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Biomedical subjects

T Yang

Publications and source records attributed to T Yang.

At least 19 recordsLinked to original sources
PubMed2003

Regulation of cyclooxygenase-2 in renal medulla.

AIM: Renal medulla is a major site for production and action of prostaglandins (PGs). The renal medullary functions as well as structural integrity are in part dependent on PGs under certain physiological or pathophysiological conditions. The two COX isoforms, COX-1 (constitutive form) and COX-2 (inducible form) are both abundantly expressed in renal inner medulla at basal state, raising a question of which COX isoform may mediate the known functions of PGs in the region. As in many other cell types, COX-1 expression in renal medulla is unlikely subject to robust regulation. In contrast, COX-2 expression in renal medulla is markedly stimulated by chronic salt loading, dehydration and endotoxaemia in vivo. At cellular levels, the signalling pathways responsible for the COX-2 stimulation in renal medullary cells seem to involve both the mitogen-activated protein kinases and NF-kappa B. It is likely that in response to various insults that are detrimental to renal medulla, the induction of PG synthesis may become more dependent on COX-2 than COX-1, and this phenomenon may be relevant to the cytoprotective response against the insults.

Animals↗
PubMed2002

Hydrogen peroxide homeostasis: activation of plant catalase by calcium/calmodulin.

Environmental stimuli such as UV, pathogen attack, and gravity can induce rapid changes in hydrogen peroxide (H(2)O(2)) levels, leading to a variety of physiological responses in plants. Catalase, which is involved in the degradation of H(2)O(2) into water and oxygen, is the major H(2)O(2)-scavenging enzyme in all aerobic organisms. A close interaction exists between intracellular H(2)O(2) and cytosolic calcium in response to biotic and abiotic stresses. Studies indicate that an increase in cytosolic calcium boosts the generation of H(2)O(2). Here we report that calmodulin (CaM), a ubiquitous calcium-binding protein, binds to and activates some plant catalases in the presence of calcium, but calcium/CaM does not have any effect on bacterial, fungal, bovine, or human catalase. These results document that calcium/CaM can down-regulate H(2)O(2) levels in plants by stimulating the catalytic activity of plant catalase. Furthermore, these results provide evidence indicating that calcium has dual functions in regulating H(2)O(2) homeostasis, which in turn influences redox signaling in response to environmental signals in plants.

Amino Acid Sequence↗
PubMed2002

Cyclooxygenase cloning in dogfish shark, Squalus acanthias, and its role in rectal gland Cl secretion.

The present studies were carried out with the aims to determine the cDNA sequence for cyclooxygenase (COX) in an elasmobranch species and to study its role in regulation of chloride secretion in the perfused shark rectal gland (SRG). With the use of long primers (43 bp) derived from regions of homology between zebrafish and rainbow trout COX-2 genes, a 600-bp product was amplified from SRG and was found to be almost equally homologous to mammalian COX-1 and COX-2 (65%). The full-length cDNA sequence was obtained by 5'-RACE and by analyzing an EST clone generated by the EST Project of the Mt. Desert Island Biological Laboratory Marine DNA Sequencing Center. The longest open reading frame encodes a 593-amino acid protein that has 68 and 64% homology to mammalian COX-1 and COX-2, respectively. The gene and its protein product is designated as shark COX (sCOX). The key residues in the active site (Try(385), His(388), and Ser(530)) are conserved between the shark and mammalian COX. sCOX contains Val(523) that has been shown to be a key residue determining the sensitivity to COX-2-specific inhibitors including NS-398. The mRNA of sCOX, detected by RT-PCR, was found in all tissues tested, including rectal gland, kidney, spleen, gill, liver, brain, and heart, but not in fin. In the perfused SRG, vasoactive intestinal peptide (VIP) at 5 nM induced rapid and marked Cl(-) secretion (basal: <250 microeq x h(-1) x g(-1); peak response: 3,108 +/- 479 microeq x h(-1) x g(-1)). In the presence of 50 microM NS-398, both the peak response (2,131 +/- 307 microeq x h(-1) x g(-1)) and the sustained response to VIP were significantly reduced. When NS-398 was removed, there was a prompt recovery of chloride secretion to control values. In conclusion, we have cloned the first COX in an elasmobranch species (sCOX) and shown that sCOX inhibition suppresses VIP-stimulated chloride secretion in the perfused SRG.

Amino Acid Sequence↗
PubMed2001

Cloning and expression of a single-chain catalytic antibody that acts as a glutathione peroxidase mimic with high catalytic efficiency.

Glutathione peroxidase (GPX) has a powerful role in scavenging reactive oxygen species. In previous papers we have developed a new strategy for generating abzymes: the monoclonal antibody with a substrate-binding site is first prepared, then a catalytic group is incorporated into the monoclonal antibody's binding site by using chemical mutation [Luo, Zhu, Ding, Gao, Sun, Liu, Yang and Shen (1994) Biochem. Biophys. Res. Commun. 198, 1240-1247; Ding, Liu, Zhu, Luo, Zhao and Ni (1998) Biochem. J. 332, 251-255]. Since then we have established a series of catalytic antibodies capable of catalysing the decomposition of hydroperoxides by GSH. The monoclonal antibody 2F3 was raised against GSH-S-2,4-dinitrophenyl t-butyl ester and exhibited high catalytic efficiency, exceeding that of rabbit liver GPX, after chemical mutation. To produce pharmaceutical proteins and to study the reason why it exhibits high catalytic efficiency, we sequenced, cloned and expressed the variable regions of 2F3 antibody as a single-chain Fv fragment (2F3-scFv) in different bacterial strains. The amounts of 2F3-scFv proteins expressed from JM109 (DE3), BL21 (DE3), and BL21 (coden plus) were 5-10%, 15-20% and 25-30% of total bacterial proteins respectively. The 2F3-scFv was expressed as inclusion bodies, purified in the presence of 8 M urea by Co(2+)-immobilized metal-affinity chromatography (IMAC) and renatured to the active form in vitro by gel filtration. The binding constants of the active 2F3-scFv for GSH and GSSG were 2.46 x 10(5) M(-1) and 1.03 x 10(5) M(-1) respectively, which were less by one order of magnitude than that of the intact 2F3 antibody. The active 2F3-scFv was converted into selenium-containing 2F3-scFv (Se-2F3-scFv) by chemical modification of the reactive serine; the GPX activity of the Se-2F3-scFv was 3394 units/micromol, which approaches the activity of rabbit liver GPX.

Amino Acid Sequence↗
PubMed2001

An improved version of embolic model of brain ischemic injury in the rat.

A modified version of focal embolic stroke model has been developed in rats. Ischemic injury was induced by injection of a pre-formed clot into the middle cerebral artery (MCA). In the first series of experiments, the model was validated. Embolizing a pre-formed clot resulted in an infarction in the territory irrigated by the MCA. At 48 h after embolization, 2,3,5-triphenyltetrazolium chloride (TTC) staining showed that infarction volume was 42.1+/-15.6% (mean+/-S.D.) when 5 microl clot was injected (n=8) and 28.4+/-8.6% in the animals received 3.5 microl clot (n=8). The infarction volume between these two groups showed a significant difference (P<0.05). In the second series of experiments, the natural dissolution of the clot in the MCA was studied. Five min after embolization (n=6), clots were observed in the MCA of all the animals. Clots in the MCA were seen in 68 and 29% of the animals at 1 and 3 h, respectively, after embolization. These results suggest that the procedure described here produces a reliable and reproducible ischemic injury. The clots injected were dissolved in the MCA in relatively short period of time. The model shows a close similarity to thromboembolic stroke in human, and it provides a useful tool to investigate mechanisms and test thrombolytic agents in ischemic brain injury.

Animals↗
PubMed2001

Requirement of two NFATc4 transactivation domains for CBP potentiation.

Recruitment of the coactivator CREB-binding protein (CBP) to transcription factors is important for gene expression. Various regions of CBP such as the KIX and CH3 domains have been shown to interact with numerous transcription factors. The NFAT group of transcription factors is involved in multiple biological processes. NFATc4/NFAT3 has been proposed to play an important role in heart hypertrophy, adipocyte differentiation, and learning and memory. We demonstrate here that two transactivation domains, located at the NH(2) and COOH termini of NFATc4, are critical for interacting with CBP. Each transactivation domain interacts with a distinct region of the CBP protein (the KIX and CH3 domains). Binding of CBP potentiates NFATc4-mediated transcription activity. Both transactivation domains of NFATc4 are required for CBP function. Removal of either NFATc4 transactivation domain abolishes CBP potentiation. Conversely, mutation of the KIX or CH3 domain prevents CBP-mediated potentiation of NFATc4 transcription activation. These data demonstrate that formation of a functional NFATc4.CBP transcription complex requires interactions at two distinct sites.

Binding Sites↗
PubMed2001

Stable ordering in Langmuir-Blodgett films.

Defects in the layering of Langmuir-Blodgett (LB) films can be eliminated by depositing from the appropriate monolayer phase at the air-water interface. LB films deposited from the hexagonal phase of cadmium arachidate (CdA2) at pH 7 spontaneously transform into the bulk soap structure, a centrosymmetric bilayer with an orthorhombic herringbone packing. A large wavelength folding mechanism accelerates the conversion between the two structures, leading to a disruption of the desired layering. At pH > 8.5, though it is more difficult to draw LB films, almost perfect layering is obtained due to the inability to convert from the as-deposited structure to the equilibrium one.

Cadmium↗
PubMed2001

Mediation of tubuloglomerular feedback by adenosine: evidence from mice lacking adenosine 1 receptors.

Adenosine is a determinant of metabolic control of organ function increasing oxygen supply through the A2 class of adenosine receptors and reducing oxygen demand through A1 adenosine receptors (A1AR). In the kidney, activation of A1AR in afferent glomerular arterioles has been suggested to contribute to tubuloglomerular feedback (TGF), the vasoconstriction elicited by elevations in [NaCl] in the macula densa region of the nephron. To further elucidate the role of A1AR in TGF, we have generated mice in which the entire A1AR coding sequence was deleted by homologous recombination. Homozygous A1AR mutants that do not express A1AR mRNA transcripts and do not respond to A1AR agonists are viable and without gross anatomical abnormalities. Plasma and urinary electrolytes were not different between genotypes. Likewise, arterial blood pressure, heart rates, and glomerular filtration rates were indistinguishable between A1AR(+/+), A1AR(+/-), and A1AR(-/-) mice. TGF responses to an increase in loop of Henle flow rate from 0 to 30 nl/min, whether determined as change of stop flow pressure or early proximal flow rate, were completely abolished in A1AR(-/-) mice (stop flow pressure response, -6.8 +/- 0.55 mmHg and -0.4 +/- 0.2 in A1AR(+/+) and A1AR(-/-) mice; early proximal flow rate response, -3.4 +/- 0.4 nl/min and +0.02 +/- 0.3 nl/min in A1AR(+/+) and A1AR(-/-) mice). Absence of TGF responses in A1AR-deficient mice suggests that adenosine is a required constituent of the juxtaglomerular signaling pathway. A1AR null mutant mice are a promising tool to study the functional role of A1AR in different target tissues.

Adenosine↗
PubMed2001

The leukocyte common antigen-related protein tyrosine phosphatase receptor regulates regenerative neurite outgrowth in vivo.

Drosophila and leech models of nervous system development demonstrate that protein tyrosine phosphatase (PTP) receptors regulate developmental neurite outgrowth. Whether PTP receptors regulate neurite outgrowth in adult systems or in regenerative states remains unknown. The leukocyte common antigen-related (LAR) receptor is known to be present in rodent dorsal root ganglion (DRG) neurons; therefore, the well established model of postcrush sciatic nerve regeneration was used to test the hypothesis that LAR is required for neurite outgrowth in the adult mammalian nervous system. In uninjured sciatic nerves, no differences in nerve morphology and sensory function were detected between wild-type and LAR-deficient littermate transgenic mice. Sciatic nerve crush resulted in increased LAR protein expression in DRG neurons. In addition, nerve injury led to an increase in the proportion of LAR protein isoforms known to have increased binding affinity to neurite-promoting laminin-nidogen complexes. Two weeks after nerve crush, morphological analysis of distal nerve segments in LAR-deficient transgenic mice demonstrated significantly decreased densities of myelinated fibers, decreased axonal areas, and increased myelin/axon area ratios compared with littermate controls. Electron microscopy analysis revealed a significant twofold reduction in the density of regenerating unmyelinated fibers in LAR-/- nerves distal to the crush site. Sensory testing at the 2 week time point revealed a corresponding 3 mm lag in the proximal-to-distal progression of functioning sensory fibers along the distal nerve segment. These studies introduce PTP receptors as a major new gene family regulating regenerative neurite outgrowth in vivo in the adult mammalian system.

Animals↗
PubMed2001

Human mdm2 mediates multiple mono-ubiquitination of p53 by a mechanism requiring enzyme isomerization.

The mdm2 gene product is an important regulator of p53 function and stability. mdm2 is an E3 ubiquitin ligase for p53 and the RING finger domain of mdm2 is critical for ligase activity. Ubiquitin (Ub) conjugation is a general targeting modification and poly-ubiquitin chains specifically target proteins to the proteasome for degradation. In this report, we show that the multistep cascade of mdm2-mediated p53 ubiquitination can be reduced to three purified recombinant proteins: ubiquitin-conjugated E2, mdm2, and p53. This simplification allows enzymatic analysis of the isolated ligase reaction. The simplified reaction recapitulates the ubiquitination of p53 observed with individual components and the p53-Ub((n)) is qualitatively similar to p53-Ub((n)) detected in lactacystin-treated cells. Surprisingly, we find that p53 is modified with multiple mono-ubiquitin moieties as opposed to a poly-ubiquitin chain. Finally, kinetic analysis indicates the transfer reaction proceeds either through a modified Ping Pong mechanism involving requisite enzyme isomerization steps, or through a Rapid Equilibrium Random Bi Bi mechanism involving very large anti-cooperative interactions between the two substrate binding pockets on the enzyme, mediated through allosteric changes in enzyme structure.

Ethylmaleimide↗
PubMed2001

Enhanced reporter gene expression in the rat brain from helper virus-free HSV-1 vectors packaged in the presence of specific mutated HSV-1 proteins that affect the virion.

Herpes simplex virus (HSV-1) gene expression is hypothesized to shut off promoters in HSV-1 vectors, but in a helper virus-free HSV-1 vector system, a number of promoters support only short-term expression. Thus, recombinant gene expression remains short-term in the absence of approximately 99% of the HSV-1 genome. To resolve this paradox, we hypothesized that specific HSV-1 proteins that affect the virion can shut off recombinant gene expression. This study evaluated expression from HSV-1 vectors, containing neuronal-specific promoters, that were packaged in the presence of specific mutated HSV-1 proteins that affect the virion. The mutated HSV-1 proteins that were examined included two protein kinases (U(L)13 and U(S)3), the virion host shut-off factor (vhs), the transactivator of immediate early promoters (VP16), and a virion protein that affects RNA metabolism (U(S)11). Helper virus-free packaging could occur in the presence of each mutated protein alone or specific combinations of two or three mutated proteins. In BHK and PC12 cells, vectors packaged in the presence of each mutated protein increased ( approximately 2-fold) the level of expression per cell, and vectors packaged in the presence of specific combinations of mutated proteins supported larger (4-7-fold) increases. In the rat striatum, vectors packaged in the presence of a mutated U(S)3 displayed enhanced gene transfer (13-18-fold increases in the number of cells at 4 days), and vectors packaged in the presence of mutated U(L)13 or VP16 enhanced long-term expression (2 months). Vectors packaged in the presence of mutated vhs or U(S)11 displayed minimal changes in expression.

Animals↗
PubMed2001

Identification and cellular localization of human PFTAIRE1.

We isolated a novel member of putative Cdc2-related serine/threonine protein kinases from a Hela cell cDNA library. The cDNA encodes a protein of 469 amino acids, sharing 95% identities with the mouse PFTAIRE1 throughout the entire protein sequence. This gene was designated human PFTAIRE1. The gene was located at human chromosome 7q21.13 with radiation hybrid polyermase chain reaction (RH-PCR) analysis. By Northern blotting analysis, an approximately 6 kb transcript is detected with varied levels of expression of the hPFTAIRE1 in 16 human tissues. The hPFTAIRE1 was highly expressed in brain, pancreas, kidney, heart, testis and ovary. The transcript was also detected at lower level in other tissues, except in spleen and thymus where the transcript was hardly detected. The protein was fused to the C-terminus of a green fluorescent protein (GFP) and ectopically expressed in Hela cells. The florescence microscope results indicated that the hPFTAIRE1 exhibits cytoplasmic distribution.

Amino Acid Sequence↗
PubMed2001

Fabrication of phospholipid bilayer-coated microchannels for on-chip immunoassays.

Herein we describe a new class of microfluidic immunoassays based upon solid supported lipid bilayers. Two-dimensionally fluid bilayer material, which can accommodate multivalent binding between surface-bound ligands and aqueous receptors, was coated on the surface of poly(dimethylsiloxane) microchannels. The bilayers contained dinitrophenyl (DNP)-conjugated lipids for binding with bivalent anti-DNP antibodies. Twelve independent data points of surface coverage versus bulk protein concentration could be made simultaneously by forming a linear array of channels and flowing fluorescently labeled antibodies into them. This enabled an entire binding curve to be obtained in a single experiment. The measured apparent binding constant for the DNP/anti-DNP system was 1.8 microM. The methodology for performing heterogeneous assays developed here not only produces rapid results but also requires much less protein than traditional procedures and eliminates some standard sources of experimental error.

Immunoassay↗
PubMed2001

Differential subtyping of depression.

We studied a group of patients with depression divided into subtypes of non-chronic major depression, chronic major depression, and pure dysthymia. The purpose of this study was to determine if clinical and family history factors separated these types of depression. We reviewed records from semi-structured clinical interviews and abstracted data regarding factors that might differentiate these three depressive subtypes. In general we found what might be predicted from the definitions of dysthymia versus major depression, that is, ratings for severity of depression were lower for dysthymic patients as compared to patients with non-chronic or chronic major depression. We also found lower ratings for social functioning (GASF) for dysthymic patients as compared to the other depressive subtypes. Our study does not provide data to sufficiently separate these three subtypes. However, in the course of reviewing the literature on this topic, very few studies have separated patients into these distinct depressive subtypes. Further studies are needed to indicate if these subtypes can be meaningfully separated.

Acute Disease↗
PubMed2001

Characterization of ultrasound-detected cerebral microemboli in patients undergoing cardiac catheterization using an in vitro middle cerebral artery model.

Cerebral embolization has been documented as one of the complications of diagnostic heart catheterization by transcranial Doppler (TCD). This study aimed to evaluate our hypothesis that the nature of embolic signals involved in different stages of catheter manipulation may be distinct. TCD-detected cerebral emboli occurring at different phases of cardiac catheterization were registered and differentiated by comparing their acoustic signatures with the Doppler signals generated from clinically frequently encountered embolic materials in an in vitro middle cerebral artery model. We found that there was a significant difference in embolic signal intensity and duration between different phases of cardiac catheterization. Our data suggest that different types of emboli may be involved in different phases of the catheterization. Cathet Cardiovasc Intervent 2001;53:323-330.

Adult↗
PubMed2001

Neurotransmitter dopamine applied in electrochemical determination of aluminum in drinking waters and biological samples.

It was demonstrated that the decrease of the differential pulse voltammetric (DPV) anodic peak current of dopamine (3,4-dihydroxyphenylethylamine, DA) was linear with the increase of aluminum (Al) concentration. Under optimum experimental conditions (pH 4.6, 1.2 x 10(-3) M DA, and 0.04 M NaAc-HAc buffer solution), the linear range is 4.0 x 10(-7)-8.0 x 10(-5) M, the detection limit is 1.4 x 10(-7) M, and the relative standard deviation for 4 x 10(-5) M Al(III) is 3.5% (n=8). Many foreign species, especially some low-molecule-weight biological molecules, were chosen for interference testing. The proposed method was applied to the determination of Al in biological samples such as synthetic renal dialysate, Ringer's solution, human blood, cerebrospinal fluid of a patient, and urine of a diabetic patient. The corresponding recoveries were generally between 95 and 105%. The basic principle of the method was determined by examining Al complexed with DA. This results in the blockage of the electroactive sites on DA, followed eventually by the reduction of the electrochemical response of DA. This result was verified by examining the behavior of DA, both in the presence and absence of Al, using electrochemical, UV-Vis, Raman, and (13)C NMR spectroscopic methods.

Aluminum↗
PubMed2001

A focal embolic model of cerebral ischemia in rats: introduction and evaluation.

Cerebral thromboembolism is the most common type of acute stroke in the clinical setting. In the present study, we have described a focal embolic model of cerebral ischemia in rat. Cerebral ischemic injury in two different sizes was induced by injection of two different volumes of pre-formed clots into the middle cerebral artery (MCA). Neurological deficits were assessed at 1 and 24 h, respectively, after the MCA embolization. The brain infarction was evaluated with 2,3,5-triphenyltetrazolium chloride (TTC) staining at 72 h following embolic stroke. The incidence of gross hemorrhage was also identified on the TTC-stained brain sections. This study consisted of two groups. In the first group 10 microl of pre-formed clot was injected into MCA (n=10), and in the second group 5 microl of clot was injected (n=10). Embolizing a pre-formed clot resulted in an infarction in the territory irrigated by the MCA. Embolization with the different volume of clots resulted in different sizes of brain infarction, 32.1+/-2.9% (mean+/-S.E.) in the 10-microl group versus 23+/-4.5% (mean+/-S.E.) in the 5-microl group (P<0.05). The infarction size as well as neurological deficits correlated well with the volume of the clot injected. These results thus show that with the procedure described here a reliable and reproducible ischemic injury is produced in the brain. The model is relevant to thromboembolic ischemia in patients, and may offer a useful tool to investigate mechanisms underlying ischemic brain injury. It may also be used to test thrombolytic agents in ischemic brain injury.

Animals↗