Biomedical subjects

R Li

Publications and source records attributed to R Li.

At least 73 records · Page 4Linked to original sources

PubMed2001

Novel differences between two human prion strains revealed by two-dimensional gel electrophoresis.

The phenotype of human sporadic prion diseases is affected by patient genotype at codon 129 of the prion protein (PrP) gene, the site of a common methionine/valine polymorphism, and by the type of the scrapie PrP (PrP(Sc)), which likely reflects the prion strain. However, two distinct disease phenotypes, identified as sporadic Creutzfeldt-Jakob disease (M/M2 sCJD) and sporadic fatal insomnia (sFI), share methionine homozygosity at codon 129 and PrP(Sc) type 2. One-dimensional gel electrophoresis and immunoblotting reveal no difference between the M/M2 sCJD and sFI species of PrP(Sc) in gel mobility and glycoform ratio. In contrast, the two-dimensional immunoblot demonstrates that in M/M2 sCJD the full-length PrP(Sc) form is overrepresented and carries glycans that are different from those present in the PrP(Sc) of sFI. Because the altered glycans are detectable only in the PrP(Sc) and not in the normal or cellular PrP (PrP(C)), they are likely to result from preferential conversion to PrP(Sc) of rare PrP(C) glycoforms. This is the first evidence that a qualitative difference in glycans contributes to prion diversity.

Electrophoresis, Gel, Two-Dimensional↗

PubMed2001

Crystal structure of the SarR protein from Staphylococcus aureus.

The expression of virulence determinants in Staphylococcus aureus is controlled by global regulatory loci (e.g., sarA and agr). The sar (Staphylococcus accessory regulator) locus is composed of three overlapping transcripts (sarA P1, P3, and P2, transcripts initiated from the P1, P3, and P2 promoters, respectively), all encoding the 124-aa SarA protein. The level of SarA, the major regulatory protein, is partially controlled by the differential activation of the sarA promoters. We previously partially purified a 13.6-kDa protein, designated SarR, that binds to the sarA promoter region to down-modulate sarA transcription from the P1 promoter and subsequently SarA expression. SarR shares sequence similarity to SarA, and another SarA homolog, SarS. Here we report the 2.3 A-resolution x-ray crystal structure of the dimeric SarR-MBP (maltose binding protein) fusion protein. The structure reveals that the SarR protein not only has a classic helix-turn-helix module for DNA binding at the major grooves, but also has an additional loop region involved in DNA recognition at the minor grooves. This interaction mode could represent a new functional class of the "winged helix" family. The dimeric SarR structure could accommodate an unusually long stretch of approximately 27 nucleotides with two or four bending points along the course, which could lead to the bending of DNA by 90 degrees or more, similar to that seen in the catabolite activator protein (CAP)-DNA complex. The structure also demonstrates the molecular basis for the stable dimerization of the SarR monomers and possible motifs for interaction with other proteins.

Amino Acid Sequence↗

PubMed2001

CD44-initiated cell spreading induces Pyk2 phosphorylation, is mediated by Src family kinases, and is negatively regulated by CD45.

CD44 is a cell adhesion molecule implicated in leukocyte adhesion and migration, co-stimulation of T cells, and tumor metastasis. CD45 is a leukocyte-specific protein tyrosine phosphatase that dephosphorylates the Src family kinases, Lck and Fyn, in T cells. Positive regulation of Lck by CD45 is required for its effective participation in T cell receptor signaling events. Here, immobilized CD44 antibody induced a distinctive cell spreading in CD45(-), but not CD45(+), T cells, and this correlated with the induction of tyrosine-phosphorylated proteins. Two focal adhesion family kinases, Pyk2 and, to a lesser extent, FAK were inducibly phosphorylated, as was a potential substrate, Cas. CD44-mediated cell spreading and induced tyrosine phosphorylation were prevented by the Src family kinase inhibitor, PP2. Furthermore, 2-fold more Lck associated with CD44 in the low density sucrose fraction from CD45(-) T cells compared with CD45(+) T cells, suggesting that CD45 may regulate the association of Lck with CD44 in this fraction. Therefore, in CD45(-) T cells, CD44 signaling is mediated by Src family kinases, and this leads to Pyk2 phosphorylation, cytoskeletal changes, and cell spreading. This implicates CD45 in the negative regulation of Src family kinase-mediated CD44 signaling leading to T cell spreading.

Animals↗

PubMed2001

Requirement for a complex array of costimulators in the negative selection of autoreactive thymocytes in vivo.

Autoreactive thymocytes can be deleted at an immature stage of their development by Ag-induced apoptosis or negative selection. In addition to Ag, negative selection also requires costimulatory signals from APC. We recently used a fetal thymus organ culture system to show that CD5, CD28, and TNF cooperatively regulate deletion of autoreactive thymocytes. Although these experiments provided strong evidence for the action of several costimulators in negative selection, we wished to demonstrate a role for these molecules in a physiologically natural model where thymocytes are deleted in vivo by endogenously expressed AGS: Accordingly, we examined thymocyte deletion in costimulator-null mice in three models of autoantigen-induced negative selection. We compared CD5(-/-) CD28(-/-) mice to CD40L(-/-) mice, which exhibited a profound block in negative selection in all three systems. Surprisingly, only one of the three models revealed a requirement for the CD5 and CD28 costimulators in autoantigen-induced deletion. These results suggest that an extraordinarily complex array of costimulators is involved in negative selection. We predict that different sets of costimulators will be required depending on the timing of negative selection, the Ag, the signal strength, the APC, and whether Ag presentation occurs on class I or class II MHC molecules.

Animals↗

PubMed2001

Physical and radiation hybrid mapping of canine chromosome 12, in a region corresponding to human chromosome 6p12-q12.

The positional cloning of the hypocretin receptor 2, the gene for autosomal recessive canine narcolepsy, has led to the development of a physical map spanning a large portion of canine chromosome 12 (CFA12), in a region corresponding to human chromosome 6p12-q13. More than 40 expressed sequence tags (ESTs) were used in homology search experiments, together with chromosome walking, to build both physical and radiation hybrid maps of the CFA12 13-21 region. The resulting map of bacterial artificial chromosome ends, ESTs, and microsatellite markers represents the longest continuous high-density map of the dog genome reported to date. These data further establish the dog as a system for studying disease genes of interest to human populations and highlight feasible approaches for positional cloning of disease genes in organisms where genomic resources are limited.

Animals↗

PubMed2001

Ganglioside GD1a enhances VEGF-induced endothelial cell proliferation and migration.

Tumor progression requires normally quiescent endothelial cells to form new vascular networks. This angiogenesis is dependent upon several soluble factors, prominent among which is vascular endothelial growth factor (VEGF). Other tumor-associated molecules, such as gangliosides, sialic acid-containing glycosphingolipids expressed by tumor cells and shed into the tumor microenvironment, may also modulate tumor angiogenesis. Here we assessed the influence of a highly purified ganglioside, G(D1a), on responses of normal human umbilical vein endothelial cells (HUVEC) to VEGF. Preincubation of HUVEC with G(D1a) enhanced VEGF-induced cell proliferation; 10 microM G(D1a) caused a twofold increase in DNA synthesis. The migration of HUVEC across a VEGF gradient was also enhanced by 50%, even with only a brief (1 h) preexposure of the cells to the same concentration of G(D1a). These findings suggest that gangliosides shed by tumor cells can promote tumor angiogenesis by enhancing the VEGF response of endothelial cells in the tumor microenvironment.

Cell Division↗

PubMed2001

Correlation of ionization potentials and HOMO energies versus relative reactivities of Cl2, of Br2, and of I2 with representative acyclic alkenes. comparison with other additions to alkenes.

Plots of log k(rel) versus IP or versus HOMO for the title reactions are presented; similarities and differences among the reactions are discussed. The Cl2 and Br2 data each show a single line of correlation with positive slope for all alkenes regardless of the steric requirements; increasing substitution at the double bond increases the reaction rate, indicating an electrophilic reaction. Each plot of the I2 data calculated for adsorption exhibits a natural separation into groups of similarly substituted alkenes, in which increased substitution reduces the rate. Within each group, a good-to-excellent correlation is observed, with a lower IP generally corresponding to a higher relative rate. The results indicate that the relative magnitude of the steric requirements about the double bond is similar to that of the electronic effects in iodination. Plot shapes for iodination are compared to those of other reactions, such as hydroboration, oxymercuration, complexation with Ag+, and complexation with MeHg+.

Journal Article↗

PubMed2001

Differential expression of cellular prion protein in mouse brain as detected with multiple anti-PrP monoclonal antibodies.

The normal cellular prion protein (PrP(C)) plays an essential role in the development of prion diseases. Indirect evidence has suggested that different PrP(C) glycoforms may be expressed in different brain regions and perform distinct functions. However, due to a lack of monoclonal antibodies (Mabs) that are specific for mouse PrP(C), the expression of PrP(C) in the mouse brain has not been studied in great detail. We used Mabs specific for either the N-terminus or the C-terminus of the mouse PrP(C) to study its expression in the mouse brain by immunoblotting and immunohistochemistry. Immunoblotting studies demonstrated that the expression of PrP(C) differed quantitatively as well as qualitatively in different regions of the brain. The anti-C-terminus Mabs reacted with all three molecular weight bands of PrP(C); the anti-N-terminus Mabs only reacted with the 39-42 kDa PrP(C). The results from immunohistochemical staining revealed the spatial distribution of PrP(C) in the mouse brain, which were consistent with that from immunoblotting. Although expression of PrP(C) has been reported to be required for long-term survival of Purkinje cells, we were unable to detect PrP(C) in the Purkinje cell layer in the cerebellum with multiple anti-PrP Mabs. Our findings suggest that PrP(C) variants, i.e. various glycoforms and truncated forms, might be specifically expressed in different regions of mouse brain and might have different functions.

Animals↗

PubMed2001

Normal cellular prion protein is preferentially expressed on subpopulations of murine hemopoietic cells.

We studied the expression of normal cellular prion protein (PrP(C)) in mouse lymphoid tissues with newly developed mAbs to PrP(C). Most of the mature T and B cells in the peripheral lymphoid organs do not express PrP(C). In contrast, most thymocytes are PrP(C+). In the bone marrow, erythroid cells and maturing granulocytes are PrP(C+). Approximately 50% of the cells in the region of small lymphocytes and progenitor cells also express PrP(C). Most of these PrP(C+) cells are CD43(+), but B220(-), surface IgM(-) (sIgM(-)), and IL-7R(-), a phenotype that belongs to cells not yet committed to the B cell lineage. Another small group of the PrP(C+) cell are B220(+), and some of these are also sIgM(+). The majority of the B220(+) cells, however, are PrP(C-). Therefore, PrP(C) is preferentially expressed in early bone marrow progenitor cells and subsets of maturing B cells. Supporting this interpretation is our observation that stimulation of bone marrow cells in vitro with PMA results in a decrease in the number of PrP(C+)B220(-) cells with a corresponding increase of sIgM(+)B220(high) mature B cells. This result suggests that the PrP(C+)B220(-) cells are potential progenitors. Furthermore, in the bone marrow of Rag-1(-/-) mice, there are an increased number of PrP(C+)B220(-) cells, and most of the developmentally arrested pro-B cells in these mice are PrP(C+). Collectively, these results suggest that PrP(C) is expressed preferentially in immature T cells in the thymus and early progenitor cells in the bone marrow, and the expression of PrP(C) is regulated during hemopoietic differentiation.

Animals↗

PubMed2001

Correlation of relative rates of PdCl(2) oxidation of functionalized acyclic alkenes versus alkene ionization potentials, HOMOs, and LUMOs.

Investigations of the title reaction, carried out by plotting logs of the relative reaction rates vs IPs, vs HOMOs, and vs LUMOs, reveal multiple nearly parallel lines of correlation with small negative slopes in each. Overall, the natural grouping into monosubstituted and disubstituted alkenes gives better correlations than that obtained by using all alkenes. Comparison with analogous plots for other reactions indicates that the mechanism for this reaction has similarities to that for hydroboration, the major difference being that the lines in the plots for hydroboration have positive slopes, indicating an electrophilic rate-determining step involving the pi electrons, while those for the title reaction have small negative slopes, indicating a nucleophilic rate-determining step. Of the two reaction mechanisms proposed for the title reaction, only one has a nucleophilic attack at the complexed alkene as the rate-determining step, and therefore, this work supports that reaction mechanism.

Journal Article↗

PubMed2001

Induction of cytochrome P450 1A1 gene expression, oxidative stress, and genotoxicity by carbaryl and thiabendazole in transfected human HepG2 and lymphoblastoid cells.

Carbaryl and thiabendazole, two widely used pesticides, have been shown to induce cytochrome P450 1A1 (CYP1A1) expression, but neither compound is capable of displacing [3H] 2,3,7,8-tetrachlorodibenzo-P-dioxin from its aryl hydrocarbon receptor binding site. In the present study, we investigated the transcriptional regulation of CYP1A1 as well as other genes in various human hepatoma HepG2 cell lines stably transfected with the chloramphenicol acetyl transferase (CAT) reporter gene and cloned under the control of each of 14 promoters or response elements from relevant stress genes. Carbaryl and thiabendazole were found to activate CYP1A1 at the level of transcription, as demonstrated by the dose-dependent increase in reporter CAT and CYP1A1 mRNAs. Moreover, this effect appeared to be mediated via the xenobiotic responsive element (XRE), because both pesticides specifically activated various fusion constructs containing XRE sequences (CYP1A, glutathione S-transferase, and XRE). Carbaryl and to a lesser extent thiabendazole also activated other stress genes such as c-fos and NF-kappaBRE, HSP70 and GRP78, and GADD153 at a transcriptional level. These data suggest that these molecules induce early alert genes, including those known to be sensitive to oxidative stress. This led us to examine the genotoxic effect of carbaryl and thiabendazole by an in vitro DNA repair solid-phase assay. Both compounds provoked a strong DNA-damaging activity in the human lymphoblastoid cell line that constitutively expresses human CYP1A1 cDNA, but not in the parental line, indicating that CYP1A1 is chiefly implicated in carbaryl and thiabendazole genotoxicity. This effect was confirmed on HepG2 cells. These observations support the notion that intracellular signals leading to CYP1A1 induction, oxidative stress, and genotoxicity are intimately related.

Carbaryl↗

PubMed2001

Interaction of the glutathione S-transferase genes and cigarette smoking on risk of lower extremity arterial disease: the Atherosclerosis Risk in Communities (ARIC) study.

Glutathione S-transferases M1 or T1 (GSTM1/GSTT1) affect the body's ability either to detoxify or to activate chemicals in cigarette smoke. Cigarette smoking increases the risk of lower extremity arterial disease (LEAD). We conducted a cross-sectional study to evaluate a hypothesized interaction of the genetic polymorphisms of GSTM1 and T1 with cigarette smoking in the risk of LEAD in the ARIC study. A stratified-random sample, including 212 LEAD cases (ankle-brachial index <0.9 in men or <0.85 in women) and 1277 non-cases, was selected from the ARIC cohort of 12041 middle-aged participants free of CHD, transient ischemic attack and stroke at baseline (1987-1989). Overall, the differences in the frequencies of GSTM1-0 and GSTT1-0 (the homozygous deletion genotype) were not statistically significant between cases and non-cases (44 vs. 41% and 28 vs. 18%). However, smoking was more prevalent among LEAD cases than non-cases. The results suggest that the non-deletion genotype GSTM1-1 interacts with smoking to increase the risk of LEAD, but this interaction was not statistically significant. The functional genotype GSTT1-1 was significantly associated with increased risk of LEAD given smoking after adjustment for other risk factors. In individuals with GSTT1-1, the odds ratios (ORs) (95% confidence intervals) of LEAD were 3.6 (1.4, 9.0) for current smoking and 5.0 (1.9, 13.0) for 20+ pack-years. However, in those with GSTT1-0, the ORs were 0.8 (0.2, 2.8) for current smoking and 0.6 (0.1, 2.1) for 20+ pack-years. The interaction was significant (P<0.05) on the additive scale for current smoking and on both the additive and multiplicative scales for 20+ pack-years. Among non-smokers, GSTT1-1 was not associated with LEAD. The results suggest that the GSTT1-1 polymorphism may be a susceptibility factor modifying the risk of LEAD associated with cigarette smoking.

Arteriosclerosis↗

PubMed2001

Lanthanide ions induce hydrolysis of hemoglobin-bound 2,3-diphosphoglycerate (2,3-DPG), conformational changes of globin and bidirectional changes of 2,3-DPG-hemoglobin's oxygen affinity.

The changes in structure and function of 2,3-diphosphoglycerate-hemoglobin (2,3-DPG-Hb) induced by Ln(3+) binding were studied by spectroscopic methods. The binding of lanthanide cations to 2,3-DPG is prior to that to Hb. Ln(3+) binding causes the hydrolysis of either one from the two phosphomonoester bonds in 2,3-DPG non-specifically. The results using the ultrafiltration method indicate that Ln(3+) binding sites for Hb can be classified into three categories: i.e. positive cooperative sites (N(I)), non-cooperative strong sites (N(S)) and non-cooperative weak sites (N(W)) with binding constants in decreasing order: K(I)>K(S)>K(W). The total number of binding sites amounts to about 65 per Hb tetramer. Information on reaction kinetics was obtained from the change of intrinsic fluorescence in Hb monitored by stopped-flow fluorometry. Fluctuation of fluorescence dependent on Ln(3+) concentration and temperature was observed and can be attributed to the successive conformational changes induced by Ln(3+) binding. The results also reveal the bidirectional changes of the oxygen affinity of Hb in the dependence on Ln(3+) concentration. At the range of [Ln(3+)]/[Hb]<2, the marked increase of oxygen affinity (P(50) decrease) with the Ln(3+) concentration can be attributed to the hydrolysis of 2,3-DPG, while the slight rebound of oxygen affinity in higher Ln(3+) concentration can be interpreted by the transition to the T-state of the Hb tetramer induced by Ln(3+) binding. This was indicated by the changes in secondary structure characterized by the decrease of alpha-helix content.

2,3-Diphosphoglycerate↗

PubMed2001

The expression and potential function of cellular prion protein in human lymphocytes.

We examined expression of the normal cellular prion protein (PrP(C)) in human peripheral blood mononuclear cells (PBMC) and in transfected neuroblastoma cells with a panel of six monoclonal antibodies (Mabs). While all six of the Mabs reacted strongly with the neuroblastoma cells, only four of the Mabs reacted with PrP(C) expressed by human PBMC. PrP(C) is expressed at high levels in human T cells, B cells, monocytes, and dendritic cells, but not in red blood cells. Immunoblotting studies revealed that the PrP(C) glycoforms and the composition of the N-linked glycans on PrP(C) in human PBMC are different from those of the brain or the neuroblastoma cells. In human PBMC and the neuroblastoma cell lines the N-terminal portion of the PrP(C) is hypersensitive to proteolytic digestion, suggesting that the N-terminus of the PrP(C) on the surface of a living cell lacks secondary structure. We found that the level of PrP(C) expressed on the surface of human T lymphocytes was up-regulated as a consequence of cellular activation. Accordingly, memory T cells express more PrP(C) than naïve T cells. In addition, the proliferation of human T lymphocytes stimulated with an anti-CD3 Mab was inhibited by anti-PrP(C) Mabs. Collectively, these results suggest that PrP(C) can participate in signal transduction in human T lymphocytes.

Antibodies, Monoclonal↗

PubMed2001

Basigin (murine EMMPRIN) stimulates matrix metalloproteinase production by fibroblasts.

Analysis of basigin-null mice has shown that basigin is involved in several important physiological processes including reproductive, immune, and neural activities (Igakura et al., 1998, Dev Biol 194:152-165). However, its molecular mechanism of action in these processes has not yet been established. Our objective here is to determine whether basigin has functional properties similar to its apparent human tumor cell homolog, EMMPRIN, i.e., the ability to stimulate matrix metalloproteinase (MMP) production in fibroblasts (Guo et al. 1997, J Biol Chem 272:24-27). Mouse cells express two major forms of basigin that differ in their degree of glycosylation (molecular weights: 45 and 58 kDa) but, in similar fashion to human EMMPRIN, mouse tumor cells express higher levels of basigin than normal cells. We have used three different methods to show that basigin stimulates MMP expression in fibroblasts. First, recombinant basigin was partially purified from transfected CHO cells by affinity chromatography. This basigin preparation stimulates production of MMPs on addition to fibroblasts in culture. Second, co-culture of basigin-transfected CHO cells with fibroblasts gives rise to increased expression of MMPs as compared to control co-cultures. Third, we employed a novel approach in which a recombinant basigin adenovirus was constructed and used to infect the target fibroblasts, so that mutual stimulation between neighboring fibroblasts would be expected to result. In this method also, basigin stimulates production of MMPs. Finally, we showed that addition of basigin or EMMPRIN antibody, respectively, to recombinant basigin or EMMPRIN adenovirus-infected cells augments stimulation of MMP synthesis, implying that cross-linking of basigin/EMMPRIN in the membrane enhances activity. We conclude that murine basigin and human EMMPRIN have similar MMP-inducing activities and are functional homologs.

3T3 Cells↗

PubMed2001

Hexamethonium-type allosteric modulators of the muscarinic receptors bearing lateral dibenzazepine moieties.

Alkane-bisammonium compounds carrying lateral phthalimido substituents are known to have a high affinity for the allosteric binding site of the acetylcholine M2 receptor. The purpose of this study was to replace the lateral phthalimido moieties with rigid tricyclic skeletons of a large volume in order to learn more about the function of the lateral heterocycles. In addition, methyl groups were introduced into the lateral connecting chains. Allosteric inhibition of the dissociation of [3H]N-methylscopolamine from the M2 receptors in porcine cardiac homogenates served to indicate binding of the test compounds to the allosteric site. The phthalimido groups could be replaced with dibenzazepine moieties without any loss in potency. Interestingly, the additional methyl group in the lateral spacer seems to have a significant influence on the allosteric behaviour.

Allosteric Regulation↗

PubMed2001

Absence of protease-resistant prion protein in the cerebrospinal fluid of Creutzfeldt-Jakob disease.

Creutzfeldt-Jakob disease (CJD), believed to be caused by a protease-resistant isoform of prion protein (PrP(Sc)), usually manifests itself as a clinically distinctive age-related dementia because of its rapid progression, occasionally accompanied by cerebellar ataxia. Recently, a variant CJD (vCJD) has been described, which has prominent early psychiatric symptoms and an earlier age of death. Although cerebrospinal fluid (CSF) is part of the extracellular fluid of the central nervous system (CNS), the bulk of its proteins are derived from the plasma and there is increasing concern about possible transmission of prion disease by blood. As investigation of CSF has played a significant role in the diagnosis and management of several neurological diseases, it was decided to characterize PrP present in the CSF of CJD individuals. Significant variation was observed in the level of PrP in the CSF from both non-CJD and CJD (including vCJD) patients, and the detected PrP forms are protease-sensitive. Using a conformation-dependent immunoassay, it was further demonstrated that the PrP detected in the CSF from CJD patients was broadly similar in conformation to that found in non-CJD patients. Taken together, the results of this study fail to demonstrate any correlation between the presence of protease-resistant PrP isoform (PrP(Sc)) in the CSF and disease manifestation.

Creutzfeldt-Jakob Syndrome↗

PubMed2001

Induction of HO-1 and NOS in doppel-expressing mice devoid of PrP: implications for doppel function.

Ectopic expression of the doppel (Dpl) protein, a homologue of the prion protein (PrP), was recently associated with cerebellar Purkinje cell degeneration observed in two aging prion protein knock-out (Prnp(0/0)) mouse lines. We investigated the possible role of Dpl in oxidative metabolism. Two Prnp(0/0) mouse lines of similar genetic background were studied. One line expresses Dpl in the brain and displays Dpl-associated cerebellar abnormalities. The other has no elevated expression of Dpl and no cerebellar abnormalities. We observed a correlation between Dpl expression and the induction of both heme oxygenase 1 (HO-1) and nitric oxide synthase systems (nNOS and iNOS). These responses are suggestive of increased oxidative stress in the brains of the Dpl-expressing Prnp(0/0) mice. No induction was observed with Hsp-60, indicating a specific response by the HO/NOS system. We proposed that Dpl expression exacerbates oxidative damage that is antagonistic to the protective function of wild-type PrP.

Animals↗