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Biomedical subjects

L L Chen

Publications and source records attributed to L L Chen.

At least 19 recordsLinked to original sources
PubMed1992

Structure/function studies on vascular cell adhesion molecule-1.

Vascular cell adhesion molecule-1 (VCAM1) is a member of the immunoglobulin (Ig) superfamily which interacts with the integrin very late antigen-4 (VLA4). The VCAM1/VLA4 interaction mediates both adhesion and signal transduction and is thought to play an important role in inflammatory and immune responses in vivo. The major form of human VCAM1 contains seven extracellular Ig-like domains, with domain 1 designated as the most N-terminal. We have examined the relationship between human VCAM1 structure and function using a combination of domain truncation mutants and proteolytic fragmentation of recombinant soluble VCAM1. We have characterized two regions of VCAM1, localized to domains 4 and 5, which are highly sensitive to proteolytic cleavage, localized the epitope of the blocking monoclonal antibody 4B9 to domain 1, and found that domains 1-3 are sufficient for both its adhesive function and its ability to initiate T cell activation.

Amino Acid Sequence↗
PubMed1992

Schistosoma mansoni p48 eggshell protein gene: characterization, developmentally regulated expression and comparison to the p14 eggshell protein gene.

Egg production by worm pairs is a major cause of pathogenesis in schistosomiasis. To further the understanding of female reproductive development, we have isolated and characterized a complete copy of an eggshell protein precursor gene, p48. Sequence analysis reveals that the gene has 3 open reading frames and does not contain an intron. One of the open reading frames, ORF1, encodes a polypeptide of 50 kDa which shows strong homology to insect chorion proteins. Determination of the position of the mRNA cap-site facilitated identification of putative regulatory elements in the 5' upstream region of the gene. Some of these elements (e.g., TCACGT) have been shown to play a role in the regulation of chorion gene expression in insects. p48 mRNA is detectable only in mature female worms and the ability to detect the mRNA coincides temporally with worm pairing. Quantitative comparisons, during female reproductive development, of p48 transcripts to those from another eggshell protein precursor gene, p14, show that the p48 mRNA is significantly less abundant than p14 mRNA. In mature female worms, p48 mRNA can only be detected in vitelline cells. Antibodies made against the polypeptide sequence deduced from ORF1 of the p48 gene recognize a 50-kDa molecule in extracts from mature female worms, but not in extracts from immature females or males.

Amino Acid Sequence↗
PubMed1992

Cytokine expression by head and neck squamous cell carcinomas.

Cytokines are known to play an important role in host defense by regulating the function, growth, and differentiation of the cells of the immune system. We hypothesize that, in the tumor microenvironment, tumor cells and resident tissue cells (e.g., fibroblasts) also produce cytokines that may regulate the local immune response to tumors. Initially, homogenates of eight head and neck squamous cell carcinomas (HNSCC) were assayed for the presence of interleukin-1 (IL-1), interleukin-4 (IL-4), interleukin-6 (IL-6), and granulocyte-macrophage colony-stimulating factor (GM-CSF) to establish the presence of these cytokines in the tumors in vivo. We detected IL-1 in all tumor homogenates and IL-4, IL-6, and GM-CSF in some homogenates. To assess the ability of HNSCC to produce these cytokines, supernatants of short-term primary cultures of HNSCC were assayed for the same cytokines. No IL-1 was detected, although baseline levels of IL-4, IL-6, and GM-CSF were present. However, the stimulation of primary tumor cultures with exogenous IL-1 induced or significantly enhanced production of IL-4 (p < 0.01), IL-6 (p < 0.001), and GM-CSF (p < 0.02). These results support our hypothesis that HNSCC secrete cytokines that may influence the response of local immune cells. Our data also suggest that IL-1 may have a central role in regulating the local immune response through the enhancement or induction of cytokine production by tumor and/or resident tissue cells.

Carcinoma, Squamous Cell↗
PubMed1992

Max: functional domains and interaction with c-Myc.

The product of the c-myc proto-oncogene is a DNA-binding protein, the deregulated expression of which is associated with a variety of malignant neoplasms. The cDNA for the max gene was recently cloned as a result of the ability of its protein product to interact with the c-Myc protein. We studied bacterially produced Max, c-Myc, and a series of truncated c-Myc proteins. Full-length c-Myc alone cannot bind DNA. However, a truncated c-Myc protein comprising the basic, helix-loop-helix, and leucine zipper regions can bind specifically to DNA bearing the sequence GGGCAC(G/A)TGCCC. Max protein, either alone or in a heteromeric complex with full-length c-Myc, binds to the same core sequence. Using a novel combination of chemical and photo-cross-linking analysis, we demonstrate that either Max or a c-Myc/Max heteromeric complex binds to DNA virtually exclusively in a dimeric structure. Using fusion proteins in cultured cells, we establish a number of functional characteristics of Max. First, we show that Max can interact with c-Myc intracellularly in a manner dependent on the integrity of the helix-loop-helix and leucine zipper motifs. Second, a nuclear localization domain that contains the sequence PQSRKKLR is mapped to the carboxy-terminal region of Max. Third, Max lacks a transcriptional activation domain that is functional in Chinese hamster ovary cells when fused to a heterologous DNA-binding domain. These data suggest that Max may serve as a cofactor for c-Myc in transcriptional activation or, by itself, as a transcriptional repressor.

Amino Acid Sequence↗
PubMed1992

General anesthetics potentiate gamma-aminobutyric acid actions on gamma-aminobutyric acidA receptors expressed by Xenopus oocytes: lack of involvement of intracellular calcium.

Potentiation of the gamma-aminobutyric acid (GABAA) receptor-gated Cl- channel response has been suggested to be a primary action of some anesthetic agents. We asked whether the GABAA receptor is a target site common for general anesthetics that are chemically and structurally diverse. This hypothesis was tested in Xenopus oocytes expressing mouse cortical mRNA, and GABA-activated Cl- currents were measured using two-electrode voltage clamping. General anesthetics, including inhalational (halothane, diethylether, enflurane and isoflurane), i.v. (3 alpha-hydroxy-5 alpha-dihydroprogesterone, ketamine and propofol) and alcohol (pentanol) anesthetics, enhanced GABA-induced currents by 56 to 1089% at concentrations that were clinically relevant. The results suggest that potentiation of the GABAA receptor/channel response may be a common action for anesthetic agents. Moreover, anesthetic effects were dependent on GABA concentrations; the enhancement was marked with low GABA concentrations and was exponentially decreased as the GABA concentration increased. Also, anesthetic effects were dependent on anesthetic concentrations. The apparent EC50 of halothane was found to be similar to the anesthetic ED50. We also investigated the role of intracellular Ca++ in mediating anesthetic enhancement of the GABA current. We found that intracellular injection of the Ca++ chelator, EGTA, did not change the enhancement by anesthetics. In addition, these anesthetics alone did not produce significant currents, suggesting that the Ca(++)-dependent Cl- current was not activated by these anesthetics per se. Thus, we found that diverse anesthetics potentiate GABA-induced Cl- currents, but this action is not mediated by a release of intracellular Ca++.

20-alpha-Dihydroprogesterone↗
PubMed1991

The increased potency of cross-linked lymphocyte function-associated antigen-3 (LFA-3) multimers is a direct consequence of changes in valency.

We have used chemically cross-linked dimers, trimers, and tetramers of lymphocyte function-associated antigen-3 (LFA-3) to study the role of multivalency in the interaction of the protein with its receptor, CD2. The cross-linked adducts showed enhanced activity in systems where LFA-3 has been shown to (i) block LFA-3/CD2 interactions in a rosetting assay and (ii) provide through the CD2 on peripheral blood lymphocytes a trigger for T-cell proliferation. The level of increase was directly related to the valency state of the multimers. In the rosetting assay, the dimers, trimers, and tetramers, by weight, exhibited 15-, 150-, and 430-fold increases in activity over monomeric LFA-3. In the proliferation assay, the tetramer produced a 6-fold increase in thymidine incorporation at 0.06 micrograms/ml, the trimer was 100 times less active than the tetramer, and the dimer and monomer were inactive. The LFA-3 multimers were generated using a three-step cross-linking chemistry that was targeted at the carbohydrates on LFA-3. With this procedure over 60% of the starting protein was converted into multimers with no effect on function. The cross-linking approach should be applicable to other surface antigens, providing a simple method for analyzing multivalent interactions.

Animals↗
PubMed1991

Production of multimeric forms of CD4 through a sugar-based cross-linking strategy.

We have developed a three-step cross-linking procedure that is specifically targeted at the carbohydrate on a protein and applied it to CD4 as a model system for studying the role of multivalent interactions in function. In the first step CD4 was oxidized with periodate, creating aldehydes that served as targets for the subsequent chemistry. Next the aldehydes were modified with cystamine, converting the reactive group into a thiol. Finally cross-linking through the thiol moiety was generated with the homobifunctional cross-linker bismaleimidohexane. With this procedure, approximately 60% of the CD4 was converted into higher molecular weight complexes that were soluble and retained function as assessed by glycoprotein gp120 binding activity. CD4 dimers and tetramers by mass were 4 and 15 times as active as CD4 monomer in blocking virus infection with HTLV-IIIB in an in vitro cellular assay. The cross-linking chemistry provides an efficient method for producing homomultimers of a glycoprotein.

Amino Acids↗
PubMed1991

Thermal response of rat fibroblasts stably transfected with the human 70-kDa heat shock protein-encoding gene.

The major heat shock protein hsp70 is synthesized by cells of a wide variety of organisms in response to heat shock or other environmental stresses and is assumed to play an important role in protecting cells from thermal stress. We have tested this hypothesis directly by transfecting a constitutively expressed recombinant human hsp70-encoding gene into rat fibroblasts and examining the relationship between the levels of human hsp70 expressed and thermal resistance of the stably transfected rat cells. Successful transfection and expression of the gene for human hsp70 were characterized by RNA hybridization analysis, two-dimensional gel electrophoresis, and immunoblot analysis. When individual cloned cell lines were exposed to 45 degrees C and their thermal survivals were determined by colony-formation assay, we found that the expression of human hsp70 conferred heat resistance to the rat cells. These results reinforce the hypothesis that hsp70 has a protective function against thermal stress.

Animals↗
PubMed1991

The remodeling of synaptic extracellular matrix and its dynamic relationship with nerve terminals at living frog neuromuscular junctions.

The question of whether the synaptic extracellular matrix undergoes remodeling and how this remodeling is related to nerve terminal plasticity was examined in living neuromuscular junctions of adult frogs. Sartorius muscles were double stained with a fluorescent nerve terminal dye 4-(4-diethylamino-styryl)-N-methylpyridinium iodide (4-Di-2-Asp) and rhodamine-tagged peanut agglutinin (PNA) which recognizes synaptic extracellular matrix. Both nerve terminals and synaptic extracellular matrix in 200 identified normal junctions were visualized in vivo two or three times over a period of 2.6-6 months. The majority of neuromuscular junctions (NMJs) showed remodeling of both nerve terminals and synaptic extracellular matrix. Only 2.5% showed no changes in either synaptic element. The most commonly seen remodeling involved correlated changes in both nerve terminals and synaptic extracellular matrix. In this large group, while some junctions (20%) showed overall proportionate changes in all branches, most junctions (68%) showed disproportionate extension and/or retraction of some but not all individual branches. Another group of NMJs (9.5%) showed mismatched changes in the nerve terminal and synaptic extracellular matrix. In this group, some NMJs showed a decrease in the nerve terminal length without a corresponding reduction in synaptic extracellular matrix length. In other junctions that displayed extension of branches, the PNA-stained matrix was longer than the distal tip of the nerve terminal. Morphometric analysis indicated an average increase of 15.6% in total nerve terminal length and 13.6% in total synaptic extracellular matrix length. Although almost all NMJs displayed remodeling in at least one branch, about 50% of the 2201 individual branches examined did not show changes. The average change was 8.9% growth in the length of individual nerve terminal branches and 8.3% growth in the length of individual branches of synaptic extracellular matrix. There was no significant difference in the morphometry between the repeatedly observed junctions and the previously unobserved control junctions. Furthermore, junctions in which the synaptic extracellular matrix was longer than the nerve terminal also were seen in control as well as in experimental muscles. Cases where the nerve terminals were longer than the synaptic extracellular matrix were never observed in newly arising junctional branches. The present study has shown extensive remodeling in not only the nerve terminal but also the synaptic extracellular matrix in adult living frog NMJs. Results suggest that nerve terminals retract before the synaptic extracellular matrix. A hypothesis that extension of synaptic extracellular matrix precedes nerve terminal growth during synaptic remodeling is proposed.

Animals↗
PubMed1991

Identification and characterization of an acyl-CoA:triterpene acyltransferase activity in rabbit and human tissues.

Rabbit and human tissues contain substantial amounts of an unusual lipid, a fatty acid ester of a pentacyclic triterpene, that is a potent in vitro inhibitor of acyl-CoA:cholesterol acyltransferase (ACAT). A possible origin of the triterpene ester is via dietary absorption of plant triterpenes (which have a similar structure to the triterpene moiety of the animal triterpene ester), followed by fatty acid esterification of the triterpene in animal tissues. To support this idea, homogenates of rabbit and human enterocytes and liver are now shown to contain an acyl-CoA:triterpene acyltransferase activity (ATAT) which esterifies triterpene to a fatty acid. The enzyme activity was stimulated by exogenous triterpene and required ATP and coenzyme A when fatty acid was used as substrate; ATP and coenzyme A were not required when fatty acyl-CoA was used. ATAT was not inhibited by two structurally different ACAT inhibitors, which may indicate that ACAT and ATAT are different enzymes. Rat enterocytes and liver contained very little ATAT activity, consistent with the finding that rat liver contained very little triterpene ester. To establish that triterpene esterification occurs in vivo, [3H]triterpene was shown to be incorporated into triterpene ester in several organs and tissues from a rabbit given a gastric bolus of the labeled triterpene. These data provide support for the hypothesis that triterpene esters in animal tissues arise from the dietary absorption of triterpenes followed by the esterification of the triterpenes by an enzymatic activity in the animal tissues.

Acyltransferases↗
PubMed1990

Rabbit and human liver contain a novel pentacyclic triterpene ester with acyl-CoA: cholesterol acyltransferase inhibitory activity.

Acyl-coenzyme A (CoA):cholesterol acyltransferase (ACAT) catalyzes the intracellular fatty acid esterification of cholesterol and is thought to play a key role in lipoprotein metabolism and atherogenesis. Herein we describe the purification and characterization of a novel pentacyclic triterpene ester from rabbit liver that has ACAT inhibitory activity. The inhibitor was purified by a combination of silicic acid chromatography and preparative thin layer chromatography. The compound inhibited both rabbit and rat liver microsomal ACAT activity with an IC50 = 20 microM. The lipid did not inhibit fatty acid incorporation into triglycerides, diglycerides, monoglycerides, or phospholipids nor did it inhibit plasma lecithin:cholesterol acyltransferase activity. However, rat liver microsomal acyl-CoA:retinol acyltransferase activity was inhibited by the terpene ester. Kinetic data are consistent with a mechanism in which ACAT is inhibited by the compound in an irreversible manner. The subcellular fractionation pattern of both ACAT activity and the ACAT inhibitor were similar in rabbit liver (both were approximately equally distributed in membranes that pelleted at 10,000 X g and 100,000 X g). A lipid with similar properties to the rabbit liver inhibitor was found in many other rabbit tissues, including adrenal and spleen, as well as in human liver. Rat liver did not contain this lipid. Structural analysis by NMR, mass spectrometry, and x-ray crystallography indicated that the rabbit liver inhibitor was a fatty acid ester (mostly stearate) of a pentacyclic triterpene acid. The carbon skeleton of the triterpene moiety is a new member of the olean-12-ene triterpene family. Both the negatively charged carboxylic acid group of the triterpene moiety and the esterified fatty acid group were necessary for the ACAT-inhibitory activity of the triterpene ester. Lastly, we present preliminary data which, together with the structural homology of the rabbit triterpene with known plant compounds, suggest the hypothesis that the triterpene moiety of the rabbit ACAT inhibitor arises from dietary absorption of a plant triterpene.

Animals↗
PubMed1989

[Studies on the immunoregulative action of polysaccharides isolated from hyphal bodies of Lentinnus edodes (PHL) on hypoimmunity in mice].

PHL possesses significant antagonist action for immunodepression in mice induced by different immunodepressors. It can reverse the hypomacrophage function and the reduction in percentage of T-lymphoid cell induced by hydrocortisone (50 or 80 mg/kg, sc), reverse the reduction of phagocytosis of reticuloendothelial system (RES) induced by hydroprednisone acetate (20 and 40 mg/kg, ip), restore the cyclophosphamide-suppressed delayed anaphylaxis reaction in mice (40 and 80 mg/kg, ip). In vitro PHL at a dose of 1 and 10 micrograms/ml accelerated lymphocyte mitosis induced by conA and PHA.

Animals↗
PubMed1989

Antileukemic effect of low-dose harringtonine.

The effect of harringtonine (HA) in various concentrations on HL-60 was evaluated. 0.10 microgram/ml HA showed a strong cell killing effect, and 0.025 microgram/ml HA a weak differentiation induction accompanied by cell growth retardation. Differentiation induction did not occur in 5 other human leukemic cell lines. We consider that antileukemic effect of HA is cytotoxicity, not differentiation induction.

Alkaloids↗
PubMed1986

Misread protein creates membrane channels: an essential step in the bactericidal action of aminoglycosides.

Among the pleiotropic effects of aminoglycosides, their irreversible uptake and their blockade of initiating ribosomes have appeared to explain their bactericidal action, while the contributions of translational misreading and membrane damage and the mechanism of that damage have remained uncertain. We now present evidence that incorporation of misread proteins into the membrane can account for the membrane damage. The bactericidal action thus appears to result from the following sequence, in which each step is essential: slight initial entry of the antibiotic; interaction with chain-elongating ribosomes, resulting in misreading; incorporation of misread protein into the membrane, creating abnormal channels; increased (and irreversible) entry through these channels, and hence increased misreading and formation of channels; and, finally, blockade of initiating ribosomes. This mechanism can account for several previously unexplained observations: that streptomycin uptake requires protein synthesis during, but not after, the lag before the membrane damage; that streptomycin-resistant cells, which fail to take up streptomycin, can do so after treatment by another aminoglycoside; and that puromycin at moderate concentrations accelerates streptomycin uptake, while high concentrations (which release shorter chains) prevent it. In addition, puromycin, prematurely releasing polypeptides of normal sequence, also evidently creates channels, since it is reported to promote streptomycin uptake even in streptomycin-resistant cells. These findings imply that normal membrane proteins must be selected not only for a hydrophobic anchoring surface, but also for a tight fit in the membrane.

Alkaline Phosphatase↗
PubMed1978

Anatomy of germinal centers in mouse spleen, with special reference to "follicular dendritic cells".

Lymphocyte proliferation in germinal centers (GC's) is thought to be triggered by antigen retained extracellularly on the surface of special "dendritic" cells. The anatomy and function of these cells have not been studied directly or in detail. We therefore examined mouse spleen GC's developing in response to sheep erythrocyte stimulation. We found that distincitve "follicular dendritic cells" (FDC's) were present in both the GC and adjacent mantle region of secondary follicles. The large, irregularly shaped nucleus, containing little heterochromatin, allowed for the light microscope (LM) identification of FDC's. By EM, the cell was stellate in shape sending out long, thin sheets of cytoplasm which could fold and coil into complex arrays. The processes were coated extracellularly by an amorphous electron-dense material of varying thickness, as well as particulates including variable numbers of virions. The FDC cytoplasm lacked organelles of active secretory and endocytic cells, such as well-developed rough endoplasmic reticulum (RER) and lysosomes. These anatomical features readily distinguished FDC's from other cell types, even those that were extended in shape. To pursue these descriptive findings, we injected three electron-dense tracers i.v. and sacrificed the mice 1 h-10 days thereafter. Colloidal carbon, colloidal thorium dioxide (cThO2), and soluble horseradish peroxidase (HRP) were actively sequestered into the vacuolar system of macrophages but were interiorized only in trace amounts by FDC's. Therefore, FDC's are not macrophages by cytologic and functional criteria. FDC's did display a unique property. Both colloidal carbon and thorium dioxide, which are nonimmunogens, could be visualized extracellularly on the cell surface for several days. The meaning of this is unclear, but the association of colloid with FDC's appeared to slow the movement of particulates through the extracellular space into the GC proper. FDC's were not readily identified in splenic white pulp lacking GC's. They must develop de novo then, possibly from novel dendritic cells that we have identified in vitro (Steinman, R. M., and Z. A. Cohn. 1973. J. Exp. Med. 137:1142-1162).

Animals↗
PubMed1978

Distribution of horseradish peroxidase (HRP)-anti-HRP immune complexes in mouse spleen with special reference to follicular dendritic cells.

The distribution of immune complexes has been studied in mouse spleen stimulated to contain many germinal centers (GC's). Horseradish peroxidase (HRP)-anti-HRP complexes were used as an appropriately precise and sensitive model. We were primarily interested in the relative abilities of three cell types to interact with complexes: lymphocytes, macrophages, and follicular dendritic cells (FDC's). The latter are distinctive, nonendocytic, stellate cells located primarily at the transition of mantle and GC zones of 2 degrees lymphoid follicles (Chen, L. L., J. C. Adams, and R. M. Steinman, 1978, J. Cell Biol. 77:148). Binding of immune complexes to lymphocytes could not be visualized in situ. Macrophages avidly interiorized complexes into lysosomes, but did not retain them extracellularly. In contrast, FDC's could retain HRP-anti-HRP extracellularly under appropriate conditions, but did not endocytose them. Cytochemical reactivity accumulated progressively on FDC's 1--6 h after administration of complexes i.v., remained stable in amount and location for 1 day, and then was progressively lost over a 1- to 5-day period. Several variables in the association of complexes with macrophages and FDC's were pursued. Only 1 microgram of complexed HRP had to be administered to visualize binding to both cell types. Macrophages interiorized complexes formed in a wide range of HRP/anti-HRP ratios, while FDC's associated with complexes formed in HRP excess only. Quantitative studies with [125I]HRP-anti-HRP demonstrated that 20% of the splenic load of HRP associated with FDC's. Complexes formed with an F(ab')2 anti-HRP were distributed primarily in macrophages. When the levels of the third component of serum complement were depleted by prior treatment with cobra venom factor, uptake of complexes by macrophages was reduced some 50% whereas association with FDC's was abolished. The fact that antigen excess complexes are retained extracellularly strengthens the idea that they are immunogenic. Finally, the association of complexes with FDC's seems to retard the entry of antigen into the GC proper.

Animals↗
PubMed1977

Synthesis and secretion of albumin by a synchronized rat hepatoma cell line.

The rat hepatoma cell H4-12 which synthesizes and secretes albumin was synchronized by growth in isoleucine-deficient medium followed by a second block with excess thymidine. Albumin synthesis and secretion was measured in the synchronized cells at different time intervals representative of early S, late S, G2, mitosis, early G1 and late G1 phases of the cell cycle. Maximal albumin synthesis occurred during G1 although significant synthesis also occurred during the other cell cyle phases. Most (75--80%) of the radioactive albumin produced during a 15 min pulse incubation with L-[4,5-3H] leucine was found in the microsomal cell fraction and this nascent albumin was secreted into the incubation medium during a 160 min chase period. Fifty percent of the nascent albumin was secreted by 50--55 min and this pattern of secretion did not change during the cell cycle. These data indicate that albumin synthesis occurs throughout the cell cycle but that it is preferred during G1. The rate of intracellular transport and secretion of albumin does not vary during the different phase of the cell cycle.

Albumins↗