979 resultados para L-stable Functions


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Stable mammalian cell lines harboring a synthetic bovine opsin gene have been derived from the suspension-adapted HEK293 cell line. The opsin gene is under the control of the immediate-early cytomegalovirus promoter/enhancer in an expression vector that also contains a selectable marker (Neo) governed by a relatively weak promoter. The cell lines expressing the opsin gene at high levels are selected by growth in the presence of high concentrations of the antibiotic geneticin. Under the conditions used for cell growth in suspension, opsin is produced at saturated culture levels of more than 2 mg/liter. After reconstitution with 11-cis-retinal, rhodopsin is purified to homogeneity in a single step by immunoaffinity column chromatography. Rhodopsin thus prepared (> 90% recovery at concentrations of up to 15 microM) is indistinguishable from rhodopsin purified from bovine rod outer segments by the following criteria: (i) UV/Vis absorption spectra in the dark and after photobleaching and the rate of metarhodopsin II decay, (ii) initial rates of transducin activation, and (iii) the rate of phosphorylation by rhodopsin kinase. Although mammalian cell opsin migrates slower than rod outer segment opsin on SDS/polyacrylamide gels, presumably due to a different N-glycosylation pattern, their mobilities after deglycosylation are identical. This method has enabled the preparation of several site-specific mutants of bovine opsin in comparable amounts.

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Proliferation of dispersed plant cells in culture is strictly dependent on cell density, and cells in a low-density culture can only grow in the presence of conditioned medium (CM). No known plant hormones have been able to substitute for CM. To quantify the mitogenic activity of CM, we examined conditions for the assay system using mechanically dispersed mesophyll cells of Asparagus officinalis L. and established a highly sensitive bioassay method. By use of this method, the mitogenic activity of CM prepared from asparagus cells was characterized: it was heat-stable, susceptible to pronase digestion, and resistant to glycosidase treatment. On the basis of these results, the mitogenic activity in CM was purified 10(7)-fold by column chromatography, and two factors named phytosulfokine-alpha and -beta (PSK-alpha and PSK-beta) were obtained. By amino acid sequence analysis and mass spectrometry, the structures of these two factors were determined to be sulfated pentapeptide (H-Tyr(SO3H)-Ile-Tyr(SO3H)-Thr-Gln-OH) and sulfated tetrapeptide (H-Tyr(SO3H)-Ile-Tyr(SO3H)-Thr-OH). PSK-alpha and PSK-beta were prepared by chemical synthesis and enzymatic sulfation. The synthetic peptides exhibited the same activity as the natural factors, confirming the structure for PSK-alpha and PSK-beta mentioned above. This is the first elucidation of the structure of a conditioned medium factor required for the growth of low-density plant cell cultures.

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Stable cationic lipid/DNA complexes were formed by solubilizing cationic liposomes with 1% octylglucoside and complexing a DNA plasmid with the lipid in the presence of detergent. Removal of the detergent by dialysis yielded a lipid/DNA suspension that was able to transfect tissue culture cells up to 90 days after formation with no loss in activity. Similar levels of gene transfer were obtained by mixing the cationic lipid in a liposome form with DNA just prior to cell addition. However, expression was completely lost 24 hr after mixing. The transfection efficiency of the stable complex in 15% fetal calf serum was 30% of that obtained in the absence of serum, whereas the transient complex was completely inactivated with 2% fetal calf serum. A 90-day stability study comparing various storage conditions showed that the stable complex could be stored frozen or as a suspension at 4 degrees C with no loss in transfection efficiency. Centrifugation of the stable complex produced a pellet that contained approximately 90% of the DNA and 10% of the lipid. Transfection of cells with the resuspended pellet and the supernatant showed that the majority of the transfection activity was in the pellet and all the toxicity was in the supernatant. Formation of a stable cationic lipid/DNA complex has produced a transfection vehicle that can be stored indefinitely, can be concentrated with no loss in transfection efficiency, and the toxicity levels can be greatly reduced when the active complex is isolated from the uncomplexed lipid.

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Resistance to virus infections in higher vertebrates is mediated in part through catalysis of RNA decay by the, interferon-regulated 2-5A system. A functional 2-5A system requires two enzymes, a 2-5A synthetase that produces 5'-phosphorylated, 2',5'-linked oligoadenylates (2-5A) in response to double-stranded RNA, and the 2-5A-dependent RNase L. We have coexpressed these human enzymes in transgenic tobacco plants by using a single plasmid containing the cDNAs for both human RNase L and a low molecular weight form of human 2-5A synthetase under control of different, constitutive promoters. Expression of the human cDNAs in the transgenic plants was demonstrated from Northern blots, by specific enzyme assays, and by immunodetection (for RNase L). Infection of leaves, detached or in planta, of the coexpressing transgenic plants by tobacco mosaic virus, alfalfa [correction of alfafa] mosaic virus, or tobacco etch virus resulted in necrotic lesions. In contrast, leaves expressing 2-5A synthetase or RNase L alone and leaves containing the plasmid vector alone produced typical systemic infections. While alfalfa mosaic virus produced lesions only in the inoculated leaves regardless of the concentration of virus in the inoculum, high, but not low, levels of tobacco etch virus inoculum resulted in escape of virus to uninoculated leaves. Nevertheless, there was a substantial reduction of tobacco etch virus yield as measured by ELISA assay in the coexpressing transgenic plants. These results indicate that expression of a mammalian 2-5A system in plants provides resistance to virus infections.

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Ca2+ influx controls multiple neuronal functions including neurotransmitter release, protein phosphorylation, gene expression, and synaptic plasticity. Brain L-type Ca2+ channels, which contain either alpha 1C or alpha 1D as their pore-forming subunits, are an important source of calcium entry into neurons. Alpha 1C exists in long and short forms, which are differentially phosphorylated, and C-terminal truncation of alpha 1C increases its activity approximately 4-fold in heterologous expression systems. Although most L-type calcium channels in brain are localized in the cell body and proximal dendrites, alpha 1C subunits in the hippocampus are also present in clusters along the dendrites of neurons. Examination by electron microscopy shows that these clusters of alpha 1C are localized in the postsynaptic membrane of excitatory synapses, which are known to contain glutamate receptors. Activation of N-methyl-D-aspartate (NMDA)-specific glutamate receptors induced the conversion of the long form of alpha 1C into the short form by proteolytic removal of the C terminus. Other classes of Ca2+ channel alpha1 subunits were unaffected. This proteolytic processing reaction required extracellular calcium and was blocked by inhibitors of the calcium-activated protease calpain, indicating that calcium entry through NMDA receptors activated proteolysis of alpha1C by calpain. Purified calpain catalyzed conversion of the long form of immunopurified alpha 1C to the short form in vitro, consistent with the hypothesis that calpain is responsible for processing of alpha 1C in hippocampal neurons. Our results suggest that NMDA receptor-induced processing of the postsynaptic class C L-type Ca2+ channel may persistently increase Ca2+ influx following intense synaptic activity and may influence Ca2+-dependent processes such as protein phosphorylation, synaptic plasticity, and gene expression.

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Positron emission tomography (PET) with L-[methyl-11C]methionine was explored as an in vivo, noninvasive, quantitative method for measuring the protein synthesis rate (PSR) in paraspinal and hind limb muscles of anesthetized dogs. Approximately 25 mCi (1 Ci = 37 GBq) of L-[methyl-11C]methionine was injected intravenously, and serial images and arterial blood samples were acquired over 90 min. Data analysis was performed by fitting tissue- and metabolite-corrected arterial blood time-activity curves to a three-compartment model and assuming insignificant transamination and transmethylation in this tissue. PSR was calculated from fitted parameter values and plasma methionine concentrations. PSRs measured by PET were compared with arterio-venous (A-V) difference measurements across the hind limb during primed constant infusion (5-6 h) of L-[1-13C, methyl-2H3]methionine. Results of PET measurements demonstrated similar PSRs for paraspinal and hind limb muscles: 0.172 +/- 0.062 vs. 0.208 +/- 0.048 nmol-1.min-1.(g of muscle)-1 (P = not significant). PSR determined by the stable isotope technique was 0.27 +/- 0.050 nmol-1.min-1.(g of leg tissue)-1 (P < 0.07 from PET) and indicated that the contribution of transmethylation to total hind limb methionine utilization was approximately 10%. High levels of L-[methyl-11C]methionine utilization by bone marrow were observed. We conclude that muscle PSR can be measured in vivo by PET and that this approach offers promise for application in human metabolic studies.

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Although the CD34 antigen is widely used in the identification and purification of hemopoietic stem and progenitor cells, its function within hemopoiesis is unknown. We have investigated this issue by ectopically expressing human (hu) CD34 on the surface of murine hemopoietic cells. Forced expression of hu-CD34 in the thymocytes of transgenic mice did not appear to affect the development, maturation, or distribution of murine T cells but did significantly increase their ability to adhere to bone marrow stromal layers of human but not mouse origin. Ectopic expression of hu-CD34 on murine 416B cells, a multipotential progenitor that expresses murine CD34, yielded similar results. In both cases hu-CD34-dependent adhesion was enhanced by molecular engagement of the hu-CD34 protein using anti-CD34 antibodies. These results provide evidence that CD34 promotes the adhesive interactions of hemopoietic cells with the stromal microenvironment of the bone marrow thereby implicating CD34 in regulation and compartmentalization of stem cells. We propose that CD34 regulates these processes in part via an indirect mechanism, signaling changes in cellular adhesion in response to molecular recognition of an as yet unidentified stromal CD34 counterreceptor or ligand.

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In conjunction with other general initiation factors, the TATA box-binding protein (TBP) can direct basal transcription by RNA polymerase II from TATA-containing promoters, but its stable interaction with TBP-associated factors (TAFs) in the TFIID complex is required both for activator-dependent transcription and for basal transcription directed by an initiator element. We have generated a TATA-binding-defective TFIID complex containing an amino acid substitution in the DNA-binding surface of its TBP subunit. This mutated TFIID is defective in both basal and activated transcription from core promoters containing only a TATA box but supports transcription from initiator-containing promoters independently of the presence or absence of a TATA sequence. Our results show that a functional initiator element is needed to bypass the requirement for an active TATA DNA-binding surface in TFIID and imply that gene-specific transcription can be achieved by modulating distinct core promoter-specific TFIID functions--e.g., TBP-TATA versus TAF-initiator interactions.

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Previous studies have failed to detect an interaction between monomeric soluble CD4 (sCD4) and class II major histocompatibility complex (MHC) proteins, suggesting that oligomerization of CD4 on the cell surface may be required to form a stable class II MHC binding site. To test this possibility, we transfected the F43I CD4 mutant, which is incapable of binding to class II MHC or human immunodeficiency virus (HIV) gp120, into COS-7 cells together with wild-type CD4 (wtCD4). Expression of F43I results in a dominant negative effect: no class II MHC binding is observed even though wtCD4 expression is preserved. Apparently, F43I associates with wtCD4 oligomers and interferes with the formation of functional class II MHC binding structures. In contrast, F43I does not affect the binding of gp120 to wtCD4, implying that gp120 binds to a CD4 monomer. By production and characterization of chimeric CD4 molecules, we show that domains 3 and/or 4 appear to be involved in oligomerization. Several models of the CD4-class II MHC interaction are offered, including the possibility that one or two CD4 molecules initially interact with class II MHC dimers and further associate to create larger complexes important for facilitating T-cell receptor crosslinking.

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We applied mutational analysis to a protein domain that functions in neither catalysis nor binding but, rather, in transmembrane signaling. The domain is part of chemoreceptor Trg from Escherichia coli. It contains four transmembrane segments, two from each subunit of the homodimer. We used cysteine scanning to investigate the functional importance of each of 54 residues in the two transmembrane segments. Cysteines at some positions resulted in subtle but significant reductions in tactic response. Those positions defined a specific helical face on each segment, implying that the segments function as helices. The functionally important faces corresponded to structural, helical packing faces identified independently by biochemical studies. All functionally impaired receptors exhibited altered signaling properties, either reduced signaling upon stimulation or induced signaling in the absence of stimulation. The distribution of substitutions creating these two phenotypes implied that conformational signaling involves movement between the two transmembrane helices within a subunit and that signaling is optimal when stable interactions are maintained across the interface between subunits.

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Adaptive reversion of a lac- frameshift mutation in Escherichia coli appears to be due to DNA polymerase errors, implying that DNA is being synthesized although the cells are not dividing. Here we report that the production of adaptive lac+ revertants (i) is much higher when the mutational target is on the F' episome than when it is on the bacterial chromosome; (ii) is enhanced by functions required for conjugation; but (iii) does not require conjugation per se. These results suggest that, in static cells, DNA synthesis is initiated from the conjugal origin of transfer. Mutations may arise as polymerase errors during this synthesis or during synthesis stimulated by recombination among the multiple gene copies.

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Infection with enterotoxigenic Escherichia coli is a leading cause of traveler's diarrhea. Many enterotoxigenic E. coli strains produce heat-stable enterotoxin (ST), a peptide that binds to the intestinal receptor guanylyl cyclase C known as STaR. The toxin-receptor interaction elevates intracellular cGMP, which then activates apical chloride secretion, resulting in secretory diarrhea. In this report, we examine how the intracellular domains of STaR participate in the propagation and regulation of signaling. We show that STaR exists as an oligomer in both the presence and the absence of toxin. We also demonstrate that deletion of the intracellular kinase-homology domain produces a constitutively active mutant, suggesting that this domain subserves an autoinhibitory function. Finally, we constructed a point mutant within a highly conserved region of the cyclase domain that completely inactivates the catalytic activity of guanylyl cyclase. Cotransfection of this point mutant with wild-type receptor causes a dominant-negative effect on receptor activation. This suggests that interaction of receptor subunits is required for toxin-induced activation and that the cyclase domain is involved in this essential interaction. We propose that the binding of ST to STaR promotes a conformational change across the cell membrane. This removes the inhibitory effects of the kinase-homology domain and promotes an interaction between cyclase domains that leads to receptor activation. The data suggest a paradigm of signal transduction that may also be relevant to other members of the guanylyl cyclase receptor family.

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Previous studies have implicated the bcl-2 protooncogene as a potential regulator of neuronal survival. However, mice lacking functional bcl-2 exhibited normal development and maintenance of the central nervous system (CNS). Since bcl-2 appears dispensable for neuronal survival, we have examined the expression and function of bcl-x, another member of the bcl-2 family of death regulatory genes. Bcl-2 is expressed in neuronal tissues during embryonic development but is down-regulated in the adult CNS. In contrast, Bcl-xL expression is retained in neurons of the adult CNS. Two different forms of bcl-x mRNA and their corresponding products, Bcl-xL and Bcl-x beta, were expressed in embryonic and adult neurons of the CNS. Microinjection of bcl-xL and bcl-x beta cDNAs into primary sympathetic neurons inhibited their death induced by nerve growth factor withdrawal. Thus, Bcl-x proteins appear to play an important role in the regulation of neuronal survival in the adult CNS.

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The proper placement of the Escherichia coli division septum requires the MinE protein. MinE accomplishes this by imparting topological specificity to a division inhibitor coded by the minC and minD genes. As a result, the division inhibitor prevents septation at potential division sites that exist at the cell poles but permits septation at the normal division site at midcell. In this paper, we define two functions of MinE that are required for this effect and present evidence that different domains within the 88-amino acid MinE protein are responsible for each of these two functions. The first domain, responsible for the ability of MinE to counteract the activity of the MinCD division inhibitor, is located in a small region near the N terminus of the protein. The second domain, required for the topological specificity of MinE function, is located in the more distal region of the protein and affects the site specificity of placement of the division septum even when separated from the domain responsible for suppression of the activity of the division inhibitor.

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We determined the distribution of 11 different transposable elements on Drosophila melanogaster mitotic chromosomes by using high-resolution fluorescent in situ hybridization (FISH) coupled with charge-coupled device camera analysis. Nine of these transposable elements (copia, gypsy, mdg-1, blood, Doc, I, F, G, and Bari-1) are preferentially clustered into one or more discrete heterochromatic regions in chromosomes of the Oregon-R laboratory stock. Moreover, FISH analysis of geographically distant strains revealed that the locations of these heterochromatic transposable element clusters are highly conserved. The P and hobo elements, which are likely to have invaded the D. melanogaster genome at the beginning of this century, are absent from Oregon-R heterochromatin but clearly exhibit heterochromatic clusters in certain natural populations. Together these data indicate that transposable elements are major structural components of Drosophila heterochromatin, and they change the current views on the role of transposable elements in host genome evolution.