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Haitao Li

Publications and source records attributed to Haitao Li.

At least 19 recordsLinked to original sources
PubMed2006

Yng1 PHD finger binding to H3 trimethylated at K4 promotes NuA3 HAT activity at K14 of H3 and transcription at a subset of targeted ORFs.

Posttranslational histone modifications participate in modulating the structure and function of chromatin. Promoters of transcribed genes are enriched with K4 trimethylation and hyperacetylation on the N-terminal tail of histone H3. Recently, PHD finger proteins, like Yng1 in the NuA3 HAT complex, were shown to interact with H3K4me3, indicating a biochemical link between K4 methylation and hyperacetylation. By using a combination of mass spectrometry, biochemistry, and NMR, we detail the Yng1 PHD-H3K4me3 interaction and the importance of NuA3-dependent acetylation at K14. Furthermore, genome-wide ChIP-Chip analysis demonstrates colocalization of Yng1 and H3K4me3 in vivo. Disrupting the K4me3 binding of Yng1 altered K14ac and transcription at certain genes, thereby demonstrating direct in vivo evidence of sequential trimethyl binding, acetyltransferase activity, and gene regulation by NuA3. Our data support a general mechanism of transcriptional control through which histone acetylation upstream of gene activation is promoted partially through availability of H3K4me3, "read" by binding modules in select subunits.

Chromatin Immunoprecipitation↗
PubMed2006

Characterization of a single molecule DNA switch in free solution.

We have studied a donor-acceptor fluorophore-labeled DNA switch where the acceptor is Alexa-647, a carbocyanine dye, in solution at the single molecule level to elucidate the fluorescence switching mechanism. The acceptor, which is in an initial high fluorescence trans state, undergoes a photoisomerization reaction resulting in two additional states during its sub-millisecond transit across the probe volume. These two states are assigned to a nonfluorescent triplet trans state that strongly quenches the donor emission and a singlet cis state that blocks the fluorescence resonance energy transfer (FRET) pathway and gives rise to donor-only fluorescence. The formation of these states is faster than the transit time, so that all three states are approximately equally populated under our experimental conditions. The acceptor dye can stick to the DNA in all these states, with the rate of unsticking determining the rate of isomerization into the other states. Measurement of the rate of change of the FRET signal therefore provides information about the fluorophore-DNA intramolecular dynamics. These results explain the large zero peak in the proximity ratio, often seen in single molecule FRET experiments, and suggest that photoinduced effects may be important in single molecule FRET experiments using carbocyanine dyes. They also suggest that for fast photoinduced switching the interactions of the acceptor dye with the DNA and other surfaces should be prevented.

Carbocyanines↗
PubMed2006

Emdogain-gel stimulates proliferation of odontoblasts and osteoblasts.

OBJECTIVE: The purpose of this study was to determine whether a premixed form of enamel matrix derivative (EMD), Emdogain-gel, has the same property as the original formula of EMD in stimulating the proliferation of osteoblasts and odontoblasts. STUDY DESIGN: Osteoblast cell line (MC3T3) and odontoblast cell line (MDPC) were cultured in the 6-well culture plates and treated in 4 different groups: (1) culture medium control, (2) 100 microg/mL Emdogain-gel directly added to the culture medium, (3) culture medium with a culture plate insert, and (4) 100 microg/mL Emdogain-gel added onto a culture plate insert. The culture plate insert prevented direct contact between Emdogain-gel and the cells. After 3-day incubation, cell morphology was examined and the total cell number per well was counted. Data were analyzed using 1-way ANOVA. RESULTS: Emdogain-gel significantly increased cell number of both osteoblasts and odontoblasts regardless the presence of the culture plate insert. CONCLUSION: Emdogain-gel stimulates cell proliferation of odontoblasts and osteoblasts. The direct contact between Emdogain-gel and cells is not required. Heat treatment of EMD and premix with propylene glycol alginate did not change its property of releasing bioactive molecules for promoting cell proliferation.

3T3 Cells↗
PubMed2006

Histone H3 recognition and presentation by the WDR5 module of the MLL1 complex.

WDR5 is a core component of SET1-family complexes that achieve transcriptional activation via methylation of histone H3 on Nzeta of Lys4 (H3K4). The role of WDR5 in the MLL1 complex has recently been described as specific recognition of dimethyl-K4 in the context of a histone H3 amino terminus; WDR5 is essential for vertebrate development, Hox gene activation and global H3K4 trimethylation. We report the high-resolution X-ray structures of WDR5 in the unliganded form and complexed with histone H3 peptides having unmodified and mono-, di- and trimethylated K4, which together provide the first comprehensive analysis of methylated histone recognition by the ubiquitous WD40-repeat fold. Contrary to predictions, the structures reveal that WDR5 does not read out the methylation state of K4 directly, but instead serves to present the K4 side chain for further methylation by SET1-family complexes.

Amino Acid Sequence↗
PubMed2006

Soybean oil methyl esters preparation using NaX zeolites loaded with KOH as a heterogeneous catalyst.

The transesterification of soybean oil with methanol to methyl esters was carried out using NaX zeolites loaded with KOH as a solid base catalyst. Best result was obtained with NaX zeolite loaded with 10% KOH, followed by heating at 393 K for 3 h. When the transesterification reaction was carried out at reflux of methanol (338 K), with a 10:1 molar ratio of methanol to soybean oil, a reaction time of 8 h and a catalyst amount of 3 wt.%, the conversion of soybean oil was 85.6%.

Catalysis↗
PubMed2006

Discovery of a novel, orally active, small molecule gonadotropin-releasing hormone (GnRH) receptor antagonist.

Gonadotropin releasing hormone (GnRH) plays an important role in the biology of reproduction. The use of GnRH receptor antagonists has been reported in the literature for the treatment of breast, ovarian, and prostate cancers. In this article, we report the synthesis, in vitro characterization, pharmacokinetics, and pharmacodynamics of an orally bioavailable, potent, small molecule GnRH receptor antagonist N-{4,6-dimethoxy-2-[(3-morpholin-4-ylpropyl)amino]pyrimidin-5-yl}-5-[3,3,6-trimthyl-2,3-dihydro-1H-inden-5-yl)oxy]-2-furamide (compound 1).

Administration, Oral↗
PubMed2006

Molecular basis for site-specific read-out of histone H3K4me3 by the BPTF PHD finger of NURF.

Mono-, di- and trimethylated states of particular histone lysine residues are selectively found in different regions of chromatin, thereby implying specialized biological functions for these marks ranging from heterochromatin formation to X-chromosome inactivation and transcriptional regulation. A major challenge in chromatin biology has centred on efforts to define the connection between specific methylation states and distinct biological read-outs impacting on function. For example, histone H3 trimethylated at lysine 4 (H3K4me3) is associated with transcription start sites of active genes, but the molecular 'effectors' involved in specific recognition of H3K4me3 tails remain poorly understood. Here we demonstrate the molecular basis for specific recognition of H3(1-15)K4me3 (residues 1-15 of histone H3 trimethylated at K4) by a plant homeodomain (PHD) finger of human BPTF (bromodomain and PHD domain transcription factor), the largest subunit of the ATP-dependent chromatin-remodelling complex, NURF (nucleosome remodelling factor). We report on crystallographic and NMR structures of the bromodomain-proximal PHD finger of BPTF in free and H3(1-15)K4me3-bound states. H3(1-15)K4me3 interacts through anti-parallel beta-sheet formation on the surface of the PHD finger, with the long side chains of arginine 2 (R2) and K4me3 fitting snugly in adjacent pre-formed surface pockets, and bracketing an invariant tryptophan. The observed stapling role by non-adjacent R2 and K4me3 provides a molecular explanation for H3K4me3 site specificity. Binding studies establish that the BPTF PHD finger exhibits a modest preference for K4me3- over K4me2-containing H3 peptides, and discriminates against monomethylated and unmodified counterparts. Furthermore, we identified key specificity-determining residues from binding studies of H3(1-15)K4me3 with PHD finger point mutants. Our findings call attention to the PHD finger as a previously uncharacterized chromatin-binding module found in a large number of chromatin-associated proteins.

Amino Acid Motifs↗
PubMed2006

Quantitative analysis of osteoclast-specific gene markers stimulated by lipopolysaccharide.

Lipopolysaccharide (LPS) in the outer layers of Gram-negative bacteria plays an important role in initiating and sustaining periapical lesions. To understand the mechanisms of osteoclastic bone resorption in periapical lesions induced by LPS, we stimulated osteoclast precursors, RAW 264.7 cells with LPS. LPS stimulated osteoclastogenesis when osteoclast precursors were primed with activator for NF-kappaB ligand (RANKL) as little as 24 h. By employing real-time PCR analysis, we have confirmed that osteoclast-like cells stimulated by LPS express high level of osteoclast-specific gene markers such as TRAP, cathepsin K, and calcitonin receptor. These results suggest that bone-resportive action by LPS is partially independent of RANKL.

Acid Phosphatase↗
PubMed2006

Evidence for resonance optical trapping of individual fluorophore-labeled antibodies using single molecule fluorescence spectroscopy.

We report single molecule fluorescence studies of the diffusion of individual multiple fluorophore-labeled antibodies in solution, which show that a trapping potential of about 3.6 k(B)T can be obtained at laser powers below 1 mW with resonant excitation. Individual antibodies can be trapped for up to 140 ms, and bound antibodies can also be used to trap a single virion for up to 1 s. Selective resonance trapping to sort and manipulate fluorophore-labeled biomolecules and complexes may be possible.

Antibodies↗
PubMed2006

Analysis of human telomerase activity and function by two color single molecule coincidence fluorescence spectroscopy.

Telomerase is a nonclassical DNA polymerase that uses its integral RNA as a template to synthesize telomeric repeats onto chromosome ends. The molecular mechanism of telomerase is unique and involves a translocation step after the synthesis of each telomeric repeat. To directly measure the enzymatic turnover of substrate and the efficiency of the translocation step we have extended our two-color single molecule fluorescence coincidence method (Anal.Chem. 2003, 75, 1664-1670). The method employs Cy5-dATP incorporation into a DNA primer that has been prelabeled with a reference fluorophore. Measurements are performed in the single molecule regime and products, which necessarily have both fluorophores, are excited by two independent lasers, and give rise to coincident events. By counting the number of coincident events and using the coincidence detection efficiency, it is possible to determine the number of the extended products generated by attomole quantities of telomerase, without separation or the use of PCR or radioactivity. Histograms of the logarithms of the ratios of the Cy5 to the reference fluorophore fluorescence can be used to determine the length distribution of the products and hence the enzyme processivity. The mean processivity obtained from the single molecule fluorescence coincidence assay is 0.32 +/- 0.04, in good agreement with the value of 0.37 +/- 0.05 derived from the direct radioactive assay approach. The function of the alignment domain of human telomerase RNA in sustaining catalytic activity in vitro has been reevaluated using this method. Together with our previous results (Nucleic Acids Res. 2002, 30, 4470-4480) these experiments identify the essential residues in the alignment domain of human telomerase RNA that contribute to the activity and processivity of telomerase.

Catalysis↗
PubMed2006

Characterization of the mechanism of cytochrome P450 reductase-cytochrome P450-mediated nitric oxide and nitrosothiol generation from organic nitrates.

Mammalian cytochrome P450 reductase (CPR) and cytochrome P450 (CP) play important roles in organic nitrate bioactivation; however, the mechanism by which they convert organic nitrate to NO remains unknown. Questions remain regarding the initial precursor of NO that serves to link organic nitrate to the activation of soluble guanylyl cyclase (sGC). To characterize the mechanism of CPR-CP-mediated organic nitrate bioactivation, EPR, chemiluminescence NO analyzer, NO electrode, and immunoassay studies were performed. With rat hepatic microsomes or purified CPR, the presence of NADPH triggered organic nitrate reduction to NO2(-). The CPR flavin site inhibitor diphenyleneiodonium inhibited this NO2(-) generation, whereas the CP inhibitor clotrimazole did not. However, clotrimazole greatly inhibited NO2(-)-dependent NO generation. Therefore, CPR catalyzes organic nitrate reduction, producing nitrite, whereas CP can mediate further nitrite reduction to NO. Nitrite-dependent NO generation contributed <10% of the CPR-CP-mediated NO generation from organic nitrates; thus, NO2(-) is not the main precursor of NO. CPR-CP-mediated NO generation was largely thiol-dependent. Studies suggested that organic nitrite (R-O-NO) was produced from organic nitrate reduction by CPR. Further reaction of organic nitrite with free or microsome-associated thiols led to NO or nitrosothiol generation and thus stimulated the activation of sGC. Thus, organic nitrite is the initial product in the process of CRP-CP-mediated organic nitrate activation and is the precursor of NO and nitrosothiols, serving as the link between organic nitrate and sGC activation.

Animals↗
PubMed2006

Electrostatic orientation of enzymes on surfaces for ligand screening probed by force spectroscopy.

In this letter, we show that electrostatic immobilization provides a simple but effective approach for the immobilization and orientation of carbonic anhydrase onto charged surfaces. The enzyme is oriented differently on oppositely charged surfaces, with the majority of active sites facing upward on a positively charged surface and downward on a negatively charged surface. An array of negatively charged microscale surface patterns within a positively charged background was prepared by microcontact printing and used as the substrate to immobilize the enzymes. This enabled the probing of the enzyme orientations on the two differently charged surface regions by force spectroscopy with the same atomic force microscopy (AFM) probe modified with a thiolated sulfonamide inhibitor. The unbinding forces between the inhibitor tip and the enzyme immobilized on the two differently charged surfaces were measured. Two control experiments, blocking of the enzyme active site with a competitive inhibitor and removal of the zinc ion from the enzyme catalytic center, were employed to distinguish between specific and nonspecific interactions and to further verify the differences in enzyme orientation. Autocorrelation analysis of the force histograms was carried out to evaluate the specific single enzyme-inhibitor interaction force.

Animals↗
PubMed2006

Prevention of rabies virus infection in dogs by a recombinant canine adenovirus type-2 encoding the rabies virus glycoprotein.

Safe and effective vaccination is important for rabies prevention in animals. Although several genetically engineered rabies vaccines have been developed, few have been licensed for use, principally due to biosafety concerns or due to poor efficacy in animal models. In this paper, we describe the construction and characterization of a replication-competent recombinant canine adenovirus type-2 expressing the rabies virus glycoprotein (SRV9 strain) by a different strategy from that reported previously, i.e., the recombinant genome carrying the glycoprotein cDNA was generated by a series of strictly gene cloning steps, infectious recombinant virus was obtained by transfecting the recombinant genome into a canine kidney cell line, MDCK. This recombinant virus, CAV-E3delta-CGS, was subcutaneously injected into dogs. All vaccinated dogs produced effective neutralizing antibodies after one inoculation and a stronger anamnestic immune response was produced after booster injection. The immunized dogs could survive the challenge of 60,000 mouse LD50 CVS-24, which is lethal to all unimmunized dogs and is comparable to the conventional vaccines. The immunity lasts for months with a protective level of neutralizing antibody. This recombinant virus would be an alternative to the attenuated and the inactivated rabies vaccines and be prospective in immunizing dogs against rabies.

Adenoviruses, Canine↗
PubMed2006

Macroscopic 2D networks self-assembled from nanometer-sized protein/DNA complexes.

We demonstrate the self-assembly of DNA and DNA binding proteins into two-dimensional networks that are then addressable by sending a second protein to a specific recognition site on the DNA network. These networks cover centimeters in area but can be addressed with nanometer precision. This hierarchical self-assembly of specific DNA protein complexes will be the basis for complex positioning of single molecules in two and three dimensions.

Base Sequence↗
PubMed2005

Xanthine oxidase catalyzes anaerobic transformation of organic nitrates to nitric oxide and nitrosothiols: characterization of this mechanism and the link between organic nitrate and guanylyl cyclase activation.

Organic nitrates have been used clinically in the treatment of ischemic heart disease for more than a century. Recently, xanthine oxidase (XO) has been reported to catalyze organic nitrate reduction under anaerobic conditions, but questions remain regarding the initial precursor of nitric oxide (NO) and the link of organic nitrate to the activation of soluble guanylyl cyclase (sGC). To characterize the mechanism of XO-mediated biotransformation of organic nitrate, studies using electron paramagnetic resonance spectroscopy, chemiluminescence NO analyzer, NO electrode, and immunoassay were performed. The XO reducing substrates xanthine, NADH, and 2,3-dihydroxybenz-aldehyde triggered the reduction of organic nitrate to nitrite anion (NO2-). Studies of the pH dependence of nitrite formation indicated that XO-mediated organic nitrate reduction occurred via an acid-catalyzed mechanism. In the absence of thiols or ascorbate, no NO generation was detected from XO-mediated organic nitrate reduction; however, addition of L-cysteine or ascorbate triggered prominent NO generation. Studies suggested that organic nitrite (R-O-NO) is produced from XO-mediated organic nitrate reduction. Further reaction of organic nitrite with thiols or ascorbate leads to the generation of NO or nitrosothiols and thus stimulates the activation of sGC. Only flavin site XO inhibitors such as diphenyleneiodonium inhibited XO-mediated organic nitrate reduction and sGC activation, indicating that organic nitrate reduction occurs at the flavin site. Thus, organic nitrite is the initial product in the process of XO-mediated organic nitrate biotransformation and is the precursor of NO and nitrosothiols, serving as the link between organic nitrate and sGC activation.

Anions↗
PubMed2005

DEPBT as an efficient coupling reagent for amide bond formation with remarkable resistance to racemization.

3-(Diethoxyphosphoryloxy)-1, 2, 3-benzotriazin-4(3H)-one (DEPBT) is an effective coupling reagent for synthesis of linear and cyclic peptides by both solution and solid-phase peptide synthesis. DEPBT mediates amide bond formation with remarkable resistance to racemization. When DEPBT is used as a coupling reagent, it is not necessary to protect the hydroxy group of the amino component (such as tyrosine, serine, and threonine) and the imidazole group in the case of histidine. The high efficiency of DEPBT and its utility have been demonstrated in the syntheses of complex natural products such as ramoplanin A2, ramoplanose aglycon, ustiloxin, and teicoplanin aglycon.

Amides↗
PubMed2005

Multifocal electroretinographic changes in patients receiving hydroxychloroquine therapy.

PURPOSE: To evaluate the longitudinal changes in multifocal electroretinography (mfERG) in patients receiving hydroxychloroquine and to assess the effects of cumulative hydroxychloroquine dose on mfERG. DESIGN: Prospective cohort study. METHODS: Twenty-four eyes in 12 patients receiving hydroxychloroquine underwent mfERG recordings at baseline and 1 to 2 years later. The first negative (N1) and first positive (P1) response amplitudes and peak latencies were compared with normal controls. Serial changes in the pattern of mfERG abnormalities and in response amplitudes and peak latencies were also compared between eyes in which hydroxychloroquine therapy was continued or stopped. Correlation analyses were performed to assess the effects of a cumulative dose of hydroxychloroquine on mfERG. RESULTS: At baseline, reductions in N1 and P1 response amplitudes were observed in patients receiving hydroxychloroquine compared with controls. At follow-up, in addition to the reductions in N1 and P1 response amplitudes, increases in P1 peak latencies compared with controls were observed. In patients who stopped hydroxychloroquine therapy, there were significant increases in N1 and P1 response amplitudes at follow-up mfERG. CONCLUSIONS: Patients receiving hydroxychloroquine showed a longitudinal decline in retinal function; patients who stopped hydroxychloroquine therapy showed improvement. Although these data are insufficient to demonstrate the sensitivity of mfERG for evaluating early hydroxychloroquine toxicity, the results suggest that serial mfERG assessment may help detect early retinal changes associated with hydroxychloroquine therapy. Further studies with long-term results will be useful in clarifying the value of mfERG in evaluating early retinal toxicity due to hydroxychloroquine.

Adult↗