR program, which allows for rapid and simultaneous typing and subtyping of influenza viruses.
PMCID: PMC2772634  PMID: 19741076

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