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Biomedical subjects

H Michel

Publications and source records attributed to H Michel.

At least 217 records · Page 12Linked to original sources
PubMed1991

Tandem mass spectrometry identifies sites of three post-translational modifications of spinach light-harvesting chlorophyll protein II. Proteolytic cleavage, acetylation, and phosphorylation.

The photosynthetic membranes of spinach (Spinacia oleracea L.) chloroplasts were incubated with [gamma-32P] ATP. When the thylakoid membrane kinase was activated with light, the 25- and 27-kDa forms of the light-harvesting chlorophyll a/b protein (LHC II) were phosphorylated on their amino termini. Treatment of the membranes with proteinase K or thermolysin released phosphopeptides which were purified by ferric ion affinity chromatography and reverse phase high performance liquid chromatography. Sequencing of the phosphopeptides was performed with tandem quadrupole mass spectrometry. Three different phosphopeptides Ac-RKTAGKPKT, Ac-RKTAGKPKN, and Ac-RKSAGKPKN originating from class I LHC II were examined after release by thermolysin. One phosphopeptide, Ac-RRTVKSAPQ, originating from class II LHC II was examined after release by proteinase K. Each of the four LHC II phosphopeptides was derived from the amino terminus of a distinct protein. Peptides were acetylated at their amino-terminal arginine and were phosphorylated on either threonine or serine in the third position. We conclude that proteolytic processing of pre-LHC II occurs at a conserved methionyl-arginyl bond and is followed by amino-terminal acetylation of the arginine and nearby phosphorylation of the mature LHC II. Eight different peptides were synthesized in acetylated and nonacetylated forms as substrates for the thylakoid membrane kinase. From a comparison of the kinetics of phosphate incorporation into the peptides, we conclude that basic residues on both sides of the phosphorylation site are important for enzyme recognition. Acetylation of the amino terminus is not required for phosphorylation.

Acetylation↗
PubMed1991

Characterization of posttranslational modifications in neuron-specific class III beta-tubulin by mass spectrometry.

Class III beta-tubulin, isolated from adult bovine brain, is resolved into at least seven charge variants on isoelectric focusing gels. To identify the posttranslational modifications responsible for this heterogeneity, a mixture of brain tubulins was treated with cyanogen bromide and the C-terminal fragments from the class III beta-tubulin isoforms were then isolated by binding them to the monoclonal antibody TuJ1. Combined use of tandem mass spectrometry and both subtractive and automated Edman degradation chemistry on the isolated peptides indicates that many of the isoforms differ by phosphorylation at Ser-444 plus attachment of one to six glutamic acid molecules to the side chain of the first glutamate residue, Glu-438, in the C-terminal sequence Tyr-Glu-Asp-Asp-Glu-Glu-Glu-Ser-glu-Ala-Gln-Gly-Pro-Lys.

Amino Acid Sequence↗
PubMed1991

Expression and cell membrane localization of rat M3 muscarinic acetylcholine receptor produced in Sf9 insect cells using the baculovirus system.

A recombinant baculovirus bearing the cDNA coding for the rat muscarinic acetylcholine receptor subtype M3 placed under the control of the Autographa californica nuclear polyhedrosis virus polyhedrin gene promoter, was constructed. Polymerase chain reaction screening was used to identify the recombinant baculovirus. Northern blot analysis of total RNA from insect cells infected with the recombinant baculovirus indicated that the transcripts were abundant. Binding assays carried out with the muscarinic antagonist [3H]quinuclidinyl benzilate indicated that more than 1 x 10(6) receptors were produced per cell. Immunofluorescence microscopy showed that the receptor is located on the cell surface.

Acetylcholine↗
PubMed1991

Purification and functional characterization of the human beta 2-adrenergic receptor produced in baculovirus-infected insect cells.

A human cDNA fragment bearing the complete coding region for the beta 2-adrenergic receptor was introduced into the genome of Autographa california nuclear polyhedrosis virus under the control of the polyhedrin promoter. Binding studies using [125I]iodocyanopindolol showed that Sf9 insect cells infected with the recombinant virus expressed approximately 1 x 10(6) beta 2-adrenergic receptors on their cell surface. Photoaffinity labeling of whole cells and membranes revealed a molecular weight of approximately 46,000 for the expressed receptor. The receptor produced in insect cells is glycosylated but the extent and pattern differ from that of the receptor from human tissue. The heterologously expressed receptor was purified by alprenolol affinity chromatography, and was able to activate isolated Gs-protein.

Adenylyl Cyclases↗
PubMed1991

Expression in Escherichia coli of the genes coding for reaction center subunits from Rhodobacter sphaeroides: wild-type proteins and fusion proteins containing one or four truncated domains from Staphylococcus aureus protein A at the carboxy-terminus.

Gene cassettes were constructed containing Rhodobacter sphaeroides puhA, pufM and pufL sequences with synthetic 5' ends for production in Escherichia coli of the H, M and L subunits of the photosynthetic reaction center. In addition, gene cassettes coding for fusion proteins with proteinase recognition site(s) between the amino-terminal part of H, M or L subunits, and the carboxy-terminal part consisting of one (B') or four (D'ABC') domains of Staphylococcus aureus protein A were constructed. A modified expression vector pDS12/RBSII containing the T5 promoter PN25, the lac operator, and a newly inserted E. coli lipoprotein ribosome-binding site was used. Inducible synthesis of plasmid-encoded polypeptides was accompanied by reduced growth. The products comigrated with R. sphaeroides reaction center subunits H, M and L. They were identified by Western blot experiments using antibodies raised against reaction center proteins. The hybrid protein containing the reaction center H subunit fused to the single domain B' was not detected by nonspecific antisera. In contrast, the three fusion proteins containing domains D'ABC' were identified using nonspecific antisera. This indicated that domains D'ABC' were sufficient to bind to the Fc part of IgG molecules, whereas domain B' was not sufficient. This property was used to purify all three fusion proteins with domains D'ABC' by affinity chromatography from the membrane fraction of E. coli cells.

Amino Acid Sequence↗
PubMed1991

[Cirrhosis secondary to chronic autoimmune hepatitis].

Cirrhosis is the usual end-result of chronic active autoimmune hepatitis. Different immunological abnormalities divide chronic active hepatitis into 3 subgroups: type I with anti-smooth muscle antibody; type II with anti-liver/kidney microsome antibody type 1 (anti-LKM1 antibody) and type III with an antibody directed against a soluble hepatic antigen. Three quarters of the patients are young women. The liver disease is often diagnosed at the stage of chronic active hepatitis, during evaluation of the subject's general condition or during investigations for an episode of jaundice; it is seldom diagnosed at the stage of constituted or decompensated cirrhosis. Fulminant forms are rare. Extrahepatic autoimmune manifestations are frequently encountered. Treatment relies on immunosuppressants, in practice corticosteroids. Corticosteroid therapy significantly prolongs the patient's life, but it does not prevent the passage to cirrhosis. In the long term these patients are exposed to all the complications of cirrhosis, notably the occurrence of hepatocellular carcinoma.

Autoimmune Diseases↗
PubMed1991

Expression in Escherichia coli of c-type cytochrome genes from Rhodopseudomonas viridis.

The genes coding for the photosynthetic reaction center cytochrome c subunit (pufC) and the soluble cytochrome c2 (cycA) from the purple non-sulfur bacterium Rhodopseudomonas viridis were expressed in Escherichia coli. Biosynthesis of the reaction center cytochrome without a signal peptide resulted in the formation of inclusion bodies in the cytoplasm amounting to 14% of the total cellular protein. A series of plasmids coding for the cytochrome subunit with varying N-terminal signal peptides was constructed in attempts to achieve translocation across the E. coli cytoplasmic membrane and heme attachment. However, the two major recombinant proteins with N-termini corresponding to the signal peptide and the cytochrome were synthesized in E. coli as non-specific aggregates without heme incorporation. An increased ratio of precursor as compared to 'processed' apo-cytochrome was obtained when expression was carried out in a proteinase-deficient strain. Cytochrome c2 from R. viridis was synthesized in E. coli as a precursor associated with the cytoplasmic membrane. An expression plasmid was designed encoding the N-terminal part of the 33 kDa precursor protein of the oxygen-evolving complex of Photosystem II from spinach followed by cytochrome c2. Two recombinant proteins without heme were found to aggregate as inclusion bodies with N-termini corresponding to the signal peptide and the mature 33 kDa protein.

Amino Acid Sequence↗
PubMed1991

Chromophore motion during the bacteriorhodopsin photocycle: polarized absorption spectroscopy of bacteriorhodopsin and its M-state in bacteriorhodopsin crystals.

The three-dimensional crystallization of bacteriorhodopsin was systematically investigated and the needle-shaped crystal form analysed. In these crystals the M-intermediate forms 10 times faster and decays 15 times more slowly than in purple membranes. Polarized absorption spectra of the crystals were measured in the dark and light adapted states. A slight decrease in the angle between the transition moment and the membrane plane was detected during dark adaptation. The crystallization of a mutated bacteriorhodopsin, in which the aspartic acid at residue 96 was replaced by asparagine, provided crystals with a long lived M-intermediate. This allowed polarized absorption measurements of the M-chromophore. The change in the polarization ratio upon formation of the M-intermediate indicates an increase in the angle between the main transition dipole and the membrane plane by 2.2 degrees +/- 0.5, corresponding to a 0.5 A displacement of one end of the chromophore out of the membrane plane of the bacteriorhodopsin molecule.

Bacteriorhodopsins↗
PubMed1991

Endogenous natriuretic and ouabain-like factors. Their roles in body fluid volume and blood pressure regulation.

An endogenous ouabain-like sodium pump inhibitor was demonstrated originally in serum or plasma of acutely extracellular fluid volume (ECFV) expanded animals and humans. Since then numerous studies have confirmed the presence of ouabain-like factor(s) (OLF) in blood, urine, cerebrospinal fluid, and various tissues including the heart and hypothalamus. Some of these OLFs represent well-known endogenous compounds, eg, free unsaturated fatty acids, which in vitro exhibit inhibition of transepithelial sodium transport, direct inhibition of the Na-K-ATPase enzyme, displacement of 3H-ouabain from its membrane receptor, and crossreaction with a digoxin antibody. Small molecular weight (MW) OLFs of yet unknown peptidic or nonpeptidic nature, which may be of hypothalamic origin, were also detected in various animal models of hypertension and in hypertensive patients. They may play a pathophysiological role especially in salt- and volume-dependent forms of hypertension. Our results show that OLFs increase basal and vasopressin-stimulated intracellular Ca2+ release in rat vascular smooth muscle cells in culture and in human platelets similar to the newly discovered endothelin. In addition, a natriuretic factor (natriuretic hormone) was detected by bioassay in plasma and urine, whose activity changes in parallel with sodium intake. We found that this natriuretic factor is associated with small peptides with a MW of less than 1,000. It is, however, unlikely that the two biological properties, ie, the ouabain-like and natriuretic activities, reside in a single compound. A number of circulating OLFs is certainly not identical with a humoral natriuretic factor. Nevertheless, there is increasing evidence for multiple interactions between OLF and the atrial natriuretic peptide (ANP).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Jejunal permeability to water and electrolytes in patients with chronic intrahepatic hypertension: evidence for a role of aldosterone.

Acute prehepatic portal hypertension induces intestinal secretion in animal models. In the course of chronic liver disease, however, these changes are not observed, despite higher portal pressures than those found in experimental studies. Eight patients without diarrhoea and with chronic alcoholic liver disease were examined for evidence of increased jejunal secretion; their suprahepatic wedge pressure was raised from 21 to 45 mmHg (mean 34.6 mmHg). Jejunal perfusion with a triple lumen catheter and a proximal occluding balloon was used to study net flows of water and chloride as well as net and unidirectional flows of sodium and potassium. No statistical difference in intestinal flows of water and electrolytes was noted between cirrhotic patients and control subjects after infusion with a 30 mmol/l glucose solution. Infusion with a 30 mmol/l mannitol solution resulted in a lower absorption of water, Na, K, and Cl than with the glucose solution. A higher rate of Na secretion was observed in cirrhotic patients than control subjects after infusion with 30 mmol/l mannitol (p less than 0.01). In addition, the rate of Na secretion was higher in cirrhotic patients than in control subjects (p less than 0.05). There was no correlation between the net flow of Na and the suprahepatic wedge pressure. A second perfusion with a 30 mmol/l glucose solution was given 75 minutes after a bolus injection of spironolactone (400 mg). Net flows of Na and Cl were lower in cirrhotic patients than in control subjects (p less than 0.05) because of a lower absorption of Na. Patients with gradually developing portal hypertension have moderate jejunal secretions of H2O and electrolytes which we assume are partly masked by increased absorption resulting from hyperaldosteronism. In contrast to animal models, this mechanism may be part of the jejunal adaptation to permeability in acute portal hypertension.

Adult↗