Biomedical subjects

X Nan

Publications and source records attributed to X Nan.

16 recordsLinked to original sources

PubMed2003

Development of an Ad7 cosmid system and generation of an Ad7deltaE1deltaE3HIV(MN) env/rev recombinant virus.

A strategy to circumvent immune responses to adenovirus (Ad) resulting from natural infection or repeated vector administrations involves sequential use of vectors from different Ad serotypes. To further develop an Ad-HIV recombinant AIDS vaccine approach, a replication-defective recombinant Ad from a non-subgroup C virus was required. Using a cosmid system, we generated an Ad7deltaE1deltaE3HIV(MN) env/rev recombinant virus and compared expression of the inserted HIV genes with a similarly constructed replication-competent Ad7deltaE3HIV(MN)env/rev recombinant. Ad7deltaE1deltaE3HIV(MN)env/rev expressed both HIV env and rev gene products. The envelope protein was correctly processed and functional, mediating syncytia formation of Ad7deltaE1deltaE3HIV(MN) env/rev-infected cells and CD4(+) T lymphocytes. Ad7deltaE1deltaE3HIV(MN)env/rev could be amplified on 293-ORF6 cells, containing the E4 ORF6 gene, shown earlier to support production of an Ad7 vector lacking the E1a gene. The utility of this cell line is now extended to the production of replication-defective Ad7 recombinants lacking E1a, E1b, and protein IX genes. Sequential immunizations with Ad-HIV recombinants based in different Ad serotypes have been shown to effectively elicit both humoral and cellular HIV-specific immune responses. The recombinant Ad7deltaE1deltaE3HIV(MN)env/rev will be useful in such AIDS vaccine strategies. Further, these studies have created new cosmid vectors that can be applied to generation of single- or double-deleted Ad7 recombinants with foreign genes inserted into the E1 and/or E3 regions.

AIDS Vaccines↗

PubMed2001

Expression and postnatal changes of adrenergic receptor subtype mRNA in rat submandibular glands.

Adrenergic receptors (ARs) are involved in regulating saliva secretion and composition in salivary glands. Nine AR subtypes, including three alpha1-ARs (alpha1a-, alpha1b- and alpha1d-ARs), three alpha2-ARs (alpha2A-, alpha2B- and alpha2C-ARs) and three beta-ARs (beta1,beta2- and beta3-ARs), have been identified through molecular cloning. The five subtype genes, alpha1a-, alpha1b-, alpha2A-, beta1-, and beta2-ARs, were expressed in rat submandibular glands. In contrast, the other four subtype mRNAs, alpha1d-, alpha2B-, alpha2C- and beta3-ARs, were not detected by reverse transcription-polymerase chain reaction (RT-PCR). The steady-state mRNA expression for the five AR subtypes in rat submandibular glands was measured by quantitative competitive RT-PCR using synthetic DNA as internal standard at different stages of postnatal development. The relative rank order of AR subtype mRNA expression was alpha1a>beta2>beta1>alpha2A>alpha1b at all stages except that beta1- and alpha2A-subtypes were reversed at 2 weeks of age. The gene expression of alpha1a-AR subtype relative to total AR was low at 2 weeks of age and increased and reached a maximum at 6 weeks of age, whereas those patterns of alpha2A-, beta1- and beta2-AR subtypes were similar to each other and their gene expressions were high at 2 weeks of age and then decreased. On the other hand, the gene expression of alpha1b-AR subtype did not change over the different stages in relation to that of a housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase, and to total AR. Although rat submandibular glands contain the five AR subtype mRNAs, distinct subtype-specific expression is evident.

Age Factors↗

PubMed2001

The biological functions of the methyl-CpG-binding protein MeCP2 and its implication in Rett syndrome.

Methylation of DNA is essential for development in the mouse and plays an important role in inactivation of the X-chromosome, genomic imprinting and gene silencing. The properties of the methyl-CpG binding proteins (MeCPs) are being proved to be the key to interpreting the connection between DNA methylation and transcriptional repression. The founder member of the family, MeCP2, consists of a single polypeptide that contains both a methyl-CpG binding domain (MBD) and transcriptional repression domain (TRD). MBD binds to a single symmetrically methylated CpG site and is responsible for chromatin localization of the protein. NMR studies have revealed that the MBD adopts a wedge-shaped molecular structure. The TRD interacts with Sin3, which is known to form complexes with histone deacetylases. MeCP2-mediated transcriptional repression may involve two distinct mechanisms, one being dependent on chromatin modification by histone deacetylation and the other being chromatin independent. Mutations in MeCP2 gene cause the X-linked neurodevelopmental disease Rett syndrome. The spectrum of mutations reflects the importance of the MBD and TRD domains. We speculate that abnormal gene expression in Rett patients leads to dysfunction of the central nervous system. We propose a genetic therapeutic approach based on activation of the wild type copy of the MeCP2 gene located in the inactive X chromosome.

Animals↗

PubMed2001

[Clinical application of three-dimensional spiral CT in the complex maxillofacial fractures].

OBJECTIVE: The aim of this study was to evaluate the clinical application of the three-dimensional spiral CT in the complex maxillofacial fractures. METHODS: A total of 26 patients with complex maxillofacial fractures were examined using spiral CT with the slice thickness of 3 to 5 mm. The surface shaded display (SSD) method was used for reconstruction of the three-dimensional (3D) images. The three-dimensional images of maxillofacial fractures were obtained by using various rotations. Before the three-dimensional images were reconstructed, the drawing and deleting way was used for these mandibular fractures. RESULTS: The method demonstrated clearly the three-dimensional anatomy and their relationships of the fractures; using the drawing and deleting way, location and move condition of mandibular fractures, especially condylar fractures, were showed clearly. CONCLUSION: The three-dimensional CT is important in evaluating complex maxillofacial fractures. It could be used to demonstrate clearly locations and move conditions of complex maxillofacial fractures and operative plans.

Adolescent↗

PubMed2000

Effects of MCI-154, a calcium sensitizer, on cardiac dysfunction in endotoxic shock in rabbits.

This study was designed to observe the effects of MCI-154, a calcium sensitizer, on cardiac dysfunction after endotoxic shock in rabbits. Ten hours after the rabbits were given injection of 1.0 mg/kg endotoxin (Escherichia coli, O111:B4) via marginal ear veins, 0.1 mg/kg MCI-154 was injected intravenously and then 50 mL/kg normal saline (NS) + 0.1 mg/kg MCI-154 was infused continuously at a rate of 0.7 mL/min. During this process, the parameters of cardiac function were measured. It was found that 10 h after the endotoxin injection, heart rate (HR) was increased significantly while the mean arterial blood pressure (MAP), left ventricular systolic pressure (LVSP), isovolumetric pressure (IP), myocardial contractility (MC), and the area of p-dp/dt(max) vector loop (Lo) all were markedly decreased. Treatment with 50 mL/kg NS alone had slight effects on these parameters. On the contrary, LVSP, IP, MC, and Lo all were increased significantly while HR was not obviously changed and left ventricular end-diastolic pressure (LVEDP) was reduced remarkably following MCI-154 administration in endotoxic shock rabbits. The parameters of myocardial contractility were improved nearly to the values in sham shock group and were markedly higher than that in NS alone-treated group. It can be concluded that MCI-154 can exert significant therapeutic effects on cardiac dysfunction after endotoxic shock, for it improves cardiac function, dilates peripheral blood vessels, and slightly affects HR.

Animals↗

PubMed2000

Effect of MCI-154, a calcium sensitizer, on calcium sensitivity of myocardial fibers in endotoxic shock rats.

This study was designed to investigate the effect of a calcium sensitizer on the Ca2+ sensitivity of myocardial fibers in endotoxic shock rats. Right ventricular papillary muscles from sham shock or endotoxic shock rats were skinned by incubation in saponin solution. Forces of the skinned muscles were recorded when they were activated sequentially by different pCa (-log[Ca2+]) activating solutions with or without positive inotropic agents. Tension-pCa relationship curve of skinned fibers delineated the affinity of troponin C(TnC) for Ca2+ and the medium value pCa50 (pCa required for producing 50% of maximal Ca2+-activated tension) was taken as the quantitative index of Ca2+ sensitivity of TNC. It was found that the maximal Ca2+ activated tension (Tmax) was lower, tension-pCa relationship curve was shifted rightward, and the pCa50 was reduced significantly in endotoxic shock group compared with that of sham shock group. Milrinone could not counteract the above abnormalities. However, when skinned right ventricular papillary fibers from endotoxic shock rats were dealt with activating solutions containing 1 x 10(-5) M MCI-154, the Tmax was significantly increased, the tension-pCa relationship curve was shifted leftward. The pCa50 in MCI-154 group was increased to an extent similar to that of sham shock group and markedly higher than the values of endotoxic shock group and milrinone group. Furthermore, such effects of MCI-154 were concentration dependent. It can been concluded that the sensitivity of cardiac contractile proteins to Ca2+ in endotoxic shock rats is decreased. MCI-154, a calcium sensitizer, can significantly reverse the decreased sensitivity and increase Tmax of myocardial muscles from endotoxic shock rats.

Animals↗

PubMed1999

The solution structure of the domain from MeCP2 that binds to methylated DNA.

MeCP2 is an abundant mammalian protein that binds methylated CpG (mCpG) sequences within double-stranded DNA, represses transcription by recruiting histone deacetylases, and is essential for embryonic development. It is one of a family of proteins which mediate the biological consequences of DNA methylation. These proteins each possess a sequence motif of about 70 residues which, in MeCP2, form a domain necessary and sufficient for binding to mCpG. The solution structure of the mCpG-binding domain (MBD) from MeCP2 has been solved and the DNA-binding surface of the domain mapped using NMR spectroscopy. Residues 95-162 of MeCP2 adopt a novel fold forming a wedge-shaped structure. An N-terminal four-stranded antiparallel beta-sheet forms one face of the wedge, while the other face is formed mainly by a C-terminal helical region. The thin end of the wedge is extended by a long loop between beta-strands B and C containing many basic residues. The B-C loop together with residues in strands B, C and D, and at the N terminus of the alpha-helix, appears to form an interface with methylated DNA. Unstructured residues at the NH2 terminus of the domain are also involved in formation of the complex. The presence of numerous arginine and lysine side-chains on the DNA-binding surface of MBD is consistent with the requirement for the mCpG site to be flanked by non-specific sequences of base-pairs. The absence of symmetry in the domain implies that recognition does not exploit the symmetry of the binding site. A conserved hydrophobic pocket containing the side-chains of Tyr123 and Ile125 on the positively charged beta-sheet face is a candidate for the region of contact with the methyl-groups of the modified cytosine residues.

Amino Acid Sequence↗

PubMed1998

Transcriptional repression by the methyl-CpG-binding protein MeCP2 involves a histone deacetylase complex.

Cytosine residues in the sequence 5'CpG (cytosine-guanine) are often postsynthetically methylated in animal genomes. CpG methylation is involved in long-term silencing of certain genes during mammalian development and in repression of viral genomes. The methyl-CpG-binding proteins MeCP1 and MeCP2 interact specifically with methylated DNA and mediate transcriptional repression. Here we study the mechanism of repression by MeCP2, an abundant nuclear protein that is essential for mouse embryogenesis. MeCP2 binds tightly to chromosomes in a methylation-dependent manner. It contains a transcriptional-repression domain (TRD) that can function at a distance in vitro and in vivo. We show that a region of MeCP2 that localizes with the TRD associates with a corepressor complex containing the transcriptional repressor mSin3A and histone deacetylases. Transcriptional repression in vivo is relieved by the deacetylase inhibitor trichostatin A, indicating that deacetylation of histones (and/or of other proteins) is an essential component of this repression mechanism. The data suggest that two global mechanisms of gene regulation, DNA methylation and histone deacetylation, can be linked by MeCP2.

Acetylation↗

PubMed1998

Gene silencing by methyl-CpG-binding proteins.

An important consequence of CpG methylation is the local silencing of gene expression. In part this can be mediated by direct interference of methylation with the binding of transcription factors. The major component of silencing, however, appears to be the binding of repressors that have an affinity for methyl-CpG. We have studied two proteins that bind to methylated DNA, methyl-CpG-binding protein 1 (MeCP1) and MeCP2. MeCP2 is a relatively abundant chromosomal protein whose localization in the nucleus is primarily dependent on CpG methylation. We find that MeCP2 is a potent transcriptional repressor with a genome-wide distribution. MeCP1 requires multiple methylated CpGs for binding and has previously been implicated as a methyl-CpG-dependent transcriptional repressor. Recent cloning of a candidate gene for a component of MeCP1 may provide clues to its mechanism of action.

Animals↗

PubMed1997

MeCP2 is a transcriptional repressor with abundant binding sites in genomic chromatin.

MeCP2 is an abundant mammalian protein that binds to methylated CpG. We have found that native and recombinant MeCP2 repress transcription in vitro from methylated promoters but do not repress nonmethylated promoters. Repression is nonlinearly dependent on the local density of methylation, becoming significant at the density found in bulk vertebrate genomic DNA. Transient transfection using fusions with the GAL4 DNA binding domain identified a region of MeCP2 that is capable of long-range repression in vivo. Moreover, MeCP2 is able to displace histone H1 from preassembled chromatin that contains methyl-CpG. These properties, together with the abundance of MeCP2 and the high frequency of its 2 bp binding site, suggest a role as a global transcriptional repressor in vertebrate genomes.

Animals↗

PubMed1997

A component of the transcriptional repressor MeCP1 shares a motif with DNA methyltransferase and HRX proteins.

Methylation of cytosines within the sequence CpG is essential for mouse development and has been linked to transcriptional suppression in vertebrate systems. Methyl-CpG binding proteins (MeCPs) 1 and 2 bind preferentially to methylated DNA and can inhibit transcription. The gene for MeCP2 has been cloned and a methyl-CpG binding domain (MBD) within it has been defined. A search of DNA sequence databases with the MBD sequence identified a human cDNA with potential to encode an MBD-like region. Sequencing of the complete cDNA revealed that the open reading frame also encodes two cysteine-rich domains that are found in animal DNA methyltransferases (DNMTs) and in the mammalian HRX protein (also known as MLL and All-1). HRX is related to Drosophila trithorax. The protein, known as Protein Containing MBD (PCM1), was expressed in bacteria and shown to bind specifically to methylated DNA. PCM1 also repressed transcription in vitro in a methylation-dependent manner. A polyclonal antibody raised against the protein was able to 'supershift' the native MeCP11 complex from HeLa cells, indicating that PCM1 is a component of mammalian MeCP1.

Amino Acid Sequence↗

PubMed1996

DNA methylation specifies chromosomal localization of MeCP2.

MeCP2 is a chromosomal protein that is concentrated in the centromeric heterochromatin of mouse cells. In vitro, the protein binds preferentially to DNA containing a single symmetrically methylated CpG. To find out whether the heterochromatic localization of MeCP2 depended on DNA methylation, we transiently expressed MeCP2-LacZ fusion proteins in cultured cells. Intact protein was targeted to heterochromatin in wild-type cells but was inefficiently localized in mutant cells with low levels of genomic DNA methylation. Deletions within MeCP2 showed that localization to heterochromatin required the 85-amino-acid methyl-CpG binding domain but not the remainder of the protein. Thus MeCP2 is a methyl-CpG-binding protein in vivo and is likely to be a major mediator of downstream consequences of DNA methylation.

Amino Acid Sequence↗

PubMed1995

Studies of DNA methylation in animals.

We have been studying the evolution and function of DNA methylation in vertebrate animals using three related approaches. The first is to further characterise proteins that bind to methylated DNA. Such proteins can be viewed as 'receptors' of the methyl-CpG 'ligand' that mediate downstream consequences of DNA modification. The second approach involves CpG islands. These patches of non-methylated DNA coincide with most gene promoters, but their origin and functional significance have only recently become the subject of intensive study. The third approach is to trace the evolution of DNA methylation. Genomic methylation patterns of vertebrates are strikingly different from those of invertebrates. By studying methylation in animals that diverged from common ancestors near to the invertebrate/vertebrate boundary, we will assess the possibility that changes in DNA methylation contributed causally to the evolution of the complex vertebrate lineage.

Animals↗

PubMed1994

Purification of CpG islands using a methylated DNA binding column.

CpG islands are short stretches of DNA containing a high density of non-methylated CpG dinucleotides, predominantly associated with coding regions. We have constructed an affinity matrix that contains the methyl-CpG binding domain from the rat chromosomal protein MeCP2, attached to a solid support. A column containing the matrix fractionates DNA according to its degree of CpG methylation, strongly retaining those sequences that are highly methylated. Using this column, we have developed a procedure for bulk isolation of CpG islands from human genomic DNA. As CpG islands overlap with approximately 60% of human genes, the resulting CpG island library can be used to isolate full-length cDNAs and to place genes on genomic maps.

Amino Acid Sequence↗

PubMed1993

Dissection of the methyl-CpG binding domain from the chromosomal protein MeCP2.

MeCP2 is a chromosomal protein which binds to DNA that is methylated at CpG. In situ immunofluorescence in mouse cells has shown that the protein is most concentrated in pericentromeric heterochromatin, suggesting that MeCP2 may play a role in the formation of inert chromatin. Here we have isolated a minimal methyl-CpG binding domain (MBD) from MeCP2. MBD is 85 amino acids in length, and binds exclusively to DNA that contains one or more symmetrically methylated CpGs. MBD has negligable non-specific affinity for DNA, confirming that non-specific and methyl-CpG specific binding domains of MeCP2 are distinct. In vitro footprinting indicates that MBD binding can protect a 12 nucleotide region surrounding a methyl-CpG pair, with an approximate dissociation constant of 10(-9) M.

Base Sequence↗

PubMed1992

Transcriptional repression by methylation of CpG.

Methylated DNA in mammals is associated with transcriptional repression and nuclease resistant chromatin. In this review we discuss how these effects may be mediated by proteins that bind to methylated DNA.

Animals↗