Biomedical subjects

Z Li

Publications and source records attributed to Z Li.

At least 451 records · Page 25Linked to original sources

PubMed2000

The function of the human homolog of Saccharomyces cerevisiae REV1 is required for mutagenesis induced by UV light.

In Saccharomyces cerevisiae, most mutations induced by a wide range of mutagens arise during translesion replication employing the REV1 gene product and DNA polymerase zeta. As part of an effort to investigate mammalian mutagenic mechanisms, we have identified cDNA clones of the human homologs of the yeast REV genes and examined their function in UV mutagenesis. Previously, we described the isolation of a human homolog of yeast REV3, the catalytic subunit of pol zeta, and here report the identification and sequence of a human homolog of yeast REV1. This gene was isolated by identifying an expressed sequence tag encoding a peptide with similarity to the C terminus of yeast Rev1p, followed by sequencing of the clone and retrieval of the remaining cDNA by 5' rapid amplification of cDNA ends. The human gene encodes an expected protein of 1,251 residues, compared with 985 residues in the yeast protein. The proteins share two amino-terminal regions of approximately 100 residues with 41% and 20% identity, a region of approximately 320 residues with 31% identity, and a central motif in which 11 of 13 residues are identical. Human cells expressing high levels of an hREV1 antisense RNA grew normally, and were not more sensitive to the cytotoxic effect of 254 nm UV radiation than cells lacking antisense RNA. However, the frequencies of 6-thioguanine resistance mutants induced by UV in the cells expressing antisense hREV1 RNA were significantly lower than in the control (P = 0.01), suggesting that the human gene has a function similar to that of the yeast homolog.

Amino Acid Sequence↗

PubMed2000

Role for cathepsin F in invariant chain processing and major histocompatibility complex class II peptide loading by macrophages.

The major histocompatibility complex (MHC) class II-associated invariant chain (Ii) regulates intracellular trafficking and peptide loading of MHC class II molecules. Such loading occurs after endosomal degradation of the invariant chain to a approximately 3-kD peptide termed CLIP (class II-associated invariant chain peptide). Cathepsins L and S have both been implicated in degradation of Ii to CLIP in thymus and peripheral lymphoid organs, respectively. However, macrophages from mice deficient in both cathepsins S and L can process Ii and load peptides onto MHC class II dimers normally. Both processes are blocked by a cysteine protease inhibitor, indicating the involvement of an additional Ii-processing enzyme(s). Comparison of cysteine proteases expressed by macrophages with those found in splenocytes and dendritic cells revealed two enzymes expressed exclusively in macrophages, cathepsins Z and F. Recombinant cathepsin Z did not generate CLIP from Ii-MHC class II complexes, whereas cathepsin F was as efficient as cathepsin S in CLIP generation. Inhibition of cathepsin F activity and MHC class II peptide loading by macrophages exhibited similar specificity and activity profiles. These experiments show that cathepsin F, in a subset of antigen presenting cells (APCs), can efficiently degrade Ii. Different APCs can thus use distinct proteases to mediate MHC class II maturation and peptide loading.

Animals↗

PubMed2000

Locus-specific contig assembly in highly-duplicated genomes, using the BAC-RF method.

Polyploidy, the presence of multiple sets of chromosomes that are similar but not identical, complicates both chromosome walking and assembly of sequence-ready contigs for many plant taxa including a large number of economically-significant crops. Traditional 'dot-blot hybridization' or PCR-based assays for identifying BAC clones corresponding to a mapped DNA landmark usually do not provide sufficient information to distinguish between allelic and non-allelic loci. A restriction fragment matching method using pools of BAC DNA in combination with dot-blots reveals the locus specificity of individual BACs that correspond to multi-locus DNA probes, in a manner that can efficiently be applied on a large scale. This approach also provides an alternative means of mapping DNA loci that exploits many advantages of 'radiation hybrid' mapping in taxa for which such hybrids are not available. The BAC-RF method is a practical and reliable approach for using high-density RFLP maps to anchor sequence-ready BAC contigs in highly-duplicated genomes, provides an alternative to high-density robotic gridding for screening BAC libraries when the necessary equipment is not available, and permits the expedient isolation of individual members of multigene or repetitive DNA families for a wide range of genetic and evolutionary investigations.

Chromosomes, Bacterial↗

PubMed2000

Interaction between the conserved region in the C-terminal domain of GRK2 and rhodopsin is necessary for GRK2 to catalyze receptor phosphorylation.

The C-terminal domain of G protein-coupled receptor kinases (GRKs) consists of a conserved region and a variable region, and the variable region has been shown to direct the membrane translocation of cytosolic enzymes. The present work has revealed that the C-terminal domain may also be involved in kinase-receptor interaction that is primarily mediated by the conserved region. Truncation of the C-terminal domain or deletion of the conserved region in this domain of GRK2 resulted in a complete loss of its ability to phosphorylate rhodopsin and in an obvious decrease in its sensitivity to receptor-mediated phosphorylation of a peptide substrate. On the contrary, deletion of the betagamma subunit binding region in the C-terminal domain of GRK2 did not significantly alter the ability of the enzyme to phosphorylate rhodopsin. In addition, the recombinant proteins that represent the C-terminal domain and the conserved region of GRK2 could inhibit GRK2-mediated phosphorylation of rhodopsin and receptor-mediated activation of GRK2 but not GRK2-mediated phosphorylation of the peptide substrate. Furthermore, the conserved region as well as the C-terminal domain could directly bind rhodopsin in vitro. These results indicate that the C-terminal domain, or more precisely, the conserved region of this domain, is important for enzyme-receptor interaction and that this interaction is required for GRK2 to catalyze receptor phosphorylation.

Adrenergic beta-Antagonists↗

PubMed2000

Comparative genomics of the eukaryotes.

A comparative analysis of the genomes of Drosophila melanogaster, Caenorhabditis elegans, and Saccharomyces cerevisiae-and the proteins they are predicted to encode-was undertaken in the context of cellular, developmental, and evolutionary processes. The nonredundant protein sets of flies and worms are similar in size and are only twice that of yeast, but different gene families are expanded in each genome, and the multidomain proteins and signaling pathways of the fly and worm are far more complex than those of yeast. The fly has orthologs to 177 of the 289 human disease genes examined and provides the foundation for rapid analysis of some of the basic processes involved in human disease.

Animals↗

PubMed2000

The effect of pentylenetetrazole-kindling on the extracellular glutamate and taurine levels in the frontal cortex of rats.

We studied the extracellular concentration of glutamate and taurine in the frontal cortex of freely-moving pentylenetetrazole (PTZ) kindled rats using an in vivo microdialysis. A significant and sustained increase in the glutamate level was observed in the kindled rats, in contrast, a slight and delayed increase was observed in the non-kindled rats when the same grade seizure was induced by PTZ. The convulsive dose of PTZ administration caused a decrease in taurine levels in the controls, however, no significant changes were found in the kindled rats.

Animals↗

PubMed2000

Helicobacter pylori-antigen-binding fragments expressed on the filamentous M13 phage prevent bacterial growth.

Colonization of the human stomach by Helicobacter pylori is associated with the development of gastritis, duodenal ulcer, mucosa-associated lymphoid tissue (MALT) lymphoma, and gastric cancer. H. pylori-antigen-binding single-chain variable fragments (ScFv) were derived from murine hybridomas producing monoclonal antibodies and expressed as a g3p-fusion protein on a filamentous M13 phage. The recombinant ScFv-phage reacted specifically with a 30-kDa monomeric protein of a H. pylori surface antigen preparation and by means of immunofluorescence microscopy the phage was shown to bind to both the spiral and coccoid forms of the bacterium. In vitro, the recombinant phage exhibited a bacteriocidal effect and inhibited specifically the growth of all the six strains of H. pylori tested. When H. pylori was pretreated with the phage 10 min before oral inoculation of mice, the colonization of the mouse stomachs by the bacterium was significantly reduced (P<0.01). The results suggest that genetic engineering may be used to generate therapy-effective phages.

Animals↗

PubMed2000

Diverse effects of 9-hydroxyellipticine on the chemosensitivity of human pancreatic cancer cells harboring p53 mutations.

Recently, it has been shown that 9-hydroxyellipticine (9-HE), an antitumor alkaloid has a unique property of restoring functional wild-type (wt) p53 activity via inhibition of mutant (mt) p53 protein phosphorylation. In the present study, we investigated the effect of 9-HE on the drug sensitivity of human pancreatic cancer cells. Exposure of cells to 9-HE at a relatively low concentration of 1 microM induced almost no cell death but was sufficient to restore wt p53 activity, as evidenced by an induction of endogenous p21WAF1/CIP1 concomitant with G1 and G2/M arrests in cell-cycle progression. Pretreatment with 1 microM 9-HE markedly enhanced cell killing when combined with cisplatin or mitomycin C. In contrast, 9-HE pretreatment protected cells from killing by 5-fluorouracil, VP-16, or vincristine. These effects of 9-HE were specific for several cell lines containing mt p53 and were not observed in p53-negative or wt p53 expressing cells. Taken together, these findings suggest that 9-HE may exert different effects on the drug sensitivity of pancreatic cancer cells displaying p53 mutations possibly through restoration of wt p53.

Antimetabolites, Antineoplastic↗

PubMed2000

Roles of PLC-beta2 and -beta3 and PI3Kgamma in chemoattractant-mediated signal transduction.

The roles of phosphoinositide 3-kinase (PI3K) and phospholipase C (PLC) in chemoattractant-elicited responses were studied in mice lacking these key enzymes. PI3Kgamma was required for chemoattractant-induced production of phosphatidylinositol 3,4,5-trisphosphate [PtdIns (3,4,5)P3] and has an important role in chemoattractant-induced superoxide production and chemotaxis in mouse neutrophils and in production of T cell-independent antigen-specific antibodies composed of the immunoglobulin lambda light chain (TI-IglambdaL). The study of the mice lacking PLC-beta2 and -beta3 revealed that the PLC pathways have an important role in chemoattractant-mediated production of superoxide and regulation of protein kinases, but not chemotaxis. The PLC pathways also appear to inhibit the chemotactic activity induced by certain chemoattractants and to suppress TI-IglambdaL production.

Animals↗

PubMed2000

Ectopic expression of fibroblast growth factor receptor 3 promotes myeloma cell proliferation and prevents apoptosis.

The t(4;14) translocation occurs in 25% of multiple myeloma (MM) and results in both the ectopic expression of fibroblast growth factor receptor 3 (FGFR3) from der4 and immunoglobulin heavy chain-MMSET hybrid messenger RNA transcripts from der14. The subsequent selection of activating mutations of the translocated FGFR3 by MM cells indicates an important role for this signaling pathway in tumor development and progression. To investigate the mechanism by which FGFR3 overexpression promotes MM development, interleukin-6 (IL-6)-dependent murine B9 cells were transduced with retroviruses expressing functional wild-type or constitutively activated mutant FGFR3. Overexpression of mutant FGFR3 resulted in IL-6 independence, decreased apoptosis, and an enhanced proliferative response to IL-6. In the presence of ligand, wild-type FGFR3-expressing cells also exhibited enhanced proliferation and survival in comparison to controls. B9 clones expressing either wild-type FGFR3 at high levels or mutant FGFR3 displayed increased phosphorylation of STAT3 and higher levels of bcl-x(L) expression than did parental B9 cells after cytokine withdrawal. The mechanism of the enhanced cell responsiveness to IL-6 is unknown at this time, but does not appear to be mediated by the mitogen-activated protein kinases SAPK, p38, or ERK. These findings provide a rational explanation for the mechanism by which FGFR3 contributes to both the viability and propagation of the myeloma clone and provide a basis for the development of therapies targeting this pathway.

Apoptosis↗

PubMed2000

[Study on genotypes of cystathionine beta-synthase in neural tube defects].

Mildly elevated maternal plasma homocysteine (Hcy) levels (hyperhomocysteinemia) have recently been observed in some neural tube defects(NTDs) pregnancies. The aetiology of NTDs is also known to have both genetic and nutritional components. The frequency of two relatively common mutations in the enzyme of cystathionine beta-synthase (CBS), which is one of the main enzymes that controls Hcy level, was examined. Among 76 nonpregnant women in the study, 40 of whom had given birth to infants with NTDs and 36 control women previously had normal offspring. The results showed that no significant difference was found between NTDs mothers and non-NTDs mothers for the frequency of T833-C and the G919-A mutations in the CBS gene. The data suggests that the mother's genotype is not the direct factor involved in the pathogenesis of NTDs.

Adult↗

PubMed2000

Collagenolytic activity of cathepsin K is specifically modulated by cartilage-resident chondroitin sulfates.

Cathepsin K is the predominant cysteine protease in osteoclast-mediated bone remodeling, and the protease is thought to be involved in the pathogenesis of diseases with excessive bone and cartilage resorption. Osteoclastic matrix degradation occurs in the extracellular resorption lacuna and upon phagocytosis within the cell's lysosomal-endosomal compartment. Since glycosaminoglycans (GAGs) are abundant in extracellular matrixes of cartilage and growing bone, we have analyzed the effect of GAGs on the activity of bone and cartilage-resident cathepsins K and L and MMP-1. GAGs, in particular chondroitin sulfates, specifically and selectively increased the stability of cathepsin K but had no effect on cathepsin L and MMP-1. GAGs strongly enhanced the stability and, to a lesser extent, the catalytic activity of cathepsin K. To combine the activity and stability parameters, we defined a novel kinetic term, named cumulative activity (CA), which reflects the total substrate turnover during the life span of the enzyme. In the presence of chondroitin-4-sulfate (C-4S), the CA value increased 200-fold for cathepsin K but only 25-fold with chondroitin-6-sulfate (C-6S). C-4S dramatically increased the hydrolysis of soluble as well insoluble type I and II collagens, whereas the effects of C-6S and hyaluronic acid were less pronounced. C-4S acts in a concentration-dependent manner but reaches saturation at approximately 0.1%, a concentration similar to that found in the synovial fluid of arthritis patients. C-4S increased the cathepsin K-mediated release of hydroxyproline from insoluble type I collagen 10-fold but had only a less than 2-fold enhancing effect on the hydrolysis of intact cartilage. The relatively small increase in the hydrolysis of cartilage by C-4S was attributed to the endogenous chondroitin sulfate content present in the cartilage. Although C-4S increased the pH stability at neutral pH, a significant increase in the collagenolytic activity of cathepsin K at this pH was not observed, thus suggesting that the unique collagenolytic activity of cathepsin K at acidic pH is mechanistically determined and not by the enzyme's instability at neutral pH. The selective and significant stabilization and activation of cathepsin K activity by C-4S may provide a rationale for a novel mechanism to regulate the enzyme's activity during bone growth and aging, two processes known for significant changes in the GAG content.

Arthritis, Rheumatoid↗

PubMed2000

Suppression of metallothionein gene expression in a rat hepatoma because of promoter-specific DNA methylation.

Metallothionein I can be induced in response to a variety of agents that include heavy metals and oxidative stress. On the contrary, its induction was suppressed in some lymphoid-derived cancer cells. The mechanism of this repression has not been elucidated. Here, we show silencing of MT-I gene in a solid transplanted rat tumor as a result of promoter methylation at all the 21 CpG dinucleotides that span the region from -225 bp to +1 bp. By contrast, none of these CpG dinucleotides were methylated in the livers from the rats bearing the tumor, which was consistent with the efficient induction of the gene in this tissue by zinc sulfate. Genomic footprinting revealed lack of access of the transcriptional activators to the respective cis-acting elements of the methylated MT-I promoter in the hepatoma. The absence of footprinting was not due to inactivation of the metal regulatory transcription factor MTF-1, because it was highly active in the hepatoma. Treatment of the hepatoma bearing rats with 5-azacytidine, a demethylating agent, induced basal as well as heavy metal-activated MT-I gene expression in the hepatoma, implying that methylation was indeed responsible for silencing the gene. Bisulfite genomic sequencing showed significant (>90%) demethylation of CpG dinucleotides spanning MT-I promoter in the hepatoma following treatment with 5-AzaC. The hypermethylation of MT-I promoter was probably caused by significantly higher (as much as 7-fold) level of DNA methyl transferase activity as well as enhanced expression of its gene in the hepatoma relative to the host liver. These data elucidated for the first time the molecular mechanism for the silencing of a highly inducible gene in a solid tumor transplanted in an animal, as compared with the robust induction in the corresponding parental tissue and have discussed the probable reasons for the suppression of this gene in some tumors.

Animals↗

PubMed2000

I-CeuI fragment analysis of the Shigella species: evidence for large-scale chromosome rearrangement in S. dysenteriae and S. flexneri.

I-CeuI fragments of four Shigella species were analyzed to investigate their taxonomic distance from Escherichia coli and to collect substantiated evidence of their genetic relatedness because their ribosomal RNA sequences and similarity values of their chromosomal DNA/DNA hybridization had proved their taxonomic identity. I-CeuI digestion of genomic DNAs yielded seven fragments in every species, indicating that all the Shigella species contained seven sets of ribosome RNA operons. To determine the fragment identities, seven genes were selected from each I-CeuI fragment of E. coli strain K-12 and used as hybridization probes. Among the four Shigella species, S. boydii and S. sonnei showed hybridization patterns similar to those observed for E. coli strains; each gene probe hybridized to the I-CeuI fragments with sizes similar to that of the corresponding E. coli fragment. In contrast, S. dysenteriae and S. flexneri showed distinct patterns; rcsF and rbsR genes that located on different I-CeuI fragments in E. coli, fragments D and E, were found to co-locate on a fragment. Further analysis using an additional three genes that located on fragment D in K-12 revealed that some chromosome rearrangements involving the fragments corresponding to fragments D and E of K-12 took place in S. dysenteriae and S. flexneri.

Blotting, Southern↗

PubMed2000

The morphogenesis of the zebrafish eye, including a fate map of the optic vesicle.

We have examined the morphogenesis of the zebrafish eye, from the flat optic vesicle at 16 hours post fertilization (hpf) to the functional hemispheric eye at 72 hpf. We have produced three-dimensional reconstructions from semithin sections, measured volumes and areas, and produced a fate map by labeling clusters of cells at 14-15 hpf and finding them in the 24 hpf eye cup. Both volume and area increased sevenfold, with different schedules. Initially (16-33 hpf), area increased but volume remained constant; later (33-72 hpf) both increased. When the volume remained constant, the presumptive pigmented epithelium (PE) shrank and the presumptive neural retina (NR) enlarged. The fate map revealed that during 14-24 hpf cells changed layers, moving from the PE into the NR, probably through involution around the margin of the eye. The transformation of the flat epithelial layers of the vesicle into their cup-shaped counterparts in the eye was also accompanied by cellular rearrangements; most cells in a cluster labeled in the vesicle remained neighbors in the eye cup, but occasionally they were separated widely. This description of normal zebrafish eye development provides explanations for some mutant phenotypes and for the effects of altered retinoic acid.

Animals↗

PubMed2000

Hydrogen-deuterium exchange reactions of cis- and trans-cyclopropane derivatives with D(2)O and CD(3)OD in the gas phase

Gas-phase hydrogen-deuterium (H/D) exchange reactions involving four isomeric cyclopropane derivatives were investigated under chemical ionization (CI) conditions, using D(2)O and CD(3)OD as reagent gases. There are abundant ions at [M + 1](+), [M + 2](+) and [M + 3](+) in the D(2)O and CD(3)OD positive-ion CI mass spectra of the two isomer pairs 1, 2 and 3, 4. Their CI mass spectra are identical with each pair, and so are the collision-induced dissociation (CID) spectra of ions [M + 1](+), [M + 2](+) and [M + 3](+) of each of the two isomer pairs. The CID spectra of [M + 1](+) ions indicate that they have common D/H exchange reactions within each pair, which take place between molecular ions and deuterium-labeling reagents to form the [M - H + D](+) ions. Those of their [M + 2](+) ions show that they have common D/H exchange reactions within each pair, which form the [M(d1) + H](+) ions. Those of their [M + 3](+) ions show that they have common D/H exchange reactions within each pair, which take place between the [M(d1)] and deuterium-labeling reagents to produce [M(d2) + H](+) for the isomer pair 1, 2 and [M(d1) + D](+) for the isomer pair 3, 4. The number and position, and active order of the active hydrogen atoms of the isomer pairs 1, 2 and 3, 4 were determined. Copyright 2000 John Wiley & Sons, Ltd.

Journal Article↗

PubMed2000

Cocaine administration decreases functional connectivity in human primary visual and motor cortex as detected by functional MRI.

Functional magnetic resonance imaging (fMRI) was conducted to observe the effects of cocaine administration on the physiological fluctuations of fMRI signal in two brain regions. Seven long-term cocaine users with an average age of 32 years and 8 years of cocaine use history were recruited for the study. A T2*-weighted fast echo-planar imaging (EPI) pulse sequence was employed at 1.5 T to acquire three sets of brain images for each subject under three conditions (at rest, after saline injection, and after cocaine injection [0.57 mg/kg]). Cross-correlation maps were constructed using the synchronous, low frequency signal from voxel time courses after filtering respiratory, cardiac, and other physiological noise. A quantitative evaluation of the changes in functional connectivity was made using spatial correlation coefficient (SCC) analysis. A marked 50% reduction in SCC values in the region of primary visual cortex and 43% reduction in SCC values in the region of primary motor cortex were observed after cocaine administration. This significant reduction in SCC values in these cortical regions is a reflection of changes in neuronal activity. It is suggested that the observed changes in low frequency components after acute cocaine administration during a resting, no-task situation may be used as a baseline reference source when assessing the effects of cocaine on task-driven activation or on mesolimbic dopamine pathways.

Adolescent↗

PubMed2000

Modulation of cell proliferation in the embryonic retina of zebrafish (Danio rerio).

We describe light-microscopically the development of the embryonic zebrafish eye with particular attention to cell number, cell proliferation, and cell death. The period from 16 to 36 hr post fertilization (hpf) comprises two phases; during the first (16-27 hpf) the optic vesicle becomes the eye cup, and during the second (27-36 hpf) the eye cup begins to differentiate into the neural retina and pigmented epithelium. All cells in the eye primordium are proliferative prior to 28 hpf, and the length of the cell cycle has been estimated to be 10 hr at 24-28 hpf (Nawrocki, 1985). Our cell counts are consistent with that estimate at that age, but not at earlier ages. A 10-hr cell cycle predicts that the cell number should increase by 7% per hr, but during 16-24 hpf the cell number increased by only 1.5% per hr. Despite the low rate of increase, all cells labeled with bromo-deoxyuridine, so all were proliferative. We considered three possible explanations for the nearly-constant cell number in the first phase: proliferation balanced by cell emigration from the eye, proliferation balanced by cell death, and low proliferation caused by a transient prolongation of the cell cycle. We excluded the first two, and found direct support for the third. Previous examinations of the cell cycle length in vertebrate central nervous system have concluded that it increases monotonically, in contrast to the modulation that we have shown. Modulation of the cell cycle length is well-known in flies, but it is generally effected by a prolonged arrest at one phase, in contrast to the general deceleration that we have shown.

Animals↗