Biomedical subjects

W Chen

Publications and source records attributed to W Chen.

At least 145 records · Page 8Linked to original sources

PubMed2001

Use of real-time polymerase chain reaction and molecular beacons for the detection of Escherichia coli O157:H7.

Molecular beacons (MBs) are oligonucleotide probes that fluoresce upon hybridization. In this paper, we described the development of a real-time PCR assay to detect the presence of Escherichia coli O157:H7 using these fluorogenic reporter molecules. MBs were designed to recognize a 26-bp region of the rfbE gene, coding for an enzyme necessary for O-antigen biosynthesis. The specificity of the MB-based PCR assay was evaluated using various enterohemorrhagic (EHEC) and Shiga-like toxin-producing (STEC) E. coli strains as well as bacteria species that cross-react with the O157 antisera. All E. coli serotype O157 tested was positively identified while all other species, including the closely related O55 were not detected by the assay. Positive detection of E. coli O157:H7 was demonstrated when >10(2) CFU/ml was present in the samples. The capability of the assay to detect E. coli O157:H7 in raw milk and apple juice was demonstrated. As few as 1 CFU/ml was detected after 6 h of enrichment. These assays could be carried out entirely in sealed PCR tubes, enabling rapid and semiautomated detection of E. coli O157:H7 in food and environmental samples.

Animals↗

PubMed2001

Palladium-catalyzed regioselective arylation of an electron-rich olefin by aryl halides in ionic liquids.

[figure: see text] Palladium-catalyzed arylation of the electron-rich olefin butyl vinyl ether has been accomplished in the ionic liquid 1-butyl-3-methylimidazolium tetrafluoroborate ([bmim][BF4]), using as the arylating agents aryl iodides and bromides instead of the commonly used, but commercially unavailable and expensive, aryl triflates. The reaction proceeds with high efficiency and remarkable regioselectivity, leading almost exclusively to substitution by various aryl groups at the olefinic carbon alpha to the heteroatom of butyl vinyl ether.

Journal Article↗

PubMed2001

Regulation of membrane targeting of the G protein-coupled receptor kinase 2 by protein kinase A and its anchoring protein AKAP79.

The beta2 adrenergic receptor (beta2AR) undergoes desensitization by a process involving its phosphorylation by both protein kinase A (PKA) and G protein-coupled receptor kinases (GRKs). The protein kinase A-anchoring protein AKAP79 influences beta2AR phosphorylation by complexing PKA with the receptor at the membrane. Here we show that AKAP79 also regulates the ability of GRK2 to phosphorylate agonist-occupied receptors. In human embryonic kidney 293 cells, overexpression of AKAP79 enhances agonist-induced phosphorylation of both the beta2AR and a mutant of the receptor that cannot be phosphorylated by PKA (beta2AR/PKA-). Mutants of AKAP79 that do not bind PKA or target to the beta2AR markedly inhibit phosphorylation of beta2AR/PKA-. We show that PKA directly phosphorylates GRK2 on serine 685. This modification increases Gbetagamma subunit binding to GRK2 and thus enhances the ability of the kinase to translocate to the membrane and phosphorylate the receptor. Abrogation of the phosphorylation of serine 685 on GRK2 by mutagenesis (S685A) or by expression of a dominant negative AKAP79 mutant reduces GRK2-mediated translocation to beta2AR and phosphorylation of agonist-occupied beta2AR, thus reducing subsequent receptor internalization. Agonist-stimulated PKA-mediated phosphorylation of GRK2 may represent a mechanism for enhancing receptor phosphorylation and desensitization.

A Kinase Anchor Proteins↗

PubMed2001

Association of serum estrogen level and ischemic neuroprotection in female rats.

Estrogen-related ischemic neuroprotection has been documented in male and ovariectomized female rats. The precise molecular mechanism underlying estrogen's neuroprotective effect remains obscure. In the present study, we examined whether endogenous estrogen levels affect post-ischemic outcomes in normal cycling female rats. Occlusion of both the common carotid arteries and the right middle cerebral artery (1.5 h) followed by reperfusion (24 h) caused cortical infarction, increased neutrophil accumulation, and elevated antioxidant enzyme and lactate dehydrogenase activities. These post-ischemic changes varied in the female rats and were inversely correlated with circulating estrogen levels. More severe post-ischemic changes and injury accompanied the decline in circulating estrogen levels in normal cycling female rats, indicating that estrogen is probably the major hormonal player in female resistance to ischemia.

Animals↗

PubMed2001

In vitro inhibition of antirecoverin immunoglobulin-mediated death of mammalian photoreceptor cells.

Cancer-associated retinopathy (CAR) is a blinding disease, which can be mediated by autoimmune reactions with a specific calcium-binding retinal protein, recoverin. A number of recent studies demonstrate that agents that mobilize intracellular calcium can protect neurons from apoptotic death induced by a variety of insults. In this study, we investigated the effect of one such agent, potassium, on the survival of mammalian rod photoreceptors exposed to antirecoverin IgG. Primary cell cultures of rat retinal neurons were grown in a chemically defined medium, and cells were exposed to antirecoverin IgG for 72 hr in various concentrations of potassium and the surviving cells counted. Rod photoreceptors were quantitated using antirhodopsin immunofluorescence microscopy, and total cell numbers were determined by 4',6-diamidino-2-phenylindole (DAPI) staining of nuclei. Apoptosis was evaluated by TdT-mediated biotin-dUTP nick-end labeling (TUNEL), cell death-detection ELISA, and DNA laddering. The present study shows that elevated extracellular K+ ([K+](o)) protects retinal neurons from antirecoverin antibody-mediated cell death. The protective effects of ([K+](o)) were shown to be time- and dose-dependent. The inhibition of antirecoverin IgG-mediated death of photoreceptors by elevated ([K+](o)) suggests that the mobilization of internal calcium stores rescues the cells by interfering with apoptotic signal transduction pathways. These data also suggest that the death of photoreceptor cells occurring in CAR possibly can be prevented by reagents and/or environmental changes that mobilize intracellular calcium.

Animals↗

PubMed2001

Elliptic flow in Au+Au collisions at square root(S)NN = 130 GeV.

Elliptic flow from nuclear collisions is a hadronic observable sensitive to the early stages of system evolution. We report first results on elliptic flow of charged particles at midrapidity in Au+Au collisions at square root(S)NN = 130 GeV using the STAR Time Projection Chamber at the Relativistic Heavy Ion Collider. The elliptic flow signal, v2, averaged over transverse momentum, reaches values of about 6% for relatively peripheral collisions and decreases for the more central collisions. This can be interpreted as the observation of a higher degree of thermalization than at lower collision energies. Pseudorapidity and transverse momentum dependence of elliptic flow are also presented.

Journal Article↗

PubMed2001

Cells adapted to the proteasome inhibitor 4-hydroxy- 5-iodo-3-nitrophenylacetyl-Leu-Leu-leucinal-vinyl sulfone require enzymatically active proteasomes for continued survival.

The proteasome is the primary protease used by cells for degrading proteins and generating peptide ligands for class I molecules of the major histocompatibility complex. Based on the properties of cells adapted to grow in the presence of the proteasome inhibitor 4-hydroxy-5-iodo-3-nitrophenylacetyl-Leu-Leu-leucinal-vinyl sulfone (NLVS), it was proposed that proteasomes can be replaced by alternative proteolytic systems, particularly a large proteolytic complex with a tripeptidyl peptidase II activity. Here we show that NLVS-adapted cells retain sensitivity to a number of highly specific proteasome inhibitors with regard to antigenic peptide generation, accumulation of polyubiquitinated proteins, degradation of p53, and cell viability. In addition, we show that in the same assays (with a single minor exception), NLVS-adapted cells are about as sensitive as nonselected cells to Ala-Ala-Phe-chloromethylketone, a specific inhibitor of tripeptidyl peptidase II activity. Based on these findings, we conclude that proteasomes still have essential proteolytic functions in adapted cells that are not replaced by Ala-Ala-Phe-chloromethylketone-sensitive proteases.

Amino Acid Chloromethyl Ketones↗

PubMed2001

Correlation of activation sizes between lateral geniculate nucleus and primary visual cortex in humans.

It is well established that the lateral geniculate nucleus (LGN) and the primary visual cortex (V1) are retinotopically organized. This similarity is expected to reflect a correlation of activation sizes between V1 and LGN during visual stimulation. However, it is a challenge to determine this functional correlation experimentally in the human brain using most neuroimaging techniques due to their low sensitivity for detecting small LGN activation in the thalamus. The purpose of this study was to examine the functional correlation of activation sizes between LGN and V1 during fullfield and hemifield visual stimulation using a large visual field and high-resolution fMRI technique at 4 Tesla. The correlation was determined by comparing the V1 and LGN activation size ratios between the left and right hemispheres of the same subjects. The results reveal 1) a linear correlation of functional activation size ratio between LGN and V1 with a correlation coefficient of 0.96, and 2) the capability of fMRI for quantifying spatial correlation between cortical and subcortical activation. Magn Reson Med 45:202-205, 2001.

Geniculate Bodies↗

PubMed2001

Study of tricarboxylic acid cycle flux changes in human visual cortex during hemifield visual stimulation using (1)H-[(13)C] MRS and fMRI.

The relationships between brain activity and accompanying hemodynamic and metabolic alterations, particularly between the cerebral metabolic rate of oxygen utilization (CMR(O2)) and cerebral blood flow (CBF), are not thoroughly established. CMR(O2) is closely coupled to the rate of tricarboxylic acid (TCA) cycle flux. In this study, the changes in glutamate labeling during (13)C labeled glucose administration were determined in the human brain as an index of alterations in neuronal TCA cycle turnover during increased neuronal activity. Two-volume (1)H-[(13)C] MR spectroscopy (MRS) of the visual cortex was combined with functional MRI (fMRI) at 4 Tesla. Hemifield visual stimulation was employed to obtain data simultaneously from activated and control regions located symmetrically in the two hemispheres of the brain. The results showed that the fractional change in the turnover rate of C4 carbon of glutamate was less than that of CBF during visual stimulation. The fractional changes in CMR(O2) (Delta CMR(O2)) induced by activation must be equal to or less than the fractional change in glutamate labeling kinetics. Therefore, the results impose an upper limit of approximately 30% for Delta CMR(O2) and demonstrate: 1) that fractional CBF increases exceed Delta CMR(O2) during elevated activity in the visual cortex, and 2) that such an unequal change would explain the observed positive blood oxygenation level dependent (BOLD) effect in fMRI. Magn Reson Med 45:349-355, 2001.

Blood Glucose↗

PubMed2001

Simultaneous degradation of organophosphorus pesticides and p-nitrophenol by a genetically engineered Moraxella sp. with surface-expressed organophosphorus hydrolase.

Moraxella sp., a native soil organism that grows on p-nitrophenol (PNP), was genetically engineered for the simultaneous degradation of organophosphorus (OP) pesticides and p-nitrophenol (PNP). The truncated ice nucleation protein (INPNC) anchor was used to target the pesticide-hydrolyzing enzyme, organophosphorus hydrolase (OPH), onto the surface of Moraxella sp., alleviating the potential substrate uptake limitation. A shuttle vector, pPNCO33, coding for INPNC-OPH was constructed and the translocation, surface display, and functionality of OPH were demonstrated in both E. coli and Moraxella sp. However, whole cell activity was 70-fold higher in Moraxella sp. than E. coli. The resulting Moraxella sp. degraded organophosphates as well as PNP rapidly, all within 10 h. The initial hydrolysis rate was 0.6 micromol/h/mg dry weight, 1.5 micromol/h/mg dry weight, and 9.0 micromol/h/mg dry weight for methyl parathion, parathion, and paraoxon, respectively. The possibility of rapidly degrading OP pesticides and their byproducts should open up new opportunities for improved remediation of OP nerve agents in the future.

Aryldialkylphosphatase↗

PubMed2001

Splicing factor SRP20 is a novel partner of BCL6 in a t(3;6)(q27;p21) translocation in transformed follicular lymphoma.

The BCL6 gene mapped at chromosome band 3q27 encodes a zinc-finger transcription factor and is frequently rearranged and deregulated in B-cell non-Hodgkin's lymphoma (NHL) by promiscuous chromosomal translocations which involve diverse genes. We identified a novel t(3;6)(q27;p21) in a follicular lymphoma (FL) with histologic evidence of transformation and, by cloning the translocation junction, determined that the SRP20 gene was the partner. In this translocation, the 5' regulatory region of the BCL6 was substituted by a putative regulatory region of SRP20. Previously, we hypothesized that substitution of BCL6 promoter by those of the partner genes that were constitutively expressed throughout B-cell development led to persistent and inappropriate expression of BCL6. We examined the expression pattern of SRP20 during B-cell development by Northern blot analysis of a panel of B-cell lines representing various stages of B-cell development and noted that SRP20 mRNA was expressed throughout B-cell development. The SRP20 gene plays an important role in regulation of pre-mRNA splicing, and is expressed specifically in lymphoid tissues. This study provides the first evidence of SRP20 gene rearrangement in human hematopoietic malignancies.

Base Sequence↗

PubMed2001

Multiple enhancers contribute to expression of the NK-2 homeobox gene ceh-22 in C. elegans pharyngeal muscle.

Gene expression in the pharyngeal muscles of C. elegans is regulated in part by the NK-2 family homeodomain factor CEH-22, which is structurally and functionally related to Drosophila Tinman and the vertebrate Nkx2-5 factors. ceh-22 is expressed exclusively in the pharyngeal muscles and is the earliest gene known to be expressed in this tissue. Here we characterize the ceh-22 promoter region in transgenic C. elegans. A 1.9-kb fragment upstream of ceh-22 is sufficient to regulate reporter gene expression in a pattern identical to the endogenous gene. Within this promoter we identified two transcriptional enhancers and characterized their cell type and temporal specificity. The distal enhancer becomes active in the pharynx near the time that ceh-22 expression initiates; however, it becomes active more broadly later in development. The proximal enhancer becomes active after the onset of ceh-22 expression, but it is active specifically in the ceh-22-expressing pharyngeal muscles. We suggest these enhancers respond to distinct signals that initiate and maintain ceh-22 gene expression. Proximal enhancer activity requires a short segment containing a CEH-22 responsive element, suggesting that CEH-22 autoregulates its own expression.

Animals↗

PubMed2001

Go!Poly: A gene-oriented polymorphism database.

Human genome polymorphisms play a key role in defining the molecular basis of phenotypic differences between individuals in aspects such as disease susceptibility and drug responses. The database requirements for supporting the study of human genetic variation have been well recognized. In order to meet these needs, several generalized databases have been built. However, it is still hard for users to find gene-related variation data from these huge and sophisticated databases. In its role as a gene-oriented directory of polymorphism data, Go!Poly (Gene Oriented Polymorphism Database; http://61.139.84.5/gopoly/) utilizes two new highly curated and non-redundant resources, LocusLink (http://www.ncbi.nlm.nih.gov/LocusLink/) and RefSeq (http://www.ncbi.nlm.nih.gov/LocusLink/refseq.html), as the standard for identifying and positioning nucleotide variations. As a generalized polymorphism database, Go!Poly extracts human gene-linked sequence variations of all common types (SNP, insertion-deletion, simple tandem repeat, and complex nucleotides variations) from various public resources including scientific journals and internet resources, such as HGBASE (http://hgbase.cgr.ki.se) and dbSNP (http://www.ncbi.nlm.nih.gov/SNP/). The polymorphism data are then categorized into different gene loci, and the reference sequences given by LocusLink are used as positioning references. Through close integration with LocusLink, Go!Poly also provides facilitated connections among sequence data, gene name, and related biological information. This feature also makes Go!Poly easy to search and navigate. Future automated annotations and internal consistency checking may also benefit from this. Extensive efforts are being taken to make the polymorphism information generated by the Chinese scientific community available from this resource.

China↗

PubMed2001

Mass spectrometric analysis of recombinant adenylate cyclase toxin from Bordetella pertussis strain 18323/pHSP9.

The adenylate cyclase toxin-hemolysin (ACT) is a key virulence factor of the whooping cough agent Bordetella pertussis (Bp). The major cytotoxic activity of this 1706-residue protein consists of its capacity to invade a variety of eukaryotic cells directly across their cytoplasmic membrane and to deliver into cells a catalytic adenylate cyclase domain. This causes impairment of immune effector cells and apoptosis of lung macrophages by uncontrolled conversion of ATP to cAMP. The adenylate cyclase toxin-hemolysin acquires biological activity upon post-translational amide-linked palmitoylation of the epsilon-amino group of lysine 983 (K983) by the accessory fatty acyltransferase, CyaC. However, an additional conserved acylation site can be identified in ACT at lysine 860 (K860) and this residue is palmitoylated when recombinant ACT is produced in Escherichia coli (r-Ec-ACT). In this paper we report the double acylation of r-Bp-ACT secreted by a recombinant Bp strain 18323/pHSP9. This strain overproduces ACT from an oligocopy plasmid carrying the entire cya locus of Bordetella pertussis 18323. Palmitoylation of both conserved lysines (K860 and K983) of r-Bp-ACT expressed by this Bp strain was found. In addition, an error in the deduced protein sequence was identified, with Leu being the real residue at position 1001 and not the Val residue given in the published gene sequence. We also discuss these results in comparison with those from recombinant ACT expressed in E. coli strain K12 XL1-Blue. The analytical approach for characterization of the fatty acylation of ACT from strain 18323/pHSP9 consisted of multiple proteolytic digestion procedures (trypsin, Asp-N), microcapillary liquid chromatography/tandem mass spectrometry and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Adenylate Cyclase Toxin↗

PubMed2001

17O relaxation time and NMR sensitivity of cerebral water and their field dependence.

17O spin relaxation times and sensitivity of detection were measured for natural abundance H(2)(17)O in the rat brain at 4.7 and 9.4 Tesla. The relaxation times were found to be magnetic field independent (T(2) = 3.03 +/- 0.08 ms, T(*)(2) = 1.79 +/- 0.04 ms, and T(1) = 4.47 +/- 0.14 ms at 4.7T (N = 5); T(2) = 3.03 +/- 0.09 ms, T(*)(2) = 1.80 +/- 0.06 ms, and T(1) = 4.84 +/- 0.18 ms at 9.4T (N = 5)), consistent with the concept that the dominant relaxation mechanism is the quadrupolar interaction for this nucleus. The (17)O NMR sensitivity was more than fourfold higher at 9.4T than at 4.7T, for both the rat brain and a sodium chloride solution. With this sensitivity gain, it was possible to obtain localized (17)O spectra with an excellent signal-to-noise ratio (SNR) within 15 s of data acquisition despite the relatively low gyromagnetic ratio of this nucleus. Such a 15-s 2D (17)O-MRS imaging data set obtained for natural abundance H(2)(17)O in the rat brain yielded an SNR greater than 40:1 for a approximately 16 microl voxel. This approach was employed to measure cerebral blood flow using a bolus injection of H(2)(17)O via one internal carotid artery. These results demonstrate the ability of (17)O-MRS imaging to reliably map the H(2)(17)O dynamics in the brain tissue, and its potential for determining tissue blood flow and oxygen consumption rate changes in vivo. Magn Reson Med 45:543-549, 2001.

Animals↗

PubMed2001

Microstrip RF surface coil design for extremely high-field MRI and spectroscopy.

A new type of high-frequency RF surface coil was developed for in vivo proton or other nuclei NMR applications at 7T. This is a purely distributed-element and transmission line design. The coil consists of a thin strip conductor (copper or silver) and a ground plane separated by a low-loss dielectric material with a thickness (H). Due to its specific semi-open transmission line structure, substantial electromagnetic energy is stored in the dielectric material between the thin conductor and the ground plane, which results in a reduced radiation loss and a reduced perturbation of sample loading to the RF coil compared to conventional surface coils. The coil is characterized by a high Q factor, no RF shielding, small physical coil size, lower cost, and easy fabrication. A brief theoretical description of the microstrip RF coil is given that can be used to guide the coil designs. A set of gradient-recalled echo images were acquired by using the single- and two-turn microstrip RF surface coils from both phantom and human brain at 7T, which show good penetration and sensitivity. The two-turn coil design significantly improves the B1 symmetry as predicted by the microstrip theory. The optimum H for microstrip surface coils is approximately 7 mm. This coil geometry yields a B1 penetration similar to that of conventional surface coils. SNR comparison was made between the microstrip coil and conventional surface coils with and without RF shielding. The results reveal that the novel surface coil design based on the microstrip concept makes very high-field MRI/MRS more convenient and efficient in research and future clinics.

Artifacts↗

PubMed2001

Observed BOLD effects on cerebral metabolite resonances in human visual cortex during visual stimulation: a functional (1)H MRS study at 4 T.

Using the localized spin-echo (1)H MRS technique, the water resonance and methyl resonance peaks of the cerebral metabolites N-acetylaspartate (NAA at 2.0 ppm) and phosphocreatine/creatine (Cr at 3.0 ppm) were studied in the human visual cortex to detect and quantify the blood oxygenation level dependent (BOLD) effect during visual stimulation at 4 T. Significant BOLD effects, which reflect the increases of spectral peak height (H) accompanied by the decreases of spectral linewidth (Deltaupsilon(1/2)), were observed in NAA (H: 2.5%; Deltaupsilon(1/2): -1.7%) and Cr (H: 3.1%; Deltaupsilon(1/2): -1.8%) as well as in water (H: 3.1%; Deltaupsilon(1/2): -2.3%). Because NAA and Cr mainly exist in the brain cells, the BOLD effects on these cerebral metabolite resonances only measure the susceptibility component spreading into the extravascular cellular compartment. In contrast, water is affected in the intra- and the extravascular compartments. Therefore, the water signal measures the BOLD effects in both compartments. BOLD responses in water were similar to those observed in metabolites. The similarity indicates that the susceptibility spreading into the extravascular parenchyma contributed significantly to the observed BOLD effects at 4 T. Finally, taking advantage of the higher NMR sensitivity at 4 T, the feasibility of measuring BOLD effects on cerebral metabolites by localized (1)H MRS is demonstrated.

Adult↗

PubMed2001

Autoimmune-mediated vasculopathy.

The use of the immunosuppressive drug cyclosporine A (CsA) in solid organ transplantation can be associated with the development of vasculopathy as part of the complex immune response involved in chronic rejection, including autoimmune recognition. Although CsA can directly affect endothelial cells, this drug alters the T cell repertoire promoting autoimmune recognition. The present studies evaluated the ability of CsA-induced autoreactive T cells to mediate vascular lesions in syngeneic heart grafts. Graft vasculopathy developed in syngeneic heart grafts following either the primary induction of autoimmunity with CsA or the adoptive transfer of CsA-induced autoreactive T cells. Initially, an inflammatory response occurred in the medial wall of the small arterial vessels, accompanied by a perivascular lymphocytic infiltrate (including a lymphocytic infiltrate into the myocardium), followed by progression of vascular disease with endothelial cell proliferation. The development and progression of vascular disease correlated with the cytokine profile of the infiltrating lymphocytes with type 1 cytokines detected early and type 2 cytokines detected as the disease progressed. Initiation of this response correlated with upregulation of the target antigen recognized by the CsA-induced autoreactive T cells, the MHC class II-invariant chain peptide complex. This antigen complex, when upregulated on endothelial cells by interferon, allowed effective targeting by the autoreactive T lymphocytes. Strategies to inhibit the upregulation of MHC class II antigens by treatment of the recipients with chloroquine truncated the disease process. The results of these studies suggest that CsA-induced autoreactive mechanisms can contribute to the development of graft vasculopathy.

Adoptive Transfer↗