Bacterial expression of an active class Ib chitinase from Castanea sativa cotyledons

Ch3, an endochitinase of 32 kDa present in Castanea sativa cotyledons, showed in vitro antifungal properties when assayed against Trichoderma viride. The characterization of a cDNA clone corresponding to this protein indicated that Ch3 is a class Ib endochitinase that is synthesized as a preprotein with a signal sequence preceding the mature polypeptide. Bacterial expression of mature Ch3 fused to the leader peptide of the periplasmic protein ompT resulted in active Ch3 enzyme. A plate assay was adapted for semi-quantitative determination of chitinase activity secreted from cultured bacteria, which should facilitate the identification of mutants with altered capacity to hydrolyse chitin.     

Theoretical basis for differential scanning calorimetric analysis of multimeric proteins

A new general equation simulating irreversible DSC transitions of multimeric proteins was developed. The equation put forward here is the result of an improved mathematical re-elaboration of the classical Lumry-Eyring models, where no restrictive a priori assumptions are made on the kinetic constraints of the denaturation process, or on the enthalpy of the final denatured state. In order to test the wide applicability of this new effective theoretical tool, a series of DSC transitions were simulated with the aim of determining the effects of all relevant thermodynamic, kinetic or experimental parameters on the shape of DSC profiles. Moreover, the classical equations used widely in DSC investigations for the calculus in both kinetic parameters and changes of molecularity, were studied in the light of the model developed here, highlighting, in each case, their rather limited applicability. The new approach proposed in this article was applied to study the thermal denaturation of an hexameric protein (Glucosamine-6-phosphate deaminase), putting in evidence the practical applicability of the theoretical equations developed.     

Increase in heart glutathione redox ratio and total antioxidant capacity and decrease in lipid peroxidation after vitamin e dietary supplementation in guinea pigs

Dietary treatment with three diets differing in vitamin E, Low E (15 mg of vitamin E/kg diet), Medium E (150 mg/kg), or High E (1,500 mg/kg), resulted in guinea pigs with low (but nondeficient), intermediate, or high heart a-tocopherol concentration. Neither the antioxidant enzymes superoxide dismutase, catalase, glutathione peroxidase, and reductase, nor the nonenzymatic antioxidants, GSH, ascorbate, and uric acid were homeostatically depressed by increases in heart a-tocopherol. Protection from both enzymatic (NADPH dependent) and nonenzymatic (ascorbate-Fe²⁺) lipid peroxidation was strongly increased by vitamin E supplementation from Low to Medium E Whereas no additional gain was obtained from the Medium E to the High E group. The GSH/GSSG and GSH/total glutathione ratios increased as a function of the vitamin E dietary concentration closely resembling the shape of the dependence of heart a-tocopherol on dietary vitamin E. The results show the capacity of dietary vitamin E to increase the global antioxidant capacity of the heart and to improve the heart redox status in both the lipid and water-soluble compartments. This capacity occurred at levels six times higher than the minimum daily requirement of vitamin E, even in the presence of optimum dietary vitamin C concentrations and basal unstressed conditions. The need for vitamin E dietary supplementation seems specially important in this tissue due to the low constitutive levels of endogenous enzymatic and nonenzymatic antioxidants present of the mammalian heart in comparison with those of other internal organs.     

Assessment of populations ofGracilaria chilensis (Graeilariales,Rhodophyta) utilizing RAPDs

Phenotypic variability and mixing of material due to massive cultivation for commercial purposes has contributed to the taxonomic confusion ofGracilaria in Chile. At least four species with cylindrical thalli and similar morphology have been recorded. However, since establishment ofG. chilensis, most of the collected thalli have been attributed to this species despite the lack of diagnostic features. In an attempt to resolve whetherGracilaria from 3 localities where it grows in natural and artificial populations belongs to the same species, gametophytic samples were compared by applying RAPD-PCR to their total DNA. This was analysed using 25 different 10-mer primers from which 21 revealed polymorphism within and between populations. Similarity matrices and cluster analyses were performed based on the presence/absence of bands representing fragments of DNA generated by random amplification. Similarity values between two of the populations were equivalent to those detected within a third, indicating the mixing of genetic material due to transplant between the two former localities. Similarities between samples of ChileanGracilaria andG. tenuistipitata from Sweden are considerably lower (0.45–0.53) than those between populations from Chile (0.74–0.88), confirming the existence of a single specific taxon,G. chilensis, in these three localities.     

Examining the relationship between secondary structure and antibody recognition in immunopeptides from foot-and-mouth disease virus

Summary The conformation of a peptide that represents antigenic site A of foot-and-mouth disease virus strain C-S8c1 (residues 136–156 of VP1; YTASARGDLAHLTTTHARHLP) has been studied by circular dichroism and compared with three analogs that reproduce amino acid substitutions at position 146 (HisArg, Gln or Asp) which affect antibody recognition. Four other peptides, incorporating replacements at position 147 predicted to maintain (LeuIle, Nle and Ala) or disrupt (LeuGly) helical structure at this site, have also been studied. In aqueous solution or in 4 M urea, the spectra of all eight peptides were typical of aperiodic conformation and independent of concentration or pH. However, upon addition of solvents such as methanol or hexafluoroisopropanol, spectral patterns evidenced significant levels (ca. 50%) of helical structure. The single residue substitutions at positions 146 and 147 caused minor to significant variations in the calculated amount of -helix of the peptides. An attempt to relate these changes in helical content to the antigenic behaviour of the peptides towards five monoclonal antibodies elicited with virus and mapping at site A could not find any straightforward correspondence between the two sets of results. The parent peptide and its His¹⁴⁶Arg analog were also analyzed by circular dichroism in the presence of the Fab fragment of SD6, a monoclonal antibody mapping at site A and much less reactive with viruses carrying the referred mutation. Although a peptide-antibody interaction was evident from spectral changes, careful inspection of the difference spectra (peptide-Fab minus Fab) of both peptides failed to detect any significant distinction between them that could be attributed to their different immunoreactivity. While these findings do not necessarily conflict with previous reports that the interaction of antigenic site A with antibodies is mediated to some extent by the adoption of a helix structure, they suggest that, at least for C-serotype viruses, other structural features in addition to a helical conformation are critically involved in antigenic recognition.     

Specific DNA probes for the identification of the fish pathogen,Renibacterium salmoninarum

To obtain specific DNA probes for the identification of the fish pathogen, Renibacterium salmoninarum, a discriminatory recombinant DNA library was constructed using selective fragments of the bacterial genome. Three renibacterial clones, pMAM29, pMAM46 and pMAM77, containing 149, 73, and 154 bp respectively, were isolated and characterized. The specificity of the probes was confirmed by dot-blot and Southern hybridization analyses. Bacterial hybridization experiments revealed that pMAM29 discriminates the R. salmoninarum genome from that of other fish pathogens such as Aeromonas salmonicida, Yersinia ruckeri, Flexibacter columnaris, Lactobacillus piscicola, Vibrio ordalii, Vibrio anguillarum and Aeromonas hydrophila. Thus, this probe may provide a new means to diagnose bacterial kidney disease in asymptomatic fish and ova.The authors are with the instituto de Bioquímica, Universidad Austral de Chile, P.O. Box 567, Valdivia, Chile     

Molecular cytogenetic analysis of a duplication Xp in a male: further delineation of a possible sex influencing region on the X chromosome

We describe a male infant with severe mental retardation and autism with a duplication of the short arm of the X chromosome. Chromosome painting confirmed the origin of this X duplication. Molecular cytogenetic analysis with fluorescence in situ hybridization (FISH) identified one copy of the zinc finger protein on the X chromosome (ZFX) and two copies of the steroid sulfatase gene (STS), further delineating the breakpoints. Based on cytogenetic and molecular comparisons of cases from the literature of sex-reversal in dup(X),Y patients and our patient, we suggest that a possible secondary sexinfluencing gene involved in the regulation of sex determination or testis morphogenesis is present at the distal Xp21.1 to p21.2 region.