Biomedical subjects

Y Li

Publications and source records attributed to Y Li.

At least 19 recordsLinked to original sources

PubMed2000

Complex regulation of calcium-binding protein D9k (calbindin-D(9k)) in the mouse uterus during early pregnancy and at the site of embryo implantation.

Establishment of receptive endometrium is essential for implantation. Our aim was to identify and characterize genes uniquely regulated at the sites of implantation in mouse uterus by RNA differential display polymerase chain reaction (DDPCR). One of the gene fragments identified was 86% homologous to rat calcium-binding protein D9k (calbindin-D(9k)); the mouse counterpart had not then been cloned, but subsequently an mRNA sequence of mouse calbindin-D(9k) became available in GenBank (accession number: AF028071). This sequence is 99% homologous to the DDPCR-derived gene tag but has a shorter 3' end. Reverse transcription-polymerase chain reaction (RT-PCR) was performed using the sequence of 3' end of the DDPCR product and the 5' end of AF028071, and a full cDNA was obtained. This gene was primarily up-regulated by progesterone, but not by estrogen. It was further increased by the combination of the two steroids. Expression of calbindin-D(9k) was overall increased in the uterus during early pregnancy, but the level was significantly lower in implantation compared to interimplantation sites on Days 4.5 and 5.5 of pregnancy, becoming barely detectable in both sites after Day 6.5. In situ hybridization localized this mRNA predominantly in the luminal epithelium of the pregnant uterus. The complex regulation of calbindin-D(9k) in mouse uterus suggests an important role for this protein during pregnancy.

Amino Acid Sequence↗

PubMed2000

Structural fingerprinting: subgrouping of comoviruses by structural studies of red clover mottle virus to 2.4-A resolution and comparisons with other comoviruses.

Red clover mottle virus (RCMV) is a member of the comoviruses, a group of picornavirus-like plant viruses. The X-ray structure of RCMV strain S has been determined and refined to 2.4 A. The overall structure of RCMV is similar to that of two other comoviruses, Cowpea mosaic virus (CPMV) and Bean pod mottle virus (BPMV). The sequence of the coat proteins of RCMV strain O were modeled into the capsid structure of strain S without causing any distortion, confirming the close resemblance between the two strains. By comparing the RCMV structure with that of other comoviruses, a structural fingerprint at the N terminus of the small subunit was identified which allowed subgrouping of comoviruses into CPMV-like and BPMV-like viruses.

Amino Acid Sequence↗

PubMed1999

Crystal structure of chondroitinase B from Flavobacterium heparinum and its complex with a disaccharide product at 1.7 A resolution.

Glycosaminoglycans (GAGs) are a family of acidic heteropolysaccharides, including such molecules as chondroitin sulfate, dermatan sulfate, heparin and keratan sulfate. Cleavage of the O-glycosidic bond within GAGs can be accomplished by hydrolases as well as lyases, yielding disaccharide and oligosaccharide products. We have determined the crystal structure of chondroitinase B, a glycosaminoglycan lyase from Flavobacterium heparinum, as well as its complex with a dermatan sulfate disaccharide product, both at 1.7 A resolution. Chondroitinase B adopts the right-handed parallel beta-helix fold, found originally in pectate lyase and subsequently in several polysaccharide lyases and hydrolases. Sequence homology between chondroitinase B and a mannuronate lyase from Pseudomonas sp. suggests this protein also adopts the beta-helix fold. Binding of the disaccharide product occurs within a positively charged cleft formed by loops extending from the surface of the beta-helix. Amino acid residues responsible for recognition of the disaccharide, as well as potential catalytic residues, have been identified. Two arginine residues, Arg318 and Arg364, are found to interact with the sulfate group attached to O-4 of N-acetylgalactosamine. Cleavage of dermatan sulfate likely occurs at the reducing end of the disaccharide, with Glu333 possibly acting as the general base.

Amino Acid Sequence↗

PubMed1999

The expression and activity of D-type cyclins in F9 embryonal carcinoma cells: modulation of growth by RXR-selective retinoids.

The growth rate of malignant F9 embryonal carcinoma cells slows considerably following all-trans-retinoic acid-induced differentiation into benign parietal endoderm. To determine the mechanism of this process, we examined the expression of cyclins D1, D2, and D3 and the activity of their associated kinases. Cyclin D1 and D3 mRNA levels decreased during complete differentiation induced by all-trans-retinoic acid and dibutyryl cAMP, while the levels of cyclin D2 and the cyclin-dependent kinase (Cdk) inhibitor p27 mRNAs increased. Ultimately, terminally differentiated cells possessed 50% of the Cdk4-associated kinase activity observed in undifferentiated cells. Since numerous genes are differentially regulated during parietal endoderm differentiation, it is difficult to determine whether retinoic acid affects cell cycle gene expression directly or if these changes are caused by differentiation. We found that the retinoid X receptor (RXR)-selective agonists LG100153 and LG100268 significantly inhibited F9 cell growth without causing overt terminal differentiation as assessed by anchorage-independent growth and differentiation-associated gene expression. As seen in cells induced to differentiate by the RAR agonist all-trans-retinoic acid, RXR activation led to an increase in the number of cells in G1 phase. RXR agonists also sharply induced the levels of the Cdk regulatory subunits, cyclin D2 and D3. However, Cdk4-dependent kinase activity was reduced by RXR-selective retinoid treatment. These observations suggest that some retinoids can directly inhibit proliferation and regulate Cdk4-dependent kinase activity without inducing terminal differentiation.

Alitretinoin↗

PubMed1999

Antigen-induced unresponsiveness results in altered T cell signaling.

Pretransplant exposure to allogeneic lymphocytes can result in donor-specific unresponsiveness and prolonged allograft survival. Intracellular signaling events have been described in anergic T cell clones, but the biochemical events underlying in vivo induced unresponsiveness have not been studied in detail. We employed a TCR transgenic mouse, bearing the 2C TCR, providing adequate numbers of homogenous peripheral T cells to study biochemical aspects of T cell unresponsiveness in vivo. 2C mice exposed to semiallogeneic lymphocytes (H-2b x H-2d) experienced prolonged H-2d cardiac allograft survival, and cells from these mice did not proliferate or make IL-2 in response to alloantigen (H-2d). Importantly, there were marked differences in TCR-associated tyrosine phosphorylation activation patterns. The targets for the unresponsive state appear to be diminished Lck activation and absent ZAP-70 and LAT (linker for activation of T cells) phosphorylation. Our study demonstrates that Ag-induced tolerance in vivo is accompanied by altered early TCR-mediated signaling events.

Animals↗

PubMed1999

Translational regulation of ribonucleotide reductase by eukaryotic initiation factor 4E links protein synthesis to the control of DNA replication.

Ribonucleotide reductase synthesizes dNDPs, a specific and limiting step in DNA synthesis, and can participate in neoplastic transformation when overexpressed. The small subunit (ribonucleotide reductase 2 (RNR2)) was cloned as a major product in a subtraction library from eukaryotic initiation factor 4E (eIF4E)-transformed cells (Chinese hamster ovary-4E (CHO-4E)). CHO-4E cells have 20-40-fold elevated RNR2 protein, reflecting an increased distribution of RNR2 mRNA to the heavy polysomes. CHO-4E cells display an altered cell cycle with shortened S phase, similar to cells selected for RNR2 overexpression with hydroxyurea. The function of ribonucleotide reductase as a checkpoint component of S progression was studied in yeast in which elevated eIF4E rescued S-arrested rnr2-68(ts) cells, by increasing recruitment of its mRNA to polysomes. Crosses between rnr2-68(ts) and mutant eIF4E (cdc33-1(ts)) engendered conditional synthetic lethality, with extreme sensitivity to hydroxyurea and the microtubule depolymerizing agent, benomyl. The double mutant (cdc33-1 rnr2-68) also identified a unique terminal phenotype, arrested with small bud and a randomly distributed single nucleus, which is distinct from those of both parental single mutants. This phenotype defines eIF4E and RNR2 as determinants in an important cell cycle checkpoint, in early/mid-S phase. These results also provide a link between protein and DNA synthesis and provide an explanation for cell cycle alterations induced by elevated eIF4E.

Animals↗

PubMed1999

Superhumps in a Peculiar SU Ursae Majoris-Type Dwarf Nova, ER Ursae Majoris.

We report the photometry of a peculiar SU Ursae Majoris-type dwarf nova, ER Ursae Majoris, for 10 nights during 1998 December and 1999 March, covering a complete rise to the supermaximum and a normal outburst cycle. Superhumps have been found during the rise to the superoutburst. A negative superhump appeared in the December 22 light curve, while the superhump on the next night became positive and had a large-amplitude waveform distinct from that of the previous night. In the normal outbursts we captured, superhumps with larger or smaller amplitudes seem to always exist, although it is not necessarily true for every normal outburst. These results show great resemblance to V1159 Ori. It is more likely that superhumps occasionally exist at essentially all phases of the eruption cycles of ER UMa stars, which should be considered in modeling.

Journal Article↗

PubMed1999

Identification of allosteric sites in rabbit phosphofructo-1-kinase.

Earlier studies indicated an evolutionary relationship between bacterial and mammalian phosphofructo-1-kinases (PFKs) that suggests duplication, tandem fusion, and divergence of catalytic and effector binding sites of a prokaryotic ancestor to yield in eukaryotes a total of six organic ligand binding sites. The identities of residues involved in the four binding sites for allosteric ligands in mammalian PFK have been inferred from this assumed relationship. In the current study of the C isozyme of rabbit PFK, two arginine residues that can be aligned with important residues in the catalytic and allosteric binding sites of bacterial PFK and that are conserved in all eukaryotic PFKs were mutated. Arg-48 was suggested previously to be part of either the ATP inhibitory or the adenine nucleotide activating site. However, the mutant enzyme showed only slightly less sensitivity to ATP inhibition and was fully activatable by adenine nucleotides. On the other hand, sensitivity to citrate and 3-phosphoglycerate inhibition was lost, indicating an important role for Arg-48 in the binding of these allosteric effectors. Mutation of Arg-481, homologous to an active site residue in bacterial PFK, prevented binding and allosteric activation by fructose 2,6-bisphosphate. A new relationship between the allosteric sites of mammalian PFK and bacterial PFK is proposed.

Adenosine Diphosphate↗

PubMed1999

A Pentacoordinated Di-N-carboxamido-dithiolato-O-sulfinato-iron(III) Complex Related to the Metal Site of Nitrile Hydratase.

Postcoordination oxidation by dioxygen of one of the thiolate groups in a pentadentate N(2)S(3) ligand results in an iron(III) complex with two N-carboxamido, two thiolato, and one O-sulfinato ligands (see the CAMERON representation). This novel mixed coordination is similar to that determined for the inactive form of the nitrile hydratase from Rhodococcus sp. N-771, but differs by the O versus S binding of the sulfinato ligand.

Journal Article↗

PubMed1999

Distinct factor requirements for exonic splicing enhancer function and binding of U2AF to the polypyrimidine tract.

Exonic splicing enhancer (ESE) sequences are important for the recognition of adjacent splice sites in pre-mRNA and for the regulation of splice site selection. It has been proposed that ESEs function by associating with one or more serine/arginine-repeat (SR) proteins which stabilize the binding of the U2 small nuclear ribonucleoprotein particle (snRNP) auxiliary factor (U2AF) to the polypyrimidine tract upstream of the 3' splice site. We have tested this model by analyzing the composition of splicing complexes assembled on an ESE-dependent pre-mRNA derived from the doublesex gene of Drosophila. Several SR proteins and hTra2beta, a human homolog of the Drosophila alternative splicing regulator Transformer-2, associate with this pre-mRNA in the presence, but not in the absence, of a purine-rich ESE. By contrast, the 65-kDa subunit of U2AF (U2AF-65 kDa) bound equally to the pre-mRNA in the presence and absence of the ESE. Time course experiments revealed differences in the levels and kinetics of association of individual SR proteins with the ESE-containing pre-mRNA, whereas U2AF-65 kDa bound prior to most SR proteins and hTra2beta and its level of binding did not change significantly during the course of the splicing reaction. Binding of U2AF-65 kDa to the ESE-dependent pre-mRNA was, however, dependent on U1 snRNP. The results indicate that an ESE promotes spliceosome formation through interactions that are distinct from those required for the binding of U2AF-65 kDa to the polypyrimidine tract.

Arginine↗

PubMed1999

The gon-1 gene is required for gonadal morphogenesis in Caenorhabditis elegans.

In wild-type Caenorhabditis elegans, the gonad is a complex epithelial tube that consists of long arms composed predominantly of germline tissue as well as somatic structures specialized for particular reproductive functions. In gon-1 mutants, the adult gonad is severely disorganized with essentially no arm extension and no recognizable somatic structure. The developmental defects in gon-1 mutants are limited to the gonad; other cells, tissues, and organs appear to develop normally. Previous work defined the regulatory "leader" cells as crucial for extension of the gonadal arms (J. E. Kimble and J. G. White, 1981, Dev. Biol. 81, 208-219). In gon-1 mutants, the leader cells are specified correctly, but they fail to migrate and gonadal arms are not generated. In addition, gon-1 is required for morphogenesis of the gonadal somatic structures. This second role appears to be independent of that required for leader migration. Parallel studies have shown that gon-1 encodes a secreted metalloprotease (R. Blelloch and J. Kimble, 1999, Nature 399, 586-590). We discuss how a metalloprotease may control two aspects of gonadal morphogenesis.

Animals↗

PubMed1999

Neuroprotective effects of progesterone after transient middle cerebral artery occlusion in rat.

Treatment of focal cerebral ischemia in the rat with intraperitoneal administration of progesterone dissolved in dimethyl sulfoxide (DMSO) has demonstrated therapeutic efficacy. In the present study we test whether iv administration of water soluble progesterone 2 h after the onset of middle cerebral artery occlusion provides therapeutic benefit for the treatment of stroke. In addition, we perform a battery of functional tests: rotarod, adhesive-backed somatosensory, and neurological score, as well as a dose-response study. The data indicate that iv administration of progesterone at a dose of 8 mg/kg significantly reduces the volume of cerebral infarction and significantly improves outcome on the array of functional measures employed. Treatment with 4 mg/kg or 32 mg/kg of progesterone failed to provide any therapeutic benefit. Progesterone, a non toxic, clinically employed, pluripotent therapeutic agent which targets both neuroprotective as well as neuroregenerative strategies, may have important therapeutic benefits for the treatment of stroke.

Animals↗

PubMed1999

Diabetes affects similarly the catalytic subunit and putative glucose-6-phosphate translocase of glucose-6-phosphatase.

The effect of streptozocin diabetes on the expression of the catalytic subunit (p36) and the putative glucose-6-phosphate translocase (p46) of the glucose-6-phosphatase system (G6Pase) was investigated in rats. In addition to the documented effect of diabetes to increase p36 mRNA and protein in the liver and kidney, a approximately 2-fold increase in the mRNA abundance of p46 was found in liver, kidney, and intestine, and a similar increase was found in the p46 protein level in liver. In HepG2 cells, glucose caused a dose-dependent (1-25 mM) increase (up to 5-fold) in p36 and p46 mRNA and a lesser increase in p46 protein, whereas insulin (1 microM) suppressed p36 mRNA, reduced p46 mRNA level by half, and decreased p46 protein by about 33%. Cyclic AMP (100 microM) increased p36 and p46 mRNA by >2- and 1.5-fold, respectively, but not p46 protein. These data suggest that insulin deficiency and hyperglycemia might each be responsible for up-regulation of G6Pase in diabetes. It is concluded that enhanced hepatic glucose output in insulin-dependent diabetes probably involves dysregulation of both the catalytic subunit and the putative glucose-6-phosphate translocase of the liver G6Pase system.

Animals↗

PubMed1999

SHIP-mediated inhibition of K562 erythroid differentiation requires an intact catalytic domain and Shc binding site.

Growing evidence supports a role for the SHIP inositol 5'-phosphatase in the negative regulation of a variety of receptor-mediated signaling pathways in hematopoietic cells. SHIP expression among cultured cell lines was examined and found to be restricted to cells of hematopoietic origin, with the exception of the K562 erythroleukemia cell line, in which SHIP protein and mRNA were undetectable. The absence of endogenous SHIP in K562 cells provided a useful system to study the role of SHIP in growth and differentiation. When stably expressed in K562 cells, SHIP was found to be constitutively tyrosine phosphorylated and associated with endogenous Shc and Grb-2. Stable expression of SHIP did not affect growth of the cells but resulted in decreased synthesis of hemoglobin protein and epsilon-globin mRNA in response to hemin, an inducer of erythroid differentiation. This effect was not due to increased cell death in the SHIP-expressing lines following hemin stimulation, but was likely the result of an impaired differentiation program in these cells. Mutational analysis indicated that SHIP must retain both an intact catalytic domain and Shc binding site to efficiently inhibit K562 erythroid differentiation.

Adaptor Proteins, Signal Transducing↗

PubMed1999

Human immunodeficiency virus infection of human placental cord blood CD34+AC133+ stem cells and their progeny.

The AC133 is a novel antigen selectively expressed on primitive CD34bright stem cells and is a valuable marker for the selection of long-term culture-initiating cells (LTC-ICs) and severe combined immunodeficiency (SCID)-repopulating cells. Human placental cord blood (HPCB) is a rich source of CD34+AC133+ cells. Since AC133 antibody is likely to be used as an alternative to CD34 for the selection of stem cells in transplant and gene therapy situations, we examined the susceptibility of HPCB-isolated CD34+AC133+ stem cells to infection with free and cell-associated HIV-1 in vitro. Freshly isolated HPCB CD34+AC133+ stem cells were not susceptible to HIV-1 infection as determined by PCR and reverse transcriptase assays. Inoculation with HIV-1 did not affect the viability and clonogenic ability of HPCB CD34+AC133+ cells. Although the highly purified HPCB CD34+AC133+ stem cells contained mRNA for CD4 and CXCR4 receptors, CD4 and CXCR4 proteins were not expressed on these cells. Similarly, CCR5 protein, the major macrophage-tropic HIV-1 coreceptor, was not expressed in freshly isolated HPCB CD34+AC133+ stem cells, although the transcript for CCR5 was identified in these cells. Expression of CD4, CXCR4, and CCR5 receptor proteins on the progeny derived from HPCB CD34+AC133+ stem cells was detected and correlated with susceptibility to HIV-1 infection in vitro. These findings suggest that freshly isolated HPCB CD34+AC133+ stem cells are not susceptible to HIV-1 infection and may not be a viral reservoir. These data have important implications for the use of AC133 antibody as a means of enriching for primitive hematopoietic stem cells from placental cord blood and in the design of stem cell or progenitor cell-based gene therapeutic strategies for perinatal HIV-1 infection.

AC133 Antigen↗

PubMed1999

Multiple solenoidal microcoil probes for high-sensitivity, high-throughput nuclear magnetic resonance spectroscopy.

Two designs for incorporating multiple solenoidal microcoils into a single probe head are presented to increase the throughput of high-resolution NMR. Through a combination of radio frequency switches and low-noise amplifiers, multiple NMR spectra can be acquired in the same time as a single spectrum from a conventional probe consisting of one coil. Since this method does not compromise sensitivity with regard to the individual microcoils, throughput increases linearly with the number of coils. Only one receiver is needed, and data acquisition parameters can be optimized for each sample. Specifically, a four-coil system has been implemented for proton NMR at 250 MHz using a wide-bore magnet, with an observe volume of 28 nL for each microcoil. Signal cross-contamination was approximately 0.2% between individual coils, and simultaneous one- and two-dimensional spectra have been obtained from samples of fructose, galactose, adenosine triphosphate, and chloroquine (7 nmol of each compound). A more compact two-coil configuration has also been designed for operation at 500 MHz, with observe volumes of 5 and 31 nL for the two coils. One- and two-dimensional spectra were acquired from samples of 1-butanol (55 nmol) and ethylbenzene (250 nmol).

Image Processing, Computer-Assisted↗

PubMed1999

Functional interaction of Fas-associated phosphatase-1 (FAP-1) with p75(NTR) and their effect on NF-kappaB activation.

The common neurotrophin receptor p75(NTR), a member of the tumor necrosis factor (TNF) receptor superfamily, plays an important role in several cellular signaling cascades, including that leading to apoptosis. FAP-1 (Fas-associated phosphatase-1), which binds to the cytoplasmic tail of Fas, was originally identified as a negative regulator of Fas-mediated apoptosis. Here we have shown by co-immunoprecipitation that FAP-1 also binds to the p75(NTR) cytoplasmic domain in vivo through the interaction between the third PDZ domain of FAP-1 and C-terminal Ser-Pro-Val residues of p75(NTR). Furthermore, cells expressing a FAP-1/green fluorescent protein showed intracellular co-localization of FAP-1 and p75(NTR) at the plasma membrane. To elucidate the functional role of this physical interaction, we examined TRAF6 (TNF receptor-associated factor 6)-mediated NF-kappaB activation and tamoxifen-induced apoptosis in 293T cells expressing p75(NTR). The results revealed that TRAF6-mediated NF-kappaB activation was suppressed by p75(NTR) and that the p75(NTR)-mediated NF-kappaB suppression was reduced by FAP-1 expression. Interestingly, a mutant of the p75(NTR) intracellular domain with a single substitution of a Met for Val in its C-terminus, which cannot interact with FAP-1, displayed enhanced pro-apoptotic activity in 293T transfected cells. Thus, similar to Fas, FAP-1 may be involved in suppressing p75(NTR)-mediated pro-apoptotic signaling through its interaction with three C-terminal amino acids (tSPV). Thus, FAP-1 may regulate p75(NTR)-mediated signal transduction by physiological interaction through its third PDZ domain.

Carrier Proteins↗

PubMed1999

Artificial senescence of bovine retinal pigment epithelial cells induced by near-ultraviolet in vitro.

RPE cells irradiated by near-ultraviolet (NUV) were characterized at cellular, biochemical and molecular levels in order to determine whether light-induced RPE changes contribute to the senescence of RPE cells in vitro. Biochemical and molecular parameters of cellular senescence were studied by using both bovine RPE cells at confluence repeatedly irradiated by NUV (peaking at 365 nm) and RPE cells at different levels of population doubling (PDL). After repeated NUV irradiation, RPE proliferation was markedly suppressed. In parallel, the BrdU index significantly reduced to a minimum level, similar to RPE cells undergoing multiple population doublings. NUV irradiation resulted in a decrease in cellular alkali-soluble melanin and an increase in lipofuscin-like fluorophores. The lipofuscin-like fluorophores, isolated from RPE cells exposing repeated NUV irradiation, represented a gradual hyperchromic change and red-shift, reaching the wavelength maxima (560-572 nm), at excitation wavelength of 365 nm, a typical range of 'age pigment'. These phenomena were substantially eliminated in oxygen-free conditions. Both the NUV-irradiated RPE cells and RPE cells at 20 Pd expressed 4 to 8-fold and 2 to 4-fold less PEDF and TIMP-3 genes, respectively. As result of experiments using chronic photochemical treatment, RPE cells represented several characteristics of cellular senescence. In addition to alterations of the melanin/lipofuscin system, DNA synthesis was greatly suppressed in NUV-irradiated RPE cells, indicating replicative senescence. The phenomena of downregulation of the possible senescence markers imply that photochemical reactions of RPE cells accelerate the process of RPE senescence.

Animals↗