Biomedical subjects

Y Liu

Publications and source records attributed to Y Liu.

At least 793 records · Page 44Linked to original sources

PubMed2000

Molecular design of crown ethers. 19.(1) synthesis of novel disulfide- and diselenide-bridged Bis(benzo-12-crown-4)s and their Ag(+)-selective electrode properties

Two novel heteroatom-bridged bis(benzo-12-crown-4 ether)s, i.e., bis(2-nitro-4,5-(1,4,7,10-tetraoxadecamethylene)phenyl) disulfide (4) and diselenide (5), have been synthesized and characterized by elemental analysis and mass, IR, UV, and (1)H NMR spectroscopy. An X-ray crystallographic structure was obtained for 4. Ion-selective electrodes (ISE) for Ag(+), containing 4 and 5 in PVC membrane as neutral carriers, were prepared, and their selectivity coefficients for Ag(+) () were determined against other heavy metal ions, alkali and alkaline-earth metal ions, and ammonium ion using the matched potential method. These ISEs showed excellent Ag(+) selectivities, log </= -4.0, against most of the interfering cations examined, except for Hg(+) (log >/= -1.2). These values are comparable to those reported for the representative Ag(+)-selective thioethers 6 and 7, revealing that both disulfide and diselenide functionalities in 4 and 5 are equally effective Ag(+)-selective binding sites as the 1,7-dithia-4-oxa functionality in 6 and 7, irrespective of the different atom type and relative position of the sulfur/selenium donors in the ligands. Also discussed are the steric and electronic effects of the nitro groups in 4 and 5 on the Nernstian slopes obtained with the 4- and 5-based ISEs.

Journal Article↗

PubMed2000

Identification of an enhancer sequence within the first intron required for cartilage-specific transcription of the alpha2(XI) collagen gene.

Type XI collagen, a heterotrimer composed of alpha1(XI), alpha2(XI) and alpha3(XI), is primarily synthesized by chondrocytes in cartilage and is also present in some other tissues. Type XI collagen plays a critical role in collagen fibril formation and skeletal morphogenesis. We investigated a tissue-specific transcriptional enhancer in the first intron of the alpha2(XI) collagen gene (Col11a2). Transient transfection assays using reporter gene constructs revealed that a 60-base pair (bp) segment within intron 1 increased promoter activity of Col11a2 in rat chondrosarcoma cells but not in either BalB/3T3 cells or undifferentiated ATDC5 cells, suggesting that it contained cell type-specific enhancer activity. In transgenic mice, this 60-bp fragment was also able to target beta-galactosidase expression to cartilage including the limbs and axial skeleton, with similar localization specificity as the full-length intron 1 fragment. Competition experiments in gel shift assays using mutated oligonucleotides showed that recombinant Sox9 bound to a 7-bp sequence, CTCAAAG, within the 60-bp segment. Anti-Sox9 antibodies supershifted the complex of the 60-bp segment with recombinant Sox9 or with rat chondrosarcoma cell extracts, confirming the binding of Sox9 to the enhancer. Moreover, a site-specific mutation within the 7-bp segment resulted in essentially complete loss of the enhancer activity in chondrosarcoma cells and transgenic mice. These results suggest that the 7-bp sequence within intron 1 plays a critical role in the cartilage-specific enhancer activity of Col11a2 through Sox9-mediated transcriptional activation.

3T3 Cells↗

PubMed2000

qBrain-2, a POU domain gene expressed in quail embryos.

We isolated a quail class III POU domain gene, qBrain-2, which was cloned from a cDNA library of E5 embryos. Northern blot and in situ hybridization analyses showed that qBrain-2 was expressed in developing central nervous system and adult brain. Moreover, qBrain-2 transcripts showed a dynamic distribution in embryonic central nervous system. Its transcripts were dominantly detected in the ventricular zone of the developing brain and spinal cord, but rarely in the differentiated region of mantle zone as well as the non-neuronal roof plate and floor plate. This suggests that qBrain-2 is involved in proliferation and differentiation of the neuroepithelial cells of quail central nervous system.

Amino Acid Sequence↗

PubMed2000

Molecular cloning and sequencing of the sodB gene from a heterocystous cyanobacterium Anabaena sp. PCC 7120.

Superoxide dismutase (Sod) plays an important role in all aerobic organisms. The sodB gene of a heterocystous cyanobacterium Anabaena sp. PCC 7120 was cloned and sequenced. The Sod protein is predicted to have 199 amino acids and a molecular mass of 22.5 kDa. Sequence comparison among SodB from cyanobacteria and chloroplasts revealed that the sodB gene indeed encodes an iron-Sod. Northern blot analysis showed that the sodB gene of Anabaena sp. PCC 7120 is transcribed as a single gene and its expression was up-regulated when the cells were subjected to a shift from a nitrogen repletion condition to a nitrogen depletion condition.

Amino Acid Sequence↗

PubMed2000

Deltamethrin induces altered expression of P53, Bax and Bcl-2 in rat brain.

In this study we investigated the effects of deltamethrin on the expression of P53, Bax and Bcl-2 in rat brain. Immunohistochemical analysis demonstrated that the immunoreactivity for P53 was markedly increased in the cerebral cortex and hippocampus at 5 h after deltamethrin treatment, and maintained at an increased level at 24 and 48 h, whereas little immunoreactivity for P53 was seen in the same brain regions of control rats. The immunostaining for Bax was also elevated in the same brain regions, showing the same time course of P53 expression after deltamethrin treatment. However, the immunolabeling for Bcl-2 was markedly decreased at 24 h after a transient increase at 5 h following deltamethrin treatment. These results indicate that deltamethrin leads to the persistent increase of P53 and Bax expression and transient elevation of Bcl-2 expression, resulting in an increased ratio of Bax to Bcl-2, which may contribute to apoptotic cell death in rat brain following deltamethrin treatment.

Animals↗

PubMed2000

Immunohistochemical distribution of the endothelin-converting enzyme-1 in the rat hypothalamo-pituitary axis.

We prepared a rabbit polyclonal antibody against an endothelin converting enzyme-1 (ECE-1) to examine the distribution of ECE-1 in the hypothalamus and the pituitary of the rat. By the Western blot analysis, the antibody specifically recognized ECE-1 proteins in membrane fractions of the rat hypothalamus. Immunohistochemical staining revealed ECE-1 immunoreactivity in both cell bodies and axons in some hypothalamic neurons. The immunopositive cell bodies were located in the paraventricullar and supraoptic nuclei; immunopositive axons were distributed in the suprachiasmatic, paraventricullar (PVN), and supraoptic (SON) nuclei. However no immunoreactivity was detected in the posterior pituitary gland. The present results indicate that ECE-1 converts the endothelin-1 (ET-1) from precursor big ET-1 to mature ET-1 in the PVN/SON. The big ET-1 existing in the posterior pituitary gland is thought to be escaped from the converting by ECE-1 in the hypothalamus.

Animals↗

PubMed2000

The role of Sp1 in the differential expression of transforming growth factor-beta receptor type II in human breast adenocarcinoma MCF-7 cells.

Progression of MCF-7 cells from early passage (MCF-7E, <200 passage) to late passage (MCF-7L, >500 passage) correlates with a loss of sensitivity to exogenous TGFbeta1. We have previously shown that loss of TGFbeta sensitivity is due to decreased expression of the transforming growth factor receptor type II (TbetaRII) and is associated with increased tumorigenicity in nude mice. Reduced TbetaRII expression in MCF-7L cells is caused by decreased TbetaRII promoter activity in this cell line. Our previous studies using 5' deletion constructs of this promoter revealed that MCF-7L cells were unable to support transcription of the minimal promoter (-47 to +2) to the same levels as the MCF-7E cells. This region of the promoter contains an Sp1 element at position -25 from the major transcription start site. In this study, we investigated the role of Sp1 in TbetaRII transcription. Mutation of the Sp1 site resulted in decreased transcription of TbetaRII in MCF-7E and MCF-7L cells, indicating that this site played a role in transcription of this promoter. Gel shift assays using the proximal Sp1 site from the TbetaRII promoter showed enhanced DNA:protein complex formation with nuclear proteins isolated from MCF-7E cells compared with MCF-7L cells. Supershift analysis identified this binding activity as Sp1. Western blot analysis of Sp1 levels demonstrated that MCF-7E cells contain increased Sp1 protein compared with MCF-7L cells, paralleling the increased binding activity. Differential Sp1 activity was also demonstrated by higher levels of transcription of an Sp1-dependent insulin-like growth factor II promoter construct in MCF-7E cells compared with MCF-7L cells. Co-transfection of an Sp1 expression vector with a TbetaRII promoter construct in MCF-7L cells induced the expression from the promoter-CAT constructs and resulted in an increase of endogenous TbetaRII protein levels. These results demonstrate that the transcriptional repression of TbetaRII in MCF-7L cells is caused, in part, by lower Sp1 levels.

Adenocarcinoma↗

PubMed2000

MRZ 2/579, a novel uncompetitive N-methyl-D-aspartate antagonist, reduces infarct volume and brain swelling and improves neurological deficit after focal cerebral ischemia in rats.

The purpose of this study was to evaluate the effects of MRZ 2/579, an uncompetitive N-methyl-D-aspartate antagonist, on infarct size, extent of swelling and neurological deficit in a model of transient middle cerebral artery occlusion in rats. Physiologically controlled Sprague-Dawley rats received 2 h MCAo by retrograde insertion of an intraluminal suture coated with poly-L-lysine. The agent (MRZ 2/579) or vehicle (sodium chloride 0.9%) was administered i.v. immediately after suture removal following a 2-h period of MCAo. Two experimental groups were studied: group A was treated by vehicle (bolus infusion:1 ml/kg for 10 min followed by infusion of 6 ml/kg/h over 6 h). Group B was treated by MRZ 2/579 (bolus infusion:10 mg/kg for 10 min followed by infusion of 6 mg/kg/h over 6 h). The neurological status was evaluated during occlusion (at 60 min) and daily for 3 days after MCAo. Brains were then perfusion-fixed, and infarct volumes and brain swelling were determined. MRZ 2/579 significantly improved the neurological score compared to vehicle-treated rats at 48 h (6.2+/-0.6 and 8.7+/-0.5, respectively; P<0.004) and 72 h after MCAo (5.2+/-0.6 and 8.4+/-0.5, respectively; P<0.001). Treatment with MRZ 2/579 also significantly reduced total infarct volume (29.3+/-11.1 and 83.2+/-16.5 mm(3), respectively; P<0. 01), cortical infarct volume (24.8+/-11.2 and 70.0+/-18.0 mm(3), respectively; P<0.04) and subcortical infarction (21.2+/-4.1 and 49. 6+/-4.5 mm(3), respectively; P<0.0002). Brain swelling was also markedly reduced compared with vehicle-treated rats (4.7+/-1.3 and 10.8+/-2.1%, respectively; P<0.02). These results demonstrate that treatment with MRZ 2/579, when administered promptly after reperfusion, confers neuroprotective effects on infarct volume, brain swelling, and neurological score compared to the vehicle group.

Animals↗

PubMed2000

A phosphorylation site regulates sorting of the vesicular acetylcholine transporter to dense core vesicles.

Vesicular transport proteins package classical neurotransmitters for regulated exocytotic release, and localize to at least two distinct types of secretory vesicles. In PC12 cells, the vesicular acetylcholine transporter (VAChT) localizes preferentially to synaptic-like microvesicles (SLMVs), whereas the closely related vesicular monoamine transporters (VMATs) localize preferentially to large dense core vesicles (LDCVs). VAChT and the VMATs contain COOH-terminal, cytoplasmic dileucine motifs required for internalization from the plasma membrane. We now show that VAChT undergoes regulated phosphorylation by protein kinase C on a serine (Ser-480) five residues upstream of the dileucine motif. Replacement of Ser-480 by glutamate, to mimic the phosphorylation event, increases the localization of VAChT to LDCVs. Conversely, the VMATs contain two glutamates upstream of their dileucine-like motif, and replacement of these residues by alanine conversely reduces sorting to LDCVs. The results provide some of the first information about sequences involved in sorting to LDCVs. Since the location of the transporters determines which vesicles store classical neurotransmitters, a change in VAChT trafficking due to phosphorylation may also influence the mode of transmitter release.

Amino Acid Substitution↗

PubMed2000

Alleviation of neuronal ganglioside storage does not improve the clinical course of the Niemann-Pick C disease mouse.

Niemann-Pick disease Type C (NP-C) is a progressive neurodegenerative disorder caused by mutations in the NPC1 gene and characterized by intracellular accumulation of cholesterol and sphingo-lipids. The major neuronal storage material in NP-C consists of gangliosides and other glycolipids, raising the possibility that the accumulation of these lipids may participate in the neurodegenerative process. To determine if ganglioside accumulation is a crucial factor in neuropathogenesis, we bred NP-C model mice with mice carrying a targeted mutation in GalNAcT, the gene encoding the beta-1-4GalNAc transferase responsible for the synthesis of GM2 and complex gangliosides. Unlike the NP-C model mice, these double mutant mice did not exhibit central nervous system (CNS) accumulation of gangliosides GM2 or of glycolipids GA1 and GA2. Histological analysis revealed that the characteristic neuronal storage pathology of NP-C disease was substantially reduced in the double mutant mice. By contrast, visceral pathology was similar in the NP-C and double mutant mice. Most notably, the clinical phenotype of the double mutant mice, in the absence of CNS ganglioside accumulation and associated neuronal pathology, did not improve. The results demonstrate that complex ganglioside storage, while responsible for much of the neuronal pathology, does not significantly influence the clinical phenotype of the NP-C model.

Animals↗

PubMed2000

Analysis of p73 in human borderline and invasive ovarian tumor.

p73 is a novel gene that has high sequence homology and similar gene structure to the tumor suppressor gene p53. We analysed p73 in seven ovarian carcinoma cell lines and a total of 63 human borderline and invasive ovarian tumor samples. Loss of heterozygosity at this locus was observed in 50% of invasive tumors but in none of the borderline tumors. Biallelic expression of the gene was observed in the heterozygous tumor tissues. Direct sequencing and single-strand conformation polymorphism analyses of the p73 cDNA sequence homologous to the highly mutatable region of p53 did not reveal any mutations. When compared to the primary cultures of normal human ovarian surface epithelial cells and immortalized cell lines, four of the seven ovarian carcinoma cell lines, 71% of the invasive tumors, and 92% of the borderline tumor tissues express elevated levels of p73 transcript. Except for the OVCA3 cell line, Western blot analysis of the nuclear extracts prepared from the cell lines showed concordant levels of p73 protein. Our analysis also demonstrated the expression of a spliced variant of p73 transcript with the omission of exon 2 solely in the cancer cell lines and invasive tumor tissues. This exon 2-spliced transcript would give rise to a truncated p73 protein without the N-terminal transactivation domain. In reminiscence of the dominant negative phenotype of the N-terminal truncated variants of another p53-related gene, p63, the expression of the truncated p73 variant form in ovarian tumors may play an important role in the pathogenesis of ovarian cancer.

Amino Acid Sequence↗

PubMed2000

Crystal structure of the cysteine-rich domain of mannose receptor complexed with a sulfated carbohydrate ligand.

The macrophage and epithelial cell mannose receptor (MR) binds carbohydrates on foreign and host molecules. Two portions of MR recognize carbohydrates: tandemly arranged C-type lectin domains facilitate carbohydrate-dependent macrophage uptake of infectious organisms, and the NH(2)-terminal cysteine-rich domain (Cys-MR) binds to sulfated glycoproteins including pituitary hormones. To elucidate the mechanism of sulfated carbohydrate recognition, we determined crystal structures of Cys-MR alone and complexed with 4-sulfated-N-acetylgalactosamine at 1.7 and 2.2 A resolution, respectively. Cys-MR folds into an approximately three-fold symmetric beta-trefoil shape resembling fibroblast growth factor. The sulfate portions of 4-sulfated-N-acetylgalactosamine and an unidentified ligand found in the native crystals bind in a neutral pocket in the third lobe. We use the structures to rationalize the carbohydrate binding specificities of Cys-MR and compare the recognition properties of Cys-MR with other beta-trefoil proteins.

Acetylgalactosamine↗

PubMed2000

Polymyxin B binds to anandamide and inhibits its cytotoxic effect.

Anandamide (ANA), an endogenous cannabinoid, can be generated by activated macrophages during endotoxin shock and is thought to be a paracrine contributor to hypotension. We discovered that ANA in saline/ethanol solution and in serum was efficiently adsorbed in a polymyxin B (PMB)-immobilized beads column and eluted with ethanol. We confirmed the direct binding of PMB to ANA by using surface plasmon resonance. The adsorption of ANA by PMB may abolish the diverse effects of ANA such as hypotension, immunosuppression, and cytotoxicity, and may suggest a new therapeutic strategy for endotoxin shock.

Adsorption↗

PubMed2000

Plasminogen enhances the secretion of plasminogen activator inhibitor-1 from cultured rat astrocytes.

To investigate the physiological significance of microglia-derived plasminogen (PGn) in the central nervous system, we determined the effects of rat PGn on the secretion of plasminogen activator inhibitor (PAI) in cultured rat astrocytes by reverse zymography and Western blotting. The cultured astrocytes normally produce only a limited amount of PAI-1. After stimulation with PGn, however, the amount of active PAI-1 in the astrocyte-conditioned medium was significantly increased in a time-dependent manner. PGn was also proved to activate p38 MAP kinase and c-Jun N-terminal kinase in these astrocytes. Taken together, these results suggest that microglia-derived PGn increases the secretion of PAI-1 in astrocytes through the activation of MAP kinases, and that enhanced PAI-1 regulates various physiological phenomena including tissue remodeling, neuronal plasticity and neurotoxicity.

Animals↗

PubMed2000

Capping DNA with DNA.

Twelve classes of deoxyribozymes that promote an ATP-dependent "self-capping" reaction were isolated by in vitro selection from a random-sequence pool of DNA. Each deoxyribozyme catalyzes the transfer of the AMP moiety of ATP to its 5'-terminal phosphate group, thereby forming a 5',5'-pyrophosphate linkage. An identical DNA adenylate structure is generated by the T4 DNA ligase during enzymatic DNA ligation. A 41-nucleotide class 1 deoxyribozyme requires Cu(2+) as a cofactor and adopts a structure that recognizes both the adenine and triphosphate moieties of ATP or dATP. The catalytic efficiency for this DNA, measured at 10(4) M(-1) x min(-1) using either ATP or dATP as substrate, is similar to other catalytic nucleic acids that use small substrates. Chemical probing and site-directed mutagenesis implicate the formation of guanine quartets as critical components of the active structure. The observation of ATP-dependent "self-charging" by DNA suggests that DNA could be made to perform the reactions typically associated with DNA cloning, but without the assistance of protein enzymes.

Adenosine Triphosphate↗

PubMed2000

SPAF, a new AAA-protein specific to early spermatogenesis and malignant conversion.

A novel spermatogenesis associated factor (SPAF) was found to be aberrantly expressed at the malignant conversion stage in a clonal epidermal model of chemical carcinogenesis. Sequence analysis revealed two ATPase modules, classifying this gene as a new member of the AAA-protein family (ATPase associated with diverse activities). Immunohistochemical staining of mouse testis sections with SPAF antibody localized expression to spermatogonia and early spermatocytes in the basal compartment of the seminiferous tubules. Northern and Western analysis of SPAF expression in testes of mice at different developmental stages confirmed its expression at early stages of spermatogenesis. In view of a mitochondrial-localization-like signal, sequence similarities to membrane-associated proteins, ATP binding properties, and intracellular expression patterns in testis, we speculate that SPAF protein may be involved in morphological and functional mitochondrial transformations during spermatogenesis. Ectopic expression of the SPAF gene in malignant epidermal cells may signify adoption of an early germ cell-like phenotype advantageous in malignant conversion.

Adenosine Triphosphatases↗

PubMed2000

2,3,4,5-Tetrakis[(2-hydroxyethyl)thio]tetrathiafulvalene tetrafluoroborate.

The title complex, C(14)H(20)O(4)S(8)(+)(.)BF(4)(-), is a charge-transfer complex with typical charges for the donor and anion of +1 and -1, respectively. Two centrosymmetrically related donors form a face-to-face pi-dimer with a strong intermolecular S.S interaction. These pi-dimers stack along the a axis to form a donor column. The structure is extensively hydrogen bonded.

Journal Article↗

PubMed2000

Effects of subunit occupancy on partitioning of an intermediate in thymidylate synthase mutants.

Experimental evidence for a 5-exocyclic methylene-dUMP intermediate in the thymidylate synthase reaction was recently obtained by demonstrating that tryptophan 82 mutants of the Lactobacillus casei enzyme produced 5-(2-hydroxyethyl)thiomethyl-dUMP (HETM-dUMP) (Barret, J. E., Maltby, D. A., Santi, D. V., and Schultz, P. G. (1998) J. Am. Chem. Soc. 120, 449-450). The unusual product was proposed to emanate from trapping of the intermediate with beta-mercaptoethanol in competition with hydride transfer from H(4)folate to form dTMP. Using mutants of the C-terminal residue of thymidylate synthase, we found that the ratio of HETM-dUMP to dTMP varies as a function of CH(2)H(4)folate concentration. This observation seemed inconsistent with the conclusion that both products arose from a common intermediate in which CH(2)H(4)folate was already bound to the enzyme. The enigma was resolved by a kinetic model that allowed for differential partitioning of the intermediate formed on each of the two subunits of the homodimeric enzyme in forming the two different products. With three C-terminal mutants of L. casei TS, HETM-dUMP formation was consistent with a model in which product formation occurs upon occupancy of the first completely bound subunit, the rate of which is unaffected by occupancy of the second subunit. With one analogous E. coli TS mutant, HETM-dUMP formation occurred upon occupancy of the first subunit, but was inhibited when both subunits were occupied. With all mutants, dTMP formation occurs from occupied forms of both subunits at different rates; here, binding of cofactor to the first subunit decreased affinity for the second, but the reaction occurred faster in the enzyme form with both subunits bound to dUMP and CH(2)H(4)folate. The model resolves the apparent enigma of the cofactor-dependent product distribution and supports the conclusion that the exocyclic methylene intermediate is common to both HETM-dUMP and dTMP formation.

Alanine↗