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Biomedical subjects

X Yang

Publications and source records attributed to X Yang.

At least 307 records · Page 17Linked to original sources
PubMed2000

The focal adhesion kinase suppresses transformation-associated, anchorage-independent apoptosis in human breast cancer cells. Involvement of death receptor-related signaling pathways.

The focal adhesion kinase (FAK) is a mediator of cell-extracellular matrix signaling events and is overexpressed in tumor cells. In order to rapidly down-regulate FAK function in normal and transformed mammary cells, we have used adenoviral gene transduction of the carboxyl-terminal domain of FAK (FAK-CD). Transduction of adenovirus containing FAK-CD in breast cancer cells caused loss of adhesion, degradation of p125(FAK), and induced apoptosis. Furthermore, breast tumor cells that were viable without matrix attachment also underwent apoptosis upon interruption of FAK function, demonstrating that FAK is a survival signal in breast tumor cells even in the absence of matrix signaling. In addition, both anchorage-dependent and anchorage-independent apoptotic signaling required Fas-associated death domain and caspase-8, suggesting that a death receptor-mediated apoptotic pathway is involved. Finally, FAK-CD had no effect on adhesion or viability in normal mammary cells, despite the loss of tyrosine phosphorylation of p125(FAK). These results indicate that FAK-mediated signaling is required for both cell adhesion and anchorage-independent survival and the disruption of FAK function involves the Fas-associated death domain and caspase-8 apoptotic pathway.

Apoptosis↗
PubMed2000

Human papillomavirus type 16-immortalized endocervical cells selected for resistance to cisplatin are malignantly transformed and have a multidrug resistance phenotype.

Cis-diamminedichloroplatinum (II) (cisplatin, CDDP) is a highly effective chemotherapeutic agent against cervical cancer, but drug resistance is a major obstacle in its clinical application. The mechanism of drug resistance in human cervical cancer is not well understood. Here, we established an in vitro endocervical, cisplatin-resistant cell system that mimics the development of cisplatin resistance in the human cervix. Human papillomavirus (HPV) type 16-immortalized human endocervical cells (HEN-16-2) were treated with cisplatin, and the cisplatin-selected cells (HEN-16-2/CDDP) were resistant to cisplatin, paclitaxel, actinomycin D, doxorubicin, etoposide, and 5-fluorouracil, thus demonstrating a multidrug resistance (MDR) phenotype. Furthermore, compared with a similar passage of drug-sensitive HEN-16-2 cells, HEN-16-2/CDDP cells exhibited the general growth characteristics of cancer cell lines: faster growth in medium containing serum and high calcium levels, higher saturation density, anchorage-independent growth, and formation of tumors in nude mice. These results provided the first in vitro evidence that cisplatin selection can transform HPV-immortalized endocervical cells and cause a phenotype of MDR.

Animals↗
PubMed2000

Culture of in vitro produced bovine zygotes in vitro vs in vivo: implications for early embryo development and quality.

The objectives of this study were to examine the effect of culture system on bovine blastocyst formation rates and quality. Presumptive IVM/IVF bovine zygotes were cultured either in vitro in synthetic oviduct fluid (SOF, 25 embryos/25 microL in 5% CO2, 5% O2, 90% N2 at 39 degrees C) or in vivo in the ewe oviduct (approximately 100 embryos per oviduct). The recovery rate after in vivo culture was 53% (813/1,530). The blastocyst rate on Day 7 was significantly higher for the in vitro system (28%, 362/1,278 vs 17%, 37/813; P< 0.0001). However, after culture in vitro for a further 24 h, there was no difference in Day 8 yields (36%, 457/1,278 vs 32%, 258/813, for in vitro and in vivo culture, respectively). There was no difference in blastocyst cell number between treatments (Day 7: 96 vs 103; Day 8: 78 vs 85 for in vitro and in vivo culture, respectively). Irrespective of culture system, Day 7 blastocysts had a significantly higher cell number than those appearing on Day 8. There was no difference in pregnancy rate at Day 35 after fresh transfer of a single Day 7 blastocyst (37.5%, 21/56 vs 45.3%/, 24/53 for in vitro and in vivo culture, respectively). After cryopreservation by freezing in 10% glycerol, VS3a vitrification or solid surface vitrification, the survival of in vitro cultured embryos was significantly lower than survival of embryos cultured in the ewe oviduct or those produced by superovulation of donors. In conclusion, these findings demonstrate that while bovine zygotes cultured in vitro are capable of rates of development similar to those of their in vivo cultured counterparts (in terms of Day 8 blastocyst yield, cell number and early pregnancy rate), there are significant differences in embryo cryosurvival. This suggests that current in vitro culture systems need to be improved to optimize embryo quality and pregnancy rates.

Animals↗
PubMed2000

Multichannel mode-filtered light detection based on an optical fiber for small-volume chemical analysis

A novel mode-filtered light detection method is described in which an unjacketed optical fiber is inserted into a transparent capillary tube and three or more detection channels are set on the capillary side for different distances from the port of the fiber. This new method is the basis of synchronization of separation and analysis, with which a modern multidimensional analysis apparatus will be constructed. For samples of different concentration, the more close to the laser incidence port of the fiber the detector has been set up, the greater the change of the intensity profile of mode-filtered light (deltaI(F)) that is obtained. Vice versa, if a parameter alpha = I0/In is established and, instead of a mode-filtered light signal, the reversed alteration trend of deltaalpha is found in comparison with the alteration trend of deltaI(F), the reason is that the background rapidly lowers with the increasing distance to the laser incidence port of the fiber; moreover, the mode-filtered light signal decreases slowly with it. With the present method and apparatus, glucose and glycerol have been determined, with good reproducibility and stability and a small sample volume. Furthermore, a real sample of glucose injection is measured for a detection volume of 5 microL, and an acceptable result is observed.

Journal Article↗
PubMed2000

myo-inositol 3,4,5,6-tetrakisphosphate inhibits an apical calcium-activated chloride conductance in polarized monolayers of a cystic fibrosis cell line.

Does inositol 3,4,5,6-tetrakisphosphate (Ins(3,4,5,6)P(4)) inhibit apical Ca(2+)-activated Cl(-) conductance (CaCC)? We studied this question using human CFPAC-1 pancreatoma cells grown in polarized monolayers. Cellular Ins(3,4,5,6)P(4) levels were acutely sensitive to purinergic receptor activation, rising 3-fold within 1 min of agonist addition. Intracellular Ins(3,4,5,6)P(4) levels were therefore specifically elevated, independently of receptor activation, by incubating cells with a cell-permeant bioactivable analogue, 1,2-di-O-butyl-myo-inositol 3,4,5,6-tetrakisphosphate octakis(acetoxymethyl)ester (Bt(2)Ins (3,4,5,6)P(4)/AM). The latter inhibited Ca(2+)-activated Cl(-) secretion by 60%. We next used nystatin to selectively permeabilize the basolateral membrane to monovalent anions and cations, thereby preventing this membrane from electrochemically dominating ion movements through the apical membrane. Thus, we studied autonomous regulation of apical Cl(-) channels in situ. The properties of Cl(-) flux across the apical membrane were those expected of CaCC: niflumic acid sensitivity, outward rectification, and 2-fold greater permeability of I(-) over Cl(-). Following nystatin-treatment, we elevated intracellular levels of Ins(3,4,5,6)P(4) with either purinergic agonists or with Bt(2)Ins(3,4,5,6)P(4)/AM. Both protocols inhibited Ca(2+)-activated Cl(-) secretion (up to 70%). These studies provide the first demonstration that, in a physiologically relevant context of a polarized monolayer, there is an apical, Ins(3,4,5,6)P(4)-inhibited CaCC.

Calcium↗
PubMed2000

A novel Golgi membrane protein is part of a GTPase-binding protein complex involved in vesicle targeting.

Through two-hybrid interactions, protein affinity and localization studies, we previously identified Yip1p, an integral yeast Golgi membrane protein able to bind the Ras-like GTPases Ypt1p and Ypt31p in their GDP-bound conformation. In a further two-hybrid screen, we identified Yif1p as an interacting factor of Yip1p. We show that Yif1p is an evolutionarily conserved, essential 35.5 kDa transmembrane protein that forms a tight complex with Yip1p on Golgi membranes. The hydrophilic N-terminal half of Yif1p faces the cytosol, and according to two-hybrid analyses can interact with the transport GTPases Ypt1p, Ypt31p and Sec4p, but in contrast to Yip1p, this interaction is dispensable for Yif1 protein function. Loss of Yif1p function in conditional-lethal mutants results in a block of endoplasmic reticulum (ER)-to-Golgi protein transport and in an accumulation of ER membranes and 40-50 nm vesicles. Genetic analyses suggest that Yif1p acts downstream of Yip1p. It is inferred that Ypt GTPase binding to the Yip1p-Yif1p complex is essential for and precedes vesicle docking and fusion.

Amino Acid Sequence↗
PubMed2000

A Quantum State-Resolved Insertion Reaction: O((1)D) + H(2)(J = 0) --> OH((2) product operator product operator product operator, v, N) + H((2)S).

The O((1)D) + H(2) --> OH + H reaction, which proceeds mainly as an insertion reaction at a collisional energy of 1.3 kilocalories per mole, has been investigated with the high-resolution H atom Rydberg "tagging" time-of-flight technique and the quasiclassical trajectory (QCT) method. Quantum state-resolved differential cross sections were measured for this prototype reaction. Different rotationally-vibrationally excited OH products have markedly different angular distributions, whereas the total reaction products are roughly forward and backward symmetric. Theoretical results obtained from QCT calculations indicate that this reaction is dominated by the insertion mechanism, with a small contribution from the collinear abstraction mechanism through quantum tunneling.

Journal Article↗
PubMed2000

[A molecular epidemiologic study of TT virus in various population].

To investigate infection and sequence divergence of TT virus in various population, a nested polymerase chain reaction (nPCR) assay with primers from TTV ORF1 genome was used to detect serum TTV DNA. TTV DNAs from various population were cloned and sequenced. Six gene sequences derived from two patients with non-A through non-E and non-G hepatitis, a hepatitis patient with HBsAg, a normal blood donor, an intravenous drug user and a woman prostitute, were compared with known sequence of TTV isolate (N22) reported by Okamoto, Japan. The nucleotide homology were 96%-98% and the whole sequences belonged to la genotype. The data indicate that TTV DNA from various population in China might chiefly be la genotype. There is no correlation between genotype and disease transmission and development.

Adult↗
PubMed2000

The osteoblast-specific transcription factor Cbfa1 contributes to the expression of osteoprotegerin, a potent inhibitor of osteoclast differentiation and function.

Bone formation and resorption are tightly coupled under normal conditions, and the interaction of osteoclast precursors with cells of the osteoblast lineage is a prerequisite for osteoclast formation. Cbfa1 is an osteoblast-specific transcription factor that is essential for osteoblast differentiation and bone formation. At present, it is not known whether Cbfa1 regulates any of the osteoblast-derived factors involved in the bone resorption pathway. Osteoprotegerin (OPG) is an osteoblast-secreted glycoprotein that functions as a potent inhibitor of osteoclast differentiation and bone resorption. Cloning and computer analysis of a 5.9-kilobase human OPG promoter sequence revealed the presence of 12 putative Cbfa1 binding elements (osteoblast-specific element 2 (OSE(2))), suggesting a possible regulation of OPG by Cbfa1. We cloned the promoter upstream of the beta-galactosidase reporter gene (pOPG5. 9betagal) and evaluated whether Cbfa1 could regulate its expression in transient transfection assays. The 5.9-kilobase promoter directed increased levels of reporter gene expression, reminiscent of OPG protein levels in osteoblastic cell lines (BALC and U2OS) as compared with the nonosteoblastic cell line COS1. Cotransfection of a Cbfa1 expression construct along with pOPG5.9betagal reporter construct led to 39-, 7-, and 16-fold increases in beta-galactosidase activity in COS1, BALC, and U2OS cells, respectively. Removal of all the putative OSE(2) elements led to an almost complete loss of transactivation. Mutational analysis demonstrated that the proximal OSE(2) element contributes to a majority of the effects of Cbfa1, and Cbfa1 bound to the proximal element in a sequence-specific manner. Further, overexpression of Cbfa1 led to a 54% increase in OPG protein levels in U2OS cells. These results indicate that Cbfa1 regulates the expression of OPG, thereby further contributing to a molecular link between bone formation and resorption.

Animals↗
PubMed2000

Substrate binding stabilizes S-adenosylhomocysteine hydrolase in a closed conformation.

Comparison of crystal structures of S-adenosylhomocysteine (AdoHcy) hydrolase in the substrate-free, NAD(+) form [Hu, Y., Komoto, J., Huang, Y., Gomi, T., Ogawa, H., Takata, Y., Fujioka, M., and Takusagawa, F. (1999) Biochemistry 38, 8323-8333] and a substrate-bound, NADH form [Turner, M. A., Yuan, C.-S., Borchardt, R. T., Hershfield, M. S., Smith, G. D., and Howell, P. L. (1998) Nat. Struct. Biol. 5, 369-376] indicates large differences in the spatial arrangement of the catalytic and NAD(+) binding domains. The substrate-free, NAD(+) form exists in an "open" form with respect to catalytic and NAD(+) binding domains, whereas the substrate-bound, NADH form exists in a closed form with respect to those domains. To address whether domain closure is induced by substrate binding or its subsequent oxidation, we have measured the rotational dynamics of spectroscopic probes covalently bound to Cys(113) and Cys(421) within the catalytic and carboxyl-terminal domains. An independent domain motion is associated with the catalytic domain prior to substrate binding, suggesting the presence of a flexible hinge element between the catalytic and NAD(+) binding domains. Following binding of substrates (i.e., adenosine or neplanocin A) or a nonsubstrate (i.e., 3'-deoxyadenosine), the independent domain motion associated with the catalytic domain is essentially abolished. Likewise, there is a substantial decrease in the average hydrodynamic volume of the protein that is consistent with a reduction in the overall dimensions of the homotetrameric enzyme following substrate binding and oxidation observed in earlier crystallographic studies. Thus, the catalytic and NAD(+) binding domains are stabilized to form a closed active site through interactions with the substrate prior to substrate oxidation.

Adenosylhomocysteinase↗
PubMed2000

Iron oxidation and hydrolysis reactions of a novel ferritin from Listeria innocua.

Iron deposition in the unusual 12-subunit ferritin from thebacterium Listeria innocua proceeds in three phases: a rapidfirst phase in which Fe(2+) binds to the apoprotein, P(Z) of charge Z, according to the postulatedreaction 2Fe(2+)+P(Z)-->[Fe(2)-P](Z+2)+2H(+), where[Fe(2)-P](Z+2) represents adinuclear iron(II) complex formed at each of the 12 ferroxidase centresof the protein; a second phase corresponding to oxidation of thisputative complex, i.e. [Fe(2)-P](Z+2)+1/2 O(2)-->[Fe(2)O-P](Z)+2H(+);and a third phase of iron(II) oxidation/mineralization, i.e. 4Fe(2+)+O(2)+8H(2)O-->8FeOOH((s))+8H(+) [where FeOOH((s)) represents the hydrous ferric oxidemineral that precipitates from the solution], which occurs when iron isadded in excess of 24Fe(2+)/protein. In contrast with otherferritins, the ferroxidation reaction in L. innocua ferritinproceeds more slowly than the oxidation/mineralization reaction. Wateris the final product of dioxygen reduction in the 12-subunit L.innocua ferritin (the present work) and in the 24-subunit Escherichia coli bacterioferritin, whereas H(2)O(2) is produced in 24-subunit mammalian ferritins. Possible reasonsfor this difference are discussed.

Ferritins↗
PubMed2000

Phosphorylation of human glutamine:fructose-6-phosphate amidotransferase by cAMP-dependent protein kinase at serine 205 blocks the enzyme activity.

Glutamine:fructose-6-phosphate amidotransferase (GFAT) is the rate-limiting enzyme in glucosamine synthesis. Prior studies from our laboratory indicated that activation of adenylate cyclase was associated with depletion of O-GlcNAc modification. This finding and evidence that human GFAT (hGFAT) might be regulated by cAMP-dependent protein kinase (PKA) led us to investigate the role of PKA in hGFAT function. We confirmed that adenylate cyclase activation by forskolin results in diminished O-GlcNAc modification of several cellular proteins which can be overcome by exposure of the cells to glucosamine but not glucose, suggesting the PKA activation results in depletion of UDP-GlcNAc for O-glycosylation. To determine if GFAT is indeed regulated by PKA, we expressed the active form of the enzyme using a vaccinia virus expression system and showed that the activity of the enzyme was to decrease to undetectable levels by PKA phosphorylation. We mapped the PKA phosphorylation sites with the aid of matrix-assisted laser desorption ionization mass spectroscopy and showed that the protein was stoichiometrically phosphorylated at serine 205 and also phosphorylated, to a lesser extent at serine 235. Mutagenesis studies indicated that the phosphorylation of serine 205 by PKA was necessary for the observed inhibition of enzyme activity while serine 235 phosphorylation played no observable role. The activity of GFAT is down-regulated by cAMP, thus placing regulation on the hexosamine pathway that is in concert with the energy requirements of the organism. During starvation, hormones acting through adenylate cyclase could direct the flux of glucose metabolism into energy production rather than into synthetic pathways that require hexosamines.

Colforsin↗
PubMed2000

Potential energy in a three-dimensional vibrated granular medium measured by NMR imaging

Fast NMR imaging was used to measure the density profile of a three-dimensional granular medium fluidized by vertical vibrations of the container. For container acceleration much larger than gravity, the rise in center of mass of the granular medium is found to scale as v(alpha)(0)/N(beta)(l) with alpha = 1.0+/-0.2 and beta = 0.5+/-0.1, where v(0) is the vibration velocity, and N(l) is the number of layers of grains in the container. This value for alpha is significantly less than found previously for experiments and simulations in one dimension ( alpha = 2) and two dimensions ( alpha = 1.3-1.5).

Journal Article↗
PubMed2000

New evidence for an extra-hepatic role of N-acetylglucosaminyltransferase III in the progression of diethylnitrosamine-induced liver tumors in mice.

N-acetylglucosaminyltransferase III (GlcNAc-TIII) is encoded by the Mgat3 gene and catalyzes the addition of the bisecting GlcNAc to the core of N-glycans. Mice lacking GlcNAc-TIII due to the insertion mutation Mgat3tmlPst (termed Mgat3neo), exhibit retarded progression of liver tumors induced by diethylnitrosamine (DEN; M. Bhaumik et al, Cancer Res., 58: 2881-2887, 1998). This phenotype seemed to be due to a reduction, in activity or amount, of a circulating glycoprotein(s) that enhances DEN-induced liver tumor progression. Here, we provide new evidence to support this hypothesis. First, we show that mice with a deletion mutation of the Mgat3 gene coding exon (Mgat3tmlJxm, termed Mgat3delta) also exhibit retarded progression of DEN-induced liver tumors. At 7 months there was a significant decrease in liver weight (approximately 27%; P < 0.01), reflecting reduced tumor burden in Mgat3delta/delta mice. In addition, tumors were generally fewer and smaller, and histological changes were less severe in Mgat3delta/delta livers. Therefore, tumor progression is retarded in mice with two different null mutations in the Mgat3 gene. Second, we show that the development of DEN-induced tumors is unaltered by high levels of GlcNAc-TIII in the liver of transgenic mice. The Mgat3 gene coding exon under the control of the major urinary protein (MUP) promoter was used to generate transgenic mice that express GlcNAc-TIII in liver. Following DEN injection and phenobarbitol treatment, however, no significant differences were observed between MUP/Mgat3 transgenic and control mice in either tumor numbers or liver weight. The combined data provide strong evidence that retarded progression of tumors in mice lacking GlcNAc-TIII is due to the absence of the bisecting GlcNAc residue on N-glycans of a circulating glycoprotein(s) from a tissue other than liver.

Alleles↗
PubMed2000

Regulation of inducible bradykinin B1 receptor gene expression through absence of internalization and resensitization.

Rapid induction and down-regulation of bradykinin B1 receptor (BKB1R) gene expression is tightly regulated at the transcriptional and mRNA levels (Zhou et al. [1998] Biochem. J. 330:361-366; Zhou et al. [1999] Mol. Cell Biol. Res. Commun. 1:29-35). Here we explore regulation of BKB1R expression at the protein level. To make this inducible gene express constitutively, we utilized a bicistronic mammalian expression vector (pCMin) for stable transfection of the BKB1R gene into human lung fibroblasts, IMR90SV40. The BKB1R displayed a high affinity and specificity (K(d) = 0.5 nM) for desArg(10)-kallidin. The receptor mediated such signaling events as arachidonic acid (ARA) release, phosphoinositide (PI) turnover and Ca(2+)-flux. The receptor function proved differentially desensitized. For example, after initial exposure to desArg(10)-kallidin, a second stimulation with desArg(10)-kallidin did not induce further Ca(2+)-flux or ARA-release while PI-turnover continued unabated. Unlike most of the G-protein coupled receptors, the BKB1R did not internalize within 60 min of exposure to 10 nM desArg(10)-kallidin. It also did not resensitize. Thus, the duration and signal capacity of the BKB1R at the protein level is regulated through lack of internalization, an absence of resensitization and a lack of desensitization for certain events such as PI turnover. In fact, the absence of BKB1R resensitization is likely a very important contributor to the rapid disappearance of this inducible receptor.

Arachidonic Acid↗
PubMed2000

Human Sug1/p45 is involved in the proteasome-dependent degradation of Sp1.

The transcription factor Sp1 was previously shown to undergo proteasome-dependent degradation when cells were glucose-starved and stimulated with the adenylate cyclase inducer, forskolin. However, the control of the Sp1 degradation process is largely unknown. Using in vitro and in vivo interaction studies, we show in the present study that Sp1 interacts with human Sug1 [hSug1, also known as p45 or thyroid-hormone-receptor interacting protein ('TRIP1')], an ATPase subunit of the 26 S proteasome and a putative transcriptional modulator. This interaction with Sp1 occurs through the C-terminus of hSug1, the region that contains the conserved ATPase domain in this protein. Both in vitro studies, in reconstituted degradation assays, and in vivo experiments, in which hSug1 is overexpressed in normal rat kidney cells, show that full-length hSug1 is able to stimulate the proteasome-dependent degradation of Sp1. However, hSug1 truncations that lack either the N- or C-terminal domain of hSug1 act as dominant negatives, inhibiting Sp1 degradation in vitro. Also, an ATPase mutant of hSug1, while still able to bind Sp1, acts as a dominant negative, blocking Sp1 degradation both in vitro and in vivo. These results demonstrate that hSug1 is involved in the degradation of Sp1 and that ATP hydrolysis by hSug1 is necessary for this process. Our findings indicate that hSug1 is an exchangeable proteasomal component that plays a critical regulatory role in the proteasome-dependent degradation of Sp1. However, hSug1 is not the factor limiting Sp1 degradation in the cells treated with glucosamine. This and other considerations suggest that hSug1 co-operation with other molecules is necessary to target Sp1 for proteasome degradation.

ATPases Associated with Diverse Cellular Activitie↗
PubMed2000

Which electron count rules are needed for four-center three-dimensional aromaticity?

A series of charged and neutral four-center n-electron (4c-ne, n = 1-4) molecules based on the adamantane framework, but which include combinations of boron, nitrogen, and phosphorus atoms at bridgehead positions, were studied computationally at the B3LYP/6-31G* level of density functional theory (DFT). The three-dimensional aromaticity, observed earlier for the 1,3,5,7-bisdehydroadamantane dication (1), is found to be general for 4c-2e electron systems. The degree of electron delocalization, evaluated by energetic, geometric, and various magnetic criteria, is quite independent of the molecular symmetry (point groups vary from Td to Cs), the degeneracy of the orbitals, the molecular charges, and the nature of the atoms participating in the delocalized bonding. Although the multiple positive (e.g., in 1 and some of the heteroatom systems) and multiple negative charges are strongly repulsive, the rigid adamantane frameworks help hold the bridgehead atoms within bonding distances with the fewer available electrons. The corresponding 4c-1e doublets are approximately half as aromatic as the 4c-2e singlets based on the same criteria. However, the three-electron systems may either adopt distorted but still four-center delocalized structures, or alternative 3c-2e two-dimensional arrangements in which the fourth bridgehead atom is more distant. There is no need to derive special rules for each point group for 4c-ne systems. Although the three-dimensional stabilization is computed to be quite appreciable, ranging between 10 and 50 kcalmol(-1), this delocalization energy is generally not sufficient to overcome distortion due to strain in higher homologues of 1 and in analogous noncage systems. Among the various 4c-2e homoadamantanedehydro dications studied, only the 1,8-dehydrohomoadamandiyl-3,6-dication forms a three-dimensional aromatic system.

Journal Article↗