Evaluation of Probiotic Properties of Novel Brazilian Lactiplantibacillus plantarum Strains

Probiotics and Antimicrobial Proteins - Beneficial effects of Lactiplantibacillus plantarum strains have been widely reported. Knowing that the effects of probiotic bacteria are strain-dependent,...     

Relationships among didelphid marsupials based on sequence variation in the mitochondrial cytochrome B gene

Variation in the mitochondrial cytochrome b gene (nucleotide and amino acid sequences) is evaluated for 9 genera and 15 species of American opossums in the family Didelphidae, using the American caenolestid rat opossumLestoros and the New Guinean peroryctid bandicootEchimypera as outgroups. Phylogenetic analyses (parsimony and distance) strongly support the monophyly of the Didelphidae and delineate two major clades; (1)Didelphis andPhilander are strongly aligned sister taxa, withMetachirus weakly but consistently associated with them, and (2)Marmosa plusMicoureus, withMonodelphis falling outside that pair. The generaMarmosops, Caluromys, andGlironia exhibit varied relationships, depending upon the method of analysis and data (DNA or amino acid sequences) used, but generally are placed individually or in combinations near or at the base of the didelphid radiation. Some aspects of these relationships are consistent with current taxonomic views, but others are in marked contrast. Specifically, a clade comprised of the mouse opossumsMarmosa, Micoureus, andMarmosops is strongly rejected by log-likelihood analysis, contrary to expectations from some current classifications. Also, the woolly opossumsCaluromys andGlironia also do not form a sister-taxon relationship, as suggested by their placement in a subfamily separate from the remaining didelphids examined. However, such a relationship cannot be rejected from log-likelihood analyses. The relationships suggested fromcyt-b sequences are strongly concordant with those based on DNA-DNA hybridization analyses. In addition to systematic and phylogenetic properties, molecular evolution of the didelphid cytochrome b gene sequence is characterized according to nucleotide bias and rate differentials at each codon position and across the entire sequence.To whom correspondence should be addressed.     

Carbon-13 nuclear magnetic resonance analysis of [1-13C]glucose metabolism in Crithidia fasciculata. Evidence of CO2 fixation by phosphoenolpyruvate carboxykinase

The non-invasive technique of 13C nuclear magnetic resonance was applied to study glucose metabolism in vivo in the insect parasite Crithidia fasciculata. It was found that under anaerobic conditions [1-13C]glucose underwent a glycolytic pathway whose main metabolic products were identified as [2-13C]ethanol, [2-13C]succinate and [1,3-13C2]glycerol. These metabolites were excreted by C. fasciculata into the incubation medium, while in the cells [3-13C]phosphoenolpyruvate was also detected in addition to the aforementioned compounds. The C3 acid is apparently the acceptor of the primary CO2 fixation reaction, which leads in Trypanosomatids to the synthesis of succinate. By addition of sodium bicarbonate to the incubation mixture L-[3-13C]malate was detected among the excretion products, while the ethanol:succinate ratio of 2.0 in the absence of bicarbonate changed to a ratio of 0.6 in the presence of the latter. This was due to a shift of the balance between carboxylation of phosphoenolpyruvate, leading to succinate, and pyruvate decarboxylation leading to ethanol. The addition of 25% 2H2O to the incubation mixture led to the formation of [2-13C, 2-2H]ethanol derived from the prior incorporation of 2H+ into pyruvate in the reactions mediated by either pyruvate kinase or malic enzyme. However, no 2H+ incorporation into L-malate was detected, excluding the possibility that the latter was formed by carboxylation of pyruvate, and lending support to the idea that L-malate results from the carboxylation of phosphoenolpyruvate to oxaloacetate by phosphoenolpyruvate carboxykinase. The formation of [2-13C, 2-2H]-succinate under the same conditions reflected the uptake of 2H+ during the reduction of fumarate. When the incubations were carried out in the presence of 100% 2H2O, several [1-13C, 1-2H]ethanol species were detected, as well as [2-13C, 2-2H]malate and [1,3-13C2, 1,3-2H2]glycerol. The former deuterated compounds reflect the existence of NAD2H species when the incubations were carried out in 100% 2H2O, while the incorporation of 2H+ into [1,3-13C2]glycerol must be attributed to the phosphoglucose-isomerase-mediated reaction during glycolysis.     

Inhibition of the polymerase chain reaction by sputum samples from tuberculosis patients after processing using a silica-guanidiniumthiocyanate DNA isolation procedure.

With the objective to evaluate PCR-mediated detection of Mycobacterium tuberculosis DNA as a diagnostic procedure for diagnosis of tuberculosis in individuals attending ambulatory services in Primary Health Units of the City Tuberculosis Program in Rio de Janeiro, Brazil, their sputum samples were collected and treated with a DNA extraction procedure using silica-guanidiniumthiocyanate. This procedure has been described to be highly efficient for extraction of different kind of nucleic acids from bacteria and clinical samples. Upon comparing PCR results with the number of acid-fast bacilli, no direct relation was observed between the number of bacilli present in the sample and PCR positivity. Part of the processed samples was therefore spiked with pure DNA of M. tuberculosis and inhibition of the PCR reaction was verified in 22 out of 36 (61%) of the samples, demonstrating that the extraction procedure as originally described should not be used for PCR analysis of sputum samples.     

Trypanosoma cruzi: Insights into naphthoquinone effects on growth and proteinase activity

In this study we compared the effects of naphthoquinones (α-lapachone, β-lapachone, nor-β-lapachone and Epoxy-α-lap) on growth of Trypanosoma cruzi epimastigotes forms, and on viability of VERO cells. In addition we also experimentally analyzed the most active compounds inhibitory profile against T. cruzi serine- and cysteine-proteinases activity and theoretically evaluated them against cruzain, the major T. cruzi cysteine proteinase by using a molecular docking approach. Our results confirmed β-lapachone and Epoxy-α-lap with a high trypanocidal activity in contrast to α-lapachone and nor-β-lapachone whereas Epoxy-α-lap presented the safest toxicity profile against VERO cells. Interestingly the evaluation of the active compounds effects against T. cruzi cysteine- and serine-proteinases activities revealed different targets for these molecules. β-Lapachone is able to inhibit the cysteine-proteinase activity of T. cruzi proteic whole extract and of cruzain, similar to E-64, a classical cysteine-proteinase inhibitor. Differently, Epoxy-α-lap inhibited the T. cruzi serine-proteinase activity, similar to PMSF, a classical serine-proteinase inhibitor. In agreement to these biological profiles in the enzymatic assays, our theoretical analysis showed that E-64 and β-lapachone interact with the cruzain specific S2 pocket and active site whereas Epoxy-α-lap showed no important interactions. Overall, our results infer that β-lapachone and Epoxy-α-lap compounds may inhibit T. cruzi epimastigotes growth by affecting T. cruzi different proteinases. Thus the present data shows the potential of these compounds as prototype of protease inhibitors on drug design studies for developing new antichagasic compounds.     

Iminodiacetyl-hydroxamate derivatives as metalloproteinase inhibitors: equilibrium complexation studies with Cu(II), Zn(II) and Ni(II)

Two new iminodiacetyl-hydroxamate derivatives, the N-benzyl-N-carboxymethyl-iminoacetohydroxamic acid (H(2)L(1)) and the N-benzyl-N'-hydroxypiperazine-2,6-dione (HL(2)), have been recently reported as very effective inhibitors against a set of zinc-containing matrix metalloproteinases (MMPs). Herein, aimed at understanding that inhibitory function, these compounds are studied in their complex formation equilibria with three biologically relevant first-row transition M(2+) metal ions (M=Cu, Zn, Ni) by using potentiometric and spectroscopic techniques. At physiological conditions, complexation of these metal ions by H(2)L(1) mostly occurs with formation of 1:1 species by tridentate co-ordination (O,N,N) (carboxylate-amino-hydroxamate), whereas complexation with HL(2) mainly involves the formation of 1:2 (M:L) species with normal (O,O) hydroxamate coordination. Moreover, at higher pH, H(2)L(1) is able to form a pentanuclear tetrameric copper complex with an interesting 12-metallacrown-4 structure.     

New cytogenetic aberrations found in a case of aggressive retinoblastoma

We describe a case of retinoblastoma with an atypical presentation and previously unreported cytogenetic aberrations. A 19-month-old girl with left intraocular retinoblastoma was treated with enucleation and chemotherapy. The disease showed aggressive evolution within a short period between diagnosis and relapse. Eight months after diagnosis, a new large tumor was present in the orbit of the right eye, with diffuse bone pain, pancytopenia and diffuse infiltration into the bone marrow and the central nervous system. The child did not respond to treatment and died. Cytogenetic studies made with G-banding, FISH and SKY analysis showed chromosomal aberrations commonly associated with retinoblastoma, including del(13q), i(6p), +1, and monosomy 16, along with others that had not been reported previously, including dup(5q), dic(15;22) and add(14q). The new chromosomal aberrations may be related to the aggressiveness of the disease in this case.