Biomedical subjects

H Lu

Publications and source records attributed to H Lu.

At least 37 records · Page 2Linked to original sources

PubMed2001

A malignant phenotype of hypertrophic cardiomyopathy caused by Arg719Gln cardiac beta-myosin heavy-chain mutation in a Chinese family.

Mutations of the cardiac beta-myosin heavy-chain (beta-MHC) gene cause hypertrophic cardiomyopathy (HCM). Recent genotype-phenotype correlation studies have shown that mutations carry prognostic significance. We studied five unrelated Chinese families with hypertrophic cardiomyopathy. Exons 3-27 and 40 of the beta-MHC gene were screened with both the polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) method and the cycle sequencing of the PCR products. A previously reported heterozygous mutation Arg719Gln (arginine-->glutamine in codon 719) in exon 19 was found in one family. The proband is a 30-year-old female diagnosed at age of 25 years when she presented with symptoms of chest pain, palpitations, and frequent incidents of dizziness and syncope. A two-dimensional echocardiogram showed moderate asymmetrical septal hypertrophy with left atrial enlargement. There was no obstruction of the left ventricular outflow tract (LVOT). The patient also developed atrial fibrillation. The proband's mother and one of her sisters had similar clinical manifestations and both died suddenly at the age of 38 years. In addition, two silent nucleotide substitutions (ACT63ACC, TTT244TTC) in the cardiac beta-MHC gene were identified in the other four families. These synonymous mutations did not cosegregate with the disease in the families and they were also present in the 60 healthy and age-matched control subjects. Of the five families studied, we did not find any missense mutation in the remaining four families. The missense mutation Arg719Gln found in the Chinese family is associated with a malignant phenotype of severe clinical symptoms and poor survival prognosis. This mutation also causes atrial enlargement and atrial fibrillation. Our study provides further evidence that the mutation, which alters the charge of the myosin heavy chain, is associated with a serious clinical outcome.

Adult↗

PubMed2001

A distance-dependent atomic knowledge-based potential for improved protein structure selection.

A heavy atom distance-dependent knowledge-based pairwise potential has been developed. This statistical potential is first evaluated and optimized with the native structure z-scores from gapless threading. The potential is then used to recognize the native and near-native structures from both published decoy test sets, as well as decoys obtained from our group's protein structure prediction program. In the gapless threading test, there is an average z-score improvement of 4 units in the optimized atomic potential over the residue-based quasichemical potential. Examination of the z-scores for individual pairwise distance shells indicates that the specificity for the native protein structure is greatest at pairwise distances of 3.5-6.5 A, i.e., in the first solvation shell. On applying the current atomic potential to test sets obtained from the web, composed of native protein and decoy structures, the current generation of the potential performs better than residue-based potentials as well as the other published atomic potentials in the task of selecting native and near-native structures. This newly developed potential is also applied to structures of varying quality generated by our group's protein structure prediction program. The current atomic potential tends to pick lower RMSD structures than do residue-based contact potentials. In particular, this atomic pairwise interaction potential has better selectivity especially for near-native structures. As such, it can be used to select near-native folds generated by structure prediction algorithms as well as for protein structure refinement.

Computational Biology↗

PubMed2001

TOUCHSTONE: an ab initio protein structure prediction method that uses threading-based tertiary restraints.

The successful prediction of protein structure from amino acid sequence requires two features: an efficient conformational search algorithm and an energy function with a global minimum in the native state. As a step toward addressing both issues, a threading-based method of secondary and tertiary restraint prediction has been developed and applied to ab initio folding. Such restraints are derived by extracting consensus contacts and local secondary structure from at least weakly scoring structures that, in some cases, can lack any global similarity to the sequence of interest. Furthermore, to generate representative protein structures, a reduced lattice-based protein model is used with replica exchange Monte Carlo to explore conformational space. We report results on the application of this methodology, termed TOUCHSTONE, to 65 proteins whose lengths range from 39 to 146 residues. For 47 (40) proteins, a cluster centroid whose rms deviation from native is below 6.5 (5) A is found in one of the five lowest energy centroids. The number of correctly predicted proteins increases to 50 when atomic detail is added and a knowledge-based atomic potential is combined with clustered and nonclustered structures for candidate selection. The combination of the ratio of the relative number of contacts to the protein length and the number of clusters generated by the folding algorithm is a reliable indicator of the likelihood of successful fold prediction, thereby opening the way for genome-scale ab initio folding.

Algorithms↗

PubMed2001

Photochemical reactions and on-line UV detection in microfabricated reactors.

This work presents an application of microfabricated reactors and detectors for photochemical reactions. Two fabrication schemes were demonstrated for the integration of the reaction and the detection modules: coupling individually packaged chips, and monolithic integration of the two functions. In the latter fabrication scheme, we have succeeded in bonding quartz wafers to patterned silicon wafers at low temperature using a Teflon-like polymer-CYTOP[trade mark sign]. Using quartz substrates allows reaction and detection with UV light of lower wavelengths than Pyrex substrates permit. The pinacol formation reaction of benzophenone in isopropanol was the model reaction to demonstrate the performance of the microreactors. The extent of reaction was controlled by varying the flow rate and therefore the on-chip residence time. Crystallization of the product inside the microreactors was avoided by the continuous-flow design. Instead, crystallization was observed in the effluent storage device. Off-chip analysis using HPLC confirms the results obtained from the on-line UV spectroscopy. The quantum yield estimated suggests that the reactor design is effective in improving the overall efficiency of the reactor unit.

Journal Article↗

PubMed2001

Antifungal activity of Artemisia annua endophyte cultures against phytopathogenic fungi.

Artemisia annua, well recognized for its production of antimalarial drug artemisinin, is seldom attacked by any of phytopathogenic fungi, which could be partially associated with the presence of endophytes. Present investigation is aiming at disclosing whether the endophytes inside A. annua produce antifungal substances. A total of 39 endophytes were isolated and fermented, and the ferment broth was evaluated in vitro for the antifungal activity against crop-threatening fungi Gaeumannomyces graminis var. tritici, Rhizoctonia cerealis, Helminthosporium sativum, Fusarium graminearum, Gerlachia nivalis and Phytophthora capsici. These plant pathogens are still causing wheat take-all, sharp eyespot, common rot, scab, snow mould, and pepper phytophthora blight, respectively. Out of 39 endophytes investigated, 21 can produce in vitro substances that are inhibitory to all or a few of the tested phytopathogens whereas the rest yielded nothing active. Moreover, the most active broth of endophyte IV403 was extracted with EtOAc and n-butanol, and comparisons of the antifungal activity of the extracts indicated that the major active metabolites were EtOAc-extractable.

Actinomycetales↗

PubMed2001

Probing the interaction of dengue virus envelope protein with heparin: assessment of glycosaminoglycan-derived inhibitors.

A structure-activity relationship study was carried out to facilitate development of inhibitors of dengue virus infectivity. Previous studies demonstrated that a highly charged heparan sulfate, a heparin-like glycosaminoglycan found on the cell surface, serves as a receptor for dengue virus by binding to its envelope protein. Interventions that disrupt this binding effectively inhibit infectivity. A competitive binding assay was developed to screen polyanionic compounds for activity in preventing binding of dengue virus envelope protein to immobilized heparin; compounds tested included drugs, excipients, and larger glycosaminoglycans and their semisynthetic derivatives. Results of this competitive binding assay were used to select agents for detailed evaluation of interactions by surface plasmon resonance spectroscopy, which afforded binding on-rates, off-rates, and dissociation constants. From these data, an understanding of the structural requirements for polyanion binding to dengue virus envelope protein has been established.

Anticoagulants↗

PubMed2001

Optical detection in microscopic domains. 2. Inner filter effects for monitoring nonfluorescent molecules with fluorescence.

In this research, we test whether optical detection techniques show different characteristics in microscopic solution volumes (nano-, pico-, and femtoliter range) compared to the usual macroscopic samples. In part 1 (Lu, H.; et al. Anal Chem. 2000, 72, 1569-1576.) absorption spectra of high quality were obtained, quantitatively obeying both Beer-Lambert's law and the law of superposition, despite the micrometer optical path lengths and the curvatures of the droplets studied. Addition and subtraction of absorbing molecules with diffusional microburets (DMBs), as well as more complex operations (simultaneous addition of one and subtraction of another molecule, and a consuming scheme), have been monitored with good spectral and temporal resolution. Despite the unexpectedly good performance of absorption microspectrometry, fluorescence-based detection schemes are considered more sensitive for microscopic studies (e.g., cell physiology). In this paper, we test whether fluorescence-based schemes can be used to indirectly measure nonfluorescent chemicals in microscopic domains. Absorption by such molecules will cause a corresponding decrease in overall fluorescence intensity of the added standard fluorescent dye. This phenomenon, the inner filter effect (IFE), was tested using Lucifer Yellow CH (LY) as the fluorescent standard dye. Its effective irradiation was absorbed by Orange G (primary IFE) or its emission by Bromophenol Blue (secondary IFE). By utilizing these phenomena, (1) we measured the concentration of absorbing molecules in microscopic samples by adding a standard amount of LY by a DMB, and (2) we monitored DMB delivery of nonfluorescent reagents into droplets preloaded with LY. The results prove that IFEs are sensitive indirect means of detection of absorbing molecules in microscopic domains. The techniques presented are expected to find applications in cellular studies where absorption spectrometry is usually not considered.

Algorithms↗

PubMed2001

Structure and function in bacteriorhodopsin: the effect of the interhelical loops on the protein folding kinetics.

The loops connecting the seven transmembrane helices of bacteriorhodopsin have each been replaced in turn by structureless linkers of Gly-Gly-Ser repeat sequences, and the effect on the protein folding kinetics has been determined. An SDS-denatured state of each loop mutant bacterio-opsin was folded in l-alpha-1,2-dihexanoylphosphatidylcholine/l-alpha-1,2-dimyristoylphosphatidylcholine micelles, containing retinal, to give functional bacteriorhodopsin. Stopped-flow mixing was used to initiate the folding reaction, giving a time resolution of milliseconds, and changes in protein fluorescence were used to monitor folding. All loop mutant proteins folded according to the same reaction scheme as wild-type protein. The folding kinetics of the AB, BC and DE loop mutants were the same as wild-type protein, despite the blue-shifted chromophore band of the BC loop mutant bR state. A partially folded apoprotein intermediate state of the AB loop mutant did however appear to decay in the absence of retinal. The most significant effects on the folding kinetics were seen for mutant protein with structureless linkers in place of the CD, EF and FG loops. The rate-limiting apoprotein folding step of the CD loop mutant was about ten times slower than wild-type, whilst that of the EF loop mutant was almost four times slower than wild-type. Wild-type behaviour was observed for the other folding and retinal binding events of the CD and EF loop mutant proteins. These effects of the CD and EF loop mutations on apoprotein folding correlate with the fact that these two loop mutants also have the least stable, partially folded apoprotein intermediate of all the loop mutants, and are the most affected by a decrease in lipid lateral pressure. In contrast, the FG loop mutant exhibited wild-type apoprotein folding, but altered covalent binding of retinal and final folding to bacteriorhodopsin. This correlates with the fact that the FG loop mutant bacteriorhodopsin is the most susceptible to denaturation by SDS of all the loop mutants, but its partially folded apoprotein intermediate is more stable than that of the CD and EF mutants. Thus the CD and EF loops may contribute to the transition state for the rate-limiting apoprotein folding step and the FG loop to that for final folding and covalent binding of retinal.

Apoproteins↗

PubMed2001

Proline residues in transmembrane alpha helices affect the folding of bacteriorhodopsin.

Proline residues occur frequently in transmembrane alpha helices, which contrasts with their behaviour as helix-breakers in water-soluble proteins. The three membrane-embedded proline residues of bacteriorhodopsin have been replaced individually by alanine and glycine to give P50A, or P50G on helix B, P91A, or P91G on helix C, and P186A or P186G on helix F, and the effect on the protein folding kinetics has been investigated. The rate-limiting apoprotein folding step, which results in formation of a seven transmembrane, alpha helical state, was slower than wild-type protein for the Pro50 and Pro91 mutants, regardless of whether they were mutated to Ala or Gly. These proline residues give rise to several inter-helix contacts, which are therefore important in folding to the seven transmembrane helix state. No evidence for cis-trans isomerisations of the peptidyl prolyl bonds was found during this rate-limiting apoprotein folding step. Mutations of all three membrane-embedded proline residues affected the subsequent retinal binding and final folding to bacteriorhodopsin, suggesting that these proline residues contribute to formation of the retinal binding pocket within the helix bundle, again via helix/helix interactions. These results point to proline residues in transmembrane alpha helices being important in the folding of integral membrane proteins. The helix/helix interactions and hydrogen bonds that arise from the presence of proline residues in transmembrane alpha helices can affect the formation of transmembrane alpha helix bundles as well as cofactor binding pockets.

Amino Acid Substitution↗

PubMed2001

Down-regulation of vascular endothelial growth factor by tissue inhibitor of metalloproteinase-2: effect on in vivo mammary tumor growth and angiogenesis.

The tissue inhibitor of metalloproteinases-2 (TIMP-2) has at least two independent functions, i.e., regulation of matrix metalloproteinases and growth promoting activity. We investigated the effects of TIMP-2 overexpression, induced by retroviral mediated gene transfer, on the in vivo development of mammary tumors in syngeneic mice inoculated with EF43.fgf-4 cells. The EF43.fgf-4 cells established by stably infecting the normal mouse mammary EF43 cells with a retroviral expression vector for the fgf-4 oncogene, are highly tumorigenic and overproduce vascular endothelial growth factor (VEGF). Despite a promotion of the in vitro growth rate of EF43.fgf-4 cells overexpressing timp-2, the in vivo tumor growth was delayed. At day 17 post-cell injection, the volume of tumor derived from TIMP-2-overexpressing cells was reduced by 80% as compared with that obtained with control cells. Overexpression of TIMP-2 was associated with a down-regulation of VEGF expression in vitro and in vivo, a reduction of vessel size, density, and blood supply in the induced tumors. In addition, TIMP-2 completely inhibited the angiogenic activity of EF43.fgf-4 cell-conditioned medium in vitro using a rat aortic ring model. Our findings suggest that overexpression of TIMP-2 delays growth and angiogenesis of mammary carcinoma in vivo and that down-regulation of VEGF expression may play an important role in this TIMP-2-mediated antitumoral and antiangiogenic effects. Finally the in vivo delivery of TIMP-2, as assessed by i.v. injection of recombinant adenoviruses vectors, significantly reduced the growth of the EF43.fgf-4-induced tumors. This effect of TIMP-2 was shown to be equally comparable with that of angiostatin, a known potent inhibitor of angiogenesis.

Adenoviridae↗

PubMed2001

[Effect of sensitized lymphocytes on rabbit calvarial osteoblasts].

OBJECTIVE: To study the effects of sensitized T lymphocytes on osteoblastic proliferation and differentiation. METHODS: Sensitized lymphocytes were obtained from a rabbit sensitized by chromium, and lymphocyte cultured mediums (LCM) were collected. Osteoblasts were obtained from new-born rabbit calvaria. The osteoblasts were identified with alkaline phosphatase staining and mineralization in vitro. By osteoblast cultured in vitro, osteoblastic proliferation, and production of alkaline phosphatase and osteocalcin were evaluated in three conditions: without LCM, with LCM not stimulated by Cr(6+) and with LCM stimulated by Cr(6+). RESULTS: The cells from rabbit calvaria were positive to ALP staining and could form calcium node in vitro. The sensitized LCM could inhibit proliferation of osteoblasts, increase alkaline phosphatase production and decrease osteocalcin production. These results all had statistically significant difference by ANOVA. CONCLUSION: Sensitized T lymphocytes could decrease the number of mature osteoblasts and impede the formation of mature mineralized bone.

Alkaline Phosphatase↗

PubMed2001

AdTIMP-2 inhibits tumor growth, angiogenesis, and metastasis, and prolongs survival in mice.

TIMP-2 is a natural matrix metalloproteinase (MMP) inhibitor that prevents the degradation of extracellular matrix proteins. It abolishes the hydrolytic activity of all activated members of the metalloproteinase family and in particular that of MT1-MMP, MMP-2, and MMP-9, which are selective for type IV collagenolysis. Since MMPs have been implicated in both cancer progression and angiogenesis, we generated a recombinant adenovirus to deliver human TIMP-2 (AdTIMP-2) and evaluated its anticancer efficiency in three murine models. Our results demonstrated that overexpression in vitro of TIMP-2 inhibited the invasion of both tumor and endothelial cells without affecting cell proliferation. Its in vivo efficiency has been evaluated in murine lung cancer LLC, and colon cancer C51 in syngeneic mice as well as in human breast cancer MDA-MB231 in athymic mice. Preinfection of tumor cells by AdTIMP-2 resulted in an inhibition of tumor establishment in more than 50% of mice in LLC and C51 models and in 100% mice in the MDA-MB231 model. A single local injection of AdTIMP-2 into preestablished tumors of these three types significantly reduced tumor growth rates by 60--80% and tumor-associated angiogenesis index by 25--75%. Lung metastasis of LLC tumor was inhibited by >90%. In addition, AdTIMP-2-treated mice showed a significantly prolonged survival in all the cancer models tested. These data demonstrate the potential of adenovirus-mediated TIMP-2 therapy in cancer treatment.

Adenoviridae↗

PubMed2001

Intracellular signal transduction pathways as targets for neurotoxicants.

The multiple cascades of signal transduction pathways that lead from receptors on the cell membrane to the nucleus, thus translating extracellular signals into changes in gene expression, may represent important targets for neurotoxic compounds. Among the biochemical steps and pathways that have been investigated are the metabolism of cyclic nucleotides, the formation of nitric oxide, the metabolism of membrane phospholipids, the activation of a multitude of protein kinases and the induction of transcription factors. This brief review will focus on the interactions of three known neurotoxicants, lead, ethanol and polychlorinated biphenyls, with signal transduction pathways, particularly the family of protein kinase C isozymes, and discusses how such effects may be involved in their neurotoxicity.

Animals↗

PubMed2001

Stabilization and activation of p53 by the coactivator protein TAFII31.

Regulation of the stability of p53 is key to its tumor-suppressing activities. mdm2 directly binds to the amino-terminal region of p53 and targets it for degradation through the ubiquitin-proteasome pathway. The coactivator protein TAF(II)31 binds to p53 at the amino-terminal region that is also required for interaction with mdm2. In this report, we demonstrate that expression of TAF(II)31 inhibits mdm2-mediated ubiquitination of p53 and increases p53 levels. TAF(II)31-mediated p53 stabilization results in activation of p53-mediated transcriptional activity and leads to p53-dependent growth arrest in fibroblasts. UV-induced stabilization of p53 coincides with an increase in p53-associated TAF(II)31 and a corresponding decrease in mdm2-p53 interaction. Non-p53 binding mutant of TAF(II)31 fails to stabilize p53. Our results suggest that direct interaction of TAF(II)31 and p53 not only mediates p53 transcriptional activation but also protects p53 from mdm2-mediated degradation, thereby resulting in activation of p53 functions.

3T3 Cells↗

PubMed2001

Helper-dependent adenoviral vector-mediated gene transfer in aged rat brain.

Transfer of the neurotrophin gene into brain can attenuate age-related deficits such as neuronal atrophy and memory loss, but a suitable vector for this procedure has been lacking. The toxicity and immunogenicity of first-generation adenoviral vectors with E1 deletion (fgAdv) prohibit the application of gene transfer in the majority of central nervous system disorders. Here, we report less toxic and persistent gene expression mediated by helper-dependent adenovirus (hdAdv) in aged rat brain. After intrahippocampal or intraventricular inoculation of the vector, transgene expression was monitored by X-Gal staining and compared with fgAdv-mediated expression. Host inflammatory and immune responses against these vectors were evaluated by immunohistochemical detection of microglia, astrocytes, and infiltrating macrophages, as well as by enzyme-linked immunosorbent assay of cytokines TNF-alpha and IL-1beta. Transgene expression mediated by hdAdv persisted for more than 183 days regardless of inoculation site, as compared with 33 and 66 days for fgAdv-mediated expression after intraventricular and intrahippocampal inoculation, respectively. Inoculation with hdAdv was also associated with reduced numbers of activated microglial cells, astrocytes, and infiltrating macrophages in brain tissue. Secretion of the proinflammatory cytokines TNF-alpha and IL-1beta was minimal after hdAdv but not after fgAdv inoculation. These findings indicate that hdAdv would provide a safe and effective means to transfer therapeutic genes into aged brain.

Adenoviridae↗

PubMed2001

p300 does not require its acetylase activity to stimulate p73 function.

We previously reported that p73, like p53, utilizes p300 or cAMP-response element-binding protein-binding protein as its coactivator. Here, we extended this work by further examining whether the intrinsic acetylase activity of p300 is necessary for stimulating p73 function. Although p300 acetylated the C-terminal fragment of p73 (amino acids 311-636) in vitro, it was unable to efficiently acetylate the full-length p73. Consistently, p300 did not acetylate p73 in vivo when both the proteins were overexpressed in cells. Also, an acetylase-defective mutant p300 named p300AT2 was able to elevate p73-dependent transcription in cells. p300 associated with p73 when forming DNA-protein complexes and stabilized p73 proteins. These results demonstrate that p300 does not need its acetylase activity to be a coactivator of p73.

Acetylation↗

PubMed2001

Activation of mitogen-activated protein kinase by muscarinic receptors in astroglial cells: role in DNA synthesis and effect of ethanol.

Mitogen-activated protein kinase (MAPK) can be phosphorylated by mitogens binding to G-protein-coupled receptors and is considered a major pathway involved in cell proliferation. In this study, we report on the activation of MAPK by muscarinic acetylcholine receptors in astroglial cells, namely the 1321N1 human astrocytoma cell line, primary rat cortical astrocytes, and fetal human astrocytes. Carbachol caused a rapid and transient phorphorylation of MAPK (ERK1/2) in all cell types, with an increase in MAPK activity, without changing the levels of MAPK proteins. Human astrocytoma cells were used to characterize the effect of carbachol on MAPK. Experiments with M2- and M3-receptor subtype-selective antagonists, and with pertussis toxin, indicated that the M3 subtype is responsible for activating MAPK in glial cells. Pretreatment of cells with the protein kinase C (PKC) inhibitor bisindolylmaleimide I, or downregulation of PKC by 24-h treatment with the phorbol ester TPA inhibited carbachol-induced MAPK activation. Additional experiments with PKC alpha- or PKC epsilon-specific compounds indicated that the epsilon isozyme of PKC is primarily involved in MAPK activation by carbachol. Chelation of calcium also inhibited MAPK activation by carbachol. Two MEK (MAPK kinase) inhibitors inhibited carbachol-induced DNA synthesis but only at concentrations that exceeded those sufficient to block carbachol-induced MAPK activation. Ethanol (< or =200 mM) had no effect on MAPK when present alone and did not affect carbachol-induced MAPK activation under various experimental conditions, although it inhibits carbachol-induced DNA synthesis at low concentrations (10-100 mM). These results suggest that activation of MAPK by carbachol may be necessary but not sufficient for its mitogenic effect in astroglial cells, and that does not represent a target for ethanol-induced inhibition of DNA synthesis elicited by muscarinic receptors.

Animals↗

PubMed2001

Replication of the gonad-specific virus Hz-2V in Ld652Y cells mimics replication in vivo.

A newly discovered, nonoccluded insect virus, known as gonad-specific virus or Hz-2V, was found to replicate differently in two insect cell lines derived from ovarian tissues (Tn-368 cells from Trichoplusia ni and Ld652Y from Lymantria dispar). Differences between these two cell lines were observed in virus plaque forming ability, rate of viral DNA replication, time course of infectious virus production, and the mechanism of virus release from infected cells. Replication of Hz-2V in Ld652Y cells was more productive and more closely resembled in vivo virus replication.

Animals↗