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Biomedical subjects

H Michel

Publications and source records attributed to H Michel.

At least 91 records · Page 5Linked to original sources
PubMed1996

Molecular cloning, DNA sequence and transcriptional analysis of the Rhodospirillum molischianum B800/850 light-harvesting genes.

The amino acid sequences of the B800/850 light-harvesting proteins from Rhodospirillum molischianum were determined by Edman degradation. On the basis of these amino acid sequences, two degenerated oligonucleotides were synthesized and used for PCR of genomic DNA. The resulting 150 bp DNA fragment was cloned, sequenced and used for subsequent Southern blot analysis of digested genomic DNA. A 2.3 kbp EcoRI fragment strongly hybridized to the probe and a size selected genomic library from genomic DNA was constructed. One clone scored positive during screening of the library with the PCR-fragment and subsequent DNA sequence analysis of the clone revealed the presence of three A-genes (A1A2A3) encoding alpha-polypeptides and of two B-genes (B1B2) encoding beta-polypeptides of the B800/850 complex. The arrangement of the different genes are B1A1, B2A2 and A3 where only B1 and B2 are preceded by typical Shine-Dalgarno sequences. In addition, typical nucleotide sequences for a rho-independent termination of transcription are located downstream of the genes A1 and A2. The deduced amino acid sequences revealed that the alpha-genes encoded for identical polypeptides, whereas the deduced beta-polypeptides differed in their amino acid sequence at four positions. Transcriptional operon analysis revealed that the genes A1B1 and A2B2 are both dicistronically transcribed, whereas the gene A3 is not.

Amino Acid Sequence↗
PubMed1996

[Painful rib syndrome (or Cyriax syndrome). Study of 100 patients].

OBJECTIVE: To analyse clinical expression and outcome of painful rib syndrome in a large series of 100 cases. METHODS: From 1978 to 1993, 100 consecutive patients with chronic anterior chest pain or supramesocolic abdominal pain of unknown origin underwent complete physical examination, laboratory tests and complementary explorations as required. RESULTS: Among the first 100 patients the sex ratio was 3.34 and mean age 50.6 years (21-80). Chronic pain had persisted for an average 41.2 months (15 days-30 years), predominantly on the right (81%) and exceptionally bilaterally (1%). No evidence of a cause could be identified from laboratory tests or complementary explorations. A past history of direct trauma was found in 71 patients and indirect trauma in 21. Seventy-three patients were given 1% lidocaine infiltrations (20 to 40 ml) including 14 who received 2 or 3 infiltrations. Six patients underwent surgical resection of a luxated cartilage with curative effect in 5. CONCLUSION: The diagnosis of painful rib syndrome is based solely on the presence of pain upon applying pressure to the anteroinferior border of the rib cage and is related to often neglected or forgotten trauma.

Adult↗
PubMed1996

Deletion of a B800-850 light-harvesting complex in Rhodospirillum molischianum DSM119 leads to "revertants" expressing a B800-820 complex: insights into pigment binding.

A B800-850 light-harvesting complex (also called LH2) deficient strain of Rhodospirillum molischianum was constructed by replacing a portion of the LH2 gene cluster by a kanamycin resistance gene cartridge. The LH2 deficient strain was characterized spectroscopically and by Southern blot analysis. Surprisingly, pseudorevertants were obtained which express a B800-820 complex which could not be observed in the wild type. This B800-820 complex was isolated and characterized. It consists of an alpha- and a beta subunit with 56 and 45 amino acid residues, respectively. The amino acid sequences of both subunits are extremely similar to those of the corresponding B800-850 complex. Resonance Raman spectroscopy shows that in the B800-820 complex the two 2-acetylcarbonyl groups of the bacteriochlo-rophyll a (BChl a) molecules absorbing at 820 nm are free from hydrogen bond interactions, whereas one of the two 2-acetylcarbonyl groups of the pair of BChl a molecules absorbing at 850 nm of the B800- 850 complex is involved in hydrogen bonds. These different protein- pigment interactions are due to the replacement of alpha Trp43 in the B800-850 complex by a Phe in the B800- 820 complex. Comparison of the amino acid sequences of the B800-850 and B800-820 complexes of Rs. molischianum and Rhodopseudomonas acidophila reveals a conserved motif comprised of three amino acid residues. Molecular modeling using the known LH2 structure of Rps. acidophila Ac 10050 indicates that this motif might be important for the precise structural arrangement of the native complex and fine tuning of its spectroscopic properties.

Amino Acid Sequence↗
PubMed1996

Structure of a water soluble fragment of the 'Rieske' iron-sulfur protein of the bovine heart mitochondrial cytochrome bc1 complex determined by MAD phasing at 1.5 A resolution.

BACKGROUND: The 'Rieske' iron-sulfur protein is the primary electron acceptor during hydroquinone oxidation in cytochrome bc complexes. The spectroscopic and electrochemical properties of the 'Rieske' [2Fe-2S] cluster differ significantly from those of other iron-sulfur clusters. A 129-residue water soluble fragment containing the intact [2Fe-2S] cluster was isolated following proteolytic digestion of the bc1 complex and used for structural studies. RESULTS: The structure of the Rieske iron-sulfur fragment containing the reduced [2Fe-2S] cluster has been determined using the multiwavelength anomalous diffraction (MAD) technique and refined at 1.5 A resolution. The fragment has a novel overall fold that includes three sheets of beta strands. The iron atoms of the [2Fe-2S] cluster are coordinated by two cysteine (Fe-1) and two histidine (Fe-2) residues, respectively, with the histidine ligands completely exposed to the solvent. This is in contrast to the four cysteine coordination pattern observed in previously characterised [2Fe-2S] ferredoxins. The cluster-binding fold is formed by two loops connected by a disulfide bridge; these loops superpose with the metal-binding loops of rubredoxins. The environment of the cluster is stabilised by an extensive hydrogen-bond network. CONCLUSIONS: The high-resolution structure supports the proposed coordination pattern involving histidine ligands and provides a basis for a detailed analysis of the spectroscopic and electrochemical properties. As the cluster is located at the tip of the protein, it might come into close contact with cytochrome b. The exposed N epsilon atoms of the histidine ligands of the cluster are readily accessible to quinones and inhibitors within the hydroquinone oxidation (QP) pocket of the bc1 complex and may undergo redox-dependent protonation/deprotonation.

Amino Acid Sequence↗
PubMed1996

The crystal structure of the light-harvesting complex II (B800-850) from Rhodospirillum molischianum.

BACKGROUND: The light-harvesting complexes II (LH-2s) are integral membrane proteins that form ring-like structures, oligomers of alpha beta-heterodimers, in the photosynthetic membranes of purple bacteria. They contain a large number of chromophores organized optimally for light absorption and rapid light energy migration. Recently, the structure of the nonameric LH-2 of Rhodopseudomonas acidophila has been determined; we report here the crystal structure of the octameric LH-2 from Rhodospirillum molischianum. The unveiling of similarities and differences in the architecture of these proteins may provide valuable insight into the efficient energy transfer mechanisms of bacterial photosynthesis. RESULTS: The crystal structure of LH-2 from Rs. molischianum has been determined by molecular replacement at 2.4 A resolution using X-ray diffraction. The crystal structure displays two concentric cylinders of sixteen membrane-spanning helical subunits, containing two rings of bacteriochlorophyll-a (BChl-a) molecules. One ring comprises sixteen B850 BChl-as perpendicular to the membrane plane and the other eight B800 BChl-as that are nearly parallel to the membrane plane; eight membrane-spanning lycopenes (the major carotenoid in this complex) stretch out between the B800 and B850 BChl-as. The B800 BChl-as exhibit a different ligation from that of Rps. acidophila (aspartate is the Mg ligand as opposed to formyl-methionine in Rps. acidophila). CONCLUSIONS: The light-harvesting complexes from different bacteria assume various ring sizes. In LH-2 of Rs. molischianum, the Qy transition dipole moments of neighbouring B850 and B800 BChl-as are nearly parallel to each other, that is, they are optimally aligned for Föster exciton transfer. Dexter energy transfer between these chlorophylls is also possible through interactions mediated by lycopenes and B850 BChl-a phytyl tails; the B800 BChl-a and one of the two B850 BChl-as associated with each heterodimeric unit are in van der Waals distance to a lycopene, such that singlet and triplet energy transfer between lycopene and the BChl-as can occur by the Dexter mechanism. The ring structure of the B850 BChl-as is optimal for light energy transfer in that it samples all spatial absorption and emission characteristics and places all oscillator strength into energetically low lying, thermally accessible exciton states.

Amino Acid Sequence↗
PubMed1996

Carboxyl group protonation upon reduction of the Paracoccus denitrificans cytochrome c oxidase: direct evidence by FTIR spectroscopy.

The redox reactions of the cytochrome c oxidase from Paracoccus denitrificans were investigated in a thin-layer cell designed for the combination of electrochemistry under anaerobic conditions with UV/VIS and IR spectroscopy. Quantitative and reversible electrochemical reactions were obtained at a surface-modified electrode for all cofactors as indicated by the optical signals in the 400-700 nm range. Fourier transform infrared (FTIR) difference spectra of reduction and oxidation (reduced-minus-oxidized and oxidized-minus-reduced, respectively) obtained in the 1800-1000 cm(-1) range reveal highly structured band features with major contributions in the amide I (1620-1680 cm(-1)) and amide II (1580-1520 cm(-1)) range which indicate structural rearrangements in the cofactor vicinity. However, the small amplitude of the IR difference signals indicates that these conformational changes are small and affect only individual peptide groups. In the spectral region above 1700 cm(-1), a positive peak in the reduced state (1733 cm(-1)) and negative peak in the oxidized st ate (1745 cm(-1)) are characteristic for the formation and decay of a COOH mode upon reduction. The most obvious interpretation of this difference signal is proton uptake by one Asp or Glu side chain carboxyl group in the reduced state and deprotonation of another Asp or Glu residue. Moreover, both residues could well be coupled as a donor-acceptor pair in the proton transfer chain. An alternative interpretation is in terms of a protonated carboxyl group which shifts to a different environment in the reduced state. The relevance of this first direct observation of protein protonation changes in the cytochrome c oxidase for vectorial proton transfer and the catalytic reaction is discussed.

Electron Transport Complex IV↗
PubMed1996

A versatile plasmid expression vector for the production of biotinylated proteins by site-specific, enzymatic modification in Escherichia coli.

A versatile plasmid vector was designed to direct the synthesis of recombinant proteins in either one of two forms that will be biotinylated in Escherichia coli with high efficiency at a single, unique site. The protein of interest can be produced with a peptide substrate for E. coli biotin holoenzyme synthetase (BirA) joined directly to its N terminus, or alternatively, as a fusion to the C terminus of a maltose-binding protein domain (MalE) with the peptide substrate on its N terminus. To maximize the yield of biotinylated protein, the vector is designed to express the substrate in a coupled translation arrangement with the enzyme.

ATP-Binding Cassette Transporters↗
PubMed1996

Crystallization and preliminary X-ray diffraction studies of formylmethanofuran: tetrahydromethanopterin formyltransferase from Methanopyrus kandleri.

Formylmethanofuran:tetrahydromethanopterin formyltransferase from the hyperthermophilic methanogenic Archaeon Methanopyrus kandleri (growth temperature optimum 98 degrees C) was crystallized by vapor diffusion methods. Crystal form M obtained with 2-methyl-2,4-pentanediol as precipitant displayed the space group P2(1) with unit cell parameters of a = 87.0 A, b = 75.4 A, c = 104.7 A, and beta = 113.9 degrees and diffracted better than 2 A resolution. Crystal form P grown from polyethylene glycol 8000 belonged to the space group I4(1)22 and had unit cell parameters of 157.5 A and 242.1 A. Diffraction data to 1.73 A were recorded. Crystal form S which was crystallized from (NH4)2SO4 in the space group I4(1)22 with unit cell parameters of 151.3 A and 249.5 A diffracted at least to 2.2 A resolution. All crystal forms probably have four molecules per asymmetric unit and are suitable for X-ray structure analysis.

Archaea↗
PubMed1996

On the role of the N-terminus of the extrinsic 33 kDa protein of Photosystem II.

The role of the N-terminus of the extrinsic 33 kDa protein of Photosystem II has been investigated by means of site-directed mutagenesis and cross-linking. Replacement of Asp-9 resulted in a dramatic increase in proteolytic sensitivity leading to the degradation of the protein forming a 31 kDa fragment with an undefined N-terminus. This fragment was unable to restore oxygen evolution. However, the variants of the 33 kDa protein which remained intact could reconstitute oxygen evolution as effectively as the wild-type protein. Cross-linking experiments with a water-soluble carbodiimide revealed that mutagenesis of residue D9 led to the disruption of an intramolecular salt bridge. Therefore we suggest that the N-terminus of the 33 kDa protein is necessary for maintaining the binding ability of the protein to Photosystem II but might not be involved in binding itself.

Amino Acid Sequence↗
PubMed1996

Calculated coupling of electron and proton transfer in the photosynthetic reaction center of Rhodopseudomonas viridis.

Based on new Rhodopseudomonas (Rp.) viridis reaction center (RC) coordinates with a reliable structure of the secondary acceptor quinone (QB) site, a continuum dielectric model and finite difference technique have been used to identify clusters of electrostatically interacting ionizable residues. Twenty-three residues within a distance of 25 A from QB (QB cluster) have been shown to be strongly electrostatically coupled to QB, either directly or indirectly. An analogous cluster of 24 residues is found to interact with QA (QA cluster). Both clusters extend to the cytoplasmic surface in at least two directions. However, the QB cluster differs from the QA cluster in that it has a surplus of acidic residues, more strong electrostatic interactions, is less solvated, and experiences a strong positive electrostatic field arising from the polypeptide backbone. Consequently, upon reduction of QA or QB, it is the QB cluster, and not the QA cluster, which is responsible for substoichiometric proton uptake at neutral pH. The bulk of the changes in the QB cluster are calculated to be due to the protonation of a tightly coupled cluster of the three Glu residues (L212, H177, and M234) within the QB cluster. If the lifetime of the doubly reduced state QB2- is long enough, Asp M43 and Ser L223 are predicted to also become protonated. The calculated complex titration behavior of the strongly interacting residues of the QB cluster and the resulting electrostatic response to electron transfer may be a common feature in proton-transferring membrane protein complexes.

Amino Acid Sequence↗
PubMed1996

Contribution of primary cultures of adult human hepatocytes to the pathophysiology of hepatocellular carcinoma.

BACKGROUND/AIMS: The mechanisms of hepatocarcinogenesis are still poorly understood. The development of hepatocellular carcinoma has recently been shown to be associated with increased DNA synthesis in cirrhosis. The aim of this work was to determine whether the high rate of hepatocyte regeneration observed in cirrhotic liver with hepatocellular carcinoma is associated with the presence of a growth factor that could be detectable in the serum. METHODS: Adult human hepatocytes in primary culture, allowing the evaluation of the release of circulating hepatotrophic factors, were used. These cultures were treated for 48 h with serum from patients with cirrhosis with and without hepatocellular carcinoma, from patients with liver metastasis, and from healthy subjects. The rate of DNA synthesis in these cultures was assessed by measuring the amount of [3H]-thymidine incorporation into genomic DNA. RESULTS: On average, the synthesis of DNA was increased 2.5-, 2.2-, 2.1-, and 2.3-fold, respectively, in response to serum from patients with cirrhosis with hepatocellular carcinoma, from patients with cirrhosis without hepatocellular carcinoma, from patients with liver metastasis, and from healthy subjects. CONCLUSIONS: We conclude that the hepatotrophic activity of the serum is not significantly different in patients with cirrhosis with or without hepatocellular carcinoma. These results suggest that the increased DNA synthesis in hepatocytes of cirrhotic liver with hepatocellular carcinoma might be due to proliferative factor(s) acting by paracrine or autocrine pathways.

Adult↗
PubMed1996

Cytochrome c oxidase.

Within the past year, the structures of the cytochrome c oxidase from the soil bacterium Paracoccus denitrificans and of the metal centers of the cytochrome c oxidase from bovine heart mitochondria, both determined at 2.8 A resolution by X-ray crystallography, have been reported. The structures form a basis for understanding the mechanism of this redox-coupled transmembrane proton pump, which is the key component of the respiratory chain of most aerobic organisms.

Animals↗
PubMed1996

Fulminant hepatitis after carbimazole and propranolol administration.

We report the case of a patient who developed jaundice, encephalopathy, a marked increase in serum aminotransferase activity and a decrease in prothrombin and proaccelerin levels, after 6 weeks' treatment with carbimazole and propranolol for hyperthyroidism. The patient ultimately underwent orthotopic liver transplantation. This case strongly suggests that carbimazole may occasionally induce fulminant hepatitis and that careful monitoring of liver enzymes may be useful during the treatment of hyperthyroidism with this drug.

Adrenergic beta-Antagonists↗
PubMed1996

Treatment of the carcinoid syndrome with the longacting somatostatin analogue lanreotide: a prospective study in 39 patients.

BACKGROUND: Somatostatin analogues effectively control flushing and diarrhoea in patients with the carcinoid syndrome. The octapeptide lanreotide is available in slow release form, which could eliminate the necessity of twice a day injections as with octreotide. PATIENTS AND METHODS: 39 patients with carcinoid syndrome were included in a prospective multicentre study. Patients received lanreotide 30 mg intramuscularly every 14 days for six months. The number and intensity of flushing episodes and bowel movements, urinary 5 hydroxy-indolacetic acid (5 HIAA) concentrations, and variations of tumour mass were recorded. RESULTS: After one month of treatment, flushing episodes (median (range)) decreased significantly (3 (0.3-24) episodes per day v 1 (0-15), p = 0.04) and completely resolved in 39% of the patients. A significant decrease was seen in the number of bowel movements and discomfort related to diarrhoea. Urinary 5 HIAA concentrations were unchanged in 57% of the patients and decreased in 18%. After six months of treatment, the actuarial proportions of patients with at least a 50% decrease in the number of flushing episodes and bowel movements were 54% and 56%, respectively. Forty two per cent of the patients who were treated for six months had at least a 50% reduction in 5 HIAA values. No clear signs of regression of tumours were seen in any of the patients. Lanreotide was well tolerated despite transient mild pain or erythema at the injection site in 25% of the patients. Biliary lithiasis appeared in two patients after six months of lanreotide. CONCLUSION: Lanreotide, 30 mg intramuscularly every other week, is an effective and convenient treatment in patients with the carcinoid syndrome.

Biomarkers, Tumor↗
PubMed1996

Use of nanogold- and fluorescent-labeled antibody Fv fragments in immunocytochemistry.

Recombinant antibody fragments are emerging as a versatile tool in both basic research and medical therapy. We describe the procedures for direct labeling of engineered antibody fragments (Fv) with fluorescein or nanogold and their use in fluorescence and immunoelectron microscopy, respectively. The Fv fragments were produced in Escherichia coli, purified by one-step Strep tag affinity chromatography, chemically labeled with the marker, and employed in microscopy to localize epitopes on the membrane protein bacteriorhodopsin in purple membranes of Halobacterium halobium and the cytochrome c oxidase of Paracoccus denitrificans. In both cases, methods involving directly labeled antibody fragments show results identical to those in which antibodies or Fv fragments are detected by a secondarily labeled conjugate. The multifunctional design of the recombinant Fv fragments, however, offers more all-around applications in immunocytochemistry. The directly labeled Fv fragments, half the size of an Fab fragment, are at the molecular level the smallest antibody fragments yet described for visualization of biomolecules in microscopy.

Escherichia coli↗