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Biomedical subjects

W Li

Publications and source records attributed to W Li.

At least 541 records · Page 30Linked to original sources
PubMed1999

Transduction of primitive human marrow and cord blood-derived hematopoietic progenitor cells with adeno-associated virus vectors.

We evaluated the capacity of adeno-associated virus (AAV) vectors to transduce primitive human myeloid progenitor cells derived from marrow and cord blood in long-term cultures and long-term culture-initiating cell (LTC-IC) assays. Single-colony analyses showed that AAV vectors transduced CD34(+) and CD34(+)38(-) clonogenic cells in long-term culture. Gene transfer was readily observed in LTC-ICs derived from 5-, 8-, and 10-week cultures. Recombinant AAV (rAAV) transduction was observed in every donor analyzed, although a wide range of gene transfer frequencies (5% to 100%) was noted. AAV transduction of LTC-ICs was stable, with week-8 and -10 LTC-ICs showing comparable or better transduction relative to week-5 LTC-ICs. Fluorescence in situ hybridization (FISH) analyses performed to determine the fate of AAV vectors in transduced cells showed that 9% to 28% of CD34(+) and CD34(+)38(-) cells showed stable vector integration as evidenced by chromosome-associated signals in metaphase spreads. Comparisons of interphase and metaphase FISH suggested that a fraction of cells also contained episomal vector at early time points after transduction. Despite the apparent loss of the episomal forms with continued culture, the number of metaphases containing integrated vector genomes remained stable long term. Transgene transcription and placental alkaline phosphatase (PLAP) expression was observed in CD34(+), CD34(+)38(-) LTC-ICs in the absence of selective pressure. These results suggest that primitive myeloid progenitors are amenable to genetic modification with AAV vectors.

Alkaline Phosphatase↗
PubMed1999

Functional interaction between retinoblastoma protein and stress-activated protein kinase in multiple myeloma cells.

Previous studies have demonstrated that gamma-irradiation (IR)-induced apoptosis in multiple myeloma (MM) is associated with activation of stress-activated protein kinase (SAPK). In the present study, we examined the molecules downstream of SAPK/C-Jun N-terminal kinase (JNK), focusing on the role of retinoblastoma protein (Rb) during IR-induced MM cell apoptosis. The results demonstrate that IR activates SAPK/JNK, which associates with Rb both in vivo and in vitro. Far Western blot analysis confirms that SAPK/JNK binds directly to Rb. IR-activated SAPK/JNK phosphorylates Rb, and deletion of the phosphorylation site in the COOH terminus domain of Rb abrogates phosphorylation of Rb by SAPK/JNK. Taken together, our results suggest that Rb is a target protein of SAPK/JNK and that the association of SAPK/JNK and Rb mediates IR-induced apoptosis in MM cells.

Apoptosis↗
PubMed1999

The Big Blue(R) transgenic mouse mutation detection assay: the mutation pattern of sectored mutant plaques.

There are mutational artifacts in the Big Blue(R) assay and it is important to characterize the source and nature of these mutations. Differences were reported in the mutation patterns of a small sample of 23 sectored and 91 circular mutant plaques derived from skin using the Big Blue(R) transgenic mouse mutation detection system [G. R. Stuart, N.J. Gorelick, J.L. Andrews, J.G. de Boer, B.W. Glickman, The genetic analysis of lacI mutations in sectored plaques from Big Blue transgenic mice, Environ. Mol. Mutagen 28 (1996) 385-392.]. We have extended these observations by analyzing 46 sectored and 224 circular mutant plaques derived from seven tissues. The frequency of sectored mutant plaques is estimated to be 16% with no significant variation with tissue type. However, the patterns of mutation for sectored mutants and mouse-derived mutations differed significantly (p=0.04). Base substitutions in sectored mutant plaques do not show the asymmetries found in circular mutants consistent with integration of a GC rich transgene into the AT rich mammalian genome. Sectored mutants have mutation patterns consistent with a mixture of mouse, in vitro and Escherichia coli-derived mutations. Data on the relative frequencies of different mutant plaque morphologies suggests that overlapped plaques are substantially contaminated by sectored plaques at recommended plating densities.

Animals↗
PubMed1999

Two different gene elements are required for glucose regulation of S14 transcription.

Carbohydrate feeding increases the transcriptional activity of the hepatic S14 gene. The region of the S14 promoter between -1384/-1275 contributes to the transcriptional regulation by carbohydrate. A previously identified element (-1303/-1289) within this region is required but is not sufficient for the carbohydrate effect. Therefore, we ligated -1384/-1275 to a heterologous promoter and created mutants in this region to identify other potential responsive sequences. We found that mutation within -1365/-1350 eliminated the response to high glucose (27.5 mM). However, three copies of this element ligated to a mouse mammary tumor virus-luciferase vector did not respond to glucose indicating the -1365/-1350 element is insufficient to confer a glucose response in isolation. Nevertheless. mutating the -1365/-1350 element in the native promoter led to a loss of response to glucose, proving this element is necessary. Electrophoretic mobility shift assays (EMSA) using three copies of the element showed significant binding to rat hepatic nuclear extracts, but no difference between the dietary states. Competition EMSA studies showed that the previously identified element at -1303/-1289 was unable to compete for proteins that bind to the -1365/-1350 element. Therefore, we have demonstrated two separate elements within the -1384/-1275 region of the S14 gene that bind different proteins and interact to elicit the carbohydrate effect.

Animals↗
PubMed1999

Prevention and restoration of second-set liver allograft rejection in presensitized mice: the role of "passenger" leukocytes, donor major histocompatibility complex antigens, and host cytotoxic effector mechanisms.

BACKGROUND: The aim was to determine whether sublethal donor total body irradiation (TBI) might be as effective as lethal TBI in preventing mouse second-set liver allograft rejection, and to evaluate the role of passenger leukocytes, donor major histocompatibility complex (MHC) antigens, and host effector mechanisms in the response to livers from sublethally irradiated donors. METHODS: B10 (H2b) donors received various doses of TBI at different times before their livers were transplanted orthotopically into normal or donor skin-presensitized C3H (H2k) recipients. The influence of irradiation on graft non-parenchymal cells (NPC) was determined by monoclonal antibody staining, and flow cytometric analysis. Hematopoietic cells within the grafts were reconstituted by intravenous infusion of syngeneic or third-party bone marrow cells. Allograft survival was determined in recipients that received no treatment, or that were given spleen cells from either normal B10 donors, or MHC class I - or class II-deficient mice syngeneic with the donors. Cytotoxic activity of graft-infiltrating cells and host spleen cells, and complement-dependent cytotoxic alloantibody titers were determined by isotype release assays. RESULTS: The protective effect of donor TBI was observed both at lethal (9.5 Gy) and sublethal doses (5 and 3 Gy; graft median survival time: >100 days). Extended delay in liver transplantation, allowing hematopoietic recovery and graft reconstitution eliminated the effect. Liver NPC were reduced about 80% within 24 hr of 3 Gy TBI, with a selective reduction in the incidence of B cells. The NPC-depleted livers underwent accelerated rejection when donor (but not third-party) spleen cells (5 x 10(7) were administered systemically to the recipient immediately after graft revascularization. Spleen cells from MHC class I-deficient (but not MHC class II-deficient) mice failed to fully restore accelerated rejection of TBI liver grafts. Freshly isolated graft NPC, or spleen cells from TBI liver recipients, harvested 4 days after transplantation, exhibited lower, donor-specific cytotoxic activity than cells from mice given normal livers. Recipients of TBI livers also showed much lower serum complement-dependent cytotoxic alloantibody titers. CONCLUSIONS: By substantially depleting "passenger leukocytes," sublethal donor TBI undermines anti-donor cell-mediated and humoral immune reactivity and inhibits second-set liver allograft rejection in presensitized recipients. The interval between irradiation and transplantation is important in conferring resistance to rejection. Expression of MHC class I on donor leukocyte infusions is important for overcoming resistance to second-set rejection induced by donor irradiation.

Animals↗
PubMed1999

Gamma interferon induces expression of Mad1 gene in macrophage, which inhibits colony-stimulating factor-1-dependent mitogenesis.

Gamma interferon (IFN-gamma) has long been known as an antiproliferative cytokine. The mechanism of its action, however, remains elusive. Monocytes and macrophages are primary targets of IFN-gamma. To understand the antiproliferative signaling of IFNgamma, we studied the effect of IFNgamma on expression of c-Myc, Mad1, Max, cyclin D1, and cyclin D2 genes in both a macrophage cell line and in primary bone marrow-derived macrophages (BMM) in response to colony-stimulating factor-1 (CSF-1). We found that whereas IFNgamma inhibits CSF-1-stimulated c-Myc gene expression, it induces Mad1 expression. Induction of Mad1 mRNA could be detected as early as 90 min following IFNgamma treatment and was maintained for at least 15 h. These results suggest that IFNgamma treatment could shift the Myc-Max complex to the Mad1-Max complex in cells. The levels of Max, cyclin D1, and cyclin D2, however, remained unchanged. Enforced ectopic expression of Mad1 in the cells results in inhibition of [3H]thymidine incorporation and proliferation in response to CSF-1. This study suggests a mechanism by which IFNgamma inhibits CSF-1-stimulated proliferation of macrophages, i.e., by elevating the Mad1 level in the cells.

Animals↗
PubMed1999

UCP4, a novel brain-specific mitochondrial protein that reduces membrane potential in mammalian cells.

Uncoupling proteins (UCPs) are a family of mitochondrial transporter proteins that have been implicated in thermoregulatory heat production and maintenance of the basal metabolic rate. We have identified and partially characterized a novel member of the human uncoupling protein family, termed uncoupling protein-4 (UCP4). Protein sequence analyses showed that UCP4 is most related to UCP3 and possesses features characteristic of mitochondrial transporter proteins. Unlike other known UCPs, UCP4 transcripts are exclusively expressed in both fetal and adult brain tissues. UCP4 maps to human chromosome 6p11.2-q12. Consistent with its potential role as an uncoupling protein, UCP4 is localized to the mitochondria and its ectopic expression in mammalian cells reduces mitochondrial membrane potential. These findings suggest that UCP4 may be involved in thermoregulatory heat production and metabolism in the brain.

Amino Acid Sequence↗
PubMed1999

Coimmobilization of glucoamylase and glucose isomerase by molecular deposition technique for one-step conversion of dextrin to fructose.

Glucose isomerase was immobilized by itself with adsorption and coimmobilized with glucoamylase by molecular deposition technique using macroporous trimethylamine polystyrene beads. Approximately 77.5% of the enzyme added was immobilized. The pH-activity curve of the immobilized glucose isomerase was broadened, resulting in 75% retention of its maximum activity at pH 6.2. The Km of the immobilized glucose isomerase was 1.28-fold that of the soluble one. When the two enzymes were immobilized together, the system was found capable of functioning at pH 6.0 to produce fructose from starch and dextrin. At this pH, the total fructose output of the coimmobilized enzyme system after 24 h was 1.9 times that of the free enzyme system.

Aldose-Ketose Isomerases↗
PubMed1999

IFN-gamma increases the severity and accelerates the onset of experimental autoimmune uveitis in transgenic rats.

Experimental autoimmune uveitis (EAU) is a predominantly Th1-mediated intraocular inflammatory disease that serves as a model for studying the immunopathogenic mechanisms of uveitis and organ-specific autoimmune diseases. Despite the well-documented role of IFN-gamma in the activation of inflammatory cells that mediate autoimmune pathology, recent studies in IFN-gamma-deficient mice paradoxically show that IFN-gamma confers protection from EAU. Because of the implications of these findings for therapeutic use of IFN-gamma, we sought to reexamine these results in the rat, another species that shares essential immunopathologic features with human uveitis and is the commonly used animal model of uveitis. We generated transgenic rats (TR) with targeted expression of IFN-gamma in the eye and examined whether constitutive ocular expression of IFN-gamma would influence the course of EAU. We show here that the onset of rat EAU is markedly accelerated and is severely exacerbated by IFN-gamma. In both wild-type and TR rats, we found that the disease onset is preceded by induction of ICAM-1 gene expression and is characterized by selective recruitment of T cells expressing a restricted TCR repertoire in the retina. In addition, these events occur 2 days earlier in TR rats. Thus, in contrast to the protective effects of IFN-gamma in mouse EAU, our data clearly show that intraocular secretion of IFN-gamma does not confer protection against EAU in the rat and suggest that IFN-gamma may activate distinct immunomodulatory pathways in mice and rats during uveitis.

Animals↗
PubMed1999

Evidence for a role of IL-17 in organ allograft rejection: IL-17 promotes the functional differentiation of dendritic cell progenitors.

IL-17 is a T cell-derived cytokine that stimulates stromal cells and macrophages to secrete proinflammatory cytokines. We hypothesized that IL-17 might play a role in alloimmune responses, and that interference with its activity might suppress allograft rejection. IL-17R:Fc or control IgG was added at the start of mouse MLR or was administered i.p. (100-500 microg/day) for different durations post-transplant to murine recipients of MHC-mismatched cardiac allografts. IL-17R:Fc (50-200 microg/ml) markedly inhibited T cell proliferation in vitro and significantly prolonged nonvascularized cardiac allograft median survival time from 13 to 20 days (100 microg/day; days 0 and 1) or to 19 days (100-300 microg/day; days 0-4). Survival of vascularized grafts was also extended significantly from 10.5 to 19 days by IL-17R:Fc (500 microg/day; days 0-6). To address a possible mechanism by which IL-17 may promote alloreactivity, we examined the influence of IL-17 on the differentiation and function of bone marrow-derived cells propagated in granulocyte-macrophage CSF with or without IL-4 to promote dendritic cell (DC) growth. A minor proportion of CD11c+ DC expressed the IL-17R. IL-17 promoted the maturation of DC progenitors, as evidenced by increased cell surface expression of CD11c, costimulatory molecules (CD40, CD80, CD86), and MHC class II Ag, and allostimulatory capacity. IL-17 had a lesser effect on the phenotype and function of more fully differentiated myeloid DC. These findings suggest a role for IL-17 in allogeneic T cell proliferation that may be mediated in part via a maturation-inducing effect on DC. IL-17 appears to be a novel target for therapeutic intervention in allograft rejection.

Adjuvants, Immunologic↗
PubMed1999

Protein loops on structurally similar scaffolds: database and conformational analysis.

A general problem in comparative modeling and protein design is the conformational evaluation of loops with a certain sequence in specific environmental protein frameworks. Loops of different sequences and structures on similar scaffolds are common in the Protein Data Bank (PDB). In order to explore both structural and sequential diversity of them, a data base of loops connecting similar secondary structure fragments is constructed by searching the data base of families of structurally similar proteins and PDB. A total of 84 loop families having 2-13 residues are found among the well-determined structures of resolution better than 2.5 A. Eight alpha-alpha, 20 alpha-beta, 19 beta-alpha, and 37 beta-beta families are identified. Every family contains more than 5 loop motifs. In each family, no loops share same sequence and all the frameworks are well superimposed. Forty-three new loop classes are distinguished in the data base. The structural variability of loops in homologous proteins are examined and shown in 44 families. Motif families are characterized with geometric parameters and sequence patterns. The conformations of loops in each family are clustered into subfamilies using average linkage cluster analysis method. Information such as geometric properties, sequence profile, sequential and structural variability in loop, structural alignment parameters, sequence similarities, and clustering results are provided. Correlations between the conformation of loops and loop sequence, motif sequence, and global sequence of PDB chain are examined in order to find how loop structures depend on their sequences and how they are affected by the local and global environment. Strong correlations (R > 0.75) are only found in 24 families. The best R value is 0.98. The data base is available through the Internet.

Amino Acid Sequence↗
PubMed1999

Quantitative assessment of pulmonary perfusion with dynamic contrast-enhanced MRI.

The feasibility of qualitative assessment of pulmonary perfusion using dynamic contrast enhanced MRI with ultra-short TE has recently been demonstrated. In the current study, quantitative analysis was attempted based on the indicator dilution principle using a pig model of pulmonary embolism. The results were compared with the absolute pulmonary perfusion obtained with colored microspheres. The inverse of apparent mean transit time (1/tau(app)), distribution volume (V), and V/tau(app) were correlated well with the absolute lung perfusion. This study demonstrates that MR has the potential to evaluate pulmonary perfusion quantitatively. Magn Reson Med 42:1033-1038, 1999.

Adult↗
PubMed1999

Design of artificial neural network and its applications to the analysis of alcoholism data.

Artificial neural networks were applied to the alcoholism data to reveal nonlinear relationships between intermediate phenotypes, marker identity-by-descent sharing, and the affection status. A variable number of hidden units were considered to achieve a balance between the minimal mean-squared error and over-fitting of the data. The predictability of the affection status based on intermediate phenotype information (event-related potential 300, monoamine oxidase, and gender) was 65% to 75%, and sensitivity/specificity ranged around 50% to 80%. The IBD approach succeeded in identifying the same marker as previous studies, but also found additional peaks.

Alcoholism↗
PubMed1999

Expression of interferon-gamma in the lens exacerbates anterior uveitis and induces retinal degenerative changes in transgenic Lewis rats.

Interferon-gamma (IFN-gamma) is a pleiotropic cytokine that has been implicated in immunopathogenic mechanisms of a number of inflammatory diseases of autoimmune or infectious disease etiology. However, its exact role is still a matter of debate. In experimental mouse models, IFN-gamma has been shown to exacerbate autoimmune thyroiditis, insulin-dependent diabetes mellitus, and autoimmune neuritis while it confers protection against experimental allergic encephalomyelitis and experimental uveitis. In this study, we generated transgenic rats with constitutive expression of IFN-gamma in the eye to study its paracrine effects and to investigate whether local production of IFN-gamma also confers protection against uveitis in the rat species. We show here that chronic exposure of ocular cells to IFN-gamma results in apoptotic death of retinal ganglion cells, development of chronic choroiditis, formation of retinal in-foldings, and activation of proinflammatory genes. In contrast to its protective systemic effect in the mouse, constitutive secretion of IFN-gamma in the rat eye was found to predispose the development of severe anterior uveitis and induction of retinal degenerative processes that impair visual acuity. Our data underscore the danger in extrapolation of cytokine effects in the mouse to humans without corroborating evidence in other species.

Animals↗
PubMed1999

g-matrix based on configuration interaction and Stone's perturbation theory

Stone's formula, which has usually been applied to the calculation of the g-matrix, is based on a single-configuration treatment. Here a limited configuration interaction is included to obtain the expressions of the principal g values for an orbitally nondegenerate molecule with spin S = 12. Copyright 1999 Academic Press.

Journal Article↗
PubMed1999

The g values of some halomolybdenyl, -vanadyl, and -chromyl complexes.

The principal g values of a series of [MOXn]m- (M = VIV, CrV, MoV; X = F-, Cl-, Br-; n = 4, 5; m = 1, 2, 3) complexes are calculated by the INDO-CI-Stone method. The results agree well with the experimental data and reveal that the traditional models have some limitations in calculating g values for some complexes of this type.

Chromates↗