Physical properties and Tris inhibition of an insect trehalase and a thermodynamic approach to the nature of its active site

The midgut from Rhynchosciara americana larvae display a trehalase (alpha,alpha'-trehalose glucohydrolase, EC 3.2.1.28) which is soluble with a molecular weight of 122 000 and pI 4.6. The optimum pH of the enzyme is 6.0, its apparent Km for trehalose is 0.67 mM and its energy of activation is 16.7 kcal/mol. Sulfhydryl reagents do not inhibit the trehalase. The results suggest the existence of two carboxyl groups in the active site, one of which has a very high (8.3) pK. The increase of the pK values of the essential groups of the free enzyme in the presence of increasing concentrations of dioxane supports the hypothesis that these groups are carboxyls. The purified enzyme hydrolyzes only alpha,alpha'-trehalose and it is competitively inhibited by several compounds.     

Trypanosoma cruzi: Immunoperoxidase antibody test for serologic diagnosis

An indirect antiglobulin immunoperoxidase test was developed for the serological diagnosis of American Trypanosomiasis. Purified rabbit antihuman IgG was labeled with the enzyme and the conjugate so obtained was characterized according to its immune and enzymatic activities, with the help of such parameters as the authors have recently described. For a maximal sensitivity in the tests, high antibody levels and a high-labeling ratio were chosen, as well as dilutions of conjugate ensuring maximal reactivity. Positive tests were found in all 90 serum samples from patients with Chagas' disease and titers did not differ significantly from those observed in immunofluorescence tests done in parallel. The specificity of both tests was also similar, as indicated by the results found for serum samples from 60 patients with other diseases, parasitic or not, showing high levels of antibodies against other infective agents or autoantibodies, and in 15 normals.     

Terpenes of Podocarpus lambertius

Sitosterol and the following terpenic compounds have been isolated from the bark of Podocarpus lambertius: 3β-hydroxytotarol, 4β-carboxynortotarol, and macrophyllic and lambertic acids. The leaves yielded sitosterol, stigmastan-3β,5α-diol-6-one, isopimaric acid, phyllocladene, isophyllocladene, 8,9-abieten-15-ol and 17-isophyllocladenol.     

Biological Aspects of Medorippe lanata (Linnaeus, 1767) (Brachyura: Dorippidae) from the Eastern Ligurian Sea (Western Mediterranean)

The aim of the present study is to investigate the demographic structure and to identify some aspects of the biology of an exploited population of Medorippe lanata (Brachyura: Dorippidae) in the eastern Ligurian Sea, western Mediterranean. 1364 specimens (639 males and 725 females) of M. lanata were collected on a monthly basis from January to December 2001, in a wide area of the eastern Ligurian Sea usually exploited by the Viareggio ‘rapido’ trawl fleet. M. lanata represented an important fraction of the discard, both in weight and in number of individuals. Maximum abundance of this species occurred in late summer-early autumn (up to 3369 ind. km⁻² and 50.6 kg km⁻² in August). The overall females:males sex-ratio was 1.13:1, while the monthly sex-ratio did not differ statistically from 1:1 in all months, except in September and October, when females significantly outnumbered males. The sampled population was composed of two cohorts from November to April. Sizes ranged from 10 to 29 mm carapace length (CL) for females and from 9 to 29 mm CL for males. The von Bertalanffy growth curve, computed for both sexes, gave a higher growth rate in males than in females. Recently moulted males and females were observed throughout the year, except in summer, when the highest number of ovigerous females was present. Females with external eggs were collected from March to November, with peaks in August and September. The monthly evolution of the ovarian maturity stages showed no clear temporal trend. At 21 mm CL, 50% of females were ovigerous or showed macroscopically mature ovaries. According to the dimorphism in chelae size, the presence of adult males (post-puberty stage) was observed all year round, from 18 to 29 mm CL, without evident temporal trends.     

Novel Tools to Analyze the Function of Salmonella Effectors Show That SvpB Ectopic Expression Induces Cell Cycle Arrest in Tumor Cells

In order to further characterize its role in pathogenesis and to establish whether its overproduction can lead to eukaryotic tumor cell death, Salmonella strains able to express its virulence factor SpvB (an ADP-ribosyl transferase enzyme) in a salicylate-inducible way have been constructed and analyzed in different eukaryotic tumor cell lines. To do so, the bacterial strains bearing the expression system have been constructed in a ∆purD background, which allows control of bacterial proliferation inside the eukaryotic cell. In the absence of bacterial proliferation, salicylate-induced SpvB production resulted in activation of caspases 3 and 7 and apoptotic cell death. The results clearly indicated that controlled SpvB production leads to F-actin depolimerization and either G1/S or G2/M phase arrest in all cell lines tested, thus shedding light on the function of SpvB in Salmonella pathogenesis. In the first place, the combined control of protein production by salicylate regulated vectors and bacterial growth by adenine concentration offers the possibility to study the role of Salmonella effectors during eukaryotic cells infection. In the second place, the salicylate-controlled expression of SpvB by the bacterium provides a way to evaluate the potential of other homologous or heterologous proteins as antitumor agents, and, eventually to construct novel potential tools for cancer therapy, given that Salmonella preferentially proliferates in tumors.     

Selection of Small Peptides, Inhibitors of Translation

Identification of small molecular weight compounds targeting specific sites in the ribosome can accelerate development of new antibiotics and provide new tools for ribosomal research. We demonstrate here that antibiotic-size short peptides capable of inhibiting protein synthesis can be selected by using specific elements of ribosomal RNA as a target. The ‘h18’ pseudoknot encompassing residues 500-545 of the small ribosomal subunit RNA was used as a target in screening a heptapeptide phage-display library. Two of the selected peptides could efficiently interfere with both bacterial and eukaryotic translation. One of these inhibitory peptides exhibited a high-affinity binding to the isolated small ribosomal subunit (K_d of 1.1 μM). Identification of inhibitory peptides that likely target a specific rRNA structure may pave new ways for validating new antibiotic sites in the ribosome. The selected peptides can be used as a tool in search of novel site-specific inhibitors of translation.