Biomedical subjects

Y Q Feng

Publications and source records attributed to Y Q Feng.

At least 19 recordsLinked to original sources

PubMed2002

Measurement of the nitric oxide metabolites nitrate and nitrite in the human brain by microdialysis.

To examine the feasibility of measuring the nitric oxide (NO) metabolites nitrate and nitrite in microdialysate samples from the human brain, microdialysis probes were placed in normal appearing cerebral cortex of severely head injured patients in the Neurosurgical Intensive Care Unit at Ben Taub General Hospital. Nitrate/nitrite analysis was performed using NO chemiluminescence. Low micromolar levels of NO metabolites were consistently and easily detected. These levels seen are comparable to levels reported in CSF but tissue tortuosity and probe recovery considerations suggest that the absolute concentrations at the probe site are probably ten fold higher. Microdialysis with measurement of nitric oxide metabolites is technically feasible and may provide valuable insights into both normal neurochemistry and neurochemical derangements in disease.

Biomarkers↗

PubMed2001

Position effects are influenced by the orientation of a transgene with respect to flanking chromatin.

We have inserted two expression cassettes at tagged reference chromosomal sites by using recombinase-mediated cassette exchange in mammalian cells. The three sites of integration displayed either stable or silencing position effects that were dominant over the different enhancers present in the cassettes. These position effects were strongly dependent on the orientation of the construct within the locus, with one orientation being permissive for expression and the other being nonpermissive. Orientation-specific silencing, which was observed at two of the three site tested, was associated with hypermethylation but not with changes in chromatin structure, as judged by DNase I hypersensitivity assays. Using CRE recombinase, we were able to switch in vivo the orientation of the transgenes from the permissive to the nonpermissive orientation and vice versa. Switching from the permissive to the nonpermissive orientation led to silencing, but switching from the nonpermissive to the permissive orientation did not lead to reactivation of the transgene. Instead, transgene expression occurred dynamically by transcriptional oscillations, with 10 to 20% of the cells expressing at any given time. This result suggested that the cassette had been imprinted (epigenetically tagged) while it was in the nonpermissive orientation. Methylation analysis revealed that the methylation state of the inverted cassettes resembled that of silenced cassettes except that the enhancer had selectively lost some of its methylation. Sorting of the expressing and nonexpressing cell populations provided evidence that the transcriptional oscillations of the epigenetically tagged cassette are associated with changes in the methylation status of regulatory elements in the transgene. This suggests that transgene methylation is more dynamic than was previously assumed.

Animals↗

PubMed2001

Sequences flanking hypersensitive sites of the beta-globin locus control region are required for synergistic enhancement.

The major distal regulatory sequence for the beta-globin gene locus, the locus control region (LCR), is composed of multiple hypersensitive sites (HSs). Different models for LCR function postulate that the HSs act either independently or synergistically. To test these possibilities, we have constructed a series of expression cassettes in which the gene encoding the enhanced green fluorescent protein (EGFP) is under the control of DNA fragments containing single and multiple HSs of the LCR. LCR DNA fragments containing only the minimal region needed for position-independent expression (HS cores) or containing cores plus flanking sequences (HS units) were compared to ascertain whether conserved sequences between the HS cores contributed to enhancement. Expression of these constructs was measured after targeted integration into three defined loci in murine erythroleukemia cells using recombinase-mediated cassette exchange. At all three marked loci, synergistic enhancement of expression was observed in cassettes containing a combination of HS2, HS3, and HS4 units. In contrast, HS2, HS3, and HS4 cores (without flanking sequences) give an activity equivalent to the sum of the activities of the individual HS cores. These data suggest a model in which an HS core plus flanking regions, bound by specific proteins, forms a structure needed for interaction with other HS units to confer strong enhancement by the LCR. The three targeted integration sites differ substantially in their permissivity for expression, but even the largest LCR construct tested could not overcome these position effects to confer equal expression at all three sites.

Binding Sites↗

PubMed2001

[Advances in solid phase microextraction coupled with high performance liquid chromatography].

Solid phase microextraction (SPME) is a solvent-free technique with high extraction efficiency and easy to perform automatically. It can be coupled with high performance liquid chromatography to perform efficient analysis of compounds with high polarity. The advances in solid phase microextraction coupled with high performance liquid chromatography, including its theory, interfaces, coating materials and applications, are reviewed with 36 references.

Chromatography, Gas↗

PubMed2000

Myocardial extracellular matrix remodeling in transgenic mice overexpressing tumor necrosis factor alpha can be modulated by anti-tumor necrosis factor alpha therapy.

Myocardial fibrosis caused by maladaptive extracellular matrix (ECM) remodeling is implicated in the dysfunction of the failing heart. Matrix metalloproteinases (MMPs) regulate ECM remodeling, and are regulated by cytokines. Transgenic mice with cardiac-specific overexpression of tumor necrosis factor alpha (TNF-alpha) (TNF1.6) develop heart failure. We hypothesized that modulation of TNF-alpha and/or MMP activity might alter the myocardial ECM remodeling process and the development of heart failure. To test this hypothesis, we took advantage of the TNF1.6 mice and studied soluble and total collagens and collagen type profiling by using hydroxyproline quantification, Sircol collagen assay, Northern blot analysis, and immunohistochemistry and studied myocardial function by using echocardiography. Progressive ventricular hypertrophy and dilation in the TNF1.6 mice were accompanied by a significant increase in MMP-2 and MMP-9 activity, an increase in collagen synthesis, deposition, and denaturation, and a decrease in undenatured collagens. In young TNF1.6 mice, these changes in the ECM were associated with marked diastolic dysfunction as demonstrated by significantly reduced transmitral Doppler echocardiographic E/A wave ratio. Anti-TNF-alpha treatment with adenoviral vector expressing soluble TNF-alpha receptor type I attenuated both MMP-2 and MMP-9 activity, prevented further collagen synthesis, deposition and denaturation, and preserved myocardial diastolic function in young, but not old, TNF1.6 mice. The results suggest a critical role of TNF-alpha and MMPs in myocardial matrix remodeling and functional regulation and support the hypothesis that both TNF-alpha and MMPs may serve as potential therapeutic targets in the treatment of heart failure.

Animals↗

PubMed2000

Beta-globin YAC transgenes exhibit uniform expression levels but position effect variegation in mice.

Expression of a construct integrated at different genomic locations often varies because of position effects that have been subcategorized as stable (decreased level of expression) and variegating (decreased proportion of expressing cells). It is well established that locus control regions (LCRs) generally overcome position effects in transgenes. However, whether stable and variegated position effects are equally overcome by an intact LCR has not been determined. We report that single-copy yeast artificial chromosome transgenes containing an unmodified human beta -globin locus were not subject to detectable stable position effects but did undergo mild to severe variegating position effects at three of the four non-centromeric integration sites tested. We also find that, at a given integration site, the distance and the orientation of the LCR relative to the regulated gene contributes to the likelihood of variegating position effects, and can affect the magnitude of its transcriptional enhancement. DNase I hypersensitive site (HSS) formation varies with the proportion of expressing cells, not the level of gene expression, suggesting that silencing of the transgene is associated with a lack of HSS formation in the LCR region. We conclude that transcriptional enhancement and variegating position effects are caused by fundamentally different but inter-dependent mechanisms.

Animals↗

PubMed2000

Deletions within the mouse beta-globin locus control region preferentially reduce beta(min) globin gene expression.

The mouse beta-globin gene cluster is regulated, at least in part, by a locus control region (LCR) composed of several developmentally stable DNase I hypersensitive sites located upstream of the genes. In this report, we examine the level of expression of the beta(min) and beta(maj) genes in adult mice in which HS2, HS3, or HS5,6 has been either deleted or replaced by a selectable marker via homologous recombination in ES cells. Primer extension analysis of RNA extracted from circulating reticulocytes and HPLC analysis of globin chains from peripheral red blood cells revealed that all mutations that reduce the overall output of the locus preferentially decrease beta(min) expression over beta(maj). The implications of these findings for the mechanism by which the LCR controls expression of the beta(maj) and beta(min) promoters are discussed.

Animals↗

PubMed2000

Genomic targeting of methylated DNA: influence of methylation on transcription, replication, chromatin structure, and histone acetylation.

We have developed a strategy to introduce in vitro-methylated DNA into defined chromosomal locations. Using this system, we examined the effects of methylation on transcription, chromatin structure, histone acetylation, and replication timing by targeting methylated and unmethylated constructs to marked genomic sites. At two sites, which support stable expression from an unmethylated enhancer-reporter construct, introduction of an in vitro-methylated but otherwise identical construct results in specific changes in transgene conformation and activity, including loss of the promoter DNase I-hypersensitive site, localized hypoacetylation of histones H3 and H4 within the reporter gene, and a block to transcriptional initiation. Insertion of methylated constructs does not alter the early replication timing of the loci and does not result in de novo methylation of flanking genomic sequences. Methylation at the promoter and gene is stable over time, as is the repression of transcription. Surprisingly, sequences within the enhancer are demethylated, the hypersensitive site forms, and the enhancer is hyperacetylated. Nevertheless, the enhancer is unable to activate the methylated and hypoacetylated reporter. Our findings suggest that CpG methylation represses transcription by interfering with RNA polymerase initiation via a mechanism that involves localized histone deacetylation. This repression is dominant over a remodeled enhancer but neither results in nor requires region-wide changes in DNA replication or chromatin structure.

Acetylation↗

PubMed2000

[Retention behavior of some polycyclic aromatic hydrocarbons on alkylphosphonate-modified magnesia-zirconia composite stationary phase for reversed-phase liquid chromatography].

An alkylphosphonate-modified magnesia-zirconia composite stationary phase (PZMS) for reversed-phase liquid chromatography was prepared. The reversed-phase chromatographic performance of the new packing material was investigated with some polycyclic aromatic hydrocarbons (PAHs) as solutes. The effect of the methanol content in the mobile phase on the retention of PAHs was examined. The logarithmic capacity factor of PAHs was decreased linearly with increasing volume percent of the methanol in the mobile phase, which is similar to that observed on Zorbax ODS. The capacity factor of PAHs on the new stationary phase was also correlated to their partition coefficient between n-octanol and water (log P). A good linear relationship between them was obtained. These results show that the new material can behave as a reversed-phase packing. The separations of phenanthrene and anthracene, and terphenyl isomers were investigated on the new stationary phase and the Zorbax ODS, respectively. The retention of the solutes on the Zorbax ODS is greater than that on the new stationary phase, but the separation selectivity of the solutes on the new stationary phase is better than that on the Zorbax ODS. The better separation selectivity on the new stationary phase may be contributed from the Lewis acid-base interaction between the solutes and the residual Lewis acid sites on the stationary phase.

Alkylation↗

PubMed2000

[Preparation and evaluation of glycine derivatized beta-cyclodextrin bonded silica for high performance liquid chromatography].

Glycine derivatized beta-cyclodextrin bonded silica (GCDS) has been prepared for high performance liquid chromatography through the reactions of beta-cyclodextrin bonded silica with tosyl chloride and glycine in sequence. The separation performance of GCDS for positional isomers, dansyl amino acids and phenylpropionic acids was investigated. It was found that nitrophenols could not be eluted with methanol as mobile phase, but could be eluted with methanol-phosphate buffer solution within appropriate time, which was much different from the behavior observed on native beta-cyclodextrin bonded silica. The effect of column temperature on the enantio-separation of dansyl D,L-Phe with GCDS was examined. The results show that the enantio-separation ability of the GCDS decreases with the increase of the column temperature. Other factors such as methanol concentration and pH of the mobile phase influencing the retention behavior of solutes on the GCDS are also discussed.

Amino Acids↗

PubMed2000

[Advances of capillary electrophoresis and capillary electrochromatography using ionic coating column].

Capillary electrochromatography (CEC) is a promising micro-separation technique, which combines the high selectivity of HPLC and the high efficiency of HPCE. In comparing to CE with fused silica capillary and CEC with ODS stationary phase, capillary electrophoresis and capillary electrochromatography with ionic coating columns can cause EOF more controllable, and provide an alternative separation mechanism. Therefore, the new CEC and CE modes are in favor of optimizing the separation and broadening the range of samples for analysis. The development of capillary electrophoresis and capillary electrochromatography using ionic coating column is reviewed with 48 references.

Amino Acids↗

PubMed1999

Site-specific chromosomal integration in mammalian cells: highly efficient CRE recombinase-mediated cassette exchange.

Expression of experimental constructs in mammalian cells or transgenic animals is difficult to control because it is markedly influenced by position effects. This has limited both the analysis of cis -DNA regulatory elements for transcription and replication, and the physiological analysis of proteins expressed from transgenes. We report here two new methods based on the concept of recombinase-mediated cassette exchange (RMCE) to perform site-specific chromosomal integration. The first method permits the exchange of a negative selectable marker pre-localized on the chromosome with a transgene via a CRE-mediated double recombination between inverted Lox sites. Integration efficiency is close to 100 % of negatively selected mouse erythroleukemia cells and ranges from 10 to 50 % in embryonic stem cells. The second method allows RMCE with no selection at all except for cells that have taken up plasmid transiently. While less efficient, this technique permits novel experimental approaches. We find that integration of a transgene at a given genomic site leads to reproducible expression. RMCE should be useful to develop artificial genetic loci that impart specific and reproducible regulation of transgenes in higher eukaryotes. This should facilitate the analysis of cis -regulatory DNA elements governing expression and position effects, improve our control over the physiological effects of transgenes, and accelerate the development of animal models for complex human diseases.

Animals↗

PubMed1999

Anti-beta s-ribozyme reduces beta s mRNA levels in transgenic mice: potential application to the gene therapy of sickle cell anemia.

Our current strategy for gene therapy of sickle cell anemia involves retroviral vectors capable of transducing "designer" globin genes that code for novel anti-sickling globins (while resisting digestion by a ribozyme), coupled with the expression of a hammerhead ribozyme that can selectively cleave the human beta s mRNA. In this report, we have tested in vivo an anti-beta s hammerhead ribozyme embedded within a cDNA coding for the luciferase reporter gene driven by the human beta-globin promoter and hyper-sensitive sites 3 and 4 of the locus control region. We have created mice transgenic for this luciferase-ribozyme construct and bred the ribozyme transgene into mice that were already transgenic for the human beta s gene. We then measured expression of the beta s transgene at the protein and RNA levels by HPLC and primer extension. The presence of the ribozyme was associated with a statistically significant reduction in the level of beta s mRNA in spleen stress reticulocytes (from 60.5 +/- 4.1% to 52.9 +/- 4.2%) and in the percentage of beta s globin chains in very young mice (from 44.5 +/- 0.6% to 40.8 +/- 0.7%). These results demonstrate that it is possible to decrease the concentration of beta s chains and mRNA with the help of a hammerhead ribozyme. While the enormous amount of globin mRNA in reticulocytes is a challenge for ribozyme technology, the exquisite dependence of the delay time for formation of Hb S nuclei on the concentration of Hb S in red blood cells suggests that even a modest reduction in Hb S concentration would have therapeutic value.

Amino Acid Sequence↗

PubMed1999

Enhancer-dependent transcriptional oscillations in mouse erythroleukemia cells.

By using recombinase-mediated cassette exchange, a method that allows integration of single copies of different constructs at the same predetermined chromosomal location, several expression cassettes have been integrated at a randomly chosen locus in the genome of mouse erythroleukemia cells. The cassettes studied contain the human beta-globin promoter fused to lacZ coding sequences either alone or linked to DNase I-hypersensitive site HS2, HS3, or HS234 (a large locus control region fragment containing HS2, HS3, and HS4) of the human beta-globin locus control region. Analysis of expression of these cassettes revealed mosaic expression patterns reminiscent of, but clearly different from, position effect variegation. Further investigations demonstrated that these mosaic expression patterns are caused by dynamic activation and inactivation of the transcription unit, resulting in oscillations of expression. These oscillations occur once in every few cell cycles at a rate specific for the enhancer present at the locus. DNase I sensitivity studies revealed that the chromatin is accessible and that DNase-hypersensitive sites were present whether or not the transcription unit is active, suggesting that the oscillations occur between transcriptionally competent and transcriptionally active chromatin conformations, rather than between open and closed chromatin conformations. Treatment of oscillating cells with trichostatin A eliminates the oscillations only after the cells have passed through late G1 or early S, suggesting that these oscillations might be caused by changes in histone acetylation patterns.

Animals↗

PubMed1994

[Immunocytochemical study of mononuclear cells in peripheral blood and cerebrospinal fluid of patients with Japanese B encephalitis].

Using the technique of monoclonal antibody-immunogold-silver staining (M-IGSS), the mononuclear cells in peripheral blood (PB) and cerebrospinal fluid (CSF) were screened for immunoreactivity against JBE virus antigen (JBEV-Ag), interleukin-2 receptor (Tac), class II human leucocyte antigen (HLA-DR) and interferon-r (IFN-r) in 17 ELISA diagnosed JEB patients and 17 control cases with non-inflammatory and non-immunological diseases. It was found that JBEV-Ag+ cells were present in PB (16/17) and CSF (12/17) of the JBE group, but absent in both the control group and another 5 cases with mumps virus meningitis. The percentages of Tac+, HLA-DR+ and IFN-r+ cells in JBE patients were higher than those in controls.

Adolescent↗

PubMed1992

[Microwave technique in hepatic surgery: report of 70 cases].

70 hepatic resections were performed using 2450 MHz microwave scalpel. Primary diseased included hepatocellular carcinoma (46 cases), hemangioma (18), hepatobiliary tract stone (2), biliary cystadenoma (1), inflammatory pseudotumor of the live (1), metastatic liver cancer (2). Hemostasis was excellent despite liver cirrhosis in all cases. The average amount of blood loss and blood transfusion was 249 ml and 294 ml respectively. Blood transfusion was not necessary in 30 patients. All cases were free from postoperative bleeding from the liver stump and abdominal infection. No complications attributable to microwave coagulation were noted. We conclude that this new operative technique can be used safely and easily in the field of hepatic surgery.

Carcinoma, Hepatocellular↗

PubMed1991

Effect of heme binding on the structure and stability of Escherichia coli apocytochrome b562.

The structure and stability of apocytochrome b562 were explored using absorption and circular dichroism spectroscopic methods. The polypeptide chain retains a well-defined structure when the prosthetic heme group is removed from cytochrome b562. Circular dichroism measurements estimate 60% helicity for apocytochrome b562, compared with 80% helicity found in holocytochrome b562. At low pH, apocytochrome b562 displays a midpoint pH of 2.9, while ferricytochrome b562 displays a midpoint pH of 2.3. The unfolding of the apoprotein by urea and heat can be well approximated by the two-state transition model. The stability of apocytochrome b562 is significantly reduced from that of the holoprotein. The free energy of stabilization (delta G degrees) and the midpoint transition temperature (Tm) for apocytochrome b562 are found to be 3.2 +/- 0.5 kcal/mol and 52.3 +/- 0.9 degrees C, respectively, compared with 6.6 +/- 0.5 kcal/mol and 67.2 +/- 0.5 degrees C for ferricytochrome b562. The smaller heat capacity change upon unfolding of apocytochrome b562 than that of ferricytochrome b562, estimated from the thermodynamic parameters, indicates that apocytochrome b562 possesses a smaller hydrophobic core than holocytochrome b562. Size-exclusion chromatography studies indicate that the apoprotein is slightly more extended in molecular dimension than ferricytochrome b562. The data suggest that apocytochrome b562 resembles a "molten globule" or a "collapsed form" of the holoprotein, in which secondary structure formation is largely complete while the global folding is either only partially complete or dynamically expanded.

Bacterial Proteins↗

PubMed1991

1H and 15N NMR resonance assignments and preliminary structural characterization of Escherichia coli apocytochrome b562.

The 1H and 15N resonances of uniformly enriched apocytochrome b562 (106 residues) have been assigned. The assignment work began with the identification of the majority of HN-H alpha-H beta subspin systems in two-dimensional DQF-COSY and TOCSY spectra of unlabeled protein in D2O and in 95% H2O/5% D2O buffer. Intraresidue and interresidue NOE connectivities were then searched for in two-dimensional homonuclear NOESY spectra recorded on unlabeled protein and in the three-dimensional NOESY-HMQC spectrum recorded on uniformly 15N-enriched protein. Those data, combined with the main-chain-directed assignment strategy (MCD), led to the assignment of the main-chain and many side-chain resonances of 103 of the 106 residues. Qualitatively, the helical conformation is found to be the dominant secondary structure in apocytochrome b562 as it is in holocytochrome b562. The helical segments in apocytochrome b562 overlap extensively with the helical regions defined in the crystal structure of ferricytochrome b562. In addition, a number of tertiary NOEs have been identified which indicate that the global fold of the apoprotein at least partially resembles the four-helix bundle of the holoprotein. The results presented here, together with the evidence obtained with other methods [Feng and Sligar (1991) Biochemistry (submitted)], support the notion that the interior of the protein is fluid and may correspond to a molten globule state.

Amino Acid Sequence↗