Antiphospholipid antibodies in patients with chronic hepatitis C virus infection or alcoholic liver disease. Preliminary results.
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Adult↗
Biomedical subjects
H Michel
Publications and source records attributed to H Michel.
[Microscopic colitis: benefit of antibiotic treatment].
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Anti-Bacterial Agents↗
Expression of functional mouse 5-HT5A serotonin receptor in the methylotrophic yeast Pichia pastoris: pharmacological characterization and localization.
The methylotrophic yeast Pichia pastoris was tested for heterologous expression of the mouse 5-HT5A receptor. Three different expression plasmids were constructed where the cDNA of the receptor was cloned under the transcriptional control of the highly inducible promoter of the P. pastoris alcohol oxidase 1 (AOX1) gen. The expression plasmids differed with respect to the signal sequences used for N-terminal fusion. In two cases the coding region was additionally fused to the c-myc tag to permit immunological detection of the receptor. Expression of functional receptor after transformation of strain GS115 was detected by radioligand binding using [3H]LSD. The construct with the best expression levels in strain GS115 was used for transformation of the protease deficient strain SMD1163. Here, the expression level was 2-8 times higher. Whole cells as well as crude membrane preparations of recombinant clones showed saturable binding of [3H]LSD with a Kd of approximately 1.9 nM. Receptor concentrations of approximately 22 pmol/mg membrane protein revealed the potential of the P. pastoris expression system for high level expression of membrane proteins. The pharmacological properties were comparable to those reported for the receptor expressed in mammalian systems.
Animals↗
[Fitz-Hugh and Curtis syndrome].
The Fitz-Hugh and Curtis syndrome is a perihepatitis following a genital infection. It usually occurs in young women but male cases have been reported. Chlamydia trachomatis is the most frequent causal agent observed in Europe and the United States. This germ is the number one cause of salpingitis in developed countries. Clinical signs include acute or recurrent pain in the right hypochondria. Liver tests are not modified and the sonographic examination is normal. The diagnosis can be suspected on the basis of serology results using either indirect immunofluorescence with live antigens specific for serotype D or K or ELISA. Positive predictive value is 80-90% and negative predictive value 96-99%. Diagnosis is formally established with laparoscopy. In our experience with more than 100 patients, the perihepatitis (glissonitis with pseudo-membranes) is frequently associated with adherences. The peritoneum is inflammatory and there are usually a small amount of liquid. Specimens are taken for culture. Polymerase chain reaction will improve the detection of Chlamydia since this germ is very difficult to culture. Antibiotic treatment with adapted drugs given for sufficient time is effective.
4-Quinolones↗
Electron transfer from the tetraheme cytochrome to the special pair in the Rhodopseudomonas viridis reaction center: effect of mutations of tyrosine L162.
The structure of the photosynthetic reaction center (RC) from Rhodopseudomonas viridis is known to high resolution. It contains a firmly bound tetraheme cytochrome from which electrons are donated to a special pair (P) of bacteriochlorophylls, which is photooxidized upon absorption of light. Tyrosine at position 162 of the L-subunit of the reaction center (L 162 Y) is a highly conserved residue positioned halfway between P and the proximal heme group (c-559) of the cytochrome. By specific mutagenesis this residue was exchanged against the amino acids phenylalanine (F), glycine (G), methionine (M), leucine (L), tryptophan (W), threonine (T), and histidine (H). All mutants were expressed in Rps. viridis using a recently established transformation system [Laussermair & Oesterhelt (1992) EMBO J. 11, 777-783]. They were shown biochemically to synthesize all four subunits of the RC (cytochrome, subunits L, M, and H) and to assemble them correctly into the membrane. The structures of two mutants (L 162 F and L 162 T) were determined and found not to differ significantly from the wild-type structure. All mutants grew photosynthetically. The absorption spectrum of all the mutants is the same as in WT, but the redox potential of P and of c-559 was changed by the mutations. The kinetics of electron transfer from the heme group to the special pair were measured in chromatophores by flash absorption. As found earlier in the wild type (Y) several exponential components were needed to fit the data.(ABSTRACT TRUNCATED AT 250 WORDS)
Circular Dichroism↗
Structure at 2.8 A resolution of cytochrome c oxidase from Paracoccus denitrificans.
The crystal structure at 2.8 A resolution of the four protein subunits containing cytochrome c oxidase from the soil bacterium Paracoccus denitrificans, complexed with antibody Fv fragment, is described. Subunit I contains 12 membrane-spanning, primarily helical segments and binds haem a and the haem a3-copper B binuclear centre where molecular oxygen is reduced to water. Two proton transfer pathways, one for protons consumed in water formation and one for 'proton pumping', could be identified. Mechanisms for proton pumping are discussed.
Computer Graphics↗
Isolation and structural characterization of trimeric cyanobacterial photosystem I complex with the help of recombinant antibody fragments.
A monoclonal antibody was derived from mice immunized with the native trimeric, photosystem I (PSI) complex from the cyanobacterium Synechococcus PCC 7002 which reacts with a conformational epitope of the PSI complex. As seen by immunoelectron microscopy, the mAb bound to the stromal side of the thylakoid membranes. The DNA sequence encoding variable regions of the mAb was cloned into recombinant plasmids, sequenced and expressed in Escherichia coli. ELISA, Western blots and immunoelectron microscopy provided evidence that the expressed paired variable domain (Fv) fragments bind to the antigen in the same way as the parent mAb. A one-step purification was applied to purify the trimeric PSI complex using an affinity tag attached to the Fv fragment. Analysis by gel electrophoresis and N-terminal sequencing revealed the presence of the psaA, psaB, psaC, psaD, psaE, psaF and psaL gene products. The antenna size of the isolated PSI/Fv was 139 +/- 9 chlorophyll a/primary electron donor. Flash-induced absorption-change measurements showed that the complex exhibited electron transfer from the primary electron donor, P700, to the Fe-S center, FA/FB. The position of the bound Fv fragment on the trimeric PSI surface was determined by high-resolution electron microscopy and digital image processing.
Amino Acid Sequence↗
Multiplicity of N-terminal structures of medium-chain alcohol dehydrogenases. Mass-spectrometric analysis of plant, lower vertebrate and higher vertebrate class I, II, and III forms of the enzyme.
Ten different alcohol dehydrogenases, representing several classes of the enzyme and a wide spread of organisms, were analyzed for patterns of N-terminal structures utilizing a combination of conventional and mass spectrometric peptide analysis. Results show all forms to be N-terminally acetylated and allow comparisons of now 40 such alcohol dehydrogenases covering a large span of forms and origins. Patterns illustrate roles of acetylation in proteins in general, define special importance of the class I N-terminal acetylation, and distinguish separate acetylated structures for all classes, as well as a common alcohol dehydrogenase motif.
Acetylation↗
Spectroscopic properties of the light-harvesting complexes from Rhodospirillum molischianum.
Spectroscopic properties, including low-temperature absorbance, linear and circular dichroism and site-selection fluorescence of the antenna complexes from Rhodospirillum molischianum have been determined. The unique 'LH1-like' character of the amino acid sequence from LH2 of this bacterium is reflected in the circular dichroism of the B850 band of this complex. The wavelength dependence of the polarization of the LH2 complex shows an unusual shape that is attributed to the octameric state of this complex. The complete amino acid sequence for the LH1 alpha-polypeptide and most of the beta-polypeptides are presented. These conform to the general features of other LH1 polypeptides. This result, in combination with spectroscopic data for LH1 imply that the organisation of the core in this bacterium is not much different from that in other purple non-sulphur bacteria.
Amino Acid Sequence↗
Interruption of the water chain in the reaction center from Rhodobacter sphaeroides reduces the rates of the proton uptake and of the second electron transfer to QB.
A chain of bound water molecules was recently identified in the photosynthetic reaction center (RC) from Rhodobacter sphaeroides by X-ray crystallography [Ermler et al. (1994) Structure 2, 925-936]. The possible role of the chain in proton transfer from the solution to the secondary quinone (QB) was investigated by site-directed mutagenesis and flash-induced absorbance spectroscopy. Pro L209, situated along the water chain about 9 A from QB, was changed into the aromatic residues Phe and Tyr in order to interrupt the chain. In the PL209Y (Pro L209-->Tyr) mutant, the very small changes in the QA-QB<==>QAQB- equilibrium constant (K2) and the first electron-transfer rates (kAB(1)) indicate that the mutation does not lead to large structural changes. In the PL209F (Pro L209-->Phe) mutant, a 7-fold decrease of kAB(1) is observed. It follows a pH dependence parallel to that of the wild type. It is consistent with no modification of the pK of the Glu L212 determined from the pH dependence of K2. The decreased kAB(1) may reflect some slight structural modification in this mutant and/or rearrangement of the cluster of charged residues close to the L209 position. The major effect of the mutations observed is a concomitant decrease of the rates of the second electron transfer, kAB(2), and of the proton uptake upon the second flash. The relative decrease of the kAB(2) rate values in the mutants is more pronounced above pH 8. Our results indicate that the mutations have specifically altered the pathway of proton transfer to QB.(ABSTRACT TRUNCATED AT 250 WORDS)
Base Sequence↗
[Secondary sclerosing cholangitis].
Secondary sclerosing cholangitis leads to slow and often irreversible destruction of the walls of both intra- and extrahepatic bile ducts. As for primary sclerosing cholangitis, clinical signs and laboratory findings reveal cholestasis. The diagnosis is confirmed by retrograde endoscopic cholangiography which shows narrowed bile ducts and rarefied ramifications of the intra-hepatic ductal system. Several causes have been identified including infectious causes with or without a relationship to bile duct obstruction and human immunodeficiency virus infection as well as ischaemic related causes after chemotherapy, arterial embolization or liver transplantation. Other causes include chemical aggression after treatment for hydatic cysts and post-surgical complications due to a damaged bile tract. Treatment is difficult and often dependent on the cause.
AIDS-Related Opportunistic Infections↗
Immunoelectron microscopy and epitope mapping with monoclonal antibodies suggest the existence of an additional N-terminal transmembrane helix in the cytochrome b subunit of bacterial ubiquinol:cytochrome-c oxidoreductases.
The topology of the ubiquinol:cytochrome-c oxidoreductase (cytochrome bc1 complex) from Paracoccus denitrificans was investigated by immunoelectron microscopy with sequence-specific murine monoclonal antibodies. Epitope mapping with synthetic peptides and enzymic proteolytic cleavage of the cytochrome bc1 complex were employed to localize precisely the respective antibody epitopes on the subunits of this membrane protein complex. Localization of defined epitopes on the cytochrome bc1 complex by immunoelectron microscopy clearly demonstrates that the N-terminus of the cytochrome b subunit is exposed to the periplasmic space. This finding is in agreement with a nine-transmembrane-helices topology model (I-IX) as predicted before for cytochrome b. However, due to other published evidence we favour the existence of an additional transmembrane helix (helix 0) complementing a more recently published eight-helices model (A-C,cd, D-H), at least for prokaryotes.
Amino Acid Sequence↗
Structural transitions during bacteriophage HK97 head assembly.
Bacteriophage HK97 builds its head shell from a 42 kDa major head protein, but neither this 42 kDa protein nor its processed, 31 kDa form is found in the mature head. Instead, each of the major head-protein subunits is covalently cross-linked into oligomers of five, six or more by a protein cross-linking reaction that occurs both in vivo and in vitro. Mutants that block prohead maturation lead to the accumulation of one of two types of proheads, termed Prohead I and Prohead II. Prohead I is assembled from about 415 copies of the 42 kDa (384 amino acids) protein subunit and accumulates in infections by mutant amU4. Following assembly, the N-terminal 102 amino acids of each subunit are removed, leaving a prohead shell constructed of 31 kDa subunits, called Prohead II, which accumulates in infections by mutant amC2. During DNA packaging, when the prohead shell expands, all of the head protein subunits become covalently cross-linked to other subunits. Purified Prohead II (or, less completely, Prohead I) becomes cross-linked in vitro in response to any of a number of conditions that induce shell expansion, including conditions commonly used for protein analysis. In vitro cross-linking occurs efficiently in the absence of added cofactors of enzymes, and we propose that cross-linking is catalyzed by shell subunits themselves. Shell expansion is easily monitored by observing a decrease in electrophoretic mobility of Prohead II in agarose gels. Using the mobility shift in agarose gel to monitor expansion and SDS/gel electrophoresis to monitor cross-linking in vitro, we find that expansion precedes and is required for cross-linking, and we propose that expansion triggers the cross-linking reaction. Comparison of peptides isolated from Prohead II and in vitro cross-linked Prohead II shows a single altered major cross-link peptide in which a lysine, originating from lysine169 of the protein sequence, is linked to asparagine356, presumably derived from the neighboring subunit. Examination of the cross-link-containing peptide by mass spectrometry shows that the cross-link bond is an amide between the side-chains of the lysine and the asparagine residues.
Amino Acid Sequence↗
Crystallographic refinement at 2.3 A resolution and refined model of the photosynthetic reaction centre from Rhodopseudomonas viridis.
The atomic model of the photosynthetic reaction centre from the purple bacterium Rhodopseudomonas viridis has been refined to an R-value of 0.193 at 2.3 A resolution. The refined model contains 10,288 non-hydrogen atoms; 10,045 of these have well defined electron density. A Luzzati-plot indicates an average co-ordinate error of 0.26 A. During refinement, the positions of a partially ordered carotenoid, a unibiquinone in the partially occupied QB site, a detergent molecule, seven putative sulphate ions, and 201 water molecules were found. More than half of these waters are bound at interfaces between protein subunits and therefore contribute significantly to subunit interactions. Water molecules also play important structural and probably functional roles in the environment of some of the cofactors. Two water molecules form hydrogen bonds to the accessory bacteriochlorophylls and to the protein in the vicinity of the special pair of bacteriophylls, the primary electron donor. A group of about 10 water molecules is bound near the binding site of the secondary quinone QB. These waters are likely to participate in the transfer of protons to the doubly reduced QB.
Amino Acid Sequence↗
Superior mesenteric vein thrombosis associated with anticardiolipin antibody without autoimmune disease.
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Adult↗
Predicting the structure of the light-harvesting complex II of Rhodospirillum molischianum.
We attempted to predict through computer modeling the structure of the light-harvesting complex II (LH-II) of Rhodospirillum molischianum, before the impending publication of the structure of a homologous protein solved by means of X-ray diffraction. The protein studied is an integral membrane protein of 16 independent polypeptides, 8 alpha-apoproteins and 8 beta-apoproteins, which aggregate and bind to 24 bacteriochlorophyll-a's and 12 lycopenes. Available diffraction data of a crystal of the protein, which could not be phased due to a lack of heavy metal derivatives, served to test the predicted structure, guiding the search. In order to determine the secondary structure, hydropathy analysis was performed to identify the putative transmembrane segments and multiple sequence alignment propensity analyses were used to pinpoint the exact sites of the 20-residue-long transmembrane segment and the 4-residue-long terminal sequence at both ends, which were independently verified and improved by homology modeling. A consensus assignment for the secondary structure was derived from a combination of all the prediction methods used. Three-dimensional structures for the alpha- and the beta-apoprotein were built by comparative modeling. The resulting tertiary structures are combined, using X-PLOR, into an alpha beta dimer pair with bacteriochlorophyll-a's attached under constraints provided by site-directed mutagenesis and spectral data. The alpha beta dimer pairs were then aggregated into a quaternary structure through further molecular dynamics simulations and energy minimization. The structure of LH-II so determined is an octamer of alpha beta heterodimers forming a ring with a diameter of 70 A.
Amino Acid Sequence↗
Crystals of an antibody Fv fragment against an integral membrane protein diffracting to 1.28 A resolution.
The Fv fragment of a monoclonal antibody, 7E2 (IgG1, kappa, murine), which is directed against the integral membrane protein cytochrome c oxidase (EC 1.9.3.1) from Paracoccus denitrificans, was cloned and produced in Escherichia coli. Crystals suitable for high-resolution X-ray analysis were obtained by microdialysis under low salt conditions. The crystals belong to the orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions of a = 51.51 A, b = 56.15 A, c = 99.86 A (1 A = 0.1 nm) and contain one Fv fragment per asymmetric unit. Using synchrotron radiation diffraction data were collected up to 1.28 A resolution. This high resolution is very unusual for a heterodimeric protein. The crystals should open the way for refining not only the atomic positions, but also for obtaining information about internal dynamics.
Amino Acid Sequence↗
Failure of ursodeoxycholic acid to prevent acute cellular rejection after liver transplantation.
BACKGROUND/AIMS: Acute rejection is still a major problem after liver transplantation. Ursodeoxycholic acid has beneficial effects in cholestasis by reducing the expression of major histocompatibility complex antigens. METHODS: We have performed a double-blind randomised study comparing ursodeoxycholic acid with placebo for the prevention of acute cellular rejection after liver transplantation. Twenty-six patients received ursodeoxycholic acid 600 mg per day and 24 patients received placebo for 2 months. RESULTS: Neither rejection incidence nor rejection severity was significantly different in the two groups (p > 0.90). CONCLUSIONS: We conclude that adjuvant ursodeoxycholic acid administration does not prevent rejection after liver transplantation.
Acute Disease↗