Biomedical subjects

Xiaodong Zhang

Publications and source records attributed to Xiaodong Zhang.

At least 19 recordsLinked to original sources

PubMed2007

Mechanism of methanol oxidation by quinoprotein methanol dehydrogenase.

At neutral pH, oxidation of CH(3)OH --> CH(2)O by an o-quinone requires general-base catalysis and the reaction is endothermic. The active-site -CO(2)(-) groups of Glu-171 and Asp-297 (Glu-171-CO(2)(-) and Asp-297-CO(2)(-)) have been considered as the required general base catalysts in the bacterial o-quinoprotein methanol dehydrogenase (MDH) reaction. Based on quantum mechanics/molecular mechanics (QM/MM) calculations, the free energy for MeOH reduction of o-PQQ when MeOH is hydrogen bonded to Glu-171-CO(2)(-) and the crystal water (Wat1) is hydrogen bonded to Asp-297-CO(2)(-) is DeltaG++ = 11.7 kcal/mol, which is comparable with the experimental value of 8.5 kcal/mol. The calculated DeltaG++ when MeOH is hydrogen bonded to Asp-297-CO(2)(-) is >50 kcal/mol. The Asp-297-CO(2)(-)...Wat1 complex is very stable. Molecular dynamics (MD) simulations on MDH.PQQ.Wat1 complex in TIP3P water for 5 ns does not result in interchange of Asp-297-CO(2)(-) bound Wat1 for a solvent water. Starting with Wat1 removed and MeOH hydrogen bonded to Asp-297-CO(2)(-), we find that MeOH returns to be hydrogen bonded to Glu-171-CO(2)(-) and Asp-297-CO(2)(-) coordinates to Ca(2+) during 3 ns simulation. The Asp-297-CO(2)(-)...Wat1 of reactant complex does play a crucial role in catalysis. By QM/MM calculation DeltaG++ = 1.1 kcal/mol for Asp-297-CO(2)(-) general-base catalysis of Wat1 hydration of the immediate CH(2)==O product --> CH(2)(OH)(2). By this means, the endothermic oxidation-reduction reaction is pulled such that the overall conversion of MeOH to CH(2)(OH)(2) is exothermic.

Alcohol Oxidoreductases↗

PubMed2007

Structural insights into the p97-Ufd1-Npl4 complex.

p97/VCP (Cdc48 in yeast) is an essential and abundant member of the AAA+ family of ATPases and is involved in a number of diverse cellular pathways through interactions with different adaptor proteins. The two most characterized adaptors for p97 are p47 and the Ufd1 (ubiquitin fusion degradation 1)-Npl4 (nuclear protein localization 4) complex. p47 directs p97 to membrane fusion events and has been shown to be involved in protein degradation. The Ufd1-Npl4 complex directs p97 to an essential role in endoplasmic reticulum-associated degradation and an important role in mitotic spindle disassembly postmitosis. Here we describe the structural features of the Ufd1-Npl4 complex and its interaction with p97 with the aid of EM and other biophysical techniques. The Ufd1-Npl4 heterodimer has an elongated bilobed structure that is approximately 80 x 30 A in dimension. One Ufd1-Npl4 heterodimer is shown to interact with one p97 hexamer to form the p97-Ufd1-Npl4 complex. The Ufd1-Npl4 heterodimer emanates from one region on the periphery of the N-D1 plane of the p97 hexamer. Intriguingly, the p97-p47 and the p97-Ufd1-Npl4 complexes are significantly different in stoichiometry, symmetry, and quaternary arrangement, reflecting their specific actions and their ability to interact with additional cofactors that cooperate with p97 in diverse cellular pathways.

Adenosine Triphosphatases↗

PubMed2006

Bacterial enhancer-binding proteins: unlocking sigma54-dependent gene transcription.

Bacterial transcription relies on the binding of dissociable sigma (sigma) factors to RNA polymerase (RNAP) for promoter specificity. The major variant sigma factor (sigma54) forms a stable closed complex with RNAP bound to DNA that rarely spontaneously isomerises to an open complex. ATP hydrolysis by bacterial enhancer-binding proteins is used to remodel the RNAP-sigma54-DNA closed complex. Recently, a wealth of structural information on bacterial enhancer-binding proteins has enabled unprecedented insights into their mechanism. These data provide a structural basis for nucleotide binding and hydrolysis, oligomerisation and the conversion of ATPase activity into remodelling events within the RNAP-sigma54 closed complex, and represent advances towards a complete understanding of the sigma54-dependent transcription activation mechanism.

Bacterial Proteins↗

PubMed2006

Antidiabetic activity of lipophilic (-)-epigallocatechin-3-gallate derivative under its role of alpha-glucosidase inhibition.

(-)-Epigallocatechin-3-gallate (EGCG), a component of catechins, has been shown to reduce blood glucose levels. In the present study, we investigated the antidiabetic activity and its mechanism of lipophilic EGCG derivative (L-EGCGd) in streptozotocin (STZ)-induced diabetic rats. L-EGCGd was chemically modified from traditional hydrophilic EGCG. After 30 days treatment, plasma levels of glucose were significantly reduced by 40.5+/-7.0% and 17.0+/-2.8% in groups administered 50 or 25 mg kg(-1)d(-1) L-EGCGd, respectively, as compared with that in the diabetic control group. Lipid metabolites, such as total cholesterol (TC), triglyceride (TG) and low-density lipoprotein cholesterol (LDLC) were effectively attenuated by L-EGCGd administration, but plasma HDLC levels did not change significantly. The oral glucose tolerance test (OGTT) greatly revealed the improved ability of glucose tolerance with treatment of L-EGCGd. L-EGCGd only retarded the postprandial rise in blood glucose with sucrose loading but not glucose loading. And activity of alpha-glucosidase was inhibited by 50% at the concentration of 246.6 microg ml(-1) L-EGCGd. As a result, we first demonstrated that the purified form of compound L-EGCGd possessed the hypoglycemic effect under its role of alpha-glucosidase inhibition, and therefore should be possibly accepted as an alternative oral medication protecting patients against postprandial hyperglycemic toxicity on the treatment of diabetes and its complications.

Animals↗

PubMed2006

Synthetic inhibitors of cytochrome P-450 2A6: inhibitory activity, difference spectra, mechanism of inhibition, and protein cocrystallization.

A series of 3-heteroaromatic analogues of nicotine were synthesized to delineate structural and mechanistic requirements for selectively inhibiting human cytochrome P450 (CYP) 2A6. Thiophene, substituted thiophene, furan, substituted furan, acetylene, imidazole, substituted imidazole, thiazole, pyrazole, substituted pyrazole, and aliphatic and isoxazol moieties were used to replace the N-methylpyrrolidine ring of nicotine. A number of potent inhibitors were identified, and several exhibited high selectivity for CYP2A6 relative to CYP2E1, -3A4, -2B6, -2C9, -2C19, and -2D6. The majority of these inhibitors elicited type II difference spectra indicating the formation of a coordinate covalent bond to the heme iron. The majority of inhibitors were reversible inhibitors although several mechanism-based inactivators were identified. Most of the inhibitors were also relatively metabolically stable. X-ray crystal structures of CYP2A6 cocrystallized with three furan analogues bearing methanamino side chains indicated that the amine side chain coordinated to the heme iron. The pyridyl moiety was positioned to accept a hydrogen bond from Asn297, and all three inhibitors exhibited orthogonal aromatic-aromatic interactions with protein side chains. For comparison, the cocrystal structure of 4,4'-dipyridyl disulfide was also obtained and showed that the pyridine moiety could assume a different orientation than that observed for the 3-heteroaromatic pyridines examined. For the 3-heteroromatic pyridines, N-methyl and N,N-dimethyl amino groups increased the apparent Ki and distorted helix I of the protein. Substitution of a phenyl ring for the pyridyl ring also increased the apparent Ki, which is likely to reflect the loss of the hydrogen bonding interaction with Asn297. In contrast, inhibitory potency for other P450s was increased, and the selectivity of the phenyl analogues for CYP2A6 was decreased relative to the pyridyl compounds. The results suggest that inhibitors that compliment the active site features of CYP2A6 can exhibit significant selectivity for CYP2A6 relative to other human liver drug-metabolizing P450s.

Acetylene↗

PubMed2006

Quantitative basal CBF and CBF fMRI of rhesus monkeys using three-coil continuous arterial spin labeling.

A three-coil continuous arterial-spin-labeling technique with a separate neck labeling coil was implemented on a Siemens 3T Trio for quantitative cerebral blood flow (CBF) and CBF fMRI measurements in non-human primates (rhesus monkeys). The optimal labeling power was 2 W, labeling efficiency was 92+/-2%, and optimal post-labeling delay was 0.8 s. Gray matter (GM) and white matter (WM) were segmented based on T1 maps. Quantitative CBF were obtained in 3 min with 1.5-mm isotropic resolution. Whole-brain average DeltaS/S was 1.0-1.5%. GM CBF was 104+/-3 ml/100 g/min (n = 6, SD) and WM CBF was 45+/-6 ml/100 g/min in isoflurane-anesthetized rhesus monkeys, with the CBF GM/WM ratio of 2.3+/-0.2. Combined CBF and BOLD (blood-oxygenation-level-dependent) fMRI associated with hypercapnia and hyperoxia were made with 8-s temporal resolution. CBF fMRI responses to 5% CO2 were 59+/-10% (GM) and 37+/-4% (WM); BOLD fMRI responses were 2.0+/-0.4% (GM) and 1.2+/-0.4% (WM). CBF fMRI responses to 100% O2 were -9.4+/-2% (GM) and -3.9+/-2.6% (WM); BOLD responses were 2.4+/-0.7% (GM) and 0.8+/-0.2% (WM). The use of a separate neck coil for spin labeling significantly increased CBF signal-to-noise ratio and the use of small receive-only surface coil significantly increased signal-to-noise ratio and spatial resolution. This study sets the stage for quantitative perfusion imaging and CBF fMRI for neurological diseases in anesthetized and awake monkeys.

Animals↗

PubMed2006

Reaction mechanism of guanidinoacetate methyltransferase, concerted or step-wise.

We describe a quantum mechanics/molecular mechanics investigation of the guanidinoacetate methyltransferase catalyzed reaction, which shows that proton transfer from guanidinoacetate (GAA) to Asp-134 and methyl transfer from S-adenosyl-L-methionine (AdoMet) to GAA are concerted. By self-consistent-charge density functional tight binding/molecular mechanics, the bond lengths in the concerted mechanism's transition state are 1.26 A for both the OD1 (Asp-134)-H(E) (GAA) and H(E) (GAA)-N(E) (GAA) bonds, and 2.47 and 2.03 A for the S8 (AdoMet)-C9 (AdoMet) and C9 (AdoMet)-N(E) (GAA) bonds, respectively. The potential-energy barrier (DeltaE++) determined by single-point B3LYP/6-31+G*//MM is 18.9 kcal/mol. The contributions of the entropy (-TDeltaS++) and zero-point energy corrections Delta(ZPE)++ by normal mode analysis are 2.3 kcal/mol and -1.7 kcal/mol, respectively. Thus, the activation enthalpy of this concerted mechanism is predicted to be DeltaH++ = DeltaE++ plus Delta(ZPE)++ = 17.2 kcal/mol. The calculated free-energy barrier for the concerted mechanism is DeltaG++ = 19.5 kcal/mol, which is in excellent agreement with the value of 19.0 kcal/mol calculated from the experimental rate constant (3.8 +/- 0.2.min(-1)).

Binding Sites↗

PubMed2006

Synthesis and evaluation of a new series of substituted acyl(thio)urea and thiadiazolo [2,3-a] pyrimidine derivatives as potent inhibitors of influenza virus neuraminidase.

A series of substituted acyl(thio)urea and 2H-1,2,4-thiadiazolo [2,3-a] pyrimidine derivatives were prepared and both of their cell culture and enzymatic activity toward influenza virus were tested. Their in vitro neuraminidase inhibitory activities were in good agreement with the corresponding activities in cultured cells and they were evaluated as potent neuraminidase inhibitors. Of the analogues that demonstrated IC(50)s<0.1microM, 16 and 60 were further investigated as candidates with the most potential for future development. The molecular docking work of the representative compound was described to provide more insight into their mechanism of action and further rationalize the observations of this new series herein, which represents a novel class of highly potent and selective inhibitors of influenza virus.

Animals↗

PubMed2006

Structure of the whole cytosolic region of ATP-dependent protease FtsH.

An ATP-dependent protease, FtsH, digests misassembled membrane proteins in order to maintain membrane integrity and digests short-lived soluble proteins in order to control their cellular regulation. This enzyme has an N-terminal transmembrane segment and a C-terminal cytosolic region consisting of an AAA+ ATPase domain and a protease domain. Here we present two crystal structures: the protease domain and the whole cytosolic region. The cytosolic region fully retains an ATP-dependent protease activity and adopts a three-fold-symmetric hexameric structure. The protease domains displayed a six-fold symmetry, while the AAA+ domains, each containing ADP, alternate two orientations relative to the protease domain, making "open" and "closed" interdomain contacts. Apparently, ATPase is active only in the closed form, and protease operates in the open form. The protease catalytic sites are accessible only through a tunnel following from the AAA+ domain of the adjacent subunit, raising a possibility of translocation of polypeptide substrate to the protease sites through this tunnel.

Adenosine Triphosphate↗

PubMed2006

Beam angle optimization and reduction for intensity-modulated radiation therapy of non-small-cell lung cancers.

PURPOSE: To optimize beam angles and reduce the number of beams used for intensity-modulated radiation therapy (IMRT) of non-small-cell lung cancer (NSCLC). METHODS AND MATERIALS: An exhaustive search scheme was used to perform beam angle optimization (BAO) for IMRT of NSCLC. This approach involved intercomparison of all possible beam angle combinations and selection of the best angles based on the scores or costs of the objective functions used in the treatment plan optimization. Ten Stage III NSCLC cases were selected to evaluate the BAO algorithm and dosimetry benefits of IMRT-BAO. IMRT plans using five or seven coplanar beams were optimized and compared with those using nine equal-spaced beams. Results of BAO were also compared between plans using different numbers of beams with or without fluence modulation. RESULTS: Each anatomic structure, e.g., tumor or lung, had its own preferred beam angles. Thus, BAO required appropriate balance of competing objective functions. Plans using fewer angles (five or seven beams) could achieve plan quality similar to those using nine equal-spaced beams, however with reduced monitor units and field segments. The number of beams used for the treatment (five vs. seven) and the fluence modulation (open or IMRT beams) did not have a significant impact on the results of the BAO. CONCLUSIONS: Use of fewer beams (e.g., five) for lung IMRT could result in acceptable plan quality but improved treatment efficiency. A multiresolution search scheme could be developed for BAO using fewer and nonmodulated beams to reduce the computation cost of BAO.

Algorithms↗

PubMed2006

Significant reduction of normal tissue dose by proton radiotherapy compared with three-dimensional conformal or intensity-modulated radiation therapy in Stage I or Stage III non-small-cell lung cancer.

PURPOSE: To compare dose-volume histograms (DVH) in patients with non-small-cell lung cancer (NSCLC) treated by photon or proton radiotherapy. METHODS AND MATERIALS: Dose-volume histograms were compared between photon, including three-dimensional conformal radiation therapy (3D-CRT), intensity-modulated radiation therapy (IMRT), and proton plans at doses of 66 Gy, 87.5 Gy in Stage I (n=10) and 60-63 Gy, and 74 Gy in Stage III (n=15). RESULTS: For Stage I, the mean total lung V5, V10, and V20 were 31.8%, 24.6%, and 15.8%, respectively, for photon 3D-CRT with 66 Gy, whereas they were 13.4%, 12.3%, and 10.9%, respectively, with proton with dose escalation to 87.5 cobalt Gray equivalents (CGE) (p=0.002). For Stage III, the mean total lung V5, V10, and V20 were 54.1%, 46.9%, and 34.8%, respectively, for photon 3D-CRT with 63 Gy, whereas they were 39.7%, 36.6%, and 31.6%, respectively, for proton with dose escalation to 74 CGE (p=0.002). In all cases, the doses to lung, spinal cord, heart, esophagus, and integral dose were lower with proton therapy even compared with IMRT. CONCLUSIONS: Proton treatment appears to reduce dose to normal tissues significantly, even with dose escalation, compared with standard-dose photon therapy, either 3D-CRT or IMRT.

Carcinoma, Non-Small-Cell Lung↗

PubMed2006

The mechanism of M.HhaI DNA C5 cytosine methyltransferase enzyme: a quantum mechanics/molecular mechanics approach.

The mechanism of DNA cytosine-5-methylation catalyzed by the bacterial M.HhaI enzyme has been considered as a stepwise nucleophilic addition of Cys-81-S- to cytosine C6 followed by C5 nucleophilic replacement of the methyl of S-adenosyl-L-methionine to produce 5-methyl-6-Cys-81-S-5,6-dihydrocytosine. In this study, we show that the reaction is concerted from a series of energy calculations by using the quantum mechanical/molecular mechanical hybrid method. Deprotonation of 5-methyl-6-Cys-81-S-5,6-dihydrocytosine and expulsion of Cys-81-S- provides the product DNA 5-methylcytosine. A required base catalyst for this deprotonation is not available as a member of the active site structure. A water channel between the active site and bulk water allows entrance of solvent to the active site. Hydroxide at 10(-7) mole fraction (pH = 7) is shown to be sufficient for the required catalysis. We also show that Glu-119-CO2H can divert the reaction by protonating cytosine N3 when Cys-81-S- attacks cytosine, to form the 6-Cys-81-S-3-hydrocytosine. The reactants and 6-Cys-81-S-3-hydrocytosine product are in rapid equilibrium, and this explains the observed hydrogen exchange of cytosine with solvent.

Binding Sites↗

PubMed2006

Conformational changes in the AAA ATPase p97-p47 adaptor complex.

The AAA+ATPase p97/VCP, helped by adaptor proteins, exerts its essential role in cellular events such as endoplasmic reticulum-associated protein degradation or the reassembly of Golgi, ER and the nuclear envelope after mitosis. Here, we report the three-dimensional cryo-electron microscopy structures at approximately 20 Angstroms resolution in two nucleotide states of the endogenous hexameric p97 in complex with a recombinant p47 trimer, one of the major p97 adaptor proteins involved in membrane fusion. Depending on the nucleotide state, we observe the p47 trimer to be in two distinct arrangements on top of the p97 hexamer. By combining the EM data with NMR and other biophysical measurements, we propose a model of ATP-dependent p97(N) domain motions that lead to a rearrangement of p47 domains, which could result in the disassembly of target protein complexes.

Adenosine Triphosphatases↗

PubMed2006

Going through the motions: the ATPase cycle of p97.

p97 (VCP, Cdc48), a type II AAA+ ATPase family member, is ubiquitous, essential, highly abundant, and involved in a diverse range of biological functions with roles in membrane fusion, endoplasmic-reticulum associated degradation, transcriptional activation, and cell cycle control. As such, dysfunction of this protein has serious pathological consequences and has been implicated in a variety of cancers and neurodegenerative diseases. p97 has a large number of adaptor proteins through which it transmits energy from ATPase activity to conformational changes which are then exerted onto target proteins. p97 has been studied by a variety of biochemical and structural techniques at various resolutions and stages throughout its ATPase cycle. From these studies, many models have been proposed and consequently a single model for p97's action cannot be suggested. Many questions about the mechanism of p97 still remain, including whether the protomers act in a concerted manner and crucially how the induced changes in p97 are transmitted to its adaptor proteins and target substrates. The elucidation of p97's mechanism is not only important in furthering our knowledge of this intriguing protein and its many functions, but subsequently in the development of potential therapies for diseases associated with p97 dysfunction.

Adenosine Triphosphatases↗

PubMed2006

Structures and organisation of AAA+ enhancer binding proteins in transcriptional activation.

Initiation of transcription is a major point of transcriptional regulation and invariably involves the transition from a closed to an open RNA polymerase (RNAP) promoter complex. In the case of the sigma(54)-RNAP, this multi step process requires energy, provided by ATP hydrolysis occurring within the AAA+ domain of enhancer binding proteins (EBPs). Typically, EBPs have an N-terminal regulatory domain, a central AAA+ domain that directly contacts sigma(54) and a C-terminal DNA binding domain. The following AAA+ EBP crystal structures have recently become available: heptameric AAA+ domains of NtrC1 and dimeric NtrC1 with its regulatory domain, hexameric AAA+ domains of ZraR with DNA binding domains, apo and nucleotide bound forms of the AAA+ domain of PspF as well as a cryo-EM structure of the AAA+ domain of PspF complexed with sigma(54). These AAA+ domains reveal the structural conservation between EBPs and other AAA+ domains. EBP specific structural features involved in substrate remodelling are located proximal to the pore of the hexameric ring. Parallels with the substrate binding elements near the central pore of other AAA+ members are drawn. We propose a structural model of EBPs in complex with a sigma(54)-RNAP-promoter complex.

Adenosine Triphosphate↗

PubMed2006

Induction of neuronal differentiation of adult human olfactory neuroepithelial-derived progenitors.

Neurosphere forming cells (NSFCs) have been established from cultures of adult olfactory neuroepithelium obtained from patients and cadavers as described previously. They remained undifferentiated in serum or defined media with or without neurotrophic factors. Many factors affect the differentiation of stem cells along a neuronal pathway. Retinoic acid (RA), forskolin (FN), and sonic hedgehog (Shh) have been reported to act as growth promoters during neurogenesis of embryonic CNS in vivo. The effect of RA, FN, and Shh on NSFCs' neuronal lineage restriction has not been described. The application of RA, FN, and Shh to NSFCs induced the expression of motoneuronal transcription factors, tyrosine hydroxylase, an indicator of dopamine production, and neurite formation. These studies further heighten the potential for using olfactory neuroepithelial progenitors for future autologous cell replacement strategies in neurodegenerative conditions and trauma as well as for use in diagnostic evaluation.

Adult↗

PubMed2006

Procedures for a dynamical system on {0,1}n with DNA molecules.

In this paper, an improved form of DNA representations of elements in {0,1}n, which was first proposed by Fujiwara et al. [Fujiwara, A., Matsumoto, K., Chen, W., 2004. Procedures for logic and arithmetic operations with DNA molecules. Int. J. Found. Comput. Sci. 15, 461-474], is given. Using this improved representations, a procedure for cycling shift is proposed, and this procedure can be implemented in O(1) lab steps theoretically. Based on the operation for cycling shift, dynamic behavior of an operator on {0,1}n is investigated by DNA molecules.

Algorithms↗

PubMed2006

Structural basis of the nucleotide driven conformational changes in the AAA+ domain of transcription activator PspF.

Bacterial enhancer-binding proteins (EBP) activate transcription by hydrolyzing ATP to restructure the sigma(54)-RNA polymerase-promoter complex. We compare six high resolution structures (<2.1 A) of the AAA(+) domain of EBP phage shock protein F (PspF) including apo, AMPPNP, Mg(2+)-ATP, and ADP forms. These structures permit a description of the atomic details underpinning the origins of the conformational changes occurring during ATP hydrolysis. Conserved regions of PspF's AAA(+) domain respond distinctively to nucleotide binding and hydrolysis, suggesting functional roles during the hydrolysis cycle, which completely agree with those derived from activities of PspF mutated at these positions. We propose a putative atomic switch that is responsible for coupling structural changes in the nucleotide-binding site to the repositioning of the sigma(54)-interacting loops. Striking similarities in nucleotide-specific conformational changes and atomic switch exist between PspF and the large T antigen helicase, suggesting conservation in the origin of those events amongst AAA(+) proteins.

Adenosine Diphosphate↗