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Biomedical subjects

J Marie

Publications and source records attributed to J Marie.

At least 19 recordsLinked to original sources
PubMed1992

Point mutation of an FGF receptor abolishes phosphatidylinositol turnover and Ca2+ flux but not mitogenesis.

Stimulation of certain receptor tyrosine kinases results in the tyrosine phosphorylation and activation of phospholipase C gamma (PLC gamma), an enzyme that catalyses the hydrolysis of phosphatidylinositol (PtdIns). This hydrolysis generates diacylglycerol and free inositol phosphate, which in turn activate protein kinase C and increase intracellular Ca2+, respectively. PLC gamma physically associates with activated receptor tyrosine kinases, suggesting that it is a substrate for direct phosphorylation by these kinases. Here we report that a fibroblast growth factor (FGF) receptor with a single point mutation at residue 766 replacing tyrosine with phenylalanine fails to associate with PLC gamma in response to FGF. This mutant receptor also failed to mediate PtdIns hydrolysis and Ca2+ mobilization after FGF stimulation. However, the mutant receptor phosphorylated itself and several other cellular proteins, and it mediated mitogenesis in response to FGF. These findings show that a point mutation in the FGF receptor selectively eliminates activation of PLC gamma and that neither Ca2+ mobilization nor PtdIns hydrolysis are required for FGF-induced mitogenesis.

Amino Acid Sequence↗
PubMed1992

Protein purification using combined streptavidin (or avidin)-Sepharose and thiopropyl-Sepharose affinity chromatography.

The major problem usually encountered in the application of the (strept)avidin-biotin system to the purification of proteins (or other biological molecules) lies in the difficult reversion of the interaction between immobilized (strept)avidin and the adsorbed biotinylated protein. Among the proposed solutions is the selective biotinylation of the entity to be purified by a disulphide-containing biotinylated reagent which allows its recovery from (strept)avidin gels by dithiothreitol (DTT) treatment. As emphasized by the example of angiotensin II receptor purification, achieved using this strategy, optimum reduction of this disulphide bridge may require improvement of its accessibility using denaturating agents such as sodium dodecyl sulphate or urea. However, these agents release important amounts of (strept)avidin. Two general ways of solving this problem are proposed. One solution takes advantage of the absence of cysteine in the streptavidin sequence: the protein to be purified is selectively readsorbed to thiopropyl-Sepharose through the thiol function generated on DTT cleavage of the biotinylated reagent. The other solution is an empirical approach to make possible the use of avidin, which possesses cysteine residues: combined avidin-Sepharose and thiopropyl-Sepharose chromatography proved efficient when carried out in the presence of urea as denaturing agent.

Animals↗
PubMed1991

RNA secondary structure repression of a muscle-specific exon in HeLa cell nuclear extracts.

The chicken beta-tropomyosin pre-messenger RNA (pre-mRNA) is spliced in a tissue-specific manner to yield messenger RNA's (mRNA's) coding for different isoforms of this protein. Exons 6A and 6B are spliced in a mutually exclusive manner; exon 6B was included in skeletal muscle, whereas exon 6A was preferred in all other tissues. The distal portion of the intron upstream of exon 6B was shown to form stable double-stranded regions with part of the intron downstream of exon 6B and with sequences in exon 6B. This structure repressed splicing of exon 6B to exon 7 in a HeLa cell extract. Derepression of splicing occurred on disruption of this structure and repression followed when the structure was re-formed, even if the structure was formed between two different RNA molecules. Repression leads to inhibition of formation of spliceosomes. Disrupting either of the two double-stranded regions could lead to derepression, whereas re-forming the helices by suppressor mutations reestablished repression. These results support a simple model of tissue-specific splicing in this region of the pre-mRNA.

Animals↗
PubMed1990

Affinity chromatography purification of angiotensin II receptor using photoactivable biotinylated probes.

We have developed biotinylated photoactivable probes that are suitable for covalent labeling of angiotensin II (AII) receptors and the subsequent purification of covalent complexes through immobilized avidin or streptavidin. One of these probes, biotin-NH(CH2)2SS(CH2)2CO-[Ala1,Phe(4N3)8]AII, which contains a cleavable disulfide bridge in its spacer arm and which displays, in its radioiodinated form, very high affinity for AII receptors (Kd approximately 1 nM), proved to be suitable for indirect affinity chromatography of rat liver receptor with facilitated recovery from avidin gels by use of reducing agents. This constituted the central step of an efficient partial purification scheme involving hydroxylapatite chromatography, streptavidin chromatography, and thiopropyl-Sepharose chromatography. SDS-PAGE analysis and autoradiography established the identity of the purified entity (molecular weight 65K) as the AII receptor. Possible ways of completing purification to homogeneity and extrapolation of the protocols to a preparative scale are discussed, as well as the potential contribution of our new probes to the study of the structural properties of angiotensin receptors.

Amino Acid Sequence↗
PubMed1990

In vitro splicing of mutually exclusive exons from the chicken beta-tropomyosin gene: role of the branch point location and very long pyrimidine stretch.

The chicken beta-tropomyosin gene contains 11 exons, two of which are spliced into mRNA only in skeletal muscle. One pair of alternative exons, 6A and 6B, is found in the middle of the gene; they are spliced in a mutually exclusive manner. The non-muscle splice 6A-7 is by far the predominant in vitro reaction in a HeLa cell nuclear extract. A minor product is the 6A-6B splice, which is excluded in all tissues. This minor product results from the use of a branch point located 105 nt upstream of the 3' end of the intron separating exons 6A and 6B. The region between the branch point sequence and the final AG contains a stretch of approximately 80 pyrimidines. We have examined the role of the distance of the branchpoint to the 3' splice site and of the sequences between these two elements. Our results suggest that at least two cis-acting elements contribute to the mutual exclusivity of exons 6A and 6B. The intron between exons 6A and 6B is intrinsically poorly 'spliceable' both because the branch point is too far upstream of the 3' end of the intron to give efficient splicing and because of the particular sequence lying between this branch point and the 3' splice site.

Animals↗
PubMed1990

Characterization of the angiotensin II receptor.

We present a brief overview of the present knowledge on the structural and molecular properties of angiotensin II receptors and the various attempts to determine their primary structures, with special reference to our strategy for receptor purification. The strategy involves covalent labeling of the receptor with synthetic biotinylated photoactivatable probes, followed by indirect affinity chromatography on immobilized streptavidin. The various applications of these probes to the study of structural and molecular properties and to the cell biology of angiotensin II receptors are discussed.

Amino Acid Sequence↗
PubMed1989

A subfragment of the beta tropomyosin gene is alternatively spliced when transfected into differentiating muscle cells.

A subgenomic fragment of the chicken beta tropomyosin gene which contains two alternative exons flanked by common exons was isolated and placed under the control of the SV 40 early promoter. This construction was subsequently used to transfect quail myoblasts together with a Neomycin resistance gene, and to isolate stable transfectants. mRNAs were isolated before and after differentiation and analyzed using a modification of the primer extension method. We show that myoblasts accumulate transcripts which contain the non muscle specific exon joined to the common exons while myotubes accumulate transcripts containing the muscle specific exon. These results, therefore demonstrate that such a subgenomic fragment contains all the necessary information to direct a correct developmentally regulated mutually exclusive splicing. They also strongly suggest that trans acting factors must be involved in the switch of the splicing pattern which takes place during the transition from myoblasts to myotubes. The same regulation cannot be faithfully reproduced during transient expression, since no difference in the use of exons 6A/6B is observed during differentiation and two aberrant minor splicing products are obtained which contain or lack both exons. We suggest that failure of exon 6A to splice to exon 6B is due to the existence of some structural constraints which lower the efficiency with which the intron between them is excised.

Animals↗
PubMed1989

Synthesis of a biotinylated, iodinatable, and photoactivatable probe for angiotensin receptors.

We propose here a biotinyl-aminohexanoyl-[Ala1, Phe(4N3)8]angiotensin II analog as a radioiodinatable and photoactivatable probe for covalent labeling, detection and isolation of angiotensin receptors. A combination of solid phase and minimum-protection segment-coupling strategy using hexafluorophosphate of (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium (BOP) as a coupling reagent is proposed for the synthesis of this probe. Optimized yields were obtained by HPLC monitoring of all reactions. A complete n.m.r. study suggests an extended conformation of this molecule, allowing a simultaneous recognition of receptor and avidin. The probe binds with high affinity (Kd = 2 nM) to angiotensin II receptors from rat liver membranes.

Amino Acid Sequence↗
PubMed1988

Covalent labeling of vasopressin receptors from LLC-PK1 cells by the use of a bifunctional reagent.

The possibility of covalently attaching vasopressin to its receptors by the use of a bifunctional reagent was explored. Plasma membranes from the LLC-PK1 pig kidney cell line were purified by Percoll density gradient centrifugation. These membranes contained a single population of high affinity (Kd = 5.2 nM) and high capacity (Bmax 3.8 pmol/mg of protein) [3H]lysine vasopressin ([3H]LVP)-binding sites. [3H]LVP-labeled receptors could be solubilized with a high yield (83%) and minimal dissociation (9%) by treatment with the non-ionic detergent, octaethylene glycol mono-n-dodecyl ether (C12E8) (0.5%, v/v) in the presence of glycerol (20%). The solubilized [3H]LVP-labeled receptors were stable upon storage at 4 degrees (5% dissociation after 24 hr). They were partially purified to a specific activity of 17 pmol/mg of protein by chromatography on a Cibacron blue-Sepharose column with a yield of 90%. The [3H]LVP-receptor complexes in both intact membranes and the partially purified preparation were almost completely dissociated by incubation at 30 degrees for 30 min in the presence of 20 mM ethylenediaminetetraacetate (EDTA). This property was used to test the effect of ethylene glycol bis (succinimidyl-succinate) (EGS) as cross-linking reagent for the covalent attachment of [3H]LVP to its receptors. After treatment of [3H]LVP-labeled membranes for 30 min with 1 mM EGS at 4 degrees, about 30% of specifically bound [3H]LVP was resistant to EDTA dissociation. The amount of EDTA-resistant binding varied as a linear function of the fractional receptor occupancy and maximal binding capacity of the different batches of membranes used. Similar results were obtained with solubilized and partially purified vasopressin receptors. Upon steric exclusion high performance liquid chromatography, the EDTA-resistant [3H]LVP-labeled material, like the native [3H]LVP-labeled receptor, was eluted as a single and apparently homogeneous peak. The covalent character of the EGS-induced [3H]LVP binding to solubilized or partially purified receptors was assessed by its resistance to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The yield of EGS-induced labeling deduced from these experiments (27%) was close to that determined by the EDTA method. SDS-PAGE analysis of the [3H]LVP-labeled cross-linked material revealed the specific labeling of a major 50-kDa component and a minor component of 30 kDa. The size of these two components was not affected by dithiothreitol.

Animals↗
PubMed1987

Vasopressin receptors from cultured mesangial cells resemble V1a type.

Mesangial cells respond to vasopressin by contraction and increased prostaglandin production. The purpose of the present study is to characterize vasopressin receptors from these cells. Glomeruli were isolated from rat kidneys and plated for explant growth of mesangial cells. Membranes were prepared from cells grown for 6 wk and tested for their ability to bind [3H]vasopressin (lysine vasopressin). These membranes contained a single class of specific vasopressin binding sites [equilibrium dissociation constant (Kd) = 10 +/- 1 nM, maximal binding capacity (Bmax) = 270 +/- 7 fmol/mg protein for 5 determinations]. Vasopressin induced a dose-dependent (apparent Kact value = 2 nM) accumulation of labeled inositol phosphates in myo[3H]inositol-prelabeled mesangial cells incubated in the presence of 10 mM of Li. Conversely, vasopressin failed to alter the adenylate cyclase activity of mesangial cell membranes. Competition experiments with a series of vasopressin structural analogues that have different degrees of affinity for V2-(renal), V1a- (vascular and hepatic), and V1b- (adenohypophyseal) receptors, indicated that vasopressin receptors from rat glomerular mesangial cells resemble the V1a- receptor subtype.

Animals↗
PubMed1986

Tissue-specific heterogeneity of the 3'-untranslated region of L-type pyruvate kinase mRNAs.

A single L-type pyruvate kinase (PK) gene seems to exist per haploid genome. It is expressed in the liver, kidney and small intestine in the form of three mRNA species of 2, 2.2 and 3.2 X 10(3) bases (kb). All three species are polyadenylated and translatable into the same L-type subunit. Primer extension experiments demonstrate that all three PK mRNAs have the same 5' ends. Nuclease S1 protection experiments with various cDNA and 3' genomic probes indicate that the different mRNA species only differ by the length of their 3' noncoding region. The mechanism responsible for the production of the three transcripts seems to be the use of alternative unusual polyadenylation sites. Run-on assays with specific probes recognizing only the 3.2-kb or all three mRNA species show that the transcription proceeds across the gene with similar rate. This means that the process involved in generation of the three transcripts is a posttranscriptional event, probably due to different sites of endonucleolytic cleavage of primary transcripts extending 3' from the gene region encoding the mature mRNAs. The ratio between the different PK mRNA species is, to a certain extent, tissue-specific and changes with development. The role of an 'identifier sequence' located in the 3' noncoding sequence of the 3.2-kb species in such a tissue-specific use of alternative polyadenylation sites is discussed.

Animals↗
PubMed1986

Complete nucleotide and deduced amino acid sequences of rat L-type pyruvate kinase.

Four overlapping cDNA clones for L-type pyruvate kinase (PK-L) were isolated from carbohydrate-induced rat liver cDNA libraries. They contained all the coding sequence of the enzyme from the 7th codon and the entire 3'-untranslated extension up to the poly(A) tail. The sequence of the first 7 codons and that of the 5'-untranslated region were determined by primer extension. The analyzed PK-L mRNA has 19 5'-untranslated bases, 1629 coding bases and 1281 3'-untranslated bases without the poly(A) tail; it corresponds to the heavier, 3.2 kb species of the L-type mRNAs. The codons for the phosphorylatable site are located at the 5'-end of the messenger. The unusually long 3'-untranslated extension contains a repetitive element complementary to the 'brain-specific' identifier sequence described by Sutcliffe et al. [(1982) Proc. Natl. Acad. Sci. USA 79, 4942-4946].

Amino Acid Sequence↗
PubMed1986

Vasopressin antagonists allow demonstration of a novel type of vasopressin receptor in the rat adenohypophysis.

The ligand specificity of rat adenohypophyseal vasopressin receptors was directly compared to that of peripheral receptors of the V1 and V2 types. For this purpose a series of 15 recently designed vasopressin antagonists was used. The affinities of these antagonists for rat adenohypophyseal membranes were deduced from the determination of the concentration-dependent inhibition of [3H]vasopressin binding. In parallel experiments the corticotropin (or anti-corticotropin)-releasing activities of the tested peptides were determined on freshly dispersed rat adenohypophyseal cells. All peptides tested which were found to be antagonists of the vasopressor and antidiuretic responses to vasopressin in vivo behaved as antagonists of vasopressin-induced corticotropin release. There was a close correlation between the relative affinities of the analogues tested for binding to adenohypophyseal membranes and their relative potencies in inhibiting vasopressin-induced corticotropin release, indicating that the detected vasopressin-binding sites are the receptors involved in the vasopressin effect on corticotropin secretion. No correlation could be demonstrated between anti-corticotropin-releasing activities and either anti-antidiuretic or antivasopressor potencies of the antagonists tested. A direct comparison of the ligand specificities of adenohypophyseal receptors on the one hand, and V1 (hepatic) and V2 (renal) receptors on the other hand, showed that most of the antagonists discriminated very efficiently between adenohypophyseal and either hepatic or renal receptors. The selectivity index reaches values as high as 260,000 for desGly(NH2)9 [1-(beta-mercapto-beta, beta-cyclopentamethylenepropionic acid), 2-D-O-ethyl-tyrosine, 4-valine] arginine vasopressin. It is concluded that adenohypophyseal receptors represent a novel type of vasopressin receptors. Based on the observation that adenohypophyseal receptors, like hepatic or vascular V1 receptors, do not appear to be coupled to adenylate cyclase, we propose that adenohypophyseal receptors could be designated as V1b receptors as opposed to the V1a receptors previously characterized on liver and blood vessels.

Adrenocorticotropic Hormone↗
PubMed1985

Characterization and metabolic regulation of a liver-specific 5.4-kilobase mRNA whose synthesis is transcriptionally induced by carbohydrates and repressed by glucagon and cyclic AMP.

Four clones derived from a carbohydrate-induced rat liver cDNA library were found to hybridize with a 5.4-kilobase mRNA species encoding a 36 kDa protein. This mRNA was abundant in the liver, barely detectable in adipocytes and kidney, and absent from the other tissues tested. In the liver, the mRNA was fully induced by a carbohydrate-rich diet, but was undetectable during both starvation and feeding with a protein-rich or lipid-rich diet. Adrenalectomized, thyroidectomized and diabetic animals did not express the mRNA in their liver when re-fed with the carbohydrate-rich diet. When these animals were given the missing hormone, the amount of hybridizable RNA returned to normal values, but administration of the hormone alone failed to induce mRNA synthesis in starved animals. Both glucagon and its second messenger, cyclic AMP, abolished the induction of the mRNA in re-fed animals. Exogenous insulin, whatever the dose, did not reverse the inhibitory action of glucagon. In an isolated nuclei transcription system, no detectable RNA transcripts were found in starved animals, whereas feeding the animals with the carbohydrate-rich diet led to a maximum rate of gene transcription. Although unidentified, this mRNA proves to be a remarkable marker of dietary and hormonal control of gene expression in vivo. It will provide a useful model for further analysis of the role of cyclic AMP in regulating the transcription of eukaryotic genes.

Animals↗
PubMed1985

Pharmacological characterization of the angiotensin receptor negatively coupled with adenylate cyclase in rat anterior pituitary gland.

Angiotensin II (AII) inhibited anterior pituitary adenylate cyclase. Whereas GTP was necessary to fully express the AII inhibitory effect, Na+ was not required. The magnitude of inhibition (42 +/- 6%) permitted a pharmacological characterization of the AII receptor involved in adenylate cyclase inhibition. Angiotensin I (AI) was less potent than AII, and deletion of aminoacids in the N-terminal position resulted in a progressive reduction of the Ki (peptide concentration producing half-maximal inhibition). The Ki values were 3 +/- 0.9, 10, and 700 nM for AII, angiotensin III (AIII), and des-Asp, des-Arg-AII, respectively. Sarcosine in position 1 [( Sar, Phe]AII) increased the potency of inhibition (Ki = 0.12 +/- 0.12 nM). Different antagonists of the AII receptors appeared to be partial agonists. There was a very close correlation (r = 0.98) between the respective potencies of a series of AII analogs to inhibit adenylate cyclase and the potencies of these analogs to elicit PRL or ACTH release or to bind to AII-binding sites. Dopamine and AII inhibition of anterior pituitary adenylate cyclase were not additive. This suggests that both receptors are on the same cell and likely on lactotrophs. This hypothesis agrees with the observation that vasoactive intestinal peptide stimulation of adenylate cyclase was inhibited by AII, whereas corticotropin-releasing factor stimulation was unaffected. Although dopamine and AII inhibited the same adenylate cyclase, they had opposing effects on PRL release (inhibition and stimulation, respectively). The possible significance of this observation is related to a model implying that PRL release can be elicited through either a Ca+2 or a cAMP pathway.

Adenylyl Cyclase Inhibitors↗