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Ming Lei

Publications and source records attributed to Ming Lei.

At least 37 records · Page 2Linked to original sources
PubMed2005

POT1 stimulates RecQ helicases WRN and BLM to unwind telomeric DNA substrates.

Defects in human RecQ helicases WRN and BLM are responsible for the cancer-prone disorders Werner syndrome and Bloom syndrome. Cellular phenotypes of Werner syndrome and Bloom syndrome, including genomic instability and premature senescence, are consistent with telomere dysfunction. RecQ helicases are proposed to function in dissociating alternative DNA structures during recombination and/or replication at telomeric ends. Here we report that the telomeric single-strand DNA-binding protein, POT1, strongly stimulates WRN and BLM to unwind long telomeric forked duplexes and D-loop structures that are otherwise poor substrates for these helicases. This stimulation is dependent on the presence of telomeric sequence in the duplex regions of the substrates. In contrast, POT1 failed to stimulate a bacterial 3'-5'-helicase. We find that purified POT1 binds to WRN and BLM in vitro and that full-length POT1 (splice variant 1) precipitates a higher amount of endogenous WRN protein, compared with BLM, from the HeLa nuclear extract. We propose roles for the cooperation of POT1 with RecQ helicases WRN and BLM in resolving DNA structures at telomeric ends, in a manner that protects the telomeric 3' tail as it is exposed during unwinding.

Adenosine Triphosphatases↗
PubMed2005

Sinus node dysfunction following targeted disruption of the murine cardiac sodium channel gene Scn5a.

We have examined sino-atrial node (SAN) function in hearts from adult mice with heterozygous targeted disruption of the Scn5a gene to clarify the role of Scn5a-encoded cardiac Na+ channels in normal SAN function and the mechanism(s) by which reduced Na+ channel function might cause sinus node dysfunction. Scn5a+/- mice showed depressed heart rates and occasional sino-atrial (SA) block. Their isolated peripheral SAN pacemaker cells showed a reduced Na+ channel expression and slowed intrinsic pacemaker rates. Wild-type (WT) and Scn5a+/- SAN preparations exhibited similar activation patterns but with significantly slower SA conduction and frequent sino-atrial conduction block in Scn5a+/- SAN preparations. Furthermore, isolated WT and Scn5a+/- SAN cells demonstrated differing correlations between cycle length, maximum upstroke velocity and action potential amplitude, and cell size. Small myocytes showed similar, but large myocytes reduced pacemaker rates, implicating the larger peripheral SAN cells in the reduced pacemaker rate that was observed in Scn5a+/- myocytes. These findings were successfully reproduced in a model that implicated i(Na) directly in action potential propagation through the SAN and from SAN to atria, and in modifying heart rate through a coupling of SAN and atrial cells. Functional alterations in the SAN following heterozygous-targeted disruption of Scn5a thus closely resemble those observed in clinical sinus node dysfunction. The findings accordingly provide a basis for understanding of the role of cardiac-type Na+ channels in normal SAN function and the pathophysiology of sinus node dysfunction and suggest new potential targets for its clinical management.

Action Potentials↗
PubMed2005

Initial conformational changes of human transthyretin under partially denaturing conditions.

Human transthyretin (TTR) is an amyloidogenic protein. The pathway of TTR amyloid formation has been proposed based on lines of evidence: TTR tetramer first dissociates into native monomers, which is shown to be a rate-limiting step in the formation of fibrils. Subsequently, the monomeric species partially unfold to form the aggregation intermediates. Once such intermediates are formed, the following self-assembly process is a downhill polymerization. Hence, tertiary structural changes within the monomers after the dissociation are essential for the amyloid formation. These tertiary structural changes can be facilitated by partial denaturation. To probe the conformational changes under the partially denaturing conditions, five independent trajectories were collected for the wild-type (WT) and its pathogenic variants at 300 and 350 K, resulting in simulations that totaled 59 ns. Under these conditions, L55P variant is more labile than the wild-type and V30M variant. We have observed that the D strand of WT-TTR is trapped in two local minima: the native conformation and the amyloidogenic fold that resembles the surface loop of residues 54-55 of L55P variant. In the tetrameric state, the F strand is bent with large separations at the F-F' interface. This strand becomes flatter in the monomeric state, which may facilitate the formation of new F-F' interface with possible prolonged hydrogen bonds and/or shift in beta-strand register in the fibril state. During the unfolding process, the anticorrelated motion between the strands H and G as well as the strands H and A pulls the H strand out of the inner sheet plane, leading to a more twisted inner sheet. Our simulation has provided important detailed structural information about the partially unfolded state of TTR that may be related to the amyloidogenic intermediates.

Amyloid↗
PubMed2005

Switching human telomerase on and off with hPOT1 protein in vitro.

POT1 (protection of telomeres 1) protein binds the G-rich single-stranded telomeric DNA at the ends of chromosomes. In human cells hPOT1 is involved in telomere length regulation, but the mechanism of this regulation remains unknown. Examination of the high-resolution crystal structure of the hPOT1-TTAGGGTTAG complex suggested that it would not be extended by telomerase, a hypothesis that we confirm by in vitro assays with recombinant telomerase. On the other hand, when hPOT1 is bound at a position one telomeric repeat before the 3'-end, leaving an 8-nucleotide 3'-tail, the complex is extended with improved activity and processivity. Thus, depending on its location relative to the DNA 3'-end, hPOT1 can either inhibit telomerase action or form a preferred substrate for telomerase. We propose that another factor catalyzes the interconversion of these states in vivo.

Alternative Splicing↗
PubMed2005

Erythropoietin prevents the acute myocardial inflammatory response induced by ischemia/reperfusion via induction of AP-1.

OBJECTIVE: Erythropoietin (EPO) prevents the myocardial dysfunction induced by ischemia/reperfusion (I/R). Since I/R-induced myocardial dysfunction is associated with an acute inflammatory response, we assessed the anti-inflammatory properties of EPO using in vitro and in vivo models of I/R. METHODS: Isolated cardiac myocytes were exposed to anoxia/reoxygenation (A/R; the in vitro counterpart to I/R). Hearts were challenged with I/R in situ. RESULTS: In vitro, A/R increased myocyte oxidant stress and converted the myocytes to a proinflammatory phenotype (these myocytes induced PMN transendothelial migration). Pretreatment of the myocytes with EPO prevented the A/R-induced proinflammatory effects. EPO increased myocyte (1) nuclear translocation of AP-1 (c-fos/c-jun), (2) eNOS, but not iNOS, protein expression, and (3) NO production. An AP-1 "decoy" oligonucleotide prevented the induction of eNOS by EPO and reversed the beneficial effect of EPO. An inhibitor of phosphatidylinostol 3 (PI3)-kinase prevented the nuclear translocation of AP-1 induced by EPO. In vivo, in wild type mice, I/R induced an increase in myocardial MPO activity (indicative of PMN infiltration); an effect prevented by pretreatment of the mice with EPO. This anti-inflammatory effect of EPO was not observed in cardiac specific c-fos(-/-) mice. CONCLUSIONS: Collectively, these findings indicate that EPO can ameliorate the myocardial inflammatory response in both in vitro and in vivo models of I/R. This beneficial effect of EPO is mediated by eNOS-derived NO via a PI3-kinase-dependent activation of AP-1.

Animals↗
PubMed2005

The active conformation of the PAK1 kinase domain.

The p21-activated kinases (PAKs) participate in cytoskeletal control networks, downstream of Rho-family GTPases. A structure of PAK1 in an autoregulated, "off" state showed that a regulatory region, N-terminal to the kinase domain, forces the latter into an inactive conformation, prevents phosphorylation of Thr423 in the activation loop, and promotes dimerization. We have now determined structures at 1.8 A resolution for the free PAK1 kinase domain, with a mutation in the active site that blocks enzymatic activity, and for the same domain with a "phosphomimetic" mutation in the activation loop. The two very similar structures show that even in the absence of a phosphorylated Thr423, the kinase has an essentially active conformation. When Cdc42 binds the regulatory region and dissociates the dimer, PAK1 will be in an "intermediate-active" state, with a capacity to phosphorylate itself or other substrates even prior to modification of its activation loop.

Adenosine Triphosphate↗
PubMed2005

Pathology of guinea pigs experimentally infected with a novel reovirus and coronavirus isolated from SARS patients.

Guinea pigs were inoculated with a reovirus (ReoV) and coronavirus (SARS-CoV) isolated from SARS patients to determine their potential role in the etiology of SARS. Animals infected with ReoV died between day 22 and day 30 postinoculation (PI) while 70% of the animals inoculated with ReoV and SARS-CoV died between day 4 to day 7 PI. The titer of neutralizing antibodies against ReoV and SARS-CoV ranged from 80 to 160 when the animals were inoculated with the two viruses, respectively, while the titer of the antibodies was just below 10 in coinfections. The animal inoculated with ReoV developed diffuse alveolar damage similar to the exudative and leakage inflammation found in SARS patients, and was characterized by diffuse hemorrhage, fibroid exudation, hyaline membrane formation, and type II pneumocytes hyperplasia in alveolar interstitia. The pulmonary epithelial necrosis, excoriation, and early fibrosis of pulmonary tissue were only observed in ReoV-SARS-CoV groups and in SARS-CoV/ReoV groups. Other typical pathological changes included hemorrhagic necrosis in lymph nodes and spleen and hydropic degeneration in the liver. On the contrary, guinea pigs infected with SARS-CoV only developed interstitial pneumonitis. Our experiment demonstrate that ReoV might be one of the primary causes of SARS, since simultaneous coinfection can duplicate the typical pathological changes similar to that of SARS patients. This guinea pig model may provide a useful animal model for SARS.

Animals↗
PubMed2005

Experimental infection of macaques with the human reovirus BYD1 strain: an animal model for the study of the severe acute respiratory syndrome.

Experimental studies were performed to determine the role of a newly isolated reovirus (ReoV) from a severe acute respiratory syndrome (SARS) patient in the etiology of this newly described serious respiratory syndrome. Four cynomologus macaques were inoculated with this reovirus (BYD1) in an attempt to replicate the infection and pathology observed in SARS. The body temperature of the infected monkeys was monitored three times a day, and blood and fecal samples were periodically collected for specific immunology determinations. On days 7 and 33 after inoculation, necropsies for pathological accessment and pathogen isolation were performed. The four infected macaques developed a fever on days 3 and 4 after inoculation, and maintainted a febrile state for 4-6 days. The highest temperature in the animals recorded was 40.4 degrees C. After a recovery phase, the macaques developed a second febrile condition. Antibody titers against the reovirus injected by the intravenous route occurred in higher number than those in the nasal cavity. Four macaque monkeys demonstrated diffuse alveolar damage, characterized by hemorrhagic pneumonia, serosanguineous exudates, formation of hyaline membranes, and type II pneumocyte hyperplasia, which were similar to those that have been noted in SARS patients. Lymphocytes decreased in the cortex of the lymph node and in the white pulp of the spleen. ReoV was detected in pneumonic tissue by virus isolation and RT-PCR. The macaques infected with the newly isolated reovirus developed a fever, diffuse alveolar damage and pulmonary interstitial inflammation similar to that noted in SARS patients. This evidence demonstrates that ReoV might have a primary role in the etiology of SARS.

Animals↗
PubMed2005

Paced electrogram fractionation analysis of arrhythmogenic tendency in DeltaKPQ Scn5a mice.

INTRODUCTION: Gain-of-function mutations within Scn5a, including the DeltaKPQ 1505-1507 deletion in the inactivation domain compromising myocardial repolarization, are implicated in human long QT 3 syndrome (LQT3), associated with ventricular arrhythmogenesis and sudden death. METHODS AND RESULTS: Patch clamp studies on isolated ventricular Scn5a+/Delta myocytes from DeltaKPQ mice produced by homologous recombination in embryonic stem (ES) cells confirmed such altered electrophysiological properties of the mutant channel. Programmed electrical stimulation (PES) with decremental pacing from the basal right ventricular epicardial surface and paced electrogram fractionation analysis (PEFA) of electrograms recorded from the basal left ventricular epicardial surface of Langendorff-perfused whole heart preparations demonstrated ventricular tachycardia (VT) in 8 of 9 Scn5a+/Delta mutant (but no Scn5a+/+ (wild-type (WT)) controls; n = 17), with increased electrogram durations (EGD) and more dispersed conduction curves. Isoproterenol (100 nM) was without effect on tachycardic Scn5a+/Delta hearts (n = 9) yet propranolol (1 microM) prevented VT in all isoproterenol-infused WT control (n = 4) but no Scn5a+/Delta hearts (n = 4). Furthermore propranolol itself increased EGD and dispersion in Scn5a+/Delta hearts. In contrast, mexiletine (10 microM) suppressed VTs in 4 of 5 Scn5a+/Delta hearts without altering EGD or dispersion. CONCLUSION: Beta-adrenoreceptor blockade does not confer an antiarrhythmic effect and may even enhance arrhythmogenesis by increasing reentrant substrate in Scn5a+/Delta hearts while mexiletine protects against VT without modifying conduction characteristics. Together these findings permit a scheme where VT in LQT3 is initiated by triggered mechanisms but propagated by reentry.

Action Potentials↗
PubMed2005

CCT chaperonin complex is required for the biogenesis of functional Plk1.

Experiments from several different organisms have demonstrated that polo-like kinases are involved in many aspects of mitosis and cytokinesis. Here, we provide evidence to show that Plk1 associates with chaperonin-containing TCP1 complex (CCT) both in vitro and in vivo. Silencing of CCT by use of RNA interference (RNAi) in mammalian cells inhibits cell proliferation, decreases cell viability, causes cell cycle arrest with 4N DNA content, and leads to apoptosis. Depletion of CCT in well-synchronized HeLa cells causes cell cycle arrest at G(2), as demonstrated by a low mitotic index and Cdc2 activity. Complete depletion of Plk1 in well-synchronized cells also leads to G(2) block, suggesting that misfolded Plk1 might be responsible for the failure of CCT-depleted cells to enter mitosis. Moreover, partial depletion of CCT or Plk1 leads to mitotic arrest. Finally, the CCT-depleted cells reenter the cell cycle upon reintroduction of the purified constitutively active form of Plk1, indicating that Plk1 might be a CCT substrate.

Animals↗
PubMed2005

The MCM complex: its role in DNA replication and implications for cancer therapy.

The MCM complex controls the once per cell cycle DNA replication in eukaryotic cells. In a process known as DNA replication licensing, it primes chromatin for DNA replication by binding origins of DNA replication during the late M to early G1 phase of the cell cycle. Activated by S phase promoting protein kinases, the origin-bound MCM complexes unwind the double stranded DNA at the origins, recruit DNA polymerases and initiate DNA synthesis. Coupled with the initiation of DNA replication in the S phase, the MCM complexes move away from replication origins as a component of the DNA replication fork, likely serving as DNA helicases. Their departure deprives replication origins the ability to re-initiate DNA replication for the reminder of the cell cycle. Because of its vital role in genome duplication in proliferating cells, deregulation of the MCM function results in chromosomal defects that may contribute to tumorigenesis. The MCM proteins are highly expressed in malignant human cancers cells and pre-cancerous cells undergoing malignant transformation. They are not expressed in differentiated somatic cells that have been withdrawn from the cell cycle. Therefore, these proteins are ideal diagnostic markers for cancer and promising targets for anti-cancer drug development. In this article, I will overview the structures and functions of the MCM complex with an effort to integrate insights from recent biochemical and structural studies. Discussions will also cover activities and structures of the complex that may be useful for the development of drug screens.

Animals↗
PubMed2004

Structure of human POT1 bound to telomeric single-stranded DNA provides a model for chromosome end-protection.

The POT1 (protection of telomeres 1) protein binds the single-stranded overhang at the ends of chromosomes in diverse eukaryotes. It is essential for chromosome end-protection in the fission yeast Schizosaccharomyces pombe, and it is involved in regulation of telomere length in human cells. Here, we report the crystal structure at a resolution of 1.73 A of the N-terminal half of human POT1 (hPOT1) protein bound to a telomeric single-stranded DNA (ssDNA) decamer, TTAGGGTTAG, the minimum tight-binding sequence indicated by in vitro binding assays. The structure reveals that hPOT1 contains two oligonucleotide/ oligosaccharide-binding (OB) folds; the N-terminal OB fold binds the first six nucleotides, resembling the structure of the S. pombe Pot1pN-ssDNA complex, whereas the second OB fold binds and protects the 3' end of the ssDNA. These results provide an atomic-resolution model for chromosome end-capping.

Base Sequence↗
PubMed2004

Modeling correlated main-chain motions in proteins for flexible molecular recognition.

We describe a new method for modeling protein and ligand main-chain flexibility, and show its ability to model flexible molecular recognition. The goal is to sample the full conformational space, including large-scale motions that typically cannot be reached in molecular dynamics simulations due to the computational intensity, as well as conformations that have not been observed yet by crystallography or NMR. A secondary goal is to assess the degree of flexibility consistent with protein-ligand recognition. Flexibility analysis of the target protein is performed using the graph-theoretic algorithm FIRST, which also identifies coupled networks of covalent and noncovalent bonds within the protein. The available conformations of the flexible regions are then explored with ROCK by random-walk sampling of the rotatable bonds. ROCK explores correlated motions by only sampling dihedral angles that preserve the coupled bond networks in the protein and generates conformers with good stereochemistry, without using a computationally expensive potential function. A representative set of the conformational ensemble generated this way can be used as targets for docking with SLIDE, which handles the flexibility of protein and ligand side-chains. The realism of this protein main-chain conformational sampling is assessed by comparison with time-resolved NMR studies of cyclophilin A motions. ROCK is also effective for modeling the flexibility of large cyclic and polycyclic ligands, as demonstrated for cyclosporin and zearalenol. The use of this combined approach to perform docking with main-chain flexibility is illustrated for the cyclophilin A-cyclosporin complex and the estrogen receptor in complex with zearalenol, while addressing the question of how much flexibility is allowed without hindering molecular recognition.

Biochemical Phenomena↗
PubMed2004

Specific pattern of ionic channel gene expression associated with pacemaker activity in the mouse heart.

Even though sequencing of the mammalian genome has led to the discovery of a large number of ionic channel genes, identification of the molecular determinants of cellular electrical properties in different regions of the heart has been rarely obtained. We developed a high-throughput approach capable of simultaneously assessing the expression pattern of ionic channel repertoires from different regions of the mouse heart. By using large-scale real-time RT-PCR, we have profiled 71 channels and related genes in the sinoatrial node (SAN), atrioventricular node (AVN), the atria (A) and ventricles (V). Hearts from 30 adult male C57BL/6 mice were microdissected and RNA was isolated from six pools of five mice each. TaqMan data were analysed using the threshold cycle (C(t)) relative quantification method. Cross-contamination of each region was checked with expression of the atrial and ventricular myosin light chains. Two-way hierarchical clustering analysis of the 71 genes successfully classified the six pools from the four distinct regions. In comparison with the A, the SAN and AVN were characterized by higher expression of Nav beta 1, Nav beta 3, Cav1.3, Cav3.1 and Cav alpha 2 delta 2, and lower expression of Kv4.2, Cx40, Cx43 and Kir3.1. In addition, the SAN was characterized by higher expression of HCN1 and HCN4, and lower expression of RYR2, Kir6.2, Cav beta 2 and Cav gamma 4. The AVN was characterized by higher expression of Nav1.1, Nav1.7, Kv1.6, Kvbeta1, MinK and Cav gamma 7. Other gene expression profiles discriminate between the ventricular and the atrial myocardium. The present study provides the first genome-scale regional ionic channel expression profile in the mouse heart.

Animals↗
PubMed2004

Sampling protein conformations and pathways.

Protein flexibility and rigidity can be analyzed using constraint theory, which views proteins as 3D networks of constraints involving covalent bonds and also including hydrophobic interactions and hydrogen bonds. This article describes an algorithm, ROCK (Rigidity Optimized Conformational Kinetics), which generates new conformations for these complex networks with many interlocked rings while maintaining the constraints. These new conformations are tracked for the flexible regions of a protein, while leaving the rigid regions undisturbed. An application to HIV protease demonstrates how large the flap motion can be. The algorithm is also used to generate conformational pathways between two distinct protein conformations. As an example, directed trajectories between the closed and the occluded conformations of the protein dihydrofolate reductase are determined.

Algorithms↗
PubMed2004

Requirement of neuronal- and cardiac-type sodium channels for murine sinoatrial node pacemaking.

The majority of Na+ channels in the heart are composed of the tetrodotoxin (TTX)-resistant (KD, 2-6 microm) Nav1.5 isoform; however, recently it has been shown that TTX-sensitive (KD, 1-10 nm) neuronal Na+ channel isoforms (Nav1.1, Nav1.3 and Nav1.6) are also present and functionally important in the myocytes of the ventricles and the sinoatrial (SA) node. In the present study, in mouse SA node pacemaker cells, we investigated Na+ currents under physiological conditions and the expression of cardiac and neuronal Na+ channel isoforms. We identified two distinct Na+ current components, TTX resistant and TTX sensitive. At 37 degrees C, TTX-resistant iNa and TTX-sensitive iNa started to activate at approximately -70 and approximately -60 mV, and peaked at -30 and -10 mV, with a current density of 22 +/- 3 and 18 +/- 1 pA pF(-1), respectively. TTX-sensitive iNa inactivated at more positive potentials as compared to TTX-resistant iNa. Using action potential clamp, TTX-sensitive iNa was observed to activate late during the pacemaker potential. Using immunocytochemistry and confocal microscopy, different distributions of the TTX-resistant cardiac isoform, Nav1.5, and the TTX-sensitive neuronal isoform, Nav1.1, were observed: Nav1.5 was absent from the centre of the SA node, but present in the periphery of the SA node, whereas Nav1.1 was present throughout the SA node. Nanomolar concentrations (10 or 100 nm) of TTX, which block TTX-sensitive iNa, slowed pacemaking in both intact SA node preparations and isolated SA node cells without a significant effect on SA node conduction. In contrast, micromolar concentrations (1-30 microm) of TTX, which block TTX-resistant iNa as well as TTX-sensitive iNa, slowed both pacemaking and SA node conduction. It is concluded that two Na+ channel isoforms are important for the functioning of the SA node: neuronal (putative Nav1.1) and cardiac Nav1.5 isoforms are involved in pacemaking, although the cardiac Nav1.5 isoform alone is involved in the propagation of the action potential from the SA node to the surrounding atrial muscle.

Action Potentials↗
PubMed2004

Upregulation of corin gene expression in hypertrophic cardiomyocytes and failing myocardium.

High levels of plasma atrial natriuretic peptides (ANP) are associated with pathological conditions such as congestive heart failure (CHF). Recently, we have identified a cardiac serine protease, corin, that is the pro-ANP convertase. In this study, we examined the regulation of corin gene expression in cultured hypertrophic cardiomyocytes and in the left ventricular (LV) myocardium of a rat model of heart failure. Quantitative RT-PCR analysis showed that both corin and ANP mRNA levels were significantly increased in phenylephrine (PE)-stimulated rat neonatal cardiomyocytes in culture. The increase in corin mRNA correlated closely with the increase in cell size and ANP mRNA expression in the PE-treated cells (r = 0.95, P < 0.01; r = 0.92, P < 0.01, respectively). The PE-treated cardiomyocytes had an increased activity in converting recombinant human pro-ANP to biologically active ANP, as determined by a pro-ANP processing assay and a cell-based cGMP assay. In a rat model of heart failure induced by ligation of the left coronary artery, corin mRNA expression in the noninfarcted LV myocardium was significantly higher than that of control heart tissues from sham-operated animals, when examined by Northern blot analysis and RT-PCR at 8 wk. These results indicate that the corin gene is upregulated in hypertrophic cardiomyocytes and failing myocardium. Increased corin expression may contribute to elevation of ANP in the setting of cardiac hypertrophy and heart failure.

Animals↗