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1
Liakopoulou, E., CA Blau, Q. Li, B. Josephson, JA Wolf, B. Fournarakis, V. Raisys, G. Dover, T. Papayannopoulou, and G. Stamatoyannopoulos. "Stimulation of fetal hemoglobin production by short chain fatty acids." Blood 86, no. 8 (October 15, 1995): 3227–35. http://dx.doi.org/10.1182/blood.v86.8.3227.3227.
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Abstract Butyrate, a four-carbon fatty acid, and its two-carbon metabolic product, acetate, are inducers of gamma-globin synthesis. To test whether other short-chain fatty acids share this property, we first examined whether propionic acid, a three-carbon fatty acid that is not catabolized to acetate, induces gamma-globin expression. Sodium propionate increased the frequency of fetal hemoglobin containing erythroblasts and the gamma/gamma + beta mRNA ratios in adult erythroid cell cultures and F reticulocyte production in a nonanemic juvenile baboon. Short-chain fatty acids containing five (pentanoic), six (hexanoic), seven (heptanoic), eight (octanoic), and nine (nonanoic) carbons induced gamma-globin expression (as measured by increase in gamma-positive erythroblasts and gamma/gamma + beta mRNA ratios) in adult erythroid burst-forming unit cultures. There was a clear-cut relationship between the concentration of fatty acids in culture and the degree of induction of gamma-globin expression. Three-, four-, and five-carbon fatty acids were better inducers of gamma globin in culture as compared with six- to nine-carbon fatty acids. These results suggest that all short-chain fatty acids share the property of gamma-globin gene inducibility. The fact that valproic acid, a derivative of pentanoic acid, also induces gamma-globin expression suggests that short-chain fatty acid derivatives that are already approved for human use may possess the property of gamma-globin inducibility and may be of therapeutic relevance to the beta-chain hemoglobinopathies.
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Liakopoulou, E., CA Blau, Q. Li, B. Josephson, JA Wolf, B. Fournarakis, V. Raisys, G. Dover, T. Papayannopoulou, and G. Stamatoyannopoulos. "Stimulation of fetal hemoglobin production by short chain fatty acids." Blood 86, no. 8 (October 15, 1995): 3227–35. http://dx.doi.org/10.1182/blood.v86.8.3227.bloodjournal8683227.
Full textAbstract:
Butyrate, a four-carbon fatty acid, and its two-carbon metabolic product, acetate, are inducers of gamma-globin synthesis. To test whether other short-chain fatty acids share this property, we first examined whether propionic acid, a three-carbon fatty acid that is not catabolized to acetate, induces gamma-globin expression. Sodium propionate increased the frequency of fetal hemoglobin containing erythroblasts and the gamma/gamma + beta mRNA ratios in adult erythroid cell cultures and F reticulocyte production in a nonanemic juvenile baboon. Short-chain fatty acids containing five (pentanoic), six (hexanoic), seven (heptanoic), eight (octanoic), and nine (nonanoic) carbons induced gamma-globin expression (as measured by increase in gamma-positive erythroblasts and gamma/gamma + beta mRNA ratios) in adult erythroid burst-forming unit cultures. There was a clear-cut relationship between the concentration of fatty acids in culture and the degree of induction of gamma-globin expression. Three-, four-, and five-carbon fatty acids were better inducers of gamma globin in culture as compared with six- to nine-carbon fatty acids. These results suggest that all short-chain fatty acids share the property of gamma-globin gene inducibility. The fact that valproic acid, a derivative of pentanoic acid, also induces gamma-globin expression suggests that short-chain fatty acid derivatives that are already approved for human use may possess the property of gamma-globin inducibility and may be of therapeutic relevance to the beta-chain hemoglobinopathies.
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Nishikawa, Koichi, and Neil L. Harrison. "The Actions of Sevoflurane and Desflurane on the γ-Aminobutyric Acid Receptor Type A." Anesthesiology 99, no. 3 (September 1, 2003): 678–84. http://dx.doi.org/10.1097/00000542-200309000-00024.
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Background Previous studies have shown that specific amino acid residues in the putative second transmembrane segment (TM2) of the gamma-aminobutyric acid receptor type A (GABAA) receptor play a critical role in the enhancement of GABAA receptor function by halothane, enflurane, and isoflurane. However, very little is known about the actions of sevoflurane and desflurane on recombinant GABAA receptors. The aim of this study was to examine the effects of sevoflurane and desflurane on potentiation of GABA-induced responses in the wild-type GABAA receptor and in receptors mutated in TM2 of the alpha1, alpha 2, or beta 2 subunits. Methods GABAA receptor alpha 1 or alpha 2, beta 2 or beta 3, and gamma 2s subunit cDNAs were expressed for pharmacologic study by transfection of human embryonic kidney 293 cells and assayed using the whole cell voltage clamp technique. Concentration-response curves and EC50 values for agonist were determined in the wild-type alpha 1 beta 2 gamma 2s and alpha 2 beta 3 gamma 2s receptors, and in receptors harboring mutations in TM2, such as alpha1(S270W)beta 2 gamma 2s, alpha 1 beta 2(N265W)gamma 2s, and alpha2(S270I)beta 3 gamma 2s. The actions of clinically relevant concentration of volatile anesthetics (isoflurane, sevoflurane, and desflurane) on GABA activated Cl- currents were compared in the wild-type and mutant GABAA receptors. Results Both sevoflurane and desflurane potentiated submaximal GABA currents in the wild-type GABAA alpha 1 beta 2 gamma 2s receptor and alpha 2 beta 3 gamma 2s receptor. Substitution of Ser270 in TM2 of the alpha subunit by a larger amino acid, tryptophan (W) or isoleucine (I), as in alpha1(S270W)beta 2 gamma 2s and alpha 2(S270I)beta 3 gamma 2s, completely abolished the potentiation of GABA-induced currents by these anesthetic agents. In contrast, mutation of Asn265 in TM2 of the beta subunit to tryptophan (W) did not prevent potentiation of GABA-induced responses. The actions of sevoflurane and desflurane in the wild-type receptor and in mutated receptors were qualitatively and quantitatively similar to those observed for isoflurane. Conclusions Positions Ser270 of the GABAA alpha1 and alpha2 subunits, but not Asn265 in the TM2 of the beta2 subunit, are critical for regulation of the GABAA receptor by sevoflurane and desflurane, as well as isoflurane, consistent with the idea that these three volatile anesthetics share a common site of actions on the alpha subunit of the GABAA receptor.
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Desai, Rooma, Dirk Ruesch, and Stuart A. Forman. "γ-Amino Butyric Acid Type A Receptor Mutations at β2N265 Alter Etomidate Efficacy While Preserving Basal and Agonist-dependent Activity." Anesthesiology 111, no. 4 (October 1, 2009): 774–84. http://dx.doi.org/10.1097/aln.0b013e3181b55fae.
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Background Etomidate acts at gamma-Aminobutyric acid type A (GABAA) receptors containing beta2 or beta3, but not beta1 subunits. Mutations at beta residue 265 (Ser in beta1; Asn in beta2 or beta3) profoundly affect etomidate sensitivity. Whether these mutations alter etomidate binding remains uncertain. Methods Heterologously expressed alpha1beta2gamma2L GABAA receptors and receptors with beta2(N265S) or beta2(N265M) mutations were studied electrophysiologically in both Xenopus oocytes and HEK293 cells. Experiments quantified the impact of beta2N265 mutations or substituting beta1 for beta2 on basal channel activation, GABA EC50, maximal GABA efficacy, etomidate-induced leftward shift in GABA responses, etomidate direct activation, and rapid macrocurrent kinetics. Results were analyzed in the context of an established allosteric co-agonist mechanism. Results Mutations produced only small changes in basal channel activity, GABA EC50, maximal GABA efficacy, and macrocurrent kinetics. Relative to wild-type, beta2(N265S) reduced etomidate enhancement of apparent GABA affinity six-fold, and it reduced etomidate direct activation efficacy 14-fold. beta2(N265M) totally eliminated both etomidate modulation of GABA responses and direct channel activation. Mechanism-based analysis showed that the function of both mutants remains consistent with the allosteric co-agonist model and that beta2(N265S) reduced etomidate allosteric efficacy five-fold, whereas etomidate-binding affinity dropped threefold. Experiments swapping beta2 subunits for beta1 indicated that etomidate efficacy is reduced 34-fold, whereas binding affinity drops less than two-fold. Conclusions Mutations at beta2N265 profoundly alter etomidate sensitivity with only small changes in basal and GABA-dependent channel activity. Mutations at the beta2N265 residue or replacement of beta2 with beta1 influence etomidate efficacy much more than binding to inactive receptors.
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Husmann, M., J. Lehmann, B. Hoffmann, T. Hermann, M. Tzukerman, and M. Pfahl. "Antagonism between retinoic acid receptors." Molecular and Cellular Biology 11, no. 8 (August 1991): 4097–103. http://dx.doi.org/10.1128/mcb.11.8.4097-4103.1991.
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In the developing mouse, retinoic acid receptors (RARs) beta and gamma 1 are expressed in characteristic spatiotemporal patterns which are correlated with different developmental fates of the respective tissues. Understanding the cues that regulate the expression of the various RARs may therefore provide insights into the process of tissue diversification. Transcription of RAR beta is rapidly upregulated through a retinoic acid-responsive element (here referred to as the beta RARE) in its promoter. Like RAR alpha and RAR beta, RAR gamma 1 has been implicated in the activation of the beta RARE. Therefore, it is puzzling that RAR beta and RAR gamma 1 appear to be expressed in reciprocal patterns. In the present report, we show that RAR gamma 1, one of the two predominant RAR gamma isoforms, can inhibit the activity of RAR gamma 2, RAR beta, and endogenous RAR on the beta RARE. In contrast, the three RAR gamma isoforms tested and RAR beta activated a palindromic thyroid hormone response element with similar levels of efficiency. The differential activity of RAR gamma 1 compared with that of RAR beta appears to reside in both the N-terminal and the C-terminal halves of RAR gamma 1. RAR gamma 1-mediated inhibition of other RARs may involve competition for the response element as well as direct interaction with other receptors and might be part of a regulatory system contributing to the characteristic tissue distribution of the various RARs.
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Husmann, M., J. Lehmann, B. Hoffmann, T. Hermann, M. Tzukerman, and M. Pfahl. "Antagonism between retinoic acid receptors." Molecular and Cellular Biology 11, no. 8 (August 1991): 4097–103. http://dx.doi.org/10.1128/mcb.11.8.4097.
Full textAbstract:
In the developing mouse, retinoic acid receptors (RARs) beta and gamma 1 are expressed in characteristic spatiotemporal patterns which are correlated with different developmental fates of the respective tissues. Understanding the cues that regulate the expression of the various RARs may therefore provide insights into the process of tissue diversification. Transcription of RAR beta is rapidly upregulated through a retinoic acid-responsive element (here referred to as the beta RARE) in its promoter. Like RAR alpha and RAR beta, RAR gamma 1 has been implicated in the activation of the beta RARE. Therefore, it is puzzling that RAR beta and RAR gamma 1 appear to be expressed in reciprocal patterns. In the present report, we show that RAR gamma 1, one of the two predominant RAR gamma isoforms, can inhibit the activity of RAR gamma 2, RAR beta, and endogenous RAR on the beta RARE. In contrast, the three RAR gamma isoforms tested and RAR beta activated a palindromic thyroid hormone response element with similar levels of efficiency. The differential activity of RAR gamma 1 compared with that of RAR beta appears to reside in both the N-terminal and the C-terminal halves of RAR gamma 1. RAR gamma 1-mediated inhibition of other RARs may involve competition for the response element as well as direct interaction with other receptors and might be part of a regulatory system contributing to the characteristic tissue distribution of the various RARs.
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Friedrich, R. J., K. S. Campbell, and J. C. Cambier. "The gamma subunit of the B cell antigen-receptor complex is a C-terminally truncated product of the B29 gene." Journal of Immunology 150, no. 7 (April 1, 1993): 2814–22. http://dx.doi.org/10.4049/jimmunol.150.7.2814.
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Abstract The predominant Ag-receptor complex of B cells consists of mIgM or mIgD noncovalently associated with glycosylated heterodimers of Ig-alpha and Ig-beta or Ig-alpha and Ig-gamma. Upon B cell stimulation the associated proteins are phosphorylated, giving rise to pp32/33 (alpha), pp37 (beta), and pp34 (previously designated gamma). Ig-alpha and Ig-beta contain extended cytoplasmic structure (61 and 48 amino acids, respectively) and associate with cytoplasmic effectors indicating that they are directly involved in signal transduction. Here we report analysis of the structural relationship of mIgM- and mIgD-associated Ig-beta and Ig-gamma chains from mice. N-terminal sequence, immunoblotting, and physicochemical analyses show that both Ig-beta and Ig-gamma are products of the B cell-specific B29 gene and demonstrate that the 37-kDa Ig-beta protein is the full length predicted product of the B29 gene. The Ig-associated protein that migrates in the 34-kDa range is actually two distinct species. The minor species is a phosphorylatable and underglycosylated form of full length Ig-beta, and the major species is a C-terminally truncated form of B29, which we now designate Ig-gamma. This conclusion is based on the observations that Ig-gamma is composed of a core protein which is 3 to 4 kDa smaller than deglycosylated Ig-beta, it is not phosphorylated, unlike Ig-beta, and it does not react with an antiserum raised against a peptide of the seven C-terminal amino acids of B29. Based on these findings we estimate that Ig-gamma is truncated by about 30 to 36 amino acid residues and hypothesize that the most 3' B29 exon, which encodes the 32 C-terminal residues, may not be expressed in Ig-gamma. All of the documented B29 products are found in association with both mIgM and mIgD. Interestingly, Ig-gamma is found in intermediate and low density splenic B cells, but is not detectable in resting B cells. This raises the possibility that it may confer some distinct signaling function on the Ag receptors of these cells.
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Pace, B., Q. Li, K. Peterson, and G. Stamatoyannopoulos. "alpha-Amino butyric acid cannot reactivate the silenced gamma gene of the beta locus YAC transgenic mouse." Blood 84, no. 12 (December 15, 1994): 4344–53. http://dx.doi.org/10.1182/blood.v84.12.4344.bloodjournal84124344.
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Butyric acid, a naturally occurring fatty acid, has been shown to increase fetal hemoglobin in BFUe cultures, in primates, and in patients with beta chain hemoglobinopathies. The precise mechanism of gamma gene induction by butyrate is unknown. Butyrate may induce fetal hemoglobin production in vivo by reactivation of silenced gamma globin genes, by inhibiting the silencing of gamma genes, or by both mechanisms. We examined the effects of butyrate on gamma gene expression in transgenic mice carrying three types of constructs: microLCRA gamma mice, which continue to express the gamma gene in the adult stage of development at a level of one-third to one-fifth of the expression in the fetus; microLCRA gamma psi beta delta beta mice, which display correct developmental regulation of gamma and beta human globin genes and have low level gamma globin expression in the adult; and beta locus YAC mice, which display correct developmental regulation of epsilon, gamma, and beta globin genes and have a totally silenced gamma gene in the adult stage. Animals were treated with a continuous infusion of alpha-amino butyric acid (alpha-ABA) for 7 days. In microLCRA gamma mice alpha-ABA produced up to a 43-fold induction of gamma and 9-fold induction of mouse alpha globin genes. In contrast, butyrate did not induce gamma globin expression in the beta locus YAC mice. However, the gamma globin genes of beta locus YAC mice were activated after administration of 5-azacytidine (5-azaC), and the level of gamma globin expression was further increased by administration of alpha-ABA. These results suggest that butyrate cannot reactivate a totally silenced gamma gene and that induction of fetal hemoglobin by this compound may require the presence of preactivated gamma globin genes.
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Munck, B. G., L. K. Munck, S. N. Rasmussen, and A. Polache. "Specificity of the imino acid carrier in rat small intestine." American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 266, no. 4 (April 1, 1994): R1154—R1161. http://dx.doi.org/10.1152/ajpregu.1994.266.4.r1154.
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The rat intestinal imino acid carrier is chloride independent, while in guinea pig and rabbit intestine it is chloride dependent. While non-alpha-amino acids do not significantly interact with guinea pig and rabbit imino acid carriers, inhibition studies had indicated that in rat small intestine beta-alanine, gamma-aminobutyric acid (GABA), and probably taurine might be transported by the imino acid carrier. The present study of rat jejunum demonstrates that the half-maximal activation concentration of beta-alanine (K1/2 beta-Ala) is identical to its inhibition constant (Ki beta-Ala) against GABA, that K1/2GABA is identical to KiGABA against beta-alanine, that proline and sarcosine have identical values of Ki against beta-alanine and GABA, and that Ki of beta-alanine and proline against sarcosine are equal to their K1/2 values. Taurine inhibits the transport of beta-alanine, and 300 mM proline and beta-alanine reduce the transport of taurine measured at 80 mM taurine to the level expected for the diffusive contribution, corresponding to Ki values equal to those against sarcosine. Thus the rat imino acid carrier is the principal carrier of taurine and the only carrier of beta-alanine and GABA. It is also demonstrated that alpha-amino-monocarboxylic acids with side chains in excess of one methyl group do not significantly interact with the imino acid carrier, and the lack of stereospecificity is confirmed.
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Gugneja, S., J. V. Virbasius, and R. C. Scarpulla. "Four structurally distinct, non-DNA-binding subunits of human nuclear respiratory factor 2 share a conserved transcriptional activation domain." Molecular and Cellular Biology 15, no. 1 (January 1995): 102–11. http://dx.doi.org/10.1128/mcb.15.1.102.
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Nuclear respiratory factor 2 (NRF-2) was previously purified to near homogeneity from HeLa cells on the basis of its ability to bind tandem recognition sites in the rat cytochrome oxidase subunit IV (RCO4) promoter. It consisted of five subunits, alpha, beta 1, beta 2, gamma 1, and gamma 2. Sequencing of tryptic peptides from alpha and from mixtures of the two beta or two gamma subunits revealed sequence identities with subunits of the mouse GA-binding protein (GABP), a ubiquitously expressed ETS domain activator composed of three subunits, alpha, beta 1, and beta 2. To understand the precise relationship between NRF-2 and GABP, cDNAs for all five NRF-2 subunits have now been cloned and their products have been overexpressed. The results establish that the two additional NRF-2 subunits are molecular variants that differ from GABP beta 1 and beta 2 by having a 12-amino-acid insertion containing two serine doublets. PCR and RNase protection assays show that mRNAs for these variants are expressed in the human but not the rodent cells and tissues examined. The insertion did not alter the ability of the beta and gamma subunits to associate with alpha, the DNA-binding subunit, nor did it affect the ability of NRF-2 beta 1 or beta 2 to direct high-affinity binding of alpha to tandem sites in the RCO4 promoter. In addition, the four NRF-2 beta and gamma subunits were equally proficient in activating transcription in transfected cells when fused to a GAL4 DNA-binding domain. The domain responsible for this transcriptional activation was localized by deletion mapping to a region of approximately 70 amino acids that is conserved in all four NRF-2 beta and gamma subunits. The repeated glutamine-containing hydrophobic clusters within this region bear a strong resemblance to those recently implicated in protein-protein interactions within the transcriptional apparatus.
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Qu, Z. X., J. Odin, J. D. Glass, and J. C. Unkeless. "Expression and characterization of a truncated murine Fc gamma receptor." Journal of Experimental Medicine 167, no. 3 (March 1, 1988): 1195–210. http://dx.doi.org/10.1084/jem.167.3.1195.
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We have isolated a recombinant secreted Fc gamma R beta molecule by deletion of the transmembrane and cytoplasmic domains encoding sequence from a Fc gamma R beta 1 cDNA clone, and insertion of the truncated cDNA into a eukaryotic expression vector, pcEXV-3. To express and amplify the production of the truncated Fc gamma R beta molecule, we transfected the truncated cDNA plasmid into a dihydrofolate reductase-minus CHO cell line along with a dhfr minigene, and amplified the gene products with methotrexate. The resulting cell line secretes 2-3 micrograms/ml/24 h of truncated Fc gamma R beta, which can be readily purified by affinity chromatography on IgG-Sepharose. The truncated Fc gamma R beta has a Mr of 31-33,000 on SDS-PAGE and is glycosylated. N-glycosidase F cleavage reduces the Mr to 19,000, consistent with the size of the truncated product, 176 amino acid residues. There are two disulfide bonds in the protein. Binding of immune complexes formed between DNP20BSA and anti-DNP mAbs reveals better binding of IgG1 aggregates than that of IgG2b and IgG2a aggregates. The binding of the immune complexes was somewhat better at more acidic pH, in contrast to previous experiments with binding of purified Fc gamma R to immune complex-coated beads. We were surprised to observe that the truncated Fc gamma R beta did not react with the anti-Fc gamma R mAb 6B7C. Previous work had shown that 6B7C reacts with Fc gamma R on immunoblots, fails to bind to the surface of resting B cells and peritoneal macrophages, but does bind to macrophage cell lines and LPS-stimulated B cells. We show, by binding of mAb 6B7C to a peptide conjugate, that the 6B7C epitope lies within residues 169-183 of the intact Fc gamma R beta, which is just outside the plasma membrane. The availability of the truncated Fc gamma R beta in microgram quantities should facilitate further analysis of structure and function of these receptors.
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Kamei, Y., T. Kawada, R. Kazuki, and E. Sugimoto. "Retinoic acid receptor γ 2 gene expression is up-regulated by retinoic acid in 3T3-L1 preadipocytes." Biochemical Journal 293, no. 3 (August 1, 1993): 807–12. http://dx.doi.org/10.1042/bj2930807.
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Retinoids, especially all-trans retinoic acid (RA), have been shown to inhibit the differentiation of preadipose cells. In the present study, the expression of retinoic acid receptors (RAR alpha, beta and gamma) and retinoid X receptors (RXR alpha, beta and gamma) was examined by Northern blot analysis in rat adipose tissue and mouse 3T3-L1 adipose cells. The adipose tissue and/or 3T3-L1 cells expressed mRNAs for a number of nuclear retinoid receptors, including RAR alpha, beta and gamma, and RXR alpha, beta and gamma. RAR alpha, RAR gamma, RXR alpha and RXR beta mRNAs were abundant in adipose tissue and 3T3-L1 cells. RXR gamma mRNA was detected in adipose tissue but not in 3T3-L1 cells. Treatment of 3T3-L1 cells with 1 microM RA led to a 4-5-fold increase in the RAR gamma mRNA level, but only a trace amount of RAR beta mRNA was detected. RAR gamma mRNA expression was rapidly (within 2 h) induced by physiological concentrations of RA in a dose-dependent manner. The response of RAR gamma mRNA expression to RA was reversible; rapid disappearance of RAR gamma mRNA occurred on RA removal. In addition, the induction of RAR gamma expression did not require de novo protein synthesis, but was completely abolished by an inhibitor of RNA synthesis. Using RAR gamma 1 and gamma 2 isoform-specific probes, the patterns of RAR gamma 1 and gamma 2 mRNA expression in 3T3-L1 cells in the presence and absence of RA were examined. RAR gamma 1 mRNA was detected in 3T3-L1 cells but was not affected by RA treatment; however, RAR gamma 2 mRNA was strongly induced by RA.
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Hill, MA, and P. Gunning. "Beta and gamma actin mRNAs are differentially located within myoblasts." Journal of Cell Biology 122, no. 4 (August 15, 1993): 825–32. http://dx.doi.org/10.1083/jcb.122.4.825.
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Actin is of fundamental importance to all eukaryotic cells. Of the six mammalian actins, beta (beta) and gamma (gamma) cytoplasmic are the isoforms found in all nonmuscle cells and differ by only four amino acids at the amino-terminal region. Both genes are regulated temporally and spatially, though no differences in protein function have been described. Using fluorescent double in situ hybridization we describe the simultaneous intracellular localization of both beta and gamma actin mRNA. This study shows that myoblasts differentially segregate the beta and gamma actin mRNAs. The distribution of gamma actin mRNA, only to perinuclear and nearby cytoplasm, suggests a distribution based on diffusion or restriction to nearby cytoplasm. The distribution of beta actin mRNA, perinuclear and at the cell periphery, implicates a peripheral localizing signal which is unique to the beta isoform. The peripheral beta actin mRNA corresponded to cellular morphologies, extending processes, and ruffling edges that reflect cell movement. Total actin and gamma actin protein steady-state distributions were identified by specific antibodies. gamma actin protein was found in both stress fibers and at the cell plasma membrane and does not correspond to its mRNA distribution. We suggest that localized protein synthesis rather than steady-state distribution functionally differentiates between the actin isoforms.
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Caspar-Bauguil, S., J. Arnaud, C. Gouaillard, X. Hou, C. Geisler, and B. Rubin. "Functionally important amino acids in the TCR revealed by immunoselection of membrane TCR-negative T cells." Journal of Immunology 152, no. 11 (June 1, 1994): 5288–98. http://dx.doi.org/10.4049/jimmunol.152.11.5288.
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Abstract A spontaneous TCR cell surface variant (3P11) of the Jurkat T cell line is described and characterized. 3P11 was selected by incubation of Jurkat cells with anti-TCR mAb followed by passage through Ig anti-Ig columns and cloning. 3P11 contained mRNA for both Ti alpha and Ti beta and CD3 gamma, delta, epsilon and zeta. Biochemical analyses demonstrated that all of the TCR components were produced in 3P11 cells. The Ti alpha beta/CD3 gamma delta epsilon zeta complex was assembled in the endoplasmic reticulum but the zeta did not associate with this complex. Epitopes recognized by the Ti beta chain specific mAb beta F1 and JOVI as well as anti-V beta 8 were affected in the 3P11 Ti beta chain indicating that the 3P11 Ti beta chain was mutated. Transfection of a wild-type Ti beta cDNA into 3P11 cells reconstituted TCR expression. Sequence analyses of the 3P11 Ti beta chain demonstrated a guanine to adenine change in the second nucleotide of the triplet coding for cysteine191 resulting in a cysteine to tyrosine exchange. Cysteine191 is the C-terminal cysteine involved in the intrachain disulfide bond in the C domain of the Ti beta chain; thus, the 3P11 Ti beta chain did not contain this disulfide bond. Transfection of a site-directed Ti beta chain containing the 3P11 mutation into a Ti beta negative variant of the Jurkat cell line resulted in a TCR phenotype identical with 3P11 demonstrating that the mutation identified in the 3P11 Ti beta chain was the sole cause for the 3P11 defect.
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Kato, S., H. Mano, T. Kumazawa, Y. Yoshizawa, R. Kojima, and S. Masushige. "Effect of retinoid status on α, β and γ retinoic acid receptor mRNA levels in various rat tissues." Biochemical Journal 286, no. 3 (September 15, 1992): 755–60. http://dx.doi.org/10.1042/bj2860755.
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We have investigated the effects of retinoids, vitamin D and thyroid hormone on the levels of retinoic acid receptor (RAR)alpha, RAR beta and RAR gamma mRNAs in intact animals. Although vitamin A deficiency caused no significant changes in the levels of RAR alpha and RAR gamma mRNAs, the level of RAR beta transcripts was greatly decreased in various tissues of vitamin A-deficient rats, but was restored rapidly to a normal level after administration of retinoic acid. Retinol also restored the RAR beta mRNA level, but the magnitude and kinetics of the induction differed from those by retinoic acid. The use of specific inhibitors demonstrated that this autoregulation of RAR beta gene expression in vivo occurred at the transcriptional level. In addition, from these results it was postulated that the maintenance of the normal RAR beta mRNA levels seemed to require a threshold serum retinol concentration (about 25 micrograms/dl). Moreover, we found that administration of retinol and retinoic acid to normal rats caused the overexpression of RAR beta transcripts (2-15-fold) when compared with the control levels of RAR beta mRNA, although the levels of RAR alpha and RAR gamma mRNAs were not affected. Vitamin D and thyroid hormone did not modulate the levels of RAR transcripts. These findings clearly indicate the specific ligand regulation of RAR beta gene expression in intact animals. The altered levels of RAR beta according to retinoid status may affect retinoid-inducible gene expression.
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Redfern, C. P., and C. Todd. "Retinoic acid receptor expression in human skin keratinocytes and dermal fibroblasts in vitro." Journal of Cell Science 102, no. 1 (May 1, 1992): 113–21. http://dx.doi.org/10.1242/jcs.102.1.113.
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Retinoic acid is essential for the normal differentiation of epithelia but its cellular function is obscure. The expression patterns of retinoic acid receptors (RARs) in skin cell types may give an insight into the role of retinoic acid in skin. We have compared the patterns of RAR expression in human keratinocytes and dermal fibroblasts in vitro, and studied the effects of retinoic acid on RAR expression. RAR-alpha and RAR-gamma were expressed in keratinocytes and fibroblasts: RAR-gamma was expressed at similar levels in both cell types but RAR-alpha was more abundant in fibroblasts. There were no differences in expression of either RAR-alpha or RAR-gamma between stratifying (high-calcium medium) and proliferating (low-calcium medium) keratinocytes and expression of these RARs was unaffected by retinoic acid. RAR-beta was undetectable in keratinocytes. In the majority of fibroblast cell lines, RAR-beta transcripts were either undetectable or expressed at a low level. Retinoic acid at low concentrations (10(−10) to 10(−9) M) rapidly induced the expression of RAR-beta. Cyclic adenosine monophosphate (cAMP) analogues inhibit RAR-beta induction in teratocarcinoma cells. However, dibutyryl-cAMP did not affect RAR-beta induction in fibroblasts. Forskolin, an adenylate cyclase activator, and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) decreased constitutive RAR-beta mRNA levels but did not block induction of RAR-beta by retinoic acid. Since intracellular cAMP levels were only increased detectably in response to forskolin, the reduction in constitutive levels of RAR-beta mRNA may be mediated by mechanisms other than via cAMP.
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Kawahara, A., Y. Minami, and T. Taniguchi. "Evidence for a critical role for the cytoplasmic region of the interleukin 2 (IL-2) receptor gamma chain in IL-2, IL-4, and IL-7 signalling." Molecular and Cellular Biology 14, no. 8 (August 1994): 5433–40. http://dx.doi.org/10.1128/mcb.14.8.5433-5440.1994.
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The high-affinity interleukin 2 receptor (IL-2R) consists of at least three distinct subunits: the IL-2R alpha chain (IL-2R alpha), beta chain (IL-2R beta), and gamma chain (IL-2R gamma). It has been shown that the cytoplasmic region of IL-2R beta, but not of IL-2R alpha, is essential for IL-2 signalling to the cell interior. In the present study, we examined the functional role of the IL-2R gamma cytoplasmic region in the IL-3-dependent mouse hematopoietic cell line BAF-B03, which expresses the endogenous IL-2R alpha and IL-2R gamma, or its subline F7, which additionally expresses human IL-2R beta cDNA. We show that overexpression of a mutant IL-2R gamma, lacking all but 7 amino acids of its cytoplasmic region, results in the selective inhibition of IL-2-induced c-fos gene activation and cellular proliferation in F7 cells. When two chimeric receptor molecules in which the cytoplasmic regions of IL-2R beta and IL-2R gamma had been swapped with each other (IL-2R beta/gamma and IL-2R gamma/beta) were coexpressed in BAF-B03, the cells responded to IL-2. These results indicate the critical importance of the IL-2-induced functional cooperation of the two cytoplasmic regions. Finally, we provide evidence that the IL-2R gamma cytoplasmic region is also critical for the IL-4 and IL-7-induced growth signal transduction in BAF-B03.
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Kawahara, A., Y. Minami, and T. Taniguchi. "Evidence for a critical role for the cytoplasmic region of the interleukin 2 (IL-2) receptor gamma chain in IL-2, IL-4, and IL-7 signalling." Molecular and Cellular Biology 14, no. 8 (August 1994): 5433–40. http://dx.doi.org/10.1128/mcb.14.8.5433.
Full textAbstract:
The high-affinity interleukin 2 receptor (IL-2R) consists of at least three distinct subunits: the IL-2R alpha chain (IL-2R alpha), beta chain (IL-2R beta), and gamma chain (IL-2R gamma). It has been shown that the cytoplasmic region of IL-2R beta, but not of IL-2R alpha, is essential for IL-2 signalling to the cell interior. In the present study, we examined the functional role of the IL-2R gamma cytoplasmic region in the IL-3-dependent mouse hematopoietic cell line BAF-B03, which expresses the endogenous IL-2R alpha and IL-2R gamma, or its subline F7, which additionally expresses human IL-2R beta cDNA. We show that overexpression of a mutant IL-2R gamma, lacking all but 7 amino acids of its cytoplasmic region, results in the selective inhibition of IL-2-induced c-fos gene activation and cellular proliferation in F7 cells. When two chimeric receptor molecules in which the cytoplasmic regions of IL-2R beta and IL-2R gamma had been swapped with each other (IL-2R beta/gamma and IL-2R gamma/beta) were coexpressed in BAF-B03, the cells responded to IL-2. These results indicate the critical importance of the IL-2-induced functional cooperation of the two cytoplasmic regions. Finally, we provide evidence that the IL-2R gamma cytoplasmic region is also critical for the IL-4 and IL-7-induced growth signal transduction in BAF-B03.
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Narayanan, A. S., R. C. Page, and J. Swanson. "Collagen synthesis by human fibroblasts. Regulation by transforming growth factor-β in the presence of other inflammatory mediators." Biochemical Journal 260, no. 2 (June 1, 1989): 463–69. http://dx.doi.org/10.1042/bj2600463.
Full textAbstract:
We have examined the combined effects of transforming growth factor-beta (TGF-beta), serum and gamma-interferon (gamma-IFN) on collagen synthesis by fibroblasts and compared the response of fibroblast subpopulations to TGF-beta. Human diploid fibroblasts were treated with TGF-beta alone and with serum of gamma-IFN. Cells were labelled with radioactive amino acids, and collagen production was measured as collagenase-digestible radioactivity. Collagen mRNA was determined by a solution-hybridization assay using procollagen-alpha 1[I] cDNA clone HF 677. The results showed that either serum or TGF-beta increased incorporation, collagen production and mRNA by fibroblasts approx. 2-fold; however, collagen synthesis relative to total protein synthesis and collagen mRNA relative to total polyadenylated [poly(A)+] RNA were not affected. Only serum activated cell growth. Collagen production increased approx. 4-fold in cells exposed to both TGF-beta and serum, and this increase was equal to that expected for an additive effect by both components. Treatment with gamma-IFN decreased collagen production and collagen mRNA to 44 and 40% respectively, whereas total incorporation and poly(A)+ RNA were affected only marginally. Cells exposed simultaneously to both gamma-IFN and TGF-beta produced less collagen and contained less mRNA than did those treated with TGF-beta alone. The gamma-IFN decreased collagen synthesis in control and TGF-beta-treated cultures to a similar extent, and TGF-beta increased collagen synthesis 2-fold in cells pre-treated with gamma-IFN. Fibroblast strains obtained in medium containing plasma-derived serum synthesized approximately half as much collagen as did cells derived from the same explant in the presence of fresh human serum, and TGF-beta stimulated collagen production and mRNA in both cell strains. We conclude that TGF-beta, serum and gamma-IFN regulate collagen synthesis by independent mechanisms, and that the combined action of these components plays a significant role in regulating collagen synthesis during wound healing and tissue repair.
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McDonald, F. J., M. P. Price, P. M. Snyder, and M. J. Welsh. "Cloning and expression of the beta- and gamma-subunits of the human epithelial sodium channel." American Journal of Physiology-Cell Physiology 268, no. 5 (May 1, 1995): C1157—C1163. http://dx.doi.org/10.1152/ajpcell.1995.268.5.c1157.
Full textAbstract:
Amiloride-sensitive Na+ channels are an important component of the Na+ reabsorption pathway in a number of epithelia. Here we report the cloning and characterization of cDNAs encoding two subunits of the human kidney epithelial Na+ channel (beta- and gamma-hENaC). Their predicted amino acid sequences were highly homologous (83-85% identical) to the corresponding subunits reported from rat colon (beta- and gamma-rENaC). Both beta- and gamma-hENaC mapped to human chromosome 16. Northern blot analysis showed high expression of beta- and gamma-hENaC in kidney and lung and differential expression of the three subunits in other tissues. Coexpression of beta- and gamma-hENaC with alpha-hENaC in Xenopus oocytes produced Na+ channels with high selectivity for Na+ and high sensitivity to amiloride. In addition, human subunits were able to substitute for the corresponding rat subunits in forming functional Na+ channels, suggesting conservation of function and suggesting that differences in sequence do not disrupt interactions between subunits. These results suggest that human alpha-, beta-, and gamma-ENaC together form Na+ channels with properties that are similar to those observed in epithelia, and will allow further investigation into the role that these channels may play in human disease.
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Wileman, T., LP Kane, J. Young, GR Carson, and C. Terhorst. "Associations between subunit ectodomains promote T cell antigen receptor assembly and protect against degradation in the ER." Journal of Cell Biology 122, no. 1 (July 1, 1993): 67–78. http://dx.doi.org/10.1083/jcb.122.1.67.
Full textAbstract:
The T cell antigen receptor (TCR) is an oligomeric protein complex made from at least six different integral membrane proteins (alpha beta gamma delta epsilon and zeta). The TCR is assembled in the ER of T cells, and correct assembly is required for transport to the cell surface. Single subunits and partial receptor complexes are retained in the ER where TCR alpha, beta, and CD3 delta chains are degraded selectively. The information required for the ER degradation of the TCR beta chain is confined to the membrane anchor of the protein (Wileman et al., 1990c; Bonifacino et al., 1990b). In this study we show that the rapid degradation of the TCR beta chain is inhibited when it assembles with single CD3 gamma, delta, or epsilon subunits in the ER, and have started to define the role played by transmembrane anchors, and receptor ectodomains, in the masking proteolytic targeting information. Acidic residues within the membrane spanning domains of CD3 subunits were essential for binding to the TCR beta chain. TCR beta chains and CD3 subunits therefore interact via transmembrane domains. However, when sites of binding were restricted to the membrane anchor of the TCR beta chain, stabilization by CD3 subunits was markedly reduced. Interactions between membrane spanning domains were not, therefore, sufficient for the protection of the beta chain from ER proteolysis. The presence of the C beta domain, containing the first 150 amino acids of the TCR ectodomain, greatly increased the stability of complexes formed in the ER. For assembly with CD3 epsilon, stability was further enhanced by the V beta amino acids. The results showed that the efficient neutralization of transmembrane proteolytic targeting information required associations between membrane spanning domains and the presence of receptor ectodomains. Interactions between receptor ectodomains may slow the dissociation of CD3 subunits from the beta chain and prolong the masking of transmembrane targeting information. In addition, the close proximity of TCR and CD3 ectodomains within the ER may provide steric protection from the action of proteases within the ER lumen.
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Page, L. J., and M. S. Robinson. "Targeting signals and subunit interactions in coated vesicle adaptor complexes." Journal of Cell Biology 131, no. 3 (November 1, 1995): 619–30. http://dx.doi.org/10.1083/jcb.131.3.619.
Full textAbstract:
There are two clathrin-coated vesicle adaptor complexes in the cell, one associated with the plasma membrane and one associated with the TGN. The subunit composition of the plasma membrane adaptor complex is alpha-adaptin, beta-adaptin, AP50, and AP17; while that of the TGN adaptor complex is gamma-adaptin, beta'-adaptin, AP47, and AP19. To search for adaptor targeting signals, we have constructed chimeras between alpha-adaptin and gamma-adaptin within their NH2-terminal domains. We have identified stretches of sequence in the two proteins between amino acids approximately 130 and 330-350 that are essential for targeting. Immunoprecipitation reveals that this region determines whether a construct coassemblies with AP50 and AP17, or with AP47 and AP19. These observations suggest that these other subunits may play an important role in targeting. In contrast, beta- and beta'-adaptins are clearly not involved in this event. Chimeras between the alpha- and gamma-adaptin COOH-terminal domains reveal the presence of a second targeting signal. We have further investigated the interactions between the adaptor subunits using the yeast two-hybrid system. Interactions can be detected between the beta/beta'-adaptins and the alpha/gamma-adaptins, between the beta/beta'-adaptins and the AP50/AP47 subunits, between alpha-adaptin and AP17, and between gamma-adaptin and AP19. These results indicate that the adaptor subunits act in concert to target the complex to the appropriate membrane.
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Ling, P. D., M. K. Warren, and S. N. Vogel. "Antagonistic effect of interferon-beta on the interferon-gamma-induced expression of Ia antigen in murine macrophages." Journal of Immunology 135, no. 3 (September 1, 1985): 1857–63. http://dx.doi.org/10.4049/jimmunol.135.3.1857.
Full textAbstract:
Abstract The cell surface expression of I region-associated (Ia) antigens by murine and human macrophages has been shown by investigators from a number of laboratories to be induced in a dose-dependent fashion by IFN-gamma, which is free of other lymphokines. The experiments described in this report demonstrate that fibroblast-derived IFN-beta exerts an antagonistic effect on IFN-gamma induced Ia expression in murine macrophages. Simultaneous addition of IFN-beta and IFN-gamma to peritoneal exudate macrophages results in decreased Ia expression when compared with macrophages treated with IFN-gamma only. Different sources of highly purified IFN-beta, as well as a recombinant human IFN-alpha (A/D Bgl; shown previously to be as active as IFN-beta in several other murine systems) acted in a similar antagonistic fashion to IFN-gamma-induced Ia induction. The down-regulation of Ia expression by IFN-beta is dose-dependent over a concentration range up to 100 U/ml. Time-course experiments indicated that for IFN-beta to down-regulate IFN-gamma-induced Ia, it had to be present either before stimulation with IFN-gamma or during the first 24 hr of simultaneous stimulation. Further experiments in which a highly specific antibody against IFN-alpha/beta was added to the cultures confirmed the findings of the time-course experiments. Inhibitors of the arachidonic acid pathway failed to reverse the effect of IFN-beta to reduce Ia antigen expression, which suggests that this inhibition is not prostaglandin mediated. Thus, these findings support a role for type I IFN as naturally occurring substances that negatively regulate the expression of class II molecules.
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Robinson, M. S. "Cloning and expression of gamma-adaptin, a component of clathrin-coated vesicles associated with the Golgi apparatus." Journal of Cell Biology 111, no. 6 (December 1, 1990): 2319–26. http://dx.doi.org/10.1083/jcb.111.6.2319.
Full textAbstract:
Adaptins are the major components of adaptors, the protein complexes that link clathrin to transmembrane proteins (e.g., receptors) in coated pits and vesicles. The plasma membrane adaptor contains an alpha-adaptin subunit and a beta-adaptin subunit, while the Golgi adaptor contains a gamma-adaptin subunit and a beta'-adaptin subunit. A partial cDNA clone encoding gamma-adaptin was isolated from a bovine brain expression library by screening with antibodies, and was used to obtain a cDNA clone from a mouse brain library containing the full coding sequence. The identity of the clones was confirmed by protein sequencing. The deduced amino acid sequence of gamma-adaptin was found to be homologous to that of alpha-adaptin, with several stretches of identical amino acids or conservative substitutions in the first approximately 70 kD, and 25% identity overall. Weaker homology was seen between gamma- and beta-adaptins. Like both alpha- and beta-adaptins, gamma-adaptin has a proline and glycine-rich hinge region, dividing it into NH2- and COOH-terminal domains. A chimeric gamma-adaptin was constructed from the mouse and bovine cDNAs and transfected into Rat 1 fibroblasts. Immunofluorescence microscopy was carried out using an mAb which recognizes an epitope present on the chimera but not found on the rodent protein. The construct was found to have a distribution typical of endogenous gamma-adaptin. Using this transfection system, it should now be possible to exchange domains between alpha- and gamma-adaptins, to try to find out how adaptors are targeted to the appropriate membrane compartment of the cell, and how they recruit the appropriate receptors into the coated vesicle.
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Liang, T., and S. Liao. "Inhibition of steroid 5α-reductase by specific aliphatic unsaturated fatty acids." Biochemical Journal 285, no. 2 (July 15, 1992): 557–62. http://dx.doi.org/10.1042/bj2850557.
Full textAbstract:
Human or rat microsomal 5 alpha-reductase activity, as measured by enzymic conversion of testosterone into 5 alpha-dihydrotestosterone or by binding of a competitive inhibitor, [3H]17 beta-NN-diethulcarbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one ([3H]4-MA) to the reductase, is inhibited by low concentrations (less than 10 microM) of certain polyunsaturated fatty acids. The relative inhibitory potencies of unsaturated fatty acids are, in decreasing order: gamma-linolenic acid greater than cis-4,7,10,13,16,19-docosahexaenoic acid = cis-6,9,12,15-octatetraenoic acid = arachidonic acid = alpha-linolenic acid greater than linoleic acid greater than palmitoleic acid greater than oleic acid greater than myristoleic acid. Other unsaturated fatty acids such as undecylenic acid, erucic acid and nervonic acid, are inactive. The methyl esters and alcohol analogues of these compounds, glycerols, phospholipids, saturated fatty acids, retinoids and carotenes were inactive even at 0.2 mM. The results of the binding assay and the enzymic assay correlated well except for elaidic acid and linolelaidic acid, the trans isomers of oleic acid and linoleic acid respectively, which were much less active than their cis isomers in the binding assay but were as potent in the enzymic assay. gamma-Linolenic acid had no effect on the activities of two other rat liver microsomal enzymes: NADH:menadione reductase and glucuronosyl transferase. gamma-Linolenic acid, the most potent inhibitor tested, decreased the Vmax. and increased Km values of substrates, NADPH and testosterone, and promoted dissociation of [3H]4-MA from the microsomal reductase. gamma-Linolenic acid, but not the corresponding saturated fatty acid (stearic acid), inhibited the 5 alpha-reductase activity, but not the 17 beta-dehydrogenase activity, of human prostate cancer cells in culture. These results suggest that unsaturated fatty acids may play an important role in regulating androgen action in target cells.
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Kage, R., L. Thim, W. Creutzfeldt, and J. M. Conlon. "Post-translational processing of preprotachykinins. Isolation of protachykinin-(1-37)-peptide from human adrenal-medullary phaeochromocytoma tissue." Biochemical Journal 253, no. 1 (July 1, 1988): 203–7. http://dx.doi.org/10.1042/bj2530203.
Full textAbstract:
The biosynthetic precursors of the mammalian tachykinins, alpha-, beta-and gamma-preprotachykinins, contain a common N-terminal region of 74 amino acids. A polyclonal antiserum was raised against a synthetic peptide representing N-tyrosylated beta-preprotachykinin-(48-56)-peptide as predicted from the nucleotide sequence of cloned DNA complementary to human beta-preprotachykinin mRNA. By using this antiserum in radioimmunoassay, a single immunoreactive peptide was identified in an extract of a human pheochromocytoma that produced substance P and neurokinin A. Partial microsequencing and determination of the amino acid composition of the peptide indicated identity with preprotachykinin-(20-56)-peptide. Thus the data demonstrate that the Ala19-Glu20 bond in preprotachykinin is the site of cleavage of the signal peptide.
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Yatani, A., K. Okabe, L. Birnbaumer, and A. M. Brown. "Detergents, dimeric G beta gamma, and eicosanoid pathways to muscarinic atrial K+ channels." American Journal of Physiology-Heart and Circulatory Physiology 258, no. 5 (May 1, 1990): H1507—H1514. http://dx.doi.org/10.1152/ajpheart.1990.258.5.h1507.
Full textAbstract:
Control experiments for the direct effects of G protein beta gamma-subunits (G beta gamma) on muscarinic atrial K+ channel [K+ (ACh)] currents have produced different results (Nature Lond. 327: 21-22, 1987; Nature Lond. 325: 296-297, 1987; Cold Spring Harbor Symp. Quant. Biol. 53: 365-373, 1989). A recent view is that stimulation is indirect via phospholipase by (PLA2) and arachidonic acid (AA) metabolites (Nature Lond. 337: 504-505, 1989). On reexamination we found that 1) the zwitterionic detergent 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) used to suspend beta gamma stimulates atrial K+ (ACh) currents by itself, and the effects are concentration and Mg2+ dependent; 2) CHAPS stimulates atrial ATP-sensitive K+ channel and inwardly rectifying K+ channel currents; 3) blockers of eicosanoid pathways have nonspecific effects on atrial K+, Ca2+, and Na+ channels. We have confirmed that detergent-free, hydrophilic G beta gamma-subunits inhibit K+ (ACh) currents. Stimulatory effects of dimeric G beta gamma could not be separated from stimulatory effects of detergent, and blockers of PLA2 or lipoxygenase pathways do not clearly establish the significance of these pathways to atrial K+ (ACh) currents.
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Yao, X., C. Chaponnier, G. Gabbiani, and J. G. Forte. "Polarized distribution of actin isoforms in gastric parietal cells." Molecular Biology of the Cell 6, no. 5 (May 1995): 541–57. http://dx.doi.org/10.1091/mbc.6.5.541.
Full textAbstract:
The actin genes encode several structurally similar, but perhaps functionally different, protein isoforms that mediate contractile function in muscle cells and determine the morphology and motility in nonmuscle cells. To reveal the isoform profile in the gastric monomeric actin pool, we purified actin from the cytosol of gastric epithelial cells by DNase I affinity chromatography followed by two-dimensional gel electrophoresis. Actin isoforms were identified by Western blotting with a monoclonal antibody against all actin isoforms and two isoform-specific antibodies against cytoplasmic beta-actin and gamma-actin. Densitometry revealed a ratio for beta-actin/gamma-actin that equaled 0.73 +/- 0.09 in the cytosol. To assess the distribution of actin isoforms in gastric glandular cells in relation to ezrin, a putative membrane-cytoskeleton linker, we carried out double immunofluorescence using actin-isoform-specific antibodies and ezrin antibody. Immunostaining confirmed that ezrin resides mainly in canaliculi and apical plasma membrane of parietal cells. Staining for the beta-actin isoform was intense along the entire gland lumen and within the canaliculi of parietal cells, thus predominantly near the apical membrane of all gastric epithelial cells, although lower levels of beta-actin were also identified near the basolateral membrane. The gamma-actin isoform was distributed heavily near the basolateral membrane of parietal cells, with much less intense staining of parietal cell canaliculi and no staining of apical membranes. Within parietal cells, the cellular localization of beta-actin, but not gamma-actin, isoform superimposed onto that of ezrin. In a search for a possible selective interaction between actin isoforms and ezrin, we carried out immunoprecipitation experiments on gastric membrane extracts in which substantial amounts of actin were co-eluted with ezrin from an anti-ezrin affinity column. The ratio of beta-actin/gamma-actin in the immunoprecipitate (beta/gamma = 2.14 +/- 0.32) was significantly greater than that found in the cytosolic fraction. In summary, we have shown that beta- and gamma-actin isoforms are differentially distributed in gastric parietal cells. Furthermore, our data suggest a preferential, but not exclusive, interaction between beta-actin and ezrin in gastric parietal cells. Finally, our results suggest that the beta- and gamma-actin-based cytoskeleton networks might function separately in response to the stimulation of acid secretion.
29
Corcoran, J., B. Shroot, J. Pizzey, and M. Maden. "The role of retinoic acid receptors in neurite outgrowth from different populations of embryonic mouse dorsal root ganglia." Journal of Cell Science 113, no. 14 (July 15, 2000): 2567–74. http://dx.doi.org/10.1242/jcs.113.14.2567.
Full textAbstract:
Dorsal root ganglion (DRG) neurons can be categorised into at least three types, based upon their neurotrophin requirement for survival. We have analysed the expression of the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs) in NGF, NT-3 and BDNF dependent neurons isolated from embryonic day (E)13.5 mouse DRG. We show that each population of neurons expressed each of the three RXRs, (alpha), (beta) and (gamma); however, whilst the NGF and NT-3 dependent neurons expressed each of the RARs (alpha), (beta) and (gamma), the BDNF dependent neurons only expressed RAR(alpha) and (beta). When retinoic acid was added to each of the neuronal classes only the NGF and NT-3 dependent neurons responded by extending neurites, and this response involved the upregulation of RAR(beta)(2). This specificity was confirmed by the use of receptor-selective agonists as only a RAR(beta)-selective compound stimulated neurite outgrowth. These results suggest a role for RA acting via RAR(beta)(2) in the outgrowth of neurites.
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Haynes, J. K., and L. Goldstein. "Volume-regulatory amino acid transport in erythrocytes of the little skate, Raja erinacea." American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 265, no. 1 (July 1, 1993): R173—R179. http://dx.doi.org/10.1152/ajpregu.1993.265.1.r173.
Full textAbstract:
Skate erythrocytes swell in a hypotonic medium and then reduce their volume mainly by releasing the beta-amino acids taurine and beta-alanine. Although these amino acids exhibit a net efflux, Na(+)-independent influx is also increased. Both the reduction in cell volume and increase in amino acid transport are inhibited by several inhibitors of band 3-mediated anion transport, including 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) [L. Goldstein and S. R. Brill, Am. J. Physiol. 260 (Regulatory Integrative Comp. Physiol. 29): R1014-R1020, 1991]. The objective of the present investigation was to further characterize the mechanism of volume-activated amino acid transport. Na(+)-independent amino acid uptake was studied because of the ease in controlling amino acid concentrations. Na(+)-independent taurine uptake was observed to be linear over a range of 0.1-15 mM and was not inhibited by 10 mM beta-alanine, suggesting that the transporter may be a channel rather than a carrier. The uptake of a variety of amino acids was examined to characterize the size of the putative channel. Glycine, beta-alanine, taurine, proline, gamma-aminobutyric acid (GABA), and threonine exhibited volume-activated transport that was DIDS inhibited, whereas aspartic acid, leucine, methionine, and ornithine were not transported. On the basis of the size of these amino acids, it appears that molecules containing eight or fewer major atoms and having a molecular mass of < 125-131 Da are transported during volume activation but larger molecules are not. We estimate the size of the channel to be 5.7-6.3 A in diameter.
31
Adachi, K., J. Pang, P. Konitzer, and S. Surrey. "Polymerization of recombinant hemoglobin F gamma E6V and hemoglobin F gamma E6V, gamma Q87T alone, and in mixtures with hemoglobin S." Blood 87, no. 4 (February 15, 1996): 1617–24. http://dx.doi.org/10.1182/blood.v87.4.1617.bloodjournal8741617.
Full textAbstract:
To further understand determinants for Hemoglobin (Hb) S polymerization, as well as the inhibitory mechanism of Hb F on Hb S polymerization, Hb F variants containing Val-gamma 6 (Hb F gamma E6V) or Val-gamma 6, Thr-gamma 87 (Hb F gamma E6V, gamma Q87T) were expressed in yeast. The oxy form of Hb F gamma E6V was about 10-fold less stable to mechanical agitation than native oxy Hb F, which is similar to stability differences comparing oxy Hb S and oxy Hb A. Deoxy Hb F gamma E6V showed approximately 20-fold decreased solubility compared with native deoxy Hb F in high phosphate buffer and formed gels like deoxy Hb S in low phosphate buffer, indicating that the Val- gamma 6 substitution decreases solubility of Hb F like Val-beta 6 in deoxy Hb S. Oversaturated deoxy Hb F gamma E6V polymerized without a delay time in low and high phosphate buffers, in contrast to deoxy Hb S, which is accompanied by a distinct delay time before polymerization. Deoxy Hb F gamma E6V, gamma Q87T also polymerized without a delay time like deoxy Hb F gamma E6V. These results suggest that deoxy Hb F gamma E6V gamma Q87T polymers are different from those of deoxy Hb S, and that contact sites differ from those of deoxy Hb S, even though both have the same primary donor (A3) and acceptor sites in the EF helix. These results also suggest that other amino acids in addition to beta 6 Val and amino acids in the F helix are critical for nucleation- controlled polymerization of deoxy Hb S. 1:1 mixtures of deoxy Hb S and either Hb F variant polymerized with a delay time when the concentrations for the Hb S/Hb F gamma E6V and Hb S/Hb F gamma E6V, gamma Q87T mixtures were about 2- and 1.5-fold, respectively, higher than that for Hb S. Logarithmic plots of delay time versus concentration for Hb S/Hb F gamma E6V mixtures showed the same straight line as the line for Hb S/Hb S beta T87Q mixtures, but values for Hb S/Hb F gamma E6V, gamma Q87T mixtures were intermediate between those for Hb S and Hb S/Hb F gamma E6V mixtures. A 1:1 mixture of deoxy Hb A and Hb F gamma E6V, gamma Q87T also polymerized, but exhibited biphasic kinetics, when the concentration was increased to more than 3.5-fold higher than that required for Hb S polymer formation. These results suggest that Gin-gamma 87 is a critical amino acid for exclusion of FS hybrids (alpha 2 beta S gamma) from nuclei formation with Hb S. Our findings also show that Val-gamma 6 in hybrids that form in mixtures of the Hb F variants with either Hb S or Hb A interacts with the hydrophobic acceptor pocket on the EF helix of an adjacent tetramer containing Thr-beta 87.
32
Nonaka, M., K. Nakayama, Y. D. Yeul, and M. Takahashi. "Complete nucleotide and derived amino acid sequences of sex-limited protein (Slp), nonfunctional isotype of the fourth component of mouse complement (C4)." Journal of Immunology 136, no. 8 (April 15, 1986): 2989–93. http://dx.doi.org/10.4049/jimmunol.136.8.2989.
Full textAbstract:
Abstract The nucleotide sequence coding for sex-limited protein (Slp), the testosterone-regulated isotype of the fourth component of mouse complement (C4), has been determined from cloned genomic DNA and cDNA fragments. The complete deduced amino acid sequence for the single chain precursor protein of Slp (pro-Slp) consists of 1716 residues. The mature beta, alpha, and gamma subunits contain 654, 763, and 291 amino acids, respectively. One potential carbohydrate attachment site is predicted from the beta-chain, five for the alpha-chain, and none for the gamma-chain. From the comparison with the mouse C4 sequences, an extensive overall sequence homology, 96.0% in nucleotides and 94.2% in amino acids, is observed. Only one deletion/insertion event is recognized between C4 and Slp sequences: three residues near the Cls cleavage site are deleted from Slp. The distribution of cysteine residues is completely conserved between pro-Slp and pro-C4.
33
BEYHAN, Omer, Akide OZCAN, Hatice OZCAN, Ebru KAFKAS, Salih KAFKAS, Mehmet SUTYEMEZ, and Sezai ERCISLI. "Fat, Fatty Acids and Tocopherol Content of Several Walnut Genotypes." Notulae Botanicae Horti Agrobotanici Cluj-Napoca 45, no. 2 (September 15, 2017): 437–41. http://dx.doi.org/10.15835/nbha45210932.
Full textAbstract:
There are seed propagated walnut (Juglans regia L.) populations with the vast genetic variation in different part of Turkey. There are also lots of monoecious and dichogamous genotypes in Turkey due to continuing sexual propagation. In this study, fruits of 19 selected walnut genotypes grown in Kahramanmaraş region were characterized based on their fat, fatty acid and tocopherol contents. The fatty acids content of genotypes were analyzed using Gas Chromatography. Tocopherol analyses such as alpha (α)-Tocopherol, gamma (γ) and beta (β) + delta (δ) were performed by HPLC technique. According to the total fat and fatty acid results, there were differences among genotypes on most of the fatty acids. Total fat ranged from 51.2 to 82.1%, stearic acid from 2.57 to 3.37%, myristic acid from 0.00 to 0.05%, palmitic acid from 6.42 to 7.92%, arachidic acid from 0.00 to 0.16%, linoleic acid from 53.23 to 63.62%, linolenic acid from 10.75 to 15.24%, oleic acid from 14.73 to 24.17% and palmitoleic acid from 0.00 to 0.16%, respectively. The same genotypes were evaluated based on their tocopherol content and (α)-Tocopherol, gamma (γ) and beta (β) + delta (δ) tocopherol were found between 23.47 and 38.04 μg/g, 161.09 and 292.56 μg/g and 16.93 and 32.34 μg/g, respectively.
34
Nowell, J., and V. Quaranta. "Chloroquine affects biosynthesis of Ia molecules by inhibiting dissociation of invariant (gamma) chains from alpha-beta dimers in B cells." Journal of Experimental Medicine 162, no. 4 (October 1, 1985): 1371–76. http://dx.doi.org/10.1084/jem.162.4.1371.
Full textAbstract:
Biosynthetic conversion of Ia oligomers from three chains (alpha, beta, gamma) to two (alpha, beta) before surface expression was inhibited in B lymphoid cells by treatment with chloroquine, resulting in the accumulation of Ia complexes composed of mature alpha and beta chains, and gamma chains at various states of sialylation. Other stages of Ia biosynthesis and processing appeared unaffected, indicating that chloroquine selectively interfered with the gamma chain dissociating mechanism itself. Similar effects were also observed with ammonium chloride. Because of the nature of such lysosomotropic agents, these results suggest that an intracellular acidic compartment may be involved in processing Ia oligomers to accomplish dissociation from gamma chains. Since chloroquine is known to inhibit Ia-restricted antigen presentation in accessory cells, our results raise the possibility that the pathways of antigen processing and Ia biosynthesis may use some common intracellular compartments.
35
Adachi, K., CH Lai, P. Konitzer, M. Donahee, A. Campbell, and S. Surrey. "Crystallization of recombinant hemoglobins with basic amino acid substitutions (Lys and Arg) at the beta 6 position." Blood 84, no. 4 (August 15, 1994): 1309–13. http://dx.doi.org/10.1182/blood.v84.4.1309.1309.
Full textAbstract:
Abstract We have produced recombinant hemoglobins (rHbs) alpha 2 beta 2(6Glu-- >Lys) (rHb beta E6K) and alpha 2 beta 2(6Glu-->Arg) (rHb beta E6R) using a yeast expression system coupled with a polymerase chain reaction (PCR)-based mutagenesis strategy for studies focused on defining determinants that facilitate crystallization of Hb C (alpha 2 beta 2(6Lys)). rHb beta E6K had the same electrophoretic mobility as native human Hb C, whereas rHb beta E6R migrated slightly slower than Hb C on cellulose acetate electrophoresis. The carbonmonoxy (CO) forms of rHb beta E6K and rHb beta E6R formed tetrahedral crystals in vitro in 2.3 mol/L phosphate buffer just like native Hb C. The Hb concentration required for crystallization of CO-rHb beta E6R was lower than that of CO-rHb beta E6K, suggesting that stronger basic amino acids at the beta 6 position accelerate crystallization of Hb. However, the size of rHb beta E6R crystals was smaller than that of rHb beta E6K. Crystallization of native Hb C and both rHbs was inhibited by Hb F. These results suggest that alpha 2 beta gamma-heterohybrids that have basic amino acids at the beta 6 position behave similarly and are unable to crystallize like Hb C.
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Adachi, K., CH Lai, P. Konitzer, M. Donahee, A. Campbell, and S. Surrey. "Crystallization of recombinant hemoglobins with basic amino acid substitutions (Lys and Arg) at the beta 6 position." Blood 84, no. 4 (August 15, 1994): 1309–13. http://dx.doi.org/10.1182/blood.v84.4.1309.bloodjournal8441309.
Full textAbstract:
We have produced recombinant hemoglobins (rHbs) alpha 2 beta 2(6Glu-- >Lys) (rHb beta E6K) and alpha 2 beta 2(6Glu-->Arg) (rHb beta E6R) using a yeast expression system coupled with a polymerase chain reaction (PCR)-based mutagenesis strategy for studies focused on defining determinants that facilitate crystallization of Hb C (alpha 2 beta 2(6Lys)). rHb beta E6K had the same electrophoretic mobility as native human Hb C, whereas rHb beta E6R migrated slightly slower than Hb C on cellulose acetate electrophoresis. The carbonmonoxy (CO) forms of rHb beta E6K and rHb beta E6R formed tetrahedral crystals in vitro in 2.3 mol/L phosphate buffer just like native Hb C. The Hb concentration required for crystallization of CO-rHb beta E6R was lower than that of CO-rHb beta E6K, suggesting that stronger basic amino acids at the beta 6 position accelerate crystallization of Hb. However, the size of rHb beta E6R crystals was smaller than that of rHb beta E6K. Crystallization of native Hb C and both rHbs was inhibited by Hb F. These results suggest that alpha 2 beta gamma-heterohybrids that have basic amino acids at the beta 6 position behave similarly and are unable to crystallize like Hb C.
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Zitnik, G., K. Peterson, G. Stamatoyannopoulos, and T. Papayannopoulou. "Effects of butyrate and glucocorticoids on gamma- to beta-globin gene switching in somatic cell hybrids." Molecular and Cellular Biology 15, no. 2 (February 1995): 790–95. http://dx.doi.org/10.1128/mcb.15.2.790.
Full textAbstract:
Butyrate and its analogs have been shown to induce fetal hemoglobin in humans and primates and in erythroid cell cultures. To obtain insights concerning the cellular mechanisms of butyrate action, we analyzed the effects of butyrate on human globin gene expression in hybrids produced by fusing mouse erythroleukemia cells (MEL) with human fetal erythroid cells (HFE). These hybrids initially express human fetal hemoglobin but subsequently switch to adult globin expression after several weeks in culture. We found that alpha-aminobutyric acid, a butyrate analog which does not induce terminal maturation, strikingly delays the rate of the gamma- to beta-globin gene (gamma-to-beta) switch in the HFE x MEL hybrids. The effect of butyrate on globin expression is transient, with the result that the delay of globin gene switching requires the continuous presence of this compound in culture. Furthermore, butyrate fails to induce fetal hemoglobin expression in hybrids which have switched, suggesting that the effect of this compound on gamma-globin expression is due to inhibition of gamma gene silencing rather than to induction of gamma gene transcription. Since in other cellular systems, glucocorticoids antagonize the action of butyrate, the effect of dexamethasone on the gamma-to-beta switch in HFE x MEL hybrids was examined. Dexamethasone strikingly accelerated the gamma-to-beta switch, and its effect was irreversible. The effects of dexamethasone and butyrate on the gamma-to-beta switch of the HFE x MEL hybrids appear to be codominant. These results indicate that steroids can have a direct effect on globin gene switching in erythroid cells.
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Higuti, Ilma Hiroko, Priscila Anunciação da Silva, and Aguinaldo José do Nascimento. "Studies on alkalophilic CGTase-producing bacteria and effect of starch on cyclodextrin-glycosyltransferase activity." Brazilian Archives of Biology and Technology 47, no. 1 (March 2004): 135–38. http://dx.doi.org/10.1590/s1516-89132004000100018.
Full textAbstract:
The production of beta- and gamma -CD by CGTase from Bacillus firmus was studied, in regard to the effect of the source and concentration of starch on the yield of CD and CGTase activity. A cyclic activity of accumulation and consumption of beta -CD and gamma -CD occurred during the bacterial growth. CGTase was more active when citrate buffer, pH 5.5 was used. No differences were found for production of gamma -CD with the use of commercial starch flour when compared with corn starch (p>0.05). The use of commercial starch flour resulted in decreased conversion of starch to beta-CD (all the 4 starch sources were statistically different, p<0.05). The best results for beta -CD production were obtained with the use of corn starch. The specific activity of CGTase was not affected by starch concentration, different source of starch nor by the presence of glutamic acid, a CD-complexing agent.
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Hibbs, M. L., M. Tolvanen, and O. Carpén. "Membrane-proximal Ig-like domain of Fc gamma RIII (CD16) contains residues critical for ligand binding." Journal of Immunology 152, no. 9 (May 1, 1994): 4466–74. http://dx.doi.org/10.4049/jimmunol.152.9.4466.
Full textAbstract:
Abstract Ag-Ab complexes are cleared from the circulation through a complex system of receptors for the Fc portion of Ig (FcRs). Fc gamma RIII (CD16) is a low affinity FcR for IgG that is composed of two highly homologous Ig-like extracellular domains. Using secondary structure predictions, we located a strongly hydrophilic region in the second Ig-like domain of Fc gamma RIII that is predicted to lie between beta-strands C and C'. Substitutions of seven out of eight amino acids in this region abolished binding to IgG. Substitution of a conformationally adjacent amino acid in a bend just before beta-strand F and an amino acid in the B-C loop also affected ligand binding. However, amino acid substitutions in two different predicted loops in the second Ig-like domain as well as substitutions to three predicted loops in the first Ig-like domain had no effect on function. A chimeric Fc gamma RIII molecule lacking the second Ig-like domain was unable to bind IgG further, suggesting the presence of the binding site in the second domain. Neutralizing mAbs that inhibit Fc gamma RIII interaction with IgG were mapped to the E-F loop in the membrane proximal domain of Fc gamma RIII, providing further evidence of the importance of this region of the molecule in ligand interaction.
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Tregaskes, C. A., F. K. Kong, E. Paramithiotis, C. L. Chen, M. J. Ratcliffe, T. F. Davison, and J. R. Young. "Identification and analysis of the expression of CD8 alpha beta and CD8 alpha alpha isoforms in chickens reveals a major TCR-gamma delta CD8 alpha beta subset of intestinal intraepithelial lymphocytes." Journal of Immunology 154, no. 9 (May 1, 1995): 4485–94. http://dx.doi.org/10.4049/jimmunol.154.9.4485.
Full textAbstract:
Abstract Expression screening has been used to clone cDNAs encoding the alpha- and beta-chains of chicken CD8. Amino acid sequence similarities with the mammalian sequences were about 30%. Many amino acid residues of structural or functional importance were more highly conserved, as were the overall structures of both chains. Like human CD8 alpha, the chicken alpha-chain lacked sites for N-linked glycosylation, but the beta-chain contained three such sites. In COS cells transfected with CD8 beta cDNA, surface expression of the beta-chain was dependent on co-transfection of the alpha-chain cDNA, indicating that, as in mammals, chicken CD8 can be expressed as a CD8 alpha alpha homodimer or as a CD8 alpha beta heterodimer. Immunofluorescence analysis with mAbs that were shown to identify the CD8 alpha- and CD8 beta-chains revealed that the vast majority of the CD8+ cells in the thymus, spleen, and blood of adult chickens express both CD8 alpha- and CD8 beta-chains. However, a relatively large proportion of the CD8+ TCR-gamma delta cells in the spleens of embryos and young chicks express only the alpha-chain of CD8. Among intestinal epithelial lymphocytes the major CD8+ T cell populations present in mice are conserved, but there is a population of TCR-gamma delta CD8 alpha beta cells that is not found in rodents. This observation is important in interpretation of experiments examining the pathways of development of intestinal intraepithelial lymphocytes in chickens.
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Devalia, V., NS Thomas, PJ Roberts, HM Jones, and DC Linch. "Down-regulation of human protein kinase C alpha is associated with terminal neutrophil differentiation." Blood 80, no. 1 (July 1, 1992): 68–76. http://dx.doi.org/10.1182/blood.v80.1.68.68.
Full textAbstract:
Abstract We have established an RNase protection method to quantify the expression of mRNA for the human protein kinase C (PK-C) isoforms alpha, beta 1, beta 2, and gamma. This was used to investigate whether each isoform is differentially expressed during the differentiation of hematopoietic cells. Myeloid and lymphoid cells express PK-C alpha, beta 1, and beta 2 mRNAs in various proportions. PK-C gamma mRNA was detected in human brain, but not in hematopoietic cells. PK-C alpha mRNA decreases as HL-60 cells mature to a neutrophil phenotype in response to retinoic acid, but its abundance does not change during monocytic differentiation in response to vitamin D3. PK-C alpha mRNA and protein were undetectable in peripheral blood neutrophils, but are present in monocytes. The mRNAs for PK-C beta 1 and beta 2 isoforms increase during HL-60 differentiation and are expressed in both neutrophils and monocytes. Therefore, the PK-C alpha isoform is specifically down-regulated during human neutrophil terminal differentiation. These data suggest that mature neutrophil functions do not require the PK-C alpha isoform.
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Devalia, V., NS Thomas, PJ Roberts, HM Jones, and DC Linch. "Down-regulation of human protein kinase C alpha is associated with terminal neutrophil differentiation." Blood 80, no. 1 (July 1, 1992): 68–76. http://dx.doi.org/10.1182/blood.v80.1.68.bloodjournal80168.
Full textAbstract:
We have established an RNase protection method to quantify the expression of mRNA for the human protein kinase C (PK-C) isoforms alpha, beta 1, beta 2, and gamma. This was used to investigate whether each isoform is differentially expressed during the differentiation of hematopoietic cells. Myeloid and lymphoid cells express PK-C alpha, beta 1, and beta 2 mRNAs in various proportions. PK-C gamma mRNA was detected in human brain, but not in hematopoietic cells. PK-C alpha mRNA decreases as HL-60 cells mature to a neutrophil phenotype in response to retinoic acid, but its abundance does not change during monocytic differentiation in response to vitamin D3. PK-C alpha mRNA and protein were undetectable in peripheral blood neutrophils, but are present in monocytes. The mRNAs for PK-C beta 1 and beta 2 isoforms increase during HL-60 differentiation and are expressed in both neutrophils and monocytes. Therefore, the PK-C alpha isoform is specifically down-regulated during human neutrophil terminal differentiation. These data suggest that mature neutrophil functions do not require the PK-C alpha isoform.
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Mabley, JG, JM Cunningham, N. John, MA Di Matteo, and IC Green. "Transforming growth factor beta 1 prevents cytokine-mediated inhibitory effects and induction of nitric oxide synthase in the RINm5F insulin-containing beta-cell line." Journal of Endocrinology 155, no. 3 (December 1, 1997): 567–75. http://dx.doi.org/10.1677/joe.0.1550567.
Full textAbstract:
The aim of this study was to examine if the growth factor, transforming growth factor beta 1 (TGF beta 1), could prevent induction of nitric oxide synthase and cytokine-mediated inhibitory effects in the insulin-containing, clonal beta cell line RINm5F. Treatment of RINm5F cells for 24 h with interleukin-1 beta (IL-1 beta) (100 pM) induced expression of nitric oxide synthase and inhibited glyceraldehyde-stimulated insulin secretion. Combinations of IL-1 beta (100 pM), tumour necrosis factor-alpha (100 pM) and interferon-gamma (100 pM) reduced RINm5F cell viability (determined by the 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium (MTT) reduction assay) and de novo protein synthesis, as measured by incorporation of radiolabelled amino acids into perchloric acid-precipitable protein. Pretreatment of RINm5F cells with TGF beta 1 (10 pM) for 18 or 24 h, prior to the addition of either IL-1 beta or combined cytokines, prevented cytokine-induced inhibition of insulin secretion, protein synthesis and the loss of cell viability. TGF beta 1 pretreatment inhibited cytokine-induced expression and activity of nitric oxide synthase in RINm5F cells as determined by Western blotting and by cytosolic conversion of radiolabelled arginine into labelled citrulline and nitric oxide. Chemically generated superoxide also induced expression of nitric oxide synthase possibly due to direct activation of the nuclear transcription factor NF kappa B, an effect prevented by both an antioxidant and TGF beta 1 pretreatment. In conclusion, the mechanism of action of TGF beta 1 in blocking cytokine inhibitory effects was by preventing induction of nitric oxide synthase.
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Hodgson, A. J., B. Penke, A. Erdei, I. W. Chubb, and P. Somogyi. "Antisera to gamma-aminobutyric acid. I. Production and characterization using a new model system." Journal of Histochemistry & Cytochemistry 33, no. 3 (March 1985): 229–39. http://dx.doi.org/10.1177/33.3.3973378.
Full textAbstract:
Antisera to the amino acid gamma-aminobutyric acid (GABA) have been developed with the aim of immunohistochemical visualization of neurons that use it as a neurotransmitter. GABA bound to bovine serum albumin was the immunogen. The reactivities of the sera to GABA and a variety of structurally related compounds were tested by coupling these compounds to nitrocellulose paper activated with polylysine and glutaraldehyde and incubating the paper with the unlabeled antibody enzyme method, thus simulating immunohistochemistry of tissue sections. The antisera did not react with L-glutamate, L-aspartate, D-aspartate, glycine, taurine, L-glutamine, L-lysine, L-threonine, L-alanine, alpha-aminobutyrate, beta-aminobutyrate, putrescine, or delta-aminolevulinate. There was cross-reaction with gamma-amino-beta-hydroxybutyrate, 1-10%, and the homologues of GABA: beta-alanine, 1-10%, delta-aminovalerate, approximately 10%, and epsilon-amino-caproate, approximately 10%. The antisera reacted slightly with the dipeptide gamma-aminobutyrylleucine, but not carnosine or homocarnosine. Immunostaining of GABA was completely abolished by adsorption of the sera to GABA coupled to polyacrylamide beads by glutaraldehyde. The immunohistochemical model is simple, amino acids and peptides are bound in the same way as in aldehyde-fixed tissue and, in contrast to radioimmunoassay, it uses an immunohistochemical detection system. This method has enabled us to define the high specificity of anti-GABA sera and to use them in some novel ways. The model should prove useful in assessing the specificity of other antisera.
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Chen, W. H., G. M. Morriss-Kay, and A. J. Copp. "Genesis and prevention of spinal neural tube defects in the curly tail mutant mouse: involvement of retinoic acid and its nuclear receptors RAR-beta and RAR-gamma." Development 121, no. 3 (March 1, 1995): 681–91. http://dx.doi.org/10.1242/dev.121.3.681.
Full textAbstract:
A role for all-trans-retinoic acid in spinal neurulation is suggested by: (1) the reciprocal domains of expression of the retinoic acid receptors RAR-beta and RAR-gamma in the region of the closed neural tube and open posterior neuropore, respectively, and (2) the preventive effect of maternally administered retinoic acid (5 mg/kg) on spinal neural tube defects in curly tail (ct/ct) mice. Using in situ hybridisation and computerised image analysis we show here that in ct/ct embryos, RAR-beta transcripts are deficient in the hindgut endoderm, a tissue whose proliferation rate is abnormal in the ct mutant, and RAR-gamma transcripts are deficient in the tail bud and posterior neuropore region. The degree of deficiency of RAR-gamma transcripts is correlated with the severity of delay of posterior neuropore closure. As early as 2 hours following RA treatment at 10 days 8 hours post coitum, i.e. well before any morphogenetic effects are detectable, RAR-beta expression is specifically upregulated in the hindgut endoderm, and the abnormal expression pattern of RAR-gamma is also altered. These results suggest that the spinal neural tube defects which characterise the curly tail phenotype may be due to interaction between the ct gene product and one or more aspects of the retinoic acid signalling pathway.
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Safaya, S., A. Ibrahim, and RF Rieder. "Augmentation of gamma-globin gene promoter activity by carboxylic acids and components of the human beta-globin locus control region." Blood 84, no. 11 (December 1, 1994): 3929–35. http://dx.doi.org/10.1182/blood.v84.11.3929.bloodjournal84113929.
Full textAbstract:
Butyric acid increases fetal hemoglobin synthesis in adult animals and in erythroid cells in culture and induces the gamma-globin gene promoter in transient expression experiments in K562 cells (McDonagh KT, Nienhuis AW, Blood 78:255a, 1992 [abstr, suppl 1]). We compared the effect of butyrate and other short-chain carboxylic acids in transient expression studies with K562 cells using an expression plasmid bearing a luciferase reporter gene driven by the normal human A gamma-globin gene promoter. Butyrate (4 carbons) increased the activity of the human A gamma-globin gene promoter up to 123 times. Marked augmentation of the normal gamma-promoter activity was also noted with 5-carbon valeric acid (up to 394 times) and 3-carbon propionic acid (up to 129 times). The branched isobutyric acid as well as phenylacetate showed less ability to increase promoter activity. Addition of the tandemly repeated AP-1/NF-E2 (AP) enhancer sequences from hypersensitive site 2 (HS2) of the locus control region (LCR) increased gamma-promoter activity up to 24 times. Addition of a nearby 16-bp conserved motif (CM) in HS2 (Safaya S, Rieder RF, Blood 78:146a, 1992 [abstr, suppl 1]) to the AP-containing plasmid construct further increased gamma-promoter activity. In the presence of butyrate, the plasmid bearing both the AP and CM sequences showed gene expression up to 477 times greater than that of the basal gamma-promoter-driven luciferase plasmid in the absence of inducer. A plasmid bearing the herpes simplex thymidine kinase promoter was also tested and gene expression was markedly increased by the same organic acids. MEL cells responded to butyrate, valerate, and propionate with induction of hemoglobin synthesis. Responses to isobutyrate and 6-carbon caproate required higher concentrations of the compounds. Thus, other short-chain organic acids as well as butyrate increase gamma-promoter activity in the transient expression system, and this activity can be further augmented by incorporating LCR elements into the expression vector. Nonglobin promoters also respond to the same carboxylic acids.
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Shihabi, Z. K., K. S. Oles, C. P. McCormick, and J. K. Penry. "Serum and Tissue Carnitine Assay Based on Dialysis." Clinical Chemistry 38, no. 8 (August 1, 1992): 1414–17. http://dx.doi.org/10.1093/clinchem/38.8.1414.
Full textAbstract:
Abstract Carnitine (L-beta-hydroxy-gamma-trimethylaminobutyric acid) aids mitochondrial energy production by transferring fatty acids across the membranes for beta-oxidation. We describe here a modified enzymatic assay for free serum and tissue carnitine based on dialysis to remove interfering substances in the serum, with subsequent conversion of carnitine to the acyl derivative by carnitine acetyltransferase (EC 2.3.1.7) in the presence of 5,5'-dithiobis-(2-nitrobenzoic acid). The method compared well with a radioenzymatic assay. The reference interval for serum is 28-70 mumol/L. Patients with advanced diabetes and those undergoing valproic acid treatment displayed lower mean values; a statistically significant number of them showed serum carnitine values below the reference interval. The method was also applied to carnitine measurement in cerebrospinal fluid and human tissues.
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Kurachi, Y. "G protein regulation of cardiac muscarinic potassium channel." American Journal of Physiology-Cell Physiology 269, no. 4 (October 1, 1995): C821—C830. http://dx.doi.org/10.1152/ajpcell.1995.269.4.c821.
Full textAbstract:
Several ion channels can be regulated by G proteins in a “membrane-delimited” manner. The cardiac muscarinic K+ (KACh) channel, which is responsible for the acetylcholine (ACh) or adenosine-induced deceleration of heart beat and atrioventricular conduction, is the prototype of this type of receptor-dependent regulation of ion channels. Because similar transduction mechanisms are utilized by various membrane receptors, such as somatostatin, 5-hydroxytryptamine-1, alpha 2-adrenergic, mu-and delta-opioid, D2-dopamine, and gamma-aminobutyric acid B receptors, in neuronal, hormone-secreting, renal, or smooth muscle cells, the G protein (GK)-KACh channel system illustrates the principles underlying one of the most important cell signaling mechanisms (B. Hille. Neuron 9: 187-195, 1992). It seems that both alpha- and beta gamma-subunits of GK may be involved in the regulation of the KACh channel of mammalian atrial muscle. A general consensus of opinion has emerged, after some years of controversy, to support the notion that physiological activation of the channel by GK is the responsibility of the beta gamma-subunits. Recent evidence suggests that the KACh channel interacts with the alpha-subunit in the terminating process of activation.
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De Benedetti, F., LA Falk, LR Ellingsworth, FW Ruscetti, and CR Faltynek. "Synergy between transforming growth factor-beta and tumor necrosis factor-alpha in the induction of monocytic differentiation of human leukemic cell lines." Blood 75, no. 3 (February 1, 1990): 626–32. http://dx.doi.org/10.1182/blood.v75.3.626.626.
Full textAbstract:
Abstract We examined the effect of transforming growth factor-beta (TGF-beta) alone and in combinations with other factors on the growth and differentiation of the human promyelocytic cell line HL60 and the human monoblastic cell line U937. Treatment with TGF-beta alone did not significantly affect growth or differentiation of HL60 cells, while it significantly inhibited proliferation and induced monocytic differentiation of a small percentage of U937 cells. Combinations of TGF-beta and tumor necrosis factor-alpha (TNF-alpha) acted in synergy to inhibit cell proliferation and to induce monocytic differentiation of both HL60 and U937 cells. In contrast, no synergy was observed when HL60 cells were treated with TGF-beta in various combinations with interferon-alpha (IFN-alpha), interferon-gamma (IFN-gamma), and retinoic acid. Examination of TNF-alpha receptor expression on HL60 and U937 cells showed that these cell lines expressed comparable levels of high-affinity TNF-alpha binding sites. Treatment of HL60 and U937 cells with TGF-beta did not induce significant changes in TNF-alpha receptor expression in either cell line. In contrast, HL60 cells expressed much lower levels of TGF-beta receptors than did U937 cells. Treatment of both HL60 and U937 cells with TNF-alpha induced a dose-dependent increase in expression of TGF-beta receptors, suggesting that the synergy between TNF-alpha and TGF-beta may result, at least in part, from upregulation of TGF-beta receptor expression by TNF-alpha.
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De Benedetti, F., LA Falk, LR Ellingsworth, FW Ruscetti, and CR Faltynek. "Synergy between transforming growth factor-beta and tumor necrosis factor-alpha in the induction of monocytic differentiation of human leukemic cell lines." Blood 75, no. 3 (February 1, 1990): 626–32. http://dx.doi.org/10.1182/blood.v75.3.626.bloodjournal753626.
Full textAbstract:
We examined the effect of transforming growth factor-beta (TGF-beta) alone and in combinations with other factors on the growth and differentiation of the human promyelocytic cell line HL60 and the human monoblastic cell line U937. Treatment with TGF-beta alone did not significantly affect growth or differentiation of HL60 cells, while it significantly inhibited proliferation and induced monocytic differentiation of a small percentage of U937 cells. Combinations of TGF-beta and tumor necrosis factor-alpha (TNF-alpha) acted in synergy to inhibit cell proliferation and to induce monocytic differentiation of both HL60 and U937 cells. In contrast, no synergy was observed when HL60 cells were treated with TGF-beta in various combinations with interferon-alpha (IFN-alpha), interferon-gamma (IFN-gamma), and retinoic acid. Examination of TNF-alpha receptor expression on HL60 and U937 cells showed that these cell lines expressed comparable levels of high-affinity TNF-alpha binding sites. Treatment of HL60 and U937 cells with TGF-beta did not induce significant changes in TNF-alpha receptor expression in either cell line. In contrast, HL60 cells expressed much lower levels of TGF-beta receptors than did U937 cells. Treatment of both HL60 and U937 cells with TNF-alpha induced a dose-dependent increase in expression of TGF-beta receptors, suggesting that the synergy between TNF-alpha and TGF-beta may result, at least in part, from upregulation of TGF-beta receptor expression by TNF-alpha.