The solution structure of the N-terminal proteinase domain of the hepatitis C virus (HCV) NS3 protein provides new insights into its activation and catalytic mechanism.

The solution structure of the hepatitis C virus (BK strain) NS3 protein N-terminal domain (186 residues) has been solved by NMR spectroscopy. The protein is a serine protease with a chymotrypsin-type fold, and is involved in the maturation of the viral polyprotein. Despite the knowledge that its activity is enhanced by the action of a viral protein cofactor, NS4A, the mechanism of activation is not yet clear. The analysis of the folding in solution and the differences from the crystallographic structures allow the formulation of a model in which, in addition to the NS4A cofactor, the substrate plays an important role in the activation of the catalytic mechanism. A unique structural feature is the presence of a zinc-binding site exposed on the surface, subject to a slow conformational exchange process.     

Structural characterization of the interactions of optimized product inhibitors with the N-terminal proteinase domain of the hepatitis C virus (HCV) NS3 protein by NMR and modelling studies

The interactions of peptide inhibitors, obtained by the optimization of N-terminal cleavage products of natural substrates, with the protease of human hepatitis C virus (HCV) are characterized by NMR and modelling studies. The S-binding region of the enzyme and the bound conformation of the ligands are experimentally determined. The NMR data are then used as the experimental basis for modelling studies of the structure of the complex. The S-binding region involves the loop connecting strands E2 and F2, and appears shallow and solvent-exposed. The ligand binds in an extended conformation, forming an antiparallel beta-sheet with strand E2 of the protein, with the P1 carboxylate group in the oxyanion hole.     

The phylogenetic relationships of Cynoglottis (Boraginaceae- Boragineae) inferred from ITS, 5.8S and trnL sequences

A molecular phylogenetic analysis of Cynoglottis was performed to evaluate previous hypotheses based on non-molecular evidence concerning the position of this genus within Boraginaceae tribe Boragineae. ITS-5.8S and trnL_UAA sequences from the nuclear and chloroplast non-coding genomes were obtained for four Cynoglottis taxa and selected members of the related genera Anchusa, Anchusella, Gastrocotyle, Brunnera and Pentaglottis. Cynoglottis is monophyletic, but neither trnL nor ITS support a close relationship with Brunnera, unlike previously supposed on morphological grounds. Brunnera is, instead, related to the southwestern European monotypic genus Pentaglottis, with which it forms a basal clade. ITS-5.8S sequences show that Anchusa thessala, a southeastern European annual species of Anchusa subg. Buglossellum, is sister to Cynoglottis and that the two taxa form a clade which also includes the Balkan endemic Gastrocotyle macedonica. Species of Anchusa subg. Anchusa form a separate lineage with high bootstrap support, suggesting that this heterogeneous genus is paraphyletic with respect to Cynoglottis. ITS sequences also discriminate between the Balkan-Apenninic diploid C. barrelieri and the Anatolian tetraploid C. chetikiana, albeit with low support. The molecular results are discussed in the light of karyological, morphological and chorological aspects.This work has been supported by M.I.U.R. 40% 2003 and the University of Firenze.     

Complement-independent, peroxide-induced antibody lysis of platelets in HIV-1-related immune thrombocytopenia

Immunologic thrombocytopenia is seen commonly in HIV-1 infection. The pathogenesis of this problem has been unclear, but it is associated with circulating immune complexes that contain platelet membrane components and anti-platelet membrane GPIIIa49-66 IgG antibodies. These antibodies cause acute thrombocytopenia when injected into mice. We now show that purified anti-GPIIIa49-66 causes platelet fragmentation, in vitro in the absence of complement, and in vivo in wild-type and C3-deficient mice. The mechanism of complement-independent platelet lysis is shown to be caused by the antibody-induced generation of H202, as indicated by in vitro experiments with inhibitors of reactive oxygen species, and in vivo studies carried out with p47phox-deficient mice. Thus, a novel mechanism of immunologic platelet clearance is described in which an anti-platelet IgG causes platelet fragmentation via the induction of reactive oxygen species.     

Casein utilization by Streptococcus thermophilus results in a diauxic growth in milk

In milk, Streptococcus thermophilus displays two distinct exponential growth phases, separated by a nonexponential one, during which proteinase synthesis was initiated. During the second exponential phase, utilization of caseins as the source of amino acids resulted in a decrease in growth rate, presumably caused by a limiting peptide transport activity.     

The EstA esterase is responsible for the main capacity of Lactococcus lactis to synthesize short chain fatty acid esters in vitro

AIMS: Esters of short-chain fatty acids and alcohols participate significantly in the overall flavour of foods. The capacity of the lactic acid bacterium Lactococcus lactis to synthesize such esters is known even though the enzymes involved in the process are not well identified. The objective of our work is to determine whether the esterase is responsible for the whole capacity of L. lactis to synthesize esters in vitro. METHODS AND RESULTS: A negative mutant for the esterase was constructed and its capacity to synthesize short chain fatty acid esters from different substrate couples was compared to that of the wild type. We observed that the esterase is responsible for the main ester synthesis activity of L. lactis in vitro. However, in the presence of some substrates, the esterase negative mutant still synthesizes low amounts of esters. CONCLUSIONS: In favourable environmental conditions, the L. lactis esterase is responsible for the main ester synthesizing activity, even though another pathway for ester synthesis probably exists. SIGNIFICANCE AND IMPACT OF THE STUDY: Since esters are potent aroma compounds, esterase is probably a key enzyme in the development of food flavour.     

Oxidative damage of the gastric mucosa in Helicobacter pylori positive chronic atrophic and nonatrophic gastritis, before and after eradication

Background. Helicobacter pylori is the main cause of gastritis and a primary carcinogen. The aim of this study was to assess oxidative damage in mucosal compartments of gastric mucosa in H. pylori positive and negative atrophic and nonatrophic gastritis. Materials and methods. Five groups of 10 patients each were identified according to H. pylori positive or negative chronic atrophic (Hp‐CAG and CAG, respectively) and nonatrophic gastritis (Hp‐CG and CG, respectively), and H. pylori negative normal mucosa (controls). Oxidative damage was evaluated by nitrotyrosine immunohistochemistry in the whole mucosa and in each compartment at baseline and at 2 and 12 months after eradication. Types of intestinal metaplasia were classified by histochemistry. Results. Total nitrotyrosine levels appeared significantly higher in H. pylori positive than in negative patients, and in Hp‐CAG than in Hp‐CG (p < .001); no differences were found between H. pylori negative gastritis and normal mucosa. Nitrotyrosine were found in foveolae and intestinal metaplasia only in Hp‐CAG. At 12 months after H. pylori eradication, total nitrotyrosine levels showed a trend toward a decrease in Hp‐CG and decreased significantly in Hp‐CAG (p = .002), disappearing from the foveolae (p = .002), but remaining unchanged in intestinal metaplasia. Type I and II of intestinal metaplasia were present with the same prevalence in Hp‐CAG and CAG, and did not change after H. pylori eradication. Conclusions. Oxidative damage of the gastric mucosa increases from Hp‐CG to Hp‐CAG, involving the foveolae and intestinal metaplasia. H. pylori eradication induces a complete healing of foveolae but not of intestinal metaplasia, reducing the overall oxidative damage in the mucosa.     

The cisproline(i - 1)-aromatic(i) interaction: folding of the Ala-cisPro-Tyr peptide characterized by NMR and theoretical approaches

Cisproline(i - 1)-aromatic(i) interactions have been detected in several short peptides in aqueous solution by analysis of anomalous chemical shifts measured by 1H-NMR spectroscopy. This formation of local structure is of importance for protein folding and binding properties. To obtain an atomic-detail characterisation of the cisproline(i - 1)-aromatic(i) interaction in terms of structure, energetics and dynamics, we studied the minimal peptide unit, blocked Ala-cisPro-Tyr, using computational and experimental techniques. Structural database analyses and a systematic search revealed two groups of conformations displaying a cisproline(i - 1)-aromatic(i) interaction. These conformations were taken as seeds for molecular dynamics simulations in explicit solvent at 278 K. During a total of 33.6 ns of simulation, all the 'folded' conformations and some 'unfolded' states were sampled. 1H- and 13C-chemical shifts and 3J-coupling constants were measured for the Ala-Pro-Tyr peptide. Excellent agreement was found between all the measured and computed NMR properties, showing the good quality of the force field. We find that under the experimental and simulation conditions, the Ala-cisPro-Tyr peptide is folded 90% of the time and displays two types of folded conformation which we denote 'a' and 'b'. The type a conformations are twice as populated as the type b conformations. The former have the tyrosine ring interacting with the alanine alpha proton and are enthalpically stabilised. The latter have the aromatic ring interacting with the proline side chain and are entropically stabilised. The combined and complementary use of computational and experimental techniques permitted derivation of a detailed scenario of the 'folding' of this peptide.     

The effects of humic substances within the Ah horizon of a Calcic Luvisol on morphological changes related to invertase and peroxidase activities in wheat roots

The chemical and biological characteristics of humus within the Ah horizon (Calcic-Luvisol) have been studied. Attention was paid to variation in the NMR spectra of humic fractions and ¹³C values and to how these changes are related to different biological humic fraction activities.The chemical changes in particular involve the decrease of the aromatic component and the increase of the non-aromatic component within the horizon and the different ¹³C value not only within the horizon but also among the humic fractions distinctive of different molecular sizes.An attempt has been made to explain the vertical chemical changes in terms of processes affecting the biological characteristics of the high and low molecular size humic fractions. The main conclusions are that the low molecular size humic fractions, in the upper part of the horizon, are of greater importance with respect to the other humic fractions in influencing the enzyme activities linked to growth metabolism. The biological role of the high molecular size humic fractions characterised by a relevant content of peptidic- and carbohydratic-C is also presented.