Biomedical subjects

L Li

Publications and source records attributed to L Li.

At least 1,225 records · Page 68Linked to original sources

PubMed1997

Fludarabine triphosphate inhibits nucleotide excision repair of cisplatin-induced DNA adducts in vitro.

Fludarabine (9-beta-arabinofuranosyl-2-fluoroadenine-5'-monophosphate) is clinically active against chronic lymphocytic leukemia and low-grade lymphomas. We reported previously that fludarabine nucleoside synergistically enhanced cisplatin (CDDP)-induced cytotoxicity in vitro, and that the synergism was concomitant with inhibition of removal of cellular CDDP-induced DNA interstrand cross-links, which are presumably repaired by homologous recombinational repair. To extend our work, we investigated whether fludarabine inhibits nucleotide excision repair (NER) of CDDP-induced DNA intrastrand adducts. The effect of fludarabine on NER was determined using a cell-free system in which a plasmid containing the DNA adducts served as the substrate for repair enzymes in whole-cell extracts from repair-competent cells. To prevent the cell-bound high mobility group box-containing proteins from interfering with repair, cell extracts were depleted with high mobility group box proteins by immunoprecipitation prior to the assay. Repair synthesis, measured by the incorporation of [(32)P]dATP or [(32)P]dCTP, was inhibited by 50% at 26 or 43 microM fludarabine triphosphate, respectively; the effect was dose dependent and may have resulted from the termination of repair-patch elongation. These results were consistent with those from pulse-chase experiments demonstrating the conversion of nicked circular plasmid to the closed circular form by cell extracts filling the repair gaps. When proliferating cell nuclear antigen-depleted cell extracts were used and aphidicolin was added in the repair assay to arrest NER at the incision/excision stage, 100 microM fludarabine triphosphate inhibited about 55% of the conversion of nicked plasmids from the closed circular damaged plasmid substrate; the inhibition was dose dependent. We conclude that fludarabine triphosphate inhibited NER at the steps of incision and repair synthesis. These results suggest that fludarabine may serve as a potential repair modulator to improve the antitumor efficacies of combination regimens containing agents that induce NER.

Adenosine Monophosphate↗

PubMed1997

Influence of changing travel patterns on child death rates from injury: trend analysis.

OBJECTIVES: To examine trends in child mortality from unintentional injury between 1985 and 1992 and to find how changes in modes of travel contributed to these trends. DESIGN: Poisson regression modelling using data from death certificates, censuses, and national travel surveys. SETTING: England and Wales. SUBJECTS: Resident children aged 0-14. MAIN OUTCOME MEASURES: Deaths from unintentional injury and poisoning. RESULTS: Child deaths from injury declined by 34% (95% confidence interval 28% to 40%) per 100,000 population between 1985 and 1992. Substantial decreases in each of the leading causes of death from injury contributed to this overall decline. On average, children walked and cycled less distance and travelled substantially more miles by car in 1992 compared with 1985. Deaths from road traffic accidents declined for pedestrians by 24% per mile walked and for cyclists by 20% per mile cycled, substantially less than the declines per 100,000 population of 37% and 38% respectively. In contrast, deaths of occupants of motor vehicles declined by 42% per mile travelled by car compared with a 21% decline per 100,000 population. CONCLUSIONS: If trends in child mortality from injury continue the government's target to reduce the rate by 33% by the year 2005 will be achieved. A substantial proportion of the decline in pedestrian traffic and pedal cycling deaths, however, seems to have been achieved at the expense of children's walking and cycling activities. Changes in travel patterns may exact a considerable price in terms of future health problems.

Accidents, Traffic↗

PubMed1997

Protein glycosylation in a heat-resistant rat fibroblast cell model expressing human HSP70.

Thermotolerance and heat resistance are frequently associated with elevated levels of heat shock proteins (HSPs). Elevated heat resistance is also found to be associated with the overexpression of high levels of HSP70, as seen in M21 cells, derived from the Rat-1 line. In the present study, we report that M21 cells also feature an increase in general protein glycosylation and specific expression of the stress glycoprotein, GP62, both of which correlate with cellular heat resistance. The expression of GP50, a major stress glycoprotein in cell lines such as CHO, however, did not correlate with cellular heat resistance in M21 cells. Protein glycosylation that occurs during acute heat stress ("prompt" glycosylation) was associated with the glycosylation of a major "prompt" stress glycoprotein, P-SG64 (M(r) of 64,000), that was identified by immunoblotting as a glycosylated form of calreticulin. The higher level of protein glycosylation in M21 cells correlated well with increased D-[2-3H]mannose incorporation into precursor pools of dolichyl phosphomannose and dolichyl pyrophosphoryl oligosaccharides and into glycoproteins. Thus, heat resistance in M21 cells is associated not only with expression of high levels of HSP70, but also with a concomitant increase in protein glycosylation. These data support the hypothesis that stress-induced protein glycosylation is a component of cellular stress response, either in association with HSPs or as an independent mechanism.

Animals↗

PubMed1997

Refinement of the NMR solution structure of the gamma-carboxyglutamic acid domain of coagulation factor IX using molecular dynamics simulation with initial Ca2+ positions determined by a genetic algorithm.

A genetic algorithm (GA) successfully identified the calcium positions in the crystal structure of bovine prothrombin fragment 1 bound with calcium ions (bf1/Ca). The same protocol was then used to determine the calcium positions in a closely related fragment, the Gla domain of coagulation factor IX, the structure of which had previously been determined by NMR spectroscopy in the presence of calcium ions. The most frequently occurring low-energy structure found by GA was used as the starting structure for a molecular dynamics refinement. The molecular dynamics simulation was performed using explicit water and the Particle-Mesh Ewald method to accommodate the long-range electrostatic forces. While the overall conformation of the NMR structure was preserved, significant refinement is apparent when comparing the simulation average structure with its NMR precursor in terms of the N-terminal (Tyr1-N) network, the total number of hydrogen bonds, the calcium ion coordinations, and the compactness of the structure. It is likely that the placement of calcium ions in the protein is critical for refinement. The calcium ions apparently induce structural changes during the course of the simulation that result in a more compact structure.

1-Carboxyglutamic Acid↗

PubMed1997

Cyclic GMP causes Ca2+ desensitization in vascular smooth muscle by activating the myosin light chain phosphatase.

Using permeabilized, arterial smooth muscle strips where membrane-associated pathways remain intact but intracellular Ca2+ stores are depleted, we investigated mechanism(s) for the Ca2+ desensitization of contractile force by cGMP. The nonhydrolyzable analog 8-bromo-cGMP, when applied to these strips with submaximal Ca2+ levels clamped, dramatically and reversibly reduced the steady state levels of phosphorylation at 20-kDa myosin light chain and contractile force, with a nanomolar concentration required to obtain 50% reduction. Supramaximal concentrations of 8-bromo-cGMP (10 microM), however, did not change the steady state relationship between phosphorylation and force. When light chain phosphatase activity was blocked at pCa 6.7, 10 microM 8-bromo-cGMP did not affect the rates of rise of light chain phosphorylation and contractile force. When light chain kinase activity was blocked, 10 microM 8-bromo-cGMP significantly accelerated light chain dephosphorylation and force relaxation from the maximal contraction steady state. The light chain phosphorylation time course of a pCa 6. 0-induced contraction in the presence of 8-bromo-cGMP exhibited kinetics that are predictable from a mathematical model in which only light chain phosphatase activity is increased. The results of this study strongly suggest that cGMP indirectly activates light chain phosphatase, the first proposed mechanism for cGMP-induced Ca2+ desensitization in vasodilatation.

Animals↗

PubMed1997

The analgesic domain of IL-2.

Human recombinant interleukin-2 (IL-2) has been showed to exert an analgesic effect and some experiments suggested that opioid receptors were involved. Because the aromatic residues at the N-termini of opioid peptides are crucial for their binding to the opioid receptors, all nine aromatic residues in IL-2 were mutated by site-directed mutagenesis and both the analgesic activity and the immune activity of the wild-type and mutated IL-2 were tested. The result indicated that there exists a putative analgesic function domain in IL-2, in which Phe44, Tyr45, Tyrl07, and Phell7 were essential. These four residues are located close together at the tertiary structure level of IL-2.

Analgesics↗

PubMed1997

Visualization of antigen-specific T cell activation and cytokine expression in vivo.

A transgenic TCR adoptive transfer system was used to visualize Ag-specific T cell activation and cytokine expression in vivo. After s.c. injection of peptide in adjuvant the entire Ag-specific population up-regulated IL-2 receptor alpha-chain expression, underwent blast transformation, and developed a memory-surface phenotype. A minority of the Ag-specific T cells produced predominantly IL-2 mRNA and localized at the T cell/B cell junction in draining lymph nodes. In the secondary response, a mixed cytokine pattern of both Th1 and Th2 cytokines was demonstrated. When peptide was administered i.v. without adjuvant, 50% of the Ag-specific cells expressed IL-2, but the peak of expression occurred before IL-2 receptor alpha-chain up-regulation, and only a minority of the Ag-specific T cells underwent blast transformation.

Adoptive Transfer↗

PubMed1997

The TSG101 tumor susceptibility gene is located in chromosome 11 band p15 and is mutated in human breast cancer.

Recent work has identified a mouse gene (tsg101) whose inactivation in fibroblasts results in cellular transformation and the ability to produce metastatic tumors in nude mice. Here, we report that the human homolog, TSG101, which we isolated and mapped to chromosome 11, bands 15.1-15.2, a region proposed to contain tumor suppressor gene(s), is mutated at high frequency in human breast cancer. In 7 of 15 uncultured primary human breast carcinomas, intragenic deletions were shown in TSG101 genomic DNA and transcripts by gel and sequence analysis, and mutations affecting two TSG101 alleles were identified in four of these cancers. No TSG101 defects were found in matched normal breast tissue from the breast cancer patients. These findings strongly implicate TSG101 mutations in human breast cancer.

Amino Acid Sequence↗

PubMed1997

Synthesis and luminescence spectral characterization of long-lifetime lipid metal-ligand probes.

We synthesized phospholipid analogues of phosphatidyl ethanolamine which contains a ruthenium metal-ligand complex (MLC) covalently bound to the amino group. Two analogues were synthesized, containing either one (Ru-PE) or two (Ru-PE2) lipid molecules covalently linked to the MLC by the amino group of the lipid. These MLC-lipid probes display intensity decay times from 682 to 357 ns, depending on temperature. Importantly, the luminescence MLC groups display polarized emission, enabling their use for studies of membrane dynamics. The long intensity decay times allowed measurement of the overall rotation correlation time of lipid vesicles to several microseconds. The spectral properties of the model membranes containing Ru-PE or Ru-PE2 were independent of the probe-to-lipid molar ratio from 1:20 to 1:100, suggesting minimal tendency for probe-probe interactions. These MLC-lipid probes can be expected to have numerous applications in studies of membrane dynamics on the microsecond timescale.

2,2'-Dipyridyl↗

PubMed1997

Impaired nonrestricted cytolytic activity in systemic lupus erythematosus: involvement of a pathway independent of Fas, tumor necrosis factor, and extracellular ATP that is associated with little detectable perforin.

OBJECTIVE: To determine the cytolytic effector pathway involved in impaired generation of nonrestricted cytolytic activity in systemic lupus erythematosus (SLE). METHODS: Peripheral blood mononuclear cells (PBMC) from normal subjects and SLE patients were stimulated in vitro with anti-CD3 monoclonal antibody (MAb) and interleukin-2 to promote the generation of nonrestricted cytolytic activity. On day 13 of culture, the PBMC were assayed for cytolytic activity against Fas-Daudi cells and Fas+ Jurkat cells. The effects on cytotoxicity of calcium chelation, antagonist anti-Fas MAb, tumor necrosis factor (TNF) alpha and beta, and ATP were measured. Intracellular perforin expression was determined by intracellular staining, and perforin messenger RNA levels were determined by quantitative competitive reverse transcription-polymerase chain reaction. RESULTS: We demonstrated the existence of a cytolytic pathway that is independent of Fas, TNF alpha, TNF beta, and ATP, but is dependent upon extracellular calcium. Despite its calcium dependence, this pathway is associated with low-to-undetectable levels of perforin. CONCLUSION: Impaired generation of nonrestricted cytolytic activity in SLE is likely due to decreased activity of this Fas-, TNF alpha-, TNF beta-, ATP-independent pathway associated with very low levels of perforin.

Adenosine Triphosphate↗

PubMed1997

Differentiation and functions of T cell subsets.

The Tc1 and Tc2 subsets of CD8+ T effector cells secrete different patterns of cytokines, but have similar functions, including perforin- and Fas-dependent cytotoxicity, and induction of delayed type hypersensitivity (DTH) reactions involving oedema and granulocytic infiltration. The characteristic cytokines of Tc1 (gamma-interferon) and Tc2 (interleukins 4 and 5) are expressed in vivo during the DTH reaction. Tc1 cells that are deficient in cytokine synthesis also induce similar levels of DTH, supporting the lack of correlation between CD8+ T cell cytokine patterns and DTH. CD8+ T cells often produce lower cytokine levels than CD4 cells because the CD8 cells kill their antigen-presenting cells before full stimulation can occur. This effect can be counteracted by increasing the frequency of stimulation, or using perforin-deficient T cells. A multiparameter analysis of cytokine effects on CD8+ T cell differentiation has been initiated, on the basis of the principle that normal immune responses involve complex cytokine mixtures. All combinations of seven cytokines were tested. In some combinations, the combined effect could not have been predicted from individual cytokine functions. Conditions were identified in which each of interleukins 4, 10 and 12 could have opposite effects on CD8+ T cell differentiation.

Animals↗

PubMed1997

Infection of gastrointestinal tract macrophages by HIV-1.

As the largest lymphoid organ and the largest reservoir of macrophages in the body, the gastrointestinal tract mucosa is probably the largest organ reservoir of macrophages infected with HIV-1. To elucidate the biology of HIV-1 infection of intestinal macrophages, we isolated lamina propria macrophages from normal human jejunum by neutral protease digestion, purified the cells by counterflow centrifugal elutriation, and then infected the cells with HIV-1. The lamina propria macrophages were permissive to macrophagetropic isolates of HIV-1 and substantially less permissive to lymphocyte-tropic isolates. Compared with blood monocytes, mucosal macrophages produced 2-3 logs less p24 antigen at peak infection. The reduced level of infection was not due to impaired macrophage viability, reduced CD4 expression, or the isolation procedure. These results confirm that macrophages isolated from the gastrointestinal tract mucosa can support HIV-1 production, albeit at a lower level than blood monocytes. The reduced level of virus production may reflect the unique biology of intestinal lamina propria macrophages.

Antigens, CD↗

PubMed1997

Low concentration of inducer favors production of active form of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase in Escherichia coli.

Expression of chicken and rat liver bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, in Escherichia coli encountered two common problems: the chicken enzyme was liable to proteolysis and the rat enzyme was prone to form inclusion bodies. Reducing the rate of protein synthesis by lowering either growth temperature or isopropyl-beta-D-thiogalactopyranoside (IPTG) concentration alleviated these two problems. Growth at 22 degrees C was optimum for expression of both enzymes. The optimum range of IPTG concentration for expression was 0.1-1 microM for the chicken liver bifunctional enzyme and 10 microM for rat liver enzyme. The components of growth medium also influenced the production. Compared with Luria-Bertani medium, an enriched medium-tryptone-phosphate medium-tripled the production of the active enzymes. Addition of glucose (0.2%) doubled the expression level of active chicken liver enzyme, but reduced the production of active rat liver enzyme to half the maximal level, while the phosphate in tryptone-phosphate medium had no effect on the production of the two enzymes.

Animals↗

PubMed1997

Functions of CD8 T-cell subsets secreting different cytokine patterns.

CD8+ T cells can differentiate into two effector phenotypes, Tc1 and Tc2, secreting different cytokine patterns. Both subsets are cytotoxic via the perforin and Fas pathways, and both kill resting and activated B cells, ruling out the possibility of cognate help, although Tc2 cells may provide bystander help. Both subsets induce inflammation with similar cellular infiltrates. Tc1 cytokine synthesis is limited by two mechanisms--IL-4 induces a permanent deficiency in cytokine secretion, and rapid killing of target cells limits CD8+ T-cell activation and cytokine production. These multiple CD8 T-cell activities provide a versatile set of immune functions.

Animals↗

PubMed1997

Acrolein-induced cell death in human alveolar macrophages.

Acrolein is an environmental air pollutant that is known to suppress respiratory host defense against infections. The mechanism of the decrease in host defense is not yet clear. In this study, the effects of acrolein on human alveolar macrophages and their function were examined. Acrolein caused dose-dependent cytotoxicity to alveolar macrophages as demonstrated by the induction of apoptosis and necrosis. In addition, at lower doses, acrolein caused induction of heme oxygenase 1 protein; however, stress protein 72 (SP72) was not induced. These findings demonstrated that acrolein caused a dose-dependent selective induction of a stress response, apoptosis, and necrosis in human alveolar macrophages. Macrophage function was assessed by release of cytokines in response to endotoxin stimulation. Acrolein caused a dose-dependent inhibition of release of IL-1beta, TNF-alpha, and IL-12. The inhibition of cytokine release and cytotoxicity to alveolar macrophages may in part be responsible for acrolein-induced immunosuppression of the lung.

Acrolein↗

PubMed1997

The effectiveness of active specific immunotherapy using interferon-gamma-gene-transduced tumor cells in a murine tumor model.

Active specific immunotherapy was examined in BALB/c mice using sonicated tumor extract(SE) from plasmacytoma MOPC104E or interferon-gamma-(IFN-gamma)-gene-transduced MOPC104E (Mu gamma), employing interleukin-1 (IL-1) as an adjuvant. Subcutaneous (s.c.) MOPC104E tumor growth was significantly suppressed in mice given a single preimmunization of IL-1 plus Mu gamma-SE, 9 days prior to inoculation, whereas the tumor growth in mice similarly pretreated with IL-1 alone or IL-1 plus MOPC104E-SE(MOPC-SE) was not affected; the mean tumor diameters on day 21 being 6.8 mm, 15.3 mm, and 13.2 mm, respectively. Two-dose preimmunization with Mu gamma-SE alone or IL-1 alone given 10 and 7 days prior to s.c. inoculation also resulted in profound suppression of tumor growth compared to the control. As postsurgical immunization, MOPC104E cells were injected into the foot pads of mice, followed by amputation of the tumor-bearing foot 20 days later, then treatment with IL-1 plus MOPC-SE or IL-1 plus Mu gamma-SE on days 4, 7, and 10 after the amputation. The mean survival of the mice treated with IL-1 plus Mu gamma-SE was significantly prolonged compared to that of the mice treated with IL-1 plus MOPC-SE, at 90.3 days vs 40.9 days, respectively (P < 0.05 by the Cox-Mantel test). These results suggest that SE prepared from IFN-gamma-gene-transduced MOPC104E is more effective for active specific immunotherapy than SE prepared from MOPC104E.

Animals↗

PubMed1997

A human homolog of the 150 kD avian osteoclast membrane antigen related to superoxide dismutase and essential for bone resorption is induced by developmental agents and opposed by estrogen in FLG 29.1 cells.

Osteoclast development from hematopoietic bone marrow precursors is associated with the expression of various enzymes, receptors, adhesion molecules, and other specialized components. Among these is a novel 150 kD superoxide dismutase-related membrane glycoprotein, originally identified by its reaction with the anti-osteoclast monoclonal antibody 121F. This antigen is uniquely restricted to osteoclasts in bone, universally present on osteoclasts from multiple species, induced during osteoclast differentiation in vitro and in ovo, and required at high levels for avian osteoclastic bone pit resorption. Expression of a comparable human antigen was investigated using human leukemic FLG 29.1 cells capable of differentiating towards an osteoclast-like phenotype. Phorbol ester, 1,25 (OH)2 vitamin D3, and osteoblast-derived soluble factors elicited dose and time-dependent inductions of this antigen as measured by enzyme-linked immunosorbent assay (ELISA) and immunocytochemical staining, coincident with their display of multiple other osteoclastic features. Synergistic interactions of these modulators led to further elevations in the ultimate expression levels of this antigen, although not to the full extent associated with in vivo-formed avian osteoclasts. The potent antiresorptive hormone 17beta-estradiol, but not its inactive alpha isomer, partially suppressed the phorbol ester-induced elevation of the 121F antibody-reactive antigen in FLG 29.1 cells as it does in avian osteoclast-like cells. Characterization of the human antigen isolated from FLG 29.1 cells by 121F immunoaffinity purification demonstrated that this regulated membrane component was synthesized by these human cells, more abundant following their differentiation into osteoclast-like cells, and similar biochemically and immunologically to the 150 kD integral membrane glycoprotein previously described from avian osteoclasts. Therefore, this report is the first documentation that human osteoclast-like FLG 29.1 cells express, in a developmentally regulated fashion, a homolog of the specific 150 kD avian osteoclast surface antigen that is related to superoxide dismutase, a protective free radical scavenging enzyme and is essential for osteoclastic bone resorption.

Acid Phosphatase↗

PubMed1997

A medium-density genetic linkage map of the bovine genome.

A cattle genetic linkage map was constructed which covers more than 95 percent of the bovine genome at medium density. Seven hundred and forty six DNA polymorphisms were genotyped in cattle families which comprise 347 individuals in full sibling pedigrees. Seven hundred and three of the loci are linked to at least one other locus. All linkage groups are assigned to chromosomes, and all are orientated with regards to the centromere. There is little overall difference in the lengths of the bull and cow linkage maps although there are individual differences between maps of chromosomes. One hundred and sixty polymorphisms are in or near genes, and the resultant genome-wide comparative analyses indicate that while there is greater conservation of synteny between cattle and humans compared with mice, the conservation of gene order between cattle and humans is much less than would be expected from the conservation of synteny. This map provides a basis for high-resolution mapping of the bovine genome with physical resources such as Yeast and Bacterial Artificial Chromosomes as well as providing the underpinning for the interpolation of information from the Human Genome Project.

Animals↗