[18FDG-PET and large vessel vasculitis: preliminary data on 25 patients]. / 18FDG-PET e vasculiti dei grandi vasi: studio preliminare su 25 pazienti.

OBJECTIVE: To evaluate the predictive value of clinical and biochemical features when compared to 18FDG-PET in the diagnostic work-up of large vessel vasculitis (LVV). METHODS: Twenty-five patients underwent 18FDG-PET for the clinical suspect of LVV. All of them presented history of systemic symptoms lasting >or=6 months and laboratoristic evidence of persistently high markers of inflammation. The patients were stratified according with: i) clinical manifestations, defined as presence of one or more ACR criteria for the classification of LVV; ii) laboratory investigations: Erythrocyte Sedimentation Rate (ESR) higher or lower than 50 mm/h, C-Reactive Protein (CRP) higher or lower than 2 mg/dl; iii) prednisone dose in the 4 weeks preceding PET examination. RESULTS: The total number of positive PET was higher in the group without clinical ACR criteria and in the group with inflammation markers under the established cut-off. The number of scans consistent with LVV was higher in the groups presenting one or more clinical criteria for LVV but in those with very high ESR and CRP. In all the cases differences between groups were not statistically significative. A clear cut negative correlation between steroid dose and number of scans suggestive for LVV has been observed. CONCLUSIONS: Diagnosis of LVV remains challenging, especially in patients presenting with a constellation of non-specific symptoms and laboratory findings. In this study, both clinical and biochemical features show low correlation with a vasculitic pattern of FDG uptake. In our experience 18FDG-PET represents an useful diagnostic tool in early stages of LVV and a powerful instrument to follow the treatment responses.

Crystal structure of a dimeric octaheme cytochrome c3 (M(r) 26,000) from Desulfovibrio desulfuricans Norway.

BACKGROUND: The octaheme cytochrome C3 (M(r) 26,000; cc3) from Desulfovibrio desulfuricans Norway is a dimeric cytochrome made up of two identical subunits, each containing four heme groups. It is involved in the redox transfer chain of sulfate-reducing bacteria, which links the periplasmic oxidation of hydrogen to the cytoplasmic reduction of sulfate. The amino-acid sequence of cc3 shows similarities to that of the tetraheme cytochrome c3 (M(r) 13,000; c3) from the same bacteria. Structural analysis of cc3 forms a basis for understanding the precise roles of the multiheme-containing redox proteins and the reason for the presence of several different multiheme cytochromes in one bacterial strain. RESULTS: The crystal structure of cytochrome cc3 has been determined at 2.16 A resolution. The subunits display the c3 structural fold with significant amino-acid substitutions, relative to the tetraheme cytochromes c3, in the regions of the dimer interface. The identical subunits are related by a crystallographic twofold axis, with one heme of each subunit in close contact. The overall structure and the environments of the different heme groups are compared with those of the tetraheme cytochromes c3. CONCLUSIONS: A common scheme for interactions between these types of cytochrome and their redox partners involves the interaction of a heme crevice, surrounded by positively charged lysine residues, with acidic residues surrounding the redox partner's functional group. Despite the relatively acidic character of cytochrome cc3, the crevice of one heme is surrounded by a high number of positively charged residues, in the same manner as has been reported for cytochromes c3. The environment of this heme is formed by four flexible surface loops which are variable in length and orientation in the different c3-type cytochromes although the overall structural folds are very similar. It has been proposed that this region, adapted in topology and charge, is the interaction site for physiological partners and is also most likely to be the interaction site in the dimeric cytochrome cc3.

Non-heme iron proteins. The amino acid sequence of rubredoxin from Desulfovibrio vulgaris.

A non-heme iron protein, rubredoxin has been isolated from the sulfate-reducing bacterium, Desulfovibrio vulgaris, strain Hildenborough. The complete amino acid sequence has been established. The 52 amino acid residues of the protein were aligned with the aid of tryptic and chymotryptic peptides and of a fragment produced by cleavage of the Asn-Gly bond (22-23) by hydroxylamine. The sequence of the first 30 residues of the molecule was determined using an automatic sequenator, after removal of the N-terminal methionine by CNBr. In comparing this sequence with those of Micrococcus aerogenes, Clostridium pasteurianum and Peptostreptococcus elsdenii rubredoxins, a high degree of mutation was observed between these homologous proteins. It has been shown that 20 amino acid residues occurred in identical positions. The locations of the four cysteine residues were found to be invariable. A crystallographic study of the Desulfovibrio vulgaris rubredoxin is in progress.

Characterization of a new type of ferredoxin from Desulfovibrio africanus.

A new ferredoxin designated ferredoxin III has been isolated from Desulfovibrio africanus grown on media high in iron. Native ferredoxin III is a dimer constituted by two identical subunits of approx. 7500. It is distinguished from the two other ferredoxins (I and II) isolated from this microorganism by its amino acids composition, N-terminal sequence, spectral properties and iron-sulfur content. The amino acid composition of D. africanus ferredoxin III is typical of ferredoxins with an excess of acidic over basic residues and the absence of histidine and arginine residues. The absorption spectrum of ferredoxin III exhibits two maxima, at 408 nm (epsilon = 58.5 . 10(3) M-1 . cm-1) and 285 nm (epsilon = 82 . 10(3) M-1 . cm-1), with a shoulder at 305 nm (epsilon = 75 . 10(3) M-1 . cm-1). Its A408/A285 absorbance ratio is 0.78. Ferredoxin III contains approx. 12--13 atoms each of iron and labile sulfur. This is in agreement with the high value of the extinction coefficient at 408 nm, which is slightly higher than 3-fold that of one [4Fe-4S] cluster. However, the number of cysteine residues of the protein (six residues), which is about the half that of iron atoms, is indicative of the presence of a new type of iron-sulfur cluster in ferredoxin III. The protein is unstable in a low ionic strength environment; the addition of neutral salts stabilizes the protein conformation. The data on the biological activity of ferredoxin III as compared to the two other ferredoxins from D. africanus show that the three iron-sulfur proteins function with equal effectiveness as electron carrier in the phosphoroclastic reaction and the H2-sulfite reductase system.

Amino-acid sequence of cytochrome c-553 from Desulfovibrio desulfuricans Norway.

The amino-acid sequence of the cytochrome c-553 from Desulfovibrio desulfuricans Norway has been determined and compared with that of two different cytochromes c-553 from D. vulgaris already described and with that cytochrome c-551 from Pseudomonas. This low-molecular-weight monohemic cytochrome comprises 80 amino acids and has the typical characteristics of small cytochromes such as mitochondrial cytochromes. Secondary-structure predictions are deduced from sequence data and are compared with X-ray three-dimensional structures of low-molecular-weight cytochrome c. The phylogenetic situation of Desulfovibrio cytochromes c-533 in the cytochrome c superfamily is discussed.

Identification of the site of interaction between cytochrome c3 and ferredoxin using peptide mapping of the cross-linked complex.

Structural studies carried out on a cross-linked complex between cytochrome c3 and ferredoxin I, both isolated from Desulfovibrio desulfuricans Norway, allowed the identification of the site of interaction between the two redox proteins. Staphylococcus aureus proteinase and chymotrypsin digestions led to characterization of peptides containing both cytochrome c3 and ferredoxin sequences. The cytochrome c3 sequences involved in the three isolated cross-linked peptides contained several lysine residues localized around the heme 4 crevice. This analysis stressed the peculiar role of lysines 100, 101, 103, 104 and 113, which could be considered as major cross-link sites, as opposed to the lysines 75, 79 and 82, which could be considered as minor cross-link sites. One cross-linked peptide, containing two ferredoxin sequences joined to one cytochrome c3 sequence, had been isolated, suggesting the possibility of more than one cross-link per covalent complex. All these results led to the identification of heme 4 of cytochrome c3 as the site of interaction for the ferredoxin I. This study confirms the proposal that could be deduced from the hypothetical structure of the complex built by computer graphics modelling (Cambillau, C., Frey, M., Mosse, J., Guerlesquin, F. and Bruschi, M. (1988) Proteins: struct., funct. genet. 4, 63-70).

Amino-acid sequence of the cytochrome c3 (M(r) 26,000) from Desulfovibrio desulfuricans Norway and a comparison with those of the other polyhemic cytochromes from Desulfovibrio.

The amino-acid sequence of an octaheme cytochrome c3 isolated from Desulfovibrio desulfuricans Norway is presented. The protein molecule (M(r) 26,000) comprises two identical subunits of 111 amino acids with the characteristics typical of tetrahemic cytochrome c3 class. Comparisons between the amino-acid sequences and physiological properties of cytochrome c3 (M(r) 26,000) and cytochromes c3 (M(r) 13,000) isolated from various species of Desulfovibrio showed the existence of considerable differences. In order to distinguish between the various subclasses in the cytochrome c3 superfamily, the amino-acid sequence of cytochrome c3 (M(r) 26,000) was compared with six known cytochrome c3 (M(r) 13,000) sequences as well as with the sequence of the four c3-like domains of a high molecular weight cytochrome c (Hmc) containing 16 hemes per molecule of 65,500 Da, isolated from Desulfovibrio vulgaris Hildenborough. The evolution and phylogenetic relationships of these various polyhemic cytochromes are discussed.

Isolation and characterization of a rubredoxin and two ferredoxins from Desulfovibrio africanus.

Rubredoxin and two distinct ferredoxins have been purified from Desulfovibrio africanus. The rubredoxin has a molecular weight of 6000 while the ferredoxins appear to be dimers of identical subunits of approximately 6000 to 7000 molecular weight. Rubredoxin contains one iron atom, no acid-labile sulfide and four cysteine residues per molecule. Its absorbance ratio A278/A490 is 2.23 and its amino acid composition is characterized by the absence of leucine and a preponderance of acidic amino acids. The two ferredoxins, designated I and II, are readily separated on DEAE-cellulose. The amino acid compositions of ferredoxins I and II show them to be different protein species; the greater number of acidic amino acid residues in ferredoxin I than in ferredoxin II appears to account for separation based on electronic charge. Both ferredoxins contain four iron atoms, four acid-labile residues per molecule. Spectra of the two ferredoxins differ from those of ferredoxins of other Desulfovibrio species by exhibiting a pronounced absorption peak at 283 nm consistent with an unusual high content of aromatic residues. The A385/A283 absorbance ratio of ferredoxins I and II are 0.56 and 0.62, respectively. The N-terminal sequencing data of the two ferredoxins clearly indicate that ferredoxins I and II are different protein species. However, the two proteins exhibit a high degree of homology.

Coordination of the heme iron in the low-potential cytochromes c-553 from Desulfovibrio vulgaris and Desulfovibrio desulfuricans. Different chirality of the axially bound methionine in the oxidized and reduced states.

The coordination geometry at the heme iron of the cytochromes c-553 from Desulfovibrio vulgaris and Desulfovibrio desulfuricans was investigated by 1H-nuclear magnetic resonance and circular dichroism spectroscopy. Individual assignments were obtained for heme c and the axial ligands. From studies of nuclear Overhauser enhancements the axial histidine imidazole ring orientation relative to the heme group was found to coincide with other c-type cytochromes. In contrast, a new structure was observed for the axial methionine in the reduced cytochromes c-553. This includes S chirality at the iron-bound sulfur atom, but compared to cytochromes c-551 from Pseudomonads and Rhodopseudomonas gelatinosa and cytochrome c5 from Pseudomonas mendocina, which also contain S-chiral methionine, a different spatial arrangement of the gamma- and beta-methylene groups and the alpha carbon of methionine prevails. For the ferricytochromes c-553 R chirality was found for the iron-bound sulfur. This is the first observation of different methionine chirality in different oxidation states of the same c-type cytochrome.

Amino-acid sequence of rusticyanin from Thiobacillus ferrooxidans and its comparison with other blue copper proteins.

Rusticyanin, a copper protein characterized by a high redox potential (+680 mV) and a high stability at acidic pH, is involved in iron oxidation in Thiobacillus ferrooxidans. It has been characterized from a new strain and its amino-acid sequence has been determined and compared to two other rusticyanin sequences isolated from different strains. It comprises 155 amino acids and the alignment of the three rusticyanins shows a high degree of homology. Comparing the rusticyanins with six blue copper proteins which have a copper-I site in common, a consensus sequence containing Cys, His and Met in the C-terminal part of the protein and His-85 is proposed to be involved in the copper coordination. Secondary structure predictions are compared to three structures of copper proteins obtained by X-ray crystallography.

Isolation and characterization of a rubredoxin and an (8Fe-8S) ferredoxin from Desulfuromonas acetoxidans.

A two cluster (4Fe-4S) ferredoxin and a rubredoxin have been isolated from the sulfur-reducing bacterium Desulfuromonas acetoxidans. Their amino acid compositions are reported and compared to those of other iron-sulfur proteins. The ferredoxin contains 8 cysteine residues, 8 atoms of iron and 8 atoms of labile sulfur per molecule; its minimum molecular weight is 6163. The protein exhibits an abosrbance ratio of A385/A283 = 0.74. Storage results in a bleaching of the chromophore; the denatured ferredoxin is reconstitutable with iron and sulfide. The instability temperature is 52 degrees C. The rubredoxin does not differ markedly from rubredoxins from other anaerobic bacteria.

Comparative studies of two ferredoxins from Desulfovibrio desulfuricans norway.

Two ferredoxins isolated from Desulfovibrio desulfuricans Norway have been purified and characterized. The less acidic, designated as ferredoxin I, contains four iron atoms, four acid-labile sulfur groups and six cysteine residues per molecule. Ferredoxin II is more acidic and abundant than ferredoxin I, but is very unstable to O2. Ferredoxin I and ferredoxin II differ according to amino acid composition but are homologous with respect to their N-terminal amino acid sequence. The absorption spectra of the two ferredoxins are similar to those of other Desulfovibrio species. Both proteins appear to be dimers of identical 6000-dalton subunits. Their activity was tested in two types of reaction in the electron transfer chain (phosphoroclastic reaction and sulfite reductase activity). The isolation of two different ferredoxins from the same organism, Desulfovibrio, has been reported in Desulfovibrio africanus but the significance of two ferredoxins functioning in the same electron transfer chain is not yet understood.

Flavodoxin and rubredoxin from Desulphovibrio salexigens.

A flavodoxin and a rubredoxin have been isolated from the sulfate-reducing bacterium Desulphovibrio salexigens (strain British Guiana, NICB 8403). Their amino acid composition and spectral characteristics did not differ markedly from the homologous proteins presented in other Desulphovibrio spp. Flavodoxin was shown to be active in the electron transport of the sulfite reductase system.

Isolation and characterization of the gene coding for human cytidine deaminase.

The human gene coding for cytidine deaminase (CD), the enzyme which catalyzes the deamination of cytidine and deoxycytidine to uridine and deoxyuridine, was isolated and structurally characterized. CD is a single copy gene with a length of 31 kb and consists of four exons. Exon-intron junctions do not bracket functional domains of the encoded protein as the boundary between exons 2 and 3 interrupts the catalytically important zinc-finger domain, which is well conserved along phylogenesis. 5'-RACE and RNase mapping experiments identify one major and multiple other minor transcription initiation sites, which are present in placenta as well as in the myeloid cell lines, HL-60 and U937. The 5'-flanking region of the gene contains an orientation-dependent functional promoter and is characterized by the presence of several potential sites for the binding of known transcriptional factors.

A comparative spectroscopic study of two non-haem iron proteins lacking labile sulphide from Desulphovibrio gigas.

The ultraviolet visible, and near infrared spectrum of a two-iron protein from Desulphovibrio gigas, a new type of non-haem iron protein lacking labile sulphide, is compared with that of D. gigas rubredoxin. The charge transfer band maxima of rubredoxin at 495 and 565 nm are less separated in the new protein implying a higher symmetry of the two iron centres. The existence of a spin-spin interaction between the two iron centres in the new protein is suggested by the magnetic susceptibility measurements of the oxidized and reduced states of both proteins, which gives a smaller value per iron centre for the new protein. The oxidized form of the two iron-protein has a complex EPR spectrum with signals at g values of 8.97, 7.72, 5.73, 4.94, and 1.84. An EPR titration gives a value of --35 +/- 15 mV for the two signals at g values of 7.72 and 5.73. Rubredoxin has the characteristic spectrum of rubredoxins with two signals at g values of 9.4 and 4.27.

NMR characterization of three forms of ferredoxin from Desulphovibrio gigas, a sulphate reducer.

A NMR and magnetic susceptibility study of the oxidized and reduced states of three different oligomers (forms) of a [4Fe-4S] ferrodoxin protein from Desulphovibrio gigas, FdI, FdI', and FdII was carried out. FdI and FdI' are different trimers and FdII a tetramer of the same basic subunit. A probable assignment of the contact shifted resonances is indicated. Since the temperature dependences of the contact shifted responances associated with each [4Fe-4S] are not all similar a delocalized model for the spin densities on the 4Fe does not apply. The exchange rate between oxidized and reduced states is slow on the NMR time scale. The three oligomers are not magnetically equivalent. Using the "three state hypothesis" terminology it is shown that FdIox is predominantly in the C2- state and changes upon reduction into the C3- state, while FdIIox is in the C- state and changes into the C2- state. FdI' does not easily fit into this classification. This study shows a similarity of magnetic behaviour between FdI and bacterial ferredoxins (e.g. Bacillus polymyxa) and between FdII and HiPIP from Chromatium sp. The influence of the quaternary structure on the stabilization of the different oxidation states of ferredoxins as well as on their redox potentials is discussed.

Cytochrome c-551.5 (c7) from Desulfuromonas acetoxidans.

Cytochrome c-551.5 of the anaerobic sulfur-reducing bacterium Desulfuromonas acetoxidans has been purified to homogeneity and characterized. It elicits absorption bands at 551.5, 522.5 and 418 nm in the reduced form; the absorptivity ratio Aalpha(red)/A280nm(ox) equals 3.8 for the pure preparation. The molecular weight was estimated to be 9800 by gel filtration. Determination of the amion acid composition and analysis of the N-terminal amino acid sequence showed the cytochrome to be identical with the threehaem cytochrome c-551.5 (c7) isolated from the syntrophic mixed culture Chloropseudomonas ethylica strain 2K. The occurrence of multihaem cytochromes c in bacteria is discussed.

A sequential electron transfer from hydrogenases to cytochromes in sulfate-reducing bacteria.

A central step in the energy metabolism of sulfate-reducing bacteria is the oxidation of molecular hydrogen, catalyzed by a periplasmic hydrogenase. The resulting electrons are then transferred to various electron transport chains and used for cytoplasmic sulfate reduction. The complex formation between [NiFeSe] hydrogenase and the soluble periplasmic polyheme cytochromes from Desulfomicrobium norvegicum was characterized by cross-linking experiments, BIAcore and kinetics analysis. Analysis of electron transfer between [NiFeSe] hydrogenase and octaheme cytochrome c(3) (M(r) 26¿ omitted¿000) pointed out that this cytochrome is reduced faster in the presence of catalytic amounts of tetraheme cytochrome c(3) (M(r) 13¿ omitted¿000) isolated from the same organism. The activation of the hydrogenase-dependent reduction of polyheme cytochromes by cytochrome c(3) (M(r) 13¿ omitted¿000), which is now described in both Desulfovibrio and Desulfomicrobium, is proposed as a general mechanism. During this process, cytochrome c(3) (M(r) 13¿ omitted¿000) would act as an electron shuttle in between hydrogenase and the polyheme cytochromes and its conductivity appears to be an important factor.

Intramolecular electron transfer in ferredoxin II from Desulfovibrio desulfuricans Norway.

In order to elucidate the role of the two (4Fe-4S) clusters in ferredoxins and to determine whether an electron-transfer mechanism may occur between the clusters, the in vitro reduction of cytochrome c3 and cytochrome c553 by Desulfovibrio desulfuricans Norway ferredoxin II was studied using spectrophotometric techniques. Ferredoxin II, covalently cross-linked with either cytochrome c3 or c553, is an obligate intermediate in cytochrome reduction by pyruvate dehydrogenase. Both titration of the complex formation under 1H-NMR spectroscopy and cross-linking experiments between ferredoxin II and either cytochrome c3 or cytochrome c553 gave a stoichiometric ratio of 1:1. Modelling the protein yielded differences between the charge distributions around the two (Fe-S) clusters. The fact that Cluster 2 is blocked in the electron-transfer domain facing the cytochrome interacting heme, indicates Cluster 1 receives electron from pyruvate dehydrogenase. Consecutively, cytochrome reduction occurs owing to an intramolecular electron exchange between the two clusters of the ferredoxin. The properties of two (Fe-S) cluster ferredoxins are compared to those of monocluster ferredoxins and discussed in evolutionary terms.

Amino acid sequence and molecular modelling of a thermostable two (4Fe-4S) ferredoxin from the archaebacterium Methanococcus thermolithotrophicus.

The amino acid sequence of a two (4Fe-4S) ferredoxin from the methanogenic bacterium Methanococcus thermolithotrophicus (FdMt) has been determined. This thermostable protein comprises 60 amino acid residues (Mr 6541) and two (4Fe-4S) clusters chelated to the protein through the eight cysteines. FdMt contains a relatively high number of lysines [5], threonines [4] and valines [10]. The three-dimensional molecular model generated from the Peptococcus aerogenes X-ray structure keeps the characteristic overall ferredoxin folding thanks to complementary substitutions of residues of the hydrophobic core. The major structural features of the model are the different environments of both clusters, and the patch of three lysines at one end of the molecule. The possible role of several structural factors in the thermostability of the protein is discussed.