Genotoxic, antigenotoxic and phytochemical assessment of Terminalia actinophylla ethanolic extract.

Terminalia actinophylla has been used for anti-diarrheic and haemostatic purposes in Brazil. The fly spot data obtained after exposure of marker-heterozygous Drosophila melanogaster larvae to T. actinophylla ethanolic extract (TAE) in the standard (ST) and high bioactivation (HB) crosses revealed that TAE did not induce any statistically significant increment in any spot categories. Differences between the two crosses are related to cytochrome P450 (CYPs) levels. In this sense, our data pointed out the absence of TAE-direct and indirect mutagenic and recombinagenic action in the Somatic Mutation and Recombination Test (SMART). When the anti-genotoxicity of TAE was analyzed, neither mitomycin C (MMC) nor ethylmethanesulfonate (EMS) genotoxicity was modified by the post-exposure to TAE, which suggests that TAE has no effect on the mechanisms involved in the processing of the lesions induced by both genotoxins. In the mwh/flr(3) genotype, co-treatment with TAE may lead to a significant protection against the genotoxicity of MMC and a weak but significant effect in the toxic genetic action of EMS. The overall findings suggested that the favorable modulations by TAE could be, at least in part, due to its antioxidative potential.

Symmetry matters for the electronic structure of core complexes from Rhodopseudomonas palustris and Rhodobacter sphaeroides PufX-.

Low-temperature (1.4 K), single-molecule fluorescence-excitation spectra have been recorded for individual reaction center-light-harvesting 1 complexes from Rhodopseudomonas palustris and the PufX(-) strain of Rhodobacter sphaeroides. More than 80% of the complexes from Rb. sphaeroides show only broad absorption bands, whereas nearly all of the complexes from Rps. palustris also have a narrow line at the low-energy end of their spectrum. We describe how the presence of this narrow feature indicates the presence of a gap in the electronic structure of the light-harvesting 1 complex from Rps. palustris, which provides strong support for the physical gap that was previously modeled in its x-ray crystal structure.

Purification and characterization of an alkaline serine endopeptidase from a feather-degrading Xanthomonas maltophilia strain.

A keratinolytic Xanthomonas maltophilia strain (POA-1), cultured on feather meal broth, using keratin as its sole source of carbon and nitrogen, secretes several extracellular peptidases. The major serine peptidase was purified to homogeneity by a five-step procedure. Its purity was evaluated by capillary zone electrophoresis. This enzyme has a molecular mass of 36 kDa, an optimum pH of 9.0, and an optimum temperature of 60 degrees C. The inhibitory profile using protease inhibitors shows that this enzyme is a serine endopeptidase. Besides keratin, the enzyme is active upon the substrates azokeratin, azocasein, and the following fluorogenic peptide substrates: Abz-Leu-Gly-Met-Ile-Ser-Leu-Met-Lys-Arg-Pro-Gln-EDDnp, Abz-Lys-Leu-Cys(SBzl)-Gly-Pro-Lys-Gln-EDDnp, and Abz-Lys-Pro-Cys(SBzl)-Phe-Ser-Lys-Gln-EDDnp.

Detection of Babesia equi (Laveran, 1901) by nested polymerase chain reaction.

We describe a nested polymerase chain reaction (PCR) for the detection of Babesia equi in equine infected erythrocytes using oligonucleotides designed on the published sequence of a B. equi merozoite antigen gene (ema-1). A 102bp DNA fragment is specifically amplified from B. equi but not from Babesia caballi, Babesia bovis or Babesia bigemina DNA. In a mock infection we were able to detect down to six infected cells in 10(8) equine erythrocytes or to detect the parasite in blood with an equivalent parasitemia of 0.000006%. Furthermore, gene polymorphism was found by performing a PCR-RFLP (PCR combined with restriction fragment length polymorphism) on both the 102bp and the entire ema-1 gene DNA amplified from two B. equi isolates, Florida (USA) and Pelotas (Southern Brazil) isolates. The polymorphism was confirmed by sequencing the entire ema-1 gene from the B. equi isolate Pelotas. Our results demonstrate that the ema-1 based nested PCR is a valuable technique for routine detection of B. equi in chronically infected horses. It may be used for epidemiological and phylogenetic studies of the parasite as well as monitoring B. equi infected horses in chemotherapeutic trials.

Improvement in the mucoadhesive properties of alginate by the covalent attachment of cysteine.

The purpose of the present study was to improve the mucoadhesive properties of alginate by the covalent attachment of cysteine. Mediated by a carbodiimide, L-cysteine was covalently linked to the polymer. The resulting thiolated alginate displayed 340.4+/-74.9 micromol thiol groups per g conjugate (means+/-S.D.; n=4). Within 2 h the viscosity of an aqueous mucus/alginate-cysteine conjugate mixture pH 7.0 increased at 37 degrees C by more than 50% compared to a mucus/alginate mixture, indicating enlarged interactions between the mucus and the thiolated polymer. Tensile studies carried out on freshly excised porcine intestinal mucosa demonstrated a total work of adhesion (TWA) of 25.8+/-0.6 and 101.6+/-36.1 microJ for alginate and the alginate-cysteine conjugate, respectively (means+/-S.D.; n=5). The maximum detachment force (MDF) was thereby in good correlation with the TWA. Due to the immobilization of cysteine, the swelling velocity of the polymer was significantly accelerated (P<0.05). In aqueous media the alginate-cysteine conjugate was capable of forming inter- and/or intramolecular disulfide bonds. Because of this crosslinking process within the polymeric network, the cohesive properties of the conjugate were also improved. Tablets comprising the unmodified polymer disintegrated within 49+/-14.5 min, whereas tablets of thiolated alginate remained stable for 148.8+/-39.1 min (means+/-S.D.; n=3). These features should render thiolated alginate useful as excipient for various drug delivery systems providing an improved stability and a prolonged residence time on certain mucosal epithelia.

Cloning and functional expression of a Boophilus microplus cathepsin L-like enzyme.

A cysteine proteinase gene homologous to cathepsins L genes was isolated from a B. microplus cDNA library. The precursor protein deduced from the nucleotide sequence contains 332 amino acid residues consisting of a signal sequence (pre-region), a pro-region and a mature proteinase. The DNA fragment coding for the proenzyme was cloned and expressed using the E. coli expression vector pMAL-p. The recombinant protein (MBP+PROCP) once activated is able to hydrolyze synthetic substrates as well as protein substrates like hemoglobin, vitellin and gelatin. Its optimal enzymatic activity on both fluorogenic and protein substrates was found to occur at an acidic pH. Expression of the proteinase gene was tested by RT-PCR with tick larvae RNA. Detection of amplified sequences indicates that the gene is expressed at this stage of the tick life cycle and the molecule is therefore potentially a target for chemotherapy or an immunogen in a vaccine.

Detection of genomic variability in different populations of the cattle tick Boophilus microplus in southern Brazil.

DNA from seven isolates of the cattle tick Boophilus microplus was analyzed by restriction fragment length polymorphism (RFLP). Three different cDNA clones, named P-9, P-25 and CP-12, isolated from a B. microplus cDNA library, were used as DNA probes. DNA sequences of P-9 have high similarity to ribosomal genes, whereas P-25 does not show significant homology with known sequences within databases. CP-12 is a cDNA clone encoding a cysteine endopeptidase gene. A limited degree of polymorphism was detected with P-9 and P-25, while CP-12 showed a different pattern of bands for each tick isolate. These findings suggest the existence of a complex genotypic diversity of the tick B. microplus population in endemic regions.

Specific contribution of Tyk2 JH regions to the binding and the expression of the interferon alpha/beta receptor component IFNAR1.

Cytokine signaling involves the activation of the Janus kinase (JAK) family of tyrosine kinases. These enzymes are physically associated with cytokine receptor components. Here, we sought to define the molecular basis of the interaction between Tyk2 and IFNAR1, a component of the interferon alpha/beta receptor, by delimiting a minimal IFNAR1 binding region in the Tyk2 protein. Using an in vitro assay system, we narrowed down the interaction domain to a region comprising the JH7 and part of the JH6 homology boxes (amino acids 22-221). When expressed in Tyk2-negative cells, the JH7-6 region was unable to stabilize IFNAR1 protein levels, a critical function that we previously attributed to the N region (amino acids 1-591) of Tyk2. Moreover, substitution of the JH7-JH6 domain in JAK1 with that of Tyk2 did not restore IFNAR1 level nor interferon alpha signaling in Tyk2-negative cells. Thus, the major interaction surface lies within JH7-6, but additional JH regions (JH5-4-3) contribute in a specific manner to the in vivo assembly of Tyk2 and IFNAR1. Evidence is also provided of the lack of specificity of the Tyk2 kinase-like and tyrosine kinase domains in interferon alpha/beta receptor signaling.

The amino-terminal region of Tyk2 sustains the level of interferon alpha receptor 1, a component of the interferon alpha/beta receptor.

Tyk2 belongs to the Janus kinase (JAK) family of receptor associated tyrosine kinases, characterized by a large N-terminal region, a kinase-like domain and a tyrosine kinase domain. It was previously shown that Tyk2 contributes to interferon-alpha (IFN-alpha) signaling not only catalytically, but also as an essential intracellular component of the receptor complex, being required for high affinity binding of IFN-alpha. For this function the tyrosine kinase domain was found to be dispensable. Here, it is shown that mutant cells lacking Tyk2 have significantly reduced IFN-alpha receptor 1 (IFNAR1) protein level, whereas the mRNA level is unaltered. Expression of the N-terminal region of Tyk2 in these cells reconstituted wild-type IFNAR1 level, but did not restore the binding activity of the receptor. Studies of mutant Tyk2 forms deleted at the N terminus indicated that the integrity of the N-terminal region is required to sustain IFNAR1. These studies also showed that the N-terminal region does not directly modulate the basal autophosphorylation activity of Tyk2, but it is required for efficient in vitro IFNAR1 phosphorylation and for rendering the enzyme activatable by IFN-alpha. Overall, these results indicate that distinct Tyk2 domains provide different functions to the receptor complex: the N-terminal region sustains IFNAR1 level, whereas the kinase-like domain provides a function toward high affinity ligand binding.

GTPase properties of the interferon-induced human guanylate-binding protein 2.

Guanylate-binding proteins (GBPs) were originally described as proteins that are strongly induced by interferons and are capable of binding to agarose-immobilized guanine nucleotides. hGBP1, the first of two members of this protein family in humans, was recently shown to represent a novel type of GTPase that hydrolyzes GTP predominantly to GMP. We now report that purified recombinant hGBP2 also hydrolyzes GTP very efficiently, although GDP rather than GMP was the major reaction product. The biochemical parameters of this reaction were as follows: Km = 313 microM, turnover number = 22 min-1. Both hGBP1 and hGBP2 failed to hydrolyze GDP, however, GDP was an effective inhibitor of the hGBP2- but not the hGBP1-catalyzed GTP hydrolysis reaction. Thus, hGBP1 and hGBP2 have similar biochemical properties, but show pronounced differences in product specificity.

Interferon-induced MxA protein. GTP binding and GTP hydrolysis properties.

MxA is a GTPase encoded by an interferon-activated human gene which inhibits the multiplication of several RNA viruses. Recombinant histidine-tagged MxA protein (His-MxA) was expressed in Escherichia coli and purified to near homogeneity. Gel filtration showed that it formed high molecular weight oligomers. Purified His-MxA exhibited specific GTP hydrolysis rates of up to 350 nmol of GTP/min/mg of protein, corresponding to a turnover number of 27 min-1. The Km for this reaction was 260 microM. Guanine nucleotides did not copurify with His-MxA. Binding experiments in solution with fluorescent-labeled nucleotides confirmed that His-MxA binds guanine nucleotides rather weakly and further showed that the fluorescent GDP analog N-methylanthraniloyl (mant)-GDP had a much lower affinity for His-MxA (Kd 20 microM, koff 8.5 s-1) than the nonhydrolyzable GTP analog mant-5'-guanylyl-beta,gamma-imidotriphosphate (mant-GMP-PNP) (Kd 0.75 microM, koff 0.012 s-1). Competitive binding studies with nonlabeled nucleotides revealed a similar binding preference of His-MxA for GTP over GDP: the Kd for GTP was 20 microM, whereas the Kd for GDP was 100 microM. Thus, a high percentage of MxA molecules may be complexed with GTP in vivo.

Unexpected structural requirements for GTPase activity of the interferon-induced MxA protein.

MxA is an interferon-induced 76-kDa GTPase that inhibits the multiplication of several RNA viruses. Deleting seven amino acids from the COOH terminus reduced the GTPase activity of purified MxA to 1.4%. MxA mutants with COOH-terminal deletions of 63 or more amino acids lost all ability to hydrolyze GTP and failed to bind guanine nucleotides. By contrast, an MxA deletion mutant consisting of 301 amino acids from the NH2 terminus and 87 amino acids from the COOH terminus retained about 9% of wild-type GTPase activity, underscoring the pivotal role of COOH-terminal sequences. Limited proteolysis of wild-type MxA with proteinase K resulted in two resistant polypeptides of 60 and 10 kDa, respectively, which copurified as a stable complex. The p60-p10 complex exhibited high GTPase activity, suggesting that it included all MxA domains required for this biochemical activity. Sequencing revealed that the NH2 terminus of the 60-kDa polypeptide mapped to leucine 41 and the NH2 terminus of the 10-kDa polypeptide to glutamine 564 of the MxA sequence. Based on these results we propose a model that suggests that the GTP-binding consensus element located in the NH2-terminal half of MxA is held in an active conformation by strong physical interactions with amino acids from the COOH-terminal region.

Vesicular stomatitis virus transcription inhibited by purified MxA protein.

MxA is a GTPase encoded by an interferon-inducible human gene. Its constitutive expression renders transfected mammalian cells resistant to infections with several different RNA viruses, including vesicular stomatitis virus (VSV). Differences in viral RNA levels of VSV-infected cells either expressing or lacking MxA indicated that VSV mRNA synthesis is the principal target of MxA action. We now used purified histidine-tagged MxA (His-MxA) that we produced in Escherichia coli to successfully inhibit VSV in vitro transcription, a reaction catalyzed by VSV ribonucleoprotein complexes isolated from virus-infected cells or from purified virions. MxA was inactive when added to preformed VSV mRNAs, arguing against the possibility that it has a negative effect on viral RNA stability. MxA inhibited both leader RNA and mRNA synthesis of VSV, suggesting that it interfered with transcription initiation. The degree of VSV inhibition correlated directly with the specific GTPase activities of the various wild-type MxA preparations. No inhibition of viral mRNA synthesis was observed when a C-terminally truncated, GTPase-inactive variant of His-MxA was added to the transcription reactions. Purified His-MxA-E645R, a mutant of MxA with normal GTPase activity whose range of antiviral activity in vivo is altered so that it no longer inhibits VSV, showed no inhibitory effect on VSV in vitro transcription. Since MxA inhibited VSV RNA synthesis in the presence of GMP-PNP or GTP gamma S, GTP analogs that are readily accepted by the viral polymerase but cannot be hydrolyzed by MxA, the possibility was excluded that MxA acts by depleting the viral polymerase for its nucleotide substrates. Thus, binding of GTP rather than its hydrolysis seems of importance for the anti-VSV activity of MxA.