Transcription factors as targets of cytokine signals.
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Acute-Phase Reaction↗
Biomedical subjects
L Li
Publications and source records attributed to L Li.
Localization of the synapsin II (SYN2) gene to human chromosome 3 and mouse chromosome 6.
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Animals↗
The GA5 locus of Arabidopsis thaliana encodes a multifunctional gibberellin 20-oxidase: molecular cloning and functional expression.
The biosynthesis of gibberellins (GAs) after GA12-aldehyde involves a series of oxidative steps that lead to the formation of bioactive GAs. Previously, a cDNA clone encoding a GA 20-oxidase [gibberellin, 2-oxoglutarate:oxygen oxidoreductase (20-hydroxylating, oxidizing), EC 1.14.11.-] was isolated by immunoscreening a cDNA library from liquid endosperm of pumpkin (Cucurbita maxima L.) with antibodies against partially purified GA 20-oxidase. Here, we report isolation of a genomic clone for GA 20-oxidase from a genomic library of the long-day species Arabidopsis thaliana Heynh., strain Columbia, by using the pumpkin cDNA clone as a heterologous probe. This genomic clone contains a GA 20-oxidase gene that consists of three exons and two introns. The three exons are 1131-bp long and encode 377 amino acid residues. A cDNA clone corresponding to the putative GA 20-oxidase genomic sequence was constructed with the reverse transcription-PCR method, and the identity of the cDNA clone was confirmed by analyzing the capability of the fusion protein expressed in Escherichia coli to convert GA53 to GA44 and GA19 to GA20. The Arabidopsis GA 20-oxidase shares 55% identity and > 80% similarity with the pumpkin GA 20-oxidase at the derived amino acid level. Both GA 20-oxidases share high homology with other 2-oxoglutarate-dependent dioxygenases (2-ODDs), but the highest homology was found between the two GA 20-oxidases. Mapping results indicated tight linkage between the cloned GA 20-oxidase and the GA5 locus of Arabidopsis. The ga5 semidwarf mutant contains a G-->A point mutation that inserts a translational stop codon in the protein-coding sequence, thus confirming that the GA5 locus encodes GA 20-oxidase. Expression of the GA5 gene in Ara-bidopsis leaves was enhanced after plants were transferred from short to long days; it was reduced by GA4 treatment, suggesting end-product repression in the GA biosynthetic pathway.
Amino Acid Sequence↗
High-resolution matrix-assisted laser desorption/ionization in a linear time-of-flight mass spectrometer.
A time-lag focusing method is developed for the improvement of mass resolution in a linear time-of-flight mass spectrometer for matrix-assisted laser desorption/ionization (MALDI). In this technique, the ions generated by the MALDI process are extracted by a pulsed voltage. A short time delay (280 ns) is inserted in between the laser desorption/ionization event and the ion extraction. The region between the repeller and extraction grid is field-free during the delay. The time-lag extraction allows the ions generated in the region between the repeller and the extraction grid to separate according to their velocity (energy). Application, to the repeller, of the appropriate pulse voltage provides the energy correction necessary to simultaneously detect all ions of the same mass/charge regardless of their initial energy, resulting in improved mass resolution. It is demonstrated that mass resolution in the range of 3000-6000 fwhm can be obtained. With this mass resolution, isotopically resolved mass spectra are observed for peptides with masses up to 3000 Da. For proteins, such as bovine insulin, cytochrome c, and apomyoglobin, resolution in the range of 800-1000 fwhm is observed with a mass measurement accuracy better than 0.01%.
Amino Acid Sequence↗
An activated mutant of the alpha subunit of G(o) increases neurite outgrowth via protein kinase C.
The GTP-binding protein, G(o), is present at very high concentration in the neuronal growth cone membrane. The expression of activated mutants of the a subunit of G(o) increases neurite outgrowth. To determine the intracellular mechanism for this outgrowth, we have examined activated alpha o-dependent outgrowth in the presence of agents which modulate different signal transduction cascades. Activation of protein kinase C with phorbol esters or with diacylglycerol prevents the alpha o-dependent increase in neurite extension. Inhibition of protein kinase C with staurosporine, with H7, or with long-term, high dose phorbol ester treatment resulted in greater neurite elongation, and no further increase after activated alpha o transfection. The protein phosphatase inhibitor, okadaic acid, also blocked the effect of activated alpha o. In contrast, tyrosine kinase inhibitors and agents which alter cAMP levels did not alter activated alpha o-dependent neurite extension. We tested a number of compounds which alter intracellular calcium levels. TMB-8 and thapsigargin prevented an increase in outgrowth by activated alpha o, but diltiazem, Bay K8644 and dantrolene had no effect on activated alpha o-dependent outgrowth. These studies suggest that activated alpha o increases neurite outgrowth by inhibiting protein kinase C and by modulating intracellular calcium release.
Alkaloids↗
Natural catalytic antibodies: peptide-hydrolyzing activities of Bence Jones proteins and VL fragment.
Monoclonal human light chains, i.e. Bence Jones proteins, and their recombinant variable fragments (VL) were screened for proteolytic activity using peptide-methylcoumarinamide (peptide-MCA) conjugates and vasoactive intestinal polypeptide (VIP) as substrates. Sixteen of 21 Bence Jones proteins and one of three VL fragments were capable of detectable cleavage of one or more substrates. The magnitude and kinetic characteristics of the activity varied with different substrates. Among the peptide-MCA substrates, the presence of tripeptide or tetrapeptide moieties with a basic residue at the scissile bond generally favored expression of the activity. The influence of N-terminal flanking residue recognition was evident from differing values of Km and kcat (turnover number) observed using different Arg-containing peptide-MCA substrates. Different light chains displayed different kinetic parameters for the same substrate, suggesting unique catalytic sites. Hydrolysis of VIP was characterized by nanomolar Michaelis-Menten constants (Km), suggesting comparatively high affinity recognition of this peptide. The 25-kDa monomer and the 50-kDa dimer forms of one light chain preparation were resolved by gel filtration in 6 M guanidine hydrochloride. Following renaturation, the monomer displayed 51-fold greater peptide-MCA-hydrolyzing activity than the dimer. A renatured VL domain prepared by gel filtration in 6 M guanidine hydrochloride displayed VIP-hydrolyzing activity in the 12.5-kDa peak fractions. These results provide evidence for the proteolytic activity of certain human light chains and imply that this phenomenon may have a pathophysiological significance.
Amino Acid Sequence↗
Continuous fluorescence assay of phytochrome assembly in vitro.
Incubation of recombinant apophytochrome with the phycobiliprotein chromophore precursor phycoerythrobilin produces a covalent adduct that exhibits a fluorescence excitation maximum at 576 nm and an emission maximum at 586 nm. Using these fluorescence parameters, we have developed a kinetic assay for quantitative analysis of the assembly of the plant photoreceptor phytochrome in real time. Kinetic measurements performed with different phycoerythrobilin concentrations confirm that bilin attachment to apophytochrome involves two steps, an initial formation of a reversible non-covalent complex followed by thioether bond formation. The kinetic constants for both steps of phycoerythrobilin attachment to apophytochrome were estimated with this assay. Methodology for determining the kinetic constants for the assembly of both the natural phytochrome chromophore precursor, phytochromobilin, and the analog phycocyanobilin is also described. Since the latter two bilins yield covalent, nonfluorescent adducts with apophytochrome, their co-incubation with phycoerythrobilin reduces the rate of formation of the fluorescent phycoerythrobilin adduct in an irreversible, competitive manner. Competition experiments were also performed with biliverdin, a structurally related bilin which does not form a covalent adduct with apophytochrome. Such measurements show that biliverdin reversibly binds to apophytochrome with a submicromolar binding constant, an affinity which is very similar to that of phytochromobilin. The utility of this fluorescence assay for identification of novel inhibitors of phytochrome assembly and for characterization of the structural features of both bilin and apophytochrome necessary for photoreceptor assembly is discussed.
Apoproteins↗
Effect of propensity of hexagonal II phase formation on the activity of mitochondrial ubiquinol-cytochrome c reductase and H(+)-ATPase.
The propensity of hexagonal II phase formation plays an important role in the activity of mitochondrial ubiquinol-cytochrome c reductase or H(+)-ATPase. The respiratory control ratio of reconstituted ubiquinol-cytochrome c reductase or the ATP-induced membrane potential of reconstituted H(+)-ATPase became higher as the non-bilayer phospholipid phosphatidylethanolamine content of proteoliposomes increased. The highest respiratory control ratio or ATP-induced membrane potential was obtained in the case of 60-80% phosphatidylethanolamine-containing proteoliposomes. Dioleoylphosphatidylethanolamine could significantly enhance the respiratory control ratio of ubiquinol-cytochrome c reductase and ATP-induced membrane potential of H(+)-ATPase, while no obvious change could be observed when dielaidoylphosphatidylethanolamine was used. The bilayer to hexagonal II phase transition temperature of ubiquinol-cytochrome c reductase-containing proteoliposomes reconstituted with phosphatidylcholine+phosphatidylethanolamine increases with decreasing content of phosphatidylethanolamine. Several additives such as the bilayer stabilizers, cholesterol 3-sulfate and carbobenzoxy-D-Phe-L-PheGly, or hexagonal II phase-forming promoters, such as diolein or eicosane, can decrease or increase the activity of these two enzyme complexes.
Adenosine Triphosphate↗
Suppression of heat-induced hsp70 expression by the 70-kDa subunit of the human Ku autoantigen.
Expression of the 70-kDa polypeptide of human Ku autoantigen in rat cells is shown to suppress specifically the induction of hsp70 upon heat shock. Thermal induction of other heat shock proteins is not significantly affected, nor is the state of phosphorylation or the DNA-binding ability of the heat shock transcription factor HSF1. These findings support a model in which hsp70 gene expression is controlled by a second regulatory factor in addition to the positive activator HSF1. The Ku autoantigen, or a protein closely related to it, is likely to be involved in the regulation of hsp70 expression.
Animals↗
Two modes of hair cell loss from the vestibular sensory epithelia of the guinea pig inner ear.
In the vestibular and auditory neurosensory epithelia of poikilothermic vertebrates and of birds, damaged sensory "hair" cells are often deleted by extrusion from the apical surface. In contrast, in the adult mammalian auditory epithelium (the organ of Corti), the bodies of damaged hair cells degenerate within the epithelium. To determine whether this apparent difference is species related or is associated with the differing structural organisation of the epithelia, hair cell deletion in the mammalian vestibular end-organs was examined. The structural organisation of these tissues is closer to that of the inner ear epithelia of lower vertebrates than to the organ of Corti. Hair cell loss was induced by chronic, systemic treatment of guinea pigs with the ototoxic aminoglycoside antibiotic gentamicin. The vestibular sensory epithelia were examined at various times after treatment via scanning electron microscopy, thin sectioning, and staining f-actin with fluorescently labelled phalloidin. Two distinct modes of hair cell loss were identified: 1) degeneration of hair cells within the epithelium, which often showed morphological features consistent with those described for apoptosis, and 2) extrusion of intact cells from the apical surface. Neither process caused the formation of obvious lesions through the epithelial surfaces. Expansion of adjacent supporting cells during hair cell deletion resulted in repair that appeared to preserve permeability barriers. There was also no evidence of inflammation accompanying hair cell removal. Thus, with both modes of hair cell loss, it appeared that deletion of hair cells was achieved without disruption of tissue architecture or integrity. This may be important for subsequent repair and regeneration processes to operate.
Animals↗
Characterization of mutants affecting the KRK sequence in the carboxyl-terminal domain of lac repressor.
The lac repressor carboxyl-terminal region is required for tetramer assembly and protein stability. To further investigate this region, especially the unusual sequence KRK, four deletion mutants eliminating the carboxyl-terminal 34, 35, 36, and 39 amino acids and five substitution mutants at the position of Arg-326, R326K, R326A, R326E, R326L, and R326W, were constructed using site-specific mutagenesis. The -34-amino-acid (aa) mutant, missing the most carboxyl-proximal lysine from the KRK sequence, exhibited lower affinity for both operator and inducer and lower protein stability than dimeric proteins studied previously. The -35-aa mutant with RK missing, as well as -36 aa and -39 aa, for which the entire KRK sequence was deleted, yielded inactive polypeptides that could be detected only by monoclonal antibody for lac repressor. In the Arg-326 mutant proteins, operator binding affinity was decreased by approximately 6-fold, the shift in inducer binding at elevated pH was diminished, and protein stability was decreased. Dramatic decreases in protein expression and stability occurred with substitution at position 326 by glutamate, leucine, or tryptophan. These results suggest that Arg-326 plays an important role in the formation of the proper tertiary structure necessary for inducer and operator affinity and for protein stability.
Amino Acid Sequence↗
Antitumor activity of 9(R)-dihydrotaxane analogs.
A novel reduced taxane, 13-acetyl-9(R)-dihydrobaccatin III (1) has been isolated from Taxus canadensis. The selective C-13 deacetylation of this isolate has allowed for the preparation of a wide variety of 9(R)-dihydrotaxane analogs. In general, this series has shown greater stability and water solubility than the 9-carbonyl series while retaining antimicrotubule and tumor cell cytotoxicity activities relative to taxol. Placement of polar functionalities at the C-7 position results in loss of activity whereas alkylation or acylation of either C-7 or C-9 hydroxyl groups ameliorate the activity.
Animals↗
Expression of chicken liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase in Escherichia coli.
Chicken liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase was expressed in E. coli by using a pET3a T7 RNA polymerase-based expression system and was purified to homogeneity. The kinase and bisphosphatase of the expressed bifunctional enzyme had kinetic properties identical to those of the native chicken liver enzyme. However, the kinase activity of the chicken liver enzyme was 7-fold higher, while the bisphosphatase activity was 50 percent lower than those of the rat liver enzyme. Cys-256 of the rat liver bisphosphatase domain is not conserved in the chicken liver enzyme. A site-directed mutation was engineered at Cys-256 of the rat liver enzyme and the results indicate that the variation of this residue is not responsible for the difference in fructose-2,6-bisphosphatase activity between the rat and chicken liver enzymes. It is postulated that the difference in the kinase/bisphosphatase activity ratios of these two enzymes results from differences in their NH2-terminal regions.
Animals↗
Inhibition of angiotensin II- and potassium-mediated aldosterone secretion by KN-62 suggests involvement of Ca(2+)-calmodulin dependent protein kinase II in aldosterone secretion.
Calcium is now acknowledged to play a major role as a cellular mediator of aldosterone secretion. However, the intricacies of the involvement of calcium in aldosterone secretion have not been clearly defined. We have shown here that calmodulin-dependent kinase II is involved in angiotensin II- and potassium-evoked aldosterone secretion as judged by the marked inhibitory effect of KN-62, a specific inhibitor of such a kinase, on aldosterone secretion and this inhibition was similar to that produced by calmodulin inhibitor, W-7. These results further strengthen the case for the participatory role of calcium-calmodulin system in aldosterone secretion.
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
Catalytic activity of anti-thyroglobulin antibodies.
Thyroglobulin (Tg)-specific autoantibodies from a patient with Hashimoto's thyroiditis hydrolyzed radiolabeled Tg, shown by production of several smaller sized products on SDS electrophoresis gels. The apparent Km value for Tg was in the nanomolar range, a property typical of an Ab combining site. The Tg antibodies also hydrolyzed tripeptide-methylcoumarinamide (MCA) substrates with lower affinity, displaying a preference for Arg-MCA and Lys-MCA containing conjugates. The hydrolysis of one of these conjugates, Pro-Phe-Arg-MCA, was inhibited competitively by Tg, suggesting a catalytic site located in the Ab combining site. In control experiments, 1) the hydrolytic activities were removed by immunoadsorption with immobilized anti-human IgG; 2) IgG depleted of the Tg-specific Abs by affinity chromatography did not display Tg and Pro-Phe-Arg-MCA hydrolyzing activities; and 3) the peptide-MCA hydrolyzing activity tracked exactly with the 150-kDa IgG peak on a gel filtration column run in denaturing solvent (6 M guanidine chloride).
Amino Acid Sequence↗
Physicochemical stability of total nutrient admixtures.
The effect of six independent factors on the stability of i.v. nutritional emulsions was studied. Forty-five i.v. nutritional admixtures were prepared, each containing the following: (1) amino acids (range, 2.5-7%), (2) hydrated glucose (range, 5-20%), (3) lipid emulsion (range, 2-5%), (4) monovalent cations (range, 0-150 meq/L), (5) divalent cations (range, 4-20 meq/L), and (6) trivalent cations (range, 0-10 mg of elemental iron/L). Stability assessments included particle-size analysis, pH determination, and visual inspection. Sizing and counting of fat particles was achieved by using light obscuration and dynamic light scatter methods. Light obscuration and visual assessments were performed at 0, 6, 12, 24, and 30 hours. Dynamic light scatter and pH determinations were performed at 0 and 30 hours. Multiple stepwise regression analysis revealed that trivalent cation concentration was the only variable that affected the stability of nutritional emulsions (p < 0.00001), accounting for approximately 60% of the potentially dangerous increases in fat particle sizes observed. In addition, a percentage of large fat particles (> 5 microns in diameter) greater than 0.4% was associated with unstable emulsions. However, this instability was visibly evident only 65% of the time. Changes in mean globule diameter, cream-layer thickness, and pH did not reveal instability in these emulsions. Emulsions in which > 0.4% of the initial fat concentration consists of particles of > 5 microns in diameter are likely to become unstable. Of the six factors studied, the trivalent cation in iron dextran was most disruptive to lipid-based parenteral nutrient admixtures.
Amino Acids↗
Targeting of the 67-kDa isoform of glutamic acid decarboxylase to intracellular organelles is mediated by its interaction with the NH2-terminal region of the 65-kDa isoform of glutamic acid decarboxylase.
The two isoforms of glutamic acid decarboxylase (GAD), GAD67 and GAD65, synthesize the neurotransmitter gamma-aminobutyric acid in neurons and pancreatic beta-cells. Previous studies suggest that GAD67 is a soluble cytosolic protein, whereas GAD65 is membrane-associated. Here, we study the intracellular distribution of GAD67 in neurons, pancreatic beta-cells, and fibroblasts transfected either with GAD65 and GAD67 together or with GAD67 alone. Neuronal GAD67 is partially recovered with GAD65 in membrane-containing pellet fractions and Triton X-114 detergent phases. The two proteins co-immunoprecipitate from extracts of brain and GAD65-GAD67 co-transfected fibroblasts, but not when extracts of GAD65 and GAD67 transfected fibroblasts were mixed and used as a starting material for immunoprecipitation. GAD67 is concentrated in the Golgi complex region in GAD65-GAD67 co-transfected fibroblasts, but not in fibroblasts transfected with GAD67 alone. A pool of neuronal GAD67 co-localizes with GAD65 in the Golgi complex region and in many synapses. The two proteins also co-localize in the perinuclear region of some pancreatic beta-cells. GAD67 interacts with the NH2-terminal region of GAD65, even in the absence of palmitoylation of this region of GAD65. Taken together, our results indicate that GAD65-GAD67 association occurs in vivo and is required for the targeting of GAD67 to membranes.
Animals↗
Conditioned media from a cell strain derived from a patient with mastocytosis induces preferential development of cells that possess high affinity IgE receptors and the granule protease phenotype of mature cutaneous mast cells.
We have demonstrated for the first time that a conditioned medium from a human cell strain can induce morphologically mature mast cells that express Fc epsilon RI and three mast cell-specific proteases from normal bone marrow progenitor cells. In contrast, recombinant human Kit ligand induced the differentiation of mast cells that were tryptase-positive but negative for chymase, carboxypeptidase, and Fc epsilon RI. This data indicates that factors other than Kit ligand are critical for inducing the differentiation and maturation of mast cells in the human. The HBM-M cell was originally derived from a patient with mastocytosis. As mastocytosis is thought to represent a reactive hyperplasia rather than a mast cell malignancy, the factor secreted by the HBM-M cell strain could well be responsible for the mast cell hyperplasia seen in some patients with mastocytosis.
Base Sequence↗