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Query: CAS:56-41-7 (alanine)
 70,945 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Various point mutations in the c-erbA thyroid hormone receptor (TR) beta gene of unrelated kindreds have been reported to be responsible for different phenotypes of generalized thyroid hormone resistance. We now report a new point mutation, Td, in one of two TR beta alleles of three affected members of one family, designated family T. In contrast to the previously described point mutations, all located in the T3-binding domain of the TR beta gene, mutation Td was identified in the carboxy-terminal part of the hinge domain. Direct sequencing of the polymerase chain reaction-amplified whole coding region of the patients' fibroblast TR beta genes displayed a single guanine to adenine transition at cDNA nucleotide position 985. This altered alanine (GCC) to threonine (ACC) in codon 229. Garnier prediction of the consequence of the mutation indicated an altered secondary structure. The G----A nucleotide substitution was not present in 80 random TR beta alleles, suggesting that this point mutation is responsible for generalized thyroid hormone resistance in family T. The in vitro expressed mutant TR beta was shown to bind with high affinity to various thyroid hormone response elements. However, the affinity of the TR beta to bind to T3 was reduced 3-fold, indicating that the hinge domain of the TR beta is important for full ligand-binding activity. Moreover, it seems that multiple subdomains of the TR beta interact cooperatively to achieve optimal T3 activity.
Mol Endocrinol 1992 Jul
PMID:A point mutation (Ala229 to Thr) in the hinge domain of the c-erbA beta thyroid hormone receptor gene in a family with generalized thyroid hormone resistance. 132 20

c-jun is a member of the family of immediate-early genes whose expression is induced by factors such as serum stimulation, phorbol ester, and differentiation signals. Here we show that increased Jun synthesis after serum stimulation is accompanied by a concomitant increase in phosphorylation. Several serine-threonine kinases were evaluated for their ability to phosphorylate Jun in vitro. p34cdc2, protein kinase C, casein kinase II, and pp44mapk phosphorylated Jun efficiently, whereas cyclic AMP-dependent protein kinase and glycogen synthase kinase III did not. The sites phosphorylated by p34cdc2 were similar to those phosphorylated in vivo after serum induction. The major sites of phosphorylation were mapped to serines 63, 73, and 246. Phosphorylation of full-length Jun with several kinases did not affect the DNA-binding activity of Jun homodimers or Fos-Jun heterodimers. Comparison of the DNA binding and in vitro transcription properties of wild-type and mutated proteins containing either alanine or aspartic acid residues in place of Ser-63, -73, and -246 revealed only minor differences among homodimeric complexes and no differences among Fos-Jun heterodimers. Thus, phosphorylation of Jun did not produce a significant change in dimerization, DNA-binding, or in vitro transcription activity. The regulatory role of phosphorylation in the modulation of Jun function is likely to be considerably more complex than previously suggested.
Mol Cell Biol 1992 Oct
PMID:Jun is phosphorylated by several protein kinases at the same sites that are modified in serum-stimulated fibroblasts. 132 60

An antibiotic, azatyrosine [L-beta-(5-hydroxy-2-pyridyl) alanine], specifically converts ras-, raf- or c-erbB2 (neu)-transformed NIH3T3 cells to apparently normal phenotype. The reversion induced by azatyrosine is permanent, and the phenotype of the revertant cells does not change even after prolonged culture in the absence of azatyrosine [N. Shindo-Okada, O. Manabe, H. Nagahara & S. Nishimura (1989). Mol. Carcinogen., 2, 159-167]. In the present study, we found that neurite outgrowth of PC12 cells induced by expression of either the ras or raf oncogenes was inhibited by addition of azatyrosine to the medium. Azatyrosine also inhibited neurite outgrowth induced by microinjection of oncogenic Ras protein into PC12 cells. The dose dependency was much the same for the two systems, inhibition of neurite outgrowth of PC12 cells and reversion of the transformed NIH3T3 cells. Microinjection of azatyrosine into the cells was as effective as addition to the medium, indicating that the target of azatyrosine is intracellular. In contrast, neurite outgrowth induced by nerve growth factor, which has been shown to be mediated by normal Ras [N. Hagag, S. Halegouna & M. Viola (1986). Nature, 319, 680-682], was found to be resistant to azatyrosine. Azatyrosine also showed no effect on neurite outgrowth induced by a membrane-permeant cyclic AMP analog through another pathway. These findings suggest that azatyrosine sensitivity is the result of abnormal signal transduction by oncogenic Ras. It was shown that azatyrosine also inhibited differentiation-associated growth arrest of PC12 cells induced by oncogenic Ras. In Ras-induced neurite outgrowth, the azatyrosine-sensitive process was found to be completed in the first 6-9 h, and is probably essential for the commitment of PC12 cells to differentiation rather than to growth.
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PMID:Azatyrosine inhibits neurite outgrowth of PC12 cells induced by oncogenic Ras. 132 88

There are at least three isozymes (C alpha, C beta, and C gamma) of the mammalian catalytic (C) subunit of cAMP-dependent protein kinase (PKA) (Beebe, S., Oyen, O., Sandberg, M., Froysa, A., Hansson, V., and Jahnsen, T. (1990) Mol. Endocrinol. 4, 465-475). To compare the C gamma and C alpha isozymes, the respective cDNAs were expressed in permanently transformed Kin-8 PKA-deficient Y1 adrenal cells using the mouse metallothionein promoter. The recombinant C subunits were characterized as immunoreactive, zinc-inducible, cAMP-dependent kinase activities. In contrast to C alpha, histone was a better substrate than Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide) for C gamma. Furthermore, C gamma histone kinase activity was not inhibited by the protein kinase inhibitor peptide (5-24 amide), which has been widely used as a PKA-specific inhibitor. The major C gamma peak (type I) eluted from DEAE-Sepharose at a higher NaCl concentration (120 mM) than the C alpha type I eluted (70 mM). C gamma and C alpha type II eluted between 220 and 240 mM NaCl. C gamma required higher concentrations of cAMP than C alpha did for dissociation from the mutant type I holoenzyme. These differences provided a basis for the separation of the mutant RI-associated isozymes on DEAE-Sepharose. Both C alpha (41-42 kDa) and C gamma (39-40 kDa) were identified by a C subunit antibody after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis. Zinc induced the PKA-mediated rounding phenotype in C gamma and C alpha clones, thereby restoring the cells to the parent Y1 adrenal cell phenotype. Collectively, these data indicate that C gamma is an active PKA C subunit but suggest that C gamma and C alpha have different protein and peptide recognition determinants.
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PMID:The C gamma subunit is a unique isozyme of the cAMP-dependent protein kinase. 133 96

The amino acid sequence of the triprotamine stelline C from mature sperm nuclei of Acipenser stellatus has been established by automated sequence analysis of the protein and from data provided by thermolysine peptides. The complete amino acid sequence of stelline C is: R-R-R-R-R-H-A-S-T-K-L-K-R-R-R-R-R-R-R-H-G-K-K-S-H-K. The comparison of the primary structure of stelline C with that of other triprotamines from Acipenser stellatus shows that they are similar except for the absence of N-terminal alanine in the stelline A molecule. The main structure difference between stelline C and other fish protamines is that the stelline C molecule begins with five arginine residues.
Mol Biol (Mosk)
PMID:[Comparison of amino acid sequences of sturgeon triprotamines using protamines from Acipenser stellatus gonads as an example]. 133 50

Ricin A chain is an N-glycosidase which removes a single adenine base from a conservative loop of 28S rRNA, thereby inactivating eukaryotic ribosomes. The mechanism of action has been proposed to include transition-state stabilization of an oxycarbonium ion on the substrate ribose by interaction with Glu 177. Conversion of Glu 177 to Gln reduces activity nearly 200-fold [Ready, M. P., Kim, Y., & Robertus, J. D. (1991) Proteins: Struct., Funct., Genet. 10, 270-278] while conversion to Ala (E177A) reduces activity only 20-fold [Schlossman, D., Withers, D., Welsh, P., Alexander, A., Robertus, J., & Frankel, A. (1989) Mol. Cell. Biol. 9, 5012-5021]. X-ray analysis of the latter mutant protein shows that a residue at the edge of the active site, Glu 208, rotates into the space left vacant by the mutation. Its rearranged carboxylate partially substitutes for that of Glu 177. This is equivalent to the rescue of enzyme activity by a second-site reversion. Kinetic analysis shows the E177A mutation affects kcat and not Km, consistent with the notion that the carboxylate serves in transition-state stabilization.
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PMID:Structure of a ricin mutant showing rescue of activity by a noncatalytic residue. 134 87

Deletions, substitutions, or mutations of the rat TSH receptor extracellular domain between residues 20 and 107 (all residue numbers are determined by counting from the methionine start site) have been made by site-directed mutagenesis of receptor cDNA. After transfection in Cos-7 cells, constructs were evaluated for their ability to bind [125I]TSH or respond to TSH and thyroid-stimulating antibodies (TSAbs) from Graves' patients in assays measuring cAMP levels of the transfected cells. Assay results were compared to results from Cos-7 cells transfected with wild-type receptor constructs or vector alone. We identify threonine-40 as a TSAb-specific site whose mutation to asparagine, but not alanine, reduces TSAb activity 10-fold, but only minimally affects TSH-increased cAMP levels. We show that thyroid-stimulating blocking antibodies (TSBAbs), which block TSH or TSAb activity and are found in hypothyroid patients with idiopathic myxedema, continue to inhibit TSH-stimulated cAMP levels when threonine-40 is mutated to asparagine or alanine, suggesting that TSBAbs interact with different TSH receptor epitopes than the TSAb autoantibodies in Graves' patients. This is confirmed by the demonstration that these TSBAbs interact with high affinity TSH-binding sites previously identified at tyrosine-385 or at residues 295-306 of the extracellular domain of the TSH receptor. This is evidenced by a loss in the ability of TSBAbs to inhibit TSAb activity when these residues are mutated or deleted, respectively. Since the TSAb and TSBAb epitopes are in regions of the extracellular domain of the TSH receptor that have no homology in gonadotropin receptors, these data explain at least in part the organ-specific nature of TSH receptor autoantibodies in autoimmune thyroid disease. Data are additionally provided which indicate that residues 30-37 and 42-45, which flank the TSAb epitope at threonine-40, appear to be ligand interaction sites more important for high affinity TSH binding than for the ability of TSH to increase cAMP levels and that cysteine-41 is critical for TSH receptor conformation and expression on the surface of the cell. Thus, despite unchanged maximal values for TSH-increased cAMP levels, substitution of residues 42-45 or deletion of residues 30-37 results in receptors, which, by comparison to wild-type constructs, exhibit significantly worsened Kd values for TSH binding than EC50 values for TSH- or TSAb-increased cAMP activity.(ABSTRACT TRUNCATED AT 400 WORDS)
Mol Endocrinol 1992 Feb
PMID:Identification of separate determinants on the thyrotropin receptor reactive with Graves' thyroid-stimulating antibodies and with thyroid-stimulating blocking antibodies in idiopathic myxedema: these determinants have no homologous sequence on gonadotropin receptors. 134 56

Using rat hepatocytes we confirmed our previous results that glucagon and beta-adrenergic agonists increased the enzyme activity of alanine aminotransferase (AAT) and propranolol abolished their effects. Only the enzyme activity was measured and other parameters like quantity of the enzyme or activation due to modification were not looked for. As in perfusion experiment phenylephrine and phenoxybenzamine (alpha-agonist and alpha-antagonist respectively) also alpha-antagonist respectively) also increased the AAT activity in isolated rat hepatocytes and propranolol reversed these effects. The additive effect of glucagon and phenoxybenzamine on AAT was also persistent in hepatocyte system. Fructose-1:6-bisphosphatase (Fru-P2-ase), another key enzyme in gluconeogenic pathway, was elevated by glucagon and other beta-adrenergic agonists both in liver perfusion and isolated hepatocyte experiments and was brought back to the normal level by propranolol. In this case also only the enzyme activity was measured and no other parameters were looked for. Unlike AAT this enzyme was not stimulated by phenylephrine or phenoxybenzamine. But AAT and Fru-P2-ase activities were increased significantly by adenylate cyclase activators like fluoride or forskolin. Thus, it appears that the regulation of fru-P2-ase by glucagon is purely a b-receptor mediated process whereas AAT activation shows a mixed type of regulation where some well known alpha-agonist and antagonists are behaving as beta-agonists. Results further indicate the presence of phosphodiesterase in hepatocyte membrane which was stimulated by glucagon and brought back to the normal level by propranolol. The different adrenergic compounds stated above, not only modified the activity of the above two enzymes but also stimulated glucose production by hepatocytes from alanine which was in turn abolished by propranolol as well as amino oxyacetate (AOA), a highly specified inhibitor of AAT. This confirm the participation of AAT in gluconeogenesis from alanine in liver. Forskolin and fluoride also increased the glucose production from alanine and showed additive effects with glucagon, phenylephrine and phenoxybenzamine.
Mol Cell Biochem 1992 Aug 18
PMID:Effect of adrenergic agonists and antagonists on alanine amino transferase, fructose-1:6-bisphosphatase and glucose production in hepatocytes. 135 93

The binding of (RS)-alpha-[3H]amino-3-hydroxy-5-methylisoxazole-4-propionic acid ([3H]AMPA), a selective ligand for non-N-methyl-D-aspartate excitatory amino acid receptors, was investigated in rat brain using an autoradiographic receptor binding technique. [3H]AMPA binding sites were widely distributed throughout the rat central nervous system, and the rank order of potency of displacers of [3H]AMPA binding was quisqualate greater than AMPA greater than 6,7-dinitroquinoxaline-2,3-dione = 6-cyano-7-nitroquinoxaline-2,3-dione greater than beta-N-oxalylamino-L-alanine greater than glutamate greater than kainate. Potassium thiocyanate (0-100 mM) exerted a 4-fold stimulation of [3H]AMPA binding, without changing the relative regional distribution of [3H]AMPA binding densities among rat brain regions. Scatchard analysis of equilibrium saturation binding revealed high affinity and low affinity components of [3H]AMPA binding, even in the absence of potassium thiocyanate. Addition of potassium thiocyanate increased the number of high affinity [3H]AMPA binding sites without a change in affinity. In addition, the number of low affinity [3H]AMPA binding sites was unchanged in the presence of potassium thiocyanate, but the affinity of low affinity [3H]AMPA binding was greatly increased. [3H]AMPA thus binds specifically to two affinity conformations of postsynaptic binding sites that appear to be interconverted with potassium thiocyanate. The pharmacologic profile of these sites is consistent with that of the ion channel-linked ("ionotropic") quisqualate/AMPA class of excitatory amino acid receptor in the rat central nervous system.
Mol Pharmacol 1992 May
PMID:Multiple states of rat brain (RS)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors as revealed by quantitative autoradiography. 137 15

Two monoclonal antibodies, designated 1F8 (IgG1) and 5B10 (IgG1), have been produced in mice against native human type III collagen. These antibodies were highly type and species specific, recognizing the triple helical domain of type III as tested by ELISA. Immunofluorescence studies using each of these antibodies resulted in a fibrous staining pattern in human skin dermis. Immunogold electron microscopy resulted in a periodic distribution of gold particulates along banded collagen fibrils. Assuming that the total contour length of pepsin digested type III collagen is 300 nm, measurements of antibody-antigen complexes visualized by rotary shadowing revealed that each antibody bound at the same two sites: one approximately at the middle of the helix (153 nm from the N-terminus), the other at a site one-quarter the triple helical length from the N-terminus (75 nm). That the one-quarter binding site was closest to the N-terminus was determined by antibody incubation following tadpole collagenase treatment, which results in a larger, N-terminus containing fragment (binding antibody) and a smaller C-terminus containing fragment (not binding antibody). Located at each antibody binding epitope is a sequence of 10 amino acids: Gly-Ala-Hyp-Gly-Leu-Arg-Gly-Gly-Ala-Gly. Renatured cyanogen bromide-cleaved(CB)-peptides, CB4 and CB8, containing these repeated sequences reacted with each antibody, whereas other renatured type III CB-peptides were unreactive as determined by Western blotting analysis and ELISA. This was further confirmed by inhibition tests using a 10 residue synthetic peptide of identical sequence, which yielded 20-30% inhibition of antibody binding to native type III collagen at 4 degrees C. However, no inhibition was noted at higher temperature. These results indicate that both monoclonal antibodies recognize a specific helical conformation of 10 or slightly fewer residues in the three identical polypeptide chains comprising type III collagen.
Mol Immunol 1992 Jun
PMID:Repeated helical epitopes of defined amino acid sequence in human type III collagen identified by monoclonal antibodies. 137 14


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