Evaluation of the genotoxicity of piplartine, an alkamide of Piper tuberculatum, in yeast and mammalian V79 cells

The genus Piper belongs to the Piperaceae family, and includes species of commercial and medicinal importance. Chemical studies on Piper species resulted in the isolation of several biologically active molecules, including alkaloid amides, such as piplartine. This molecule, isolated from Piper tuberculatum, has significant cytotoxic activity against tumor cell lines, and presents antifungal, anti-platelet aggregation, anxiolytic, and antidepressant effects. In order to understand the biological properties of piplartine, this study investigated the genotoxicity and the induction of apoptosis by piplartine in V79 cells and its mutagenic and recombinogenic potential in Saccharomyces cerevisiae. Piplartine induced dose-dependent cytotoxicity in S. cerevisiae cultures in either stationary -- or exponential growth phase. In addition, piplartine was not mutagenic when cells were treated during exponential-growth phase and kept in buffer solution, but it increased the frequencies of point, frameshift, and forward mutations when cells were treated in medium during growth. Piplartine treatment induced DNA strand breaks in V79 cells, as detected by neutral and alkaline comet assay. In cell cycle analysis, piplartine induced G2/M cell cycle arrest, probably as a consequence of the DNA damage induced and repair. Moreover, piplartine treatment induced apoptosis in a dose-dependent manner, as observed by a decrease in mitochondrial membrane potential and an increase in internucleosomal DNA fragmentation. These data suggest that the DNA damage caused by piplartine induces G2/M cell cycle arrest, followed by apoptosis. Moreover, we suggest that cells surviving piplartine-induced DNA damage can accumulate mutations, since this alkaloid was mutagenic and recombinogenic in S. cerevisiae assays.     

Description of by-product inhibiton effects on biodesulfurization of dibenzothiophene in biphasic media

As several authors have reported previously, the Biodesulfurization of hydrodesulfurization recalcitrants, such as dibenzothiophene, is not yet commercially viable because mass transfer limitations and feedback inhibition effects are produced during the conversion. This work has been focused to investigate the inhibition process in aqueous and oil-water systems with two different aerobic biocatalysts types, Rhodococcus erythropolis IGTS8 and Pseudomonas putida CECT 5279. The results obtained have proven that global DBT desulfurization process using CECT 5279 was not clearly deactivated due to final product accumulation, under the experimental conditions assayed. Consistently, the desulfurization pattern has been described with the Michaelis-Menten equation, determining the kinetic parameters. On other hand, the assays have shown that important mass transfer limitations produced the decrease of the yields obtained with this Gram(-) strain in biphasic media. With strain IGTS8 it was observed lower mass transfer problems, but contrary the reaction was severely affected by the final product accumulation, in both aqueous and biphasic systems. Therefore it has been proposed an enzymatic kinetic model with competitive inhibition to describe the BDS evolution pattern when this Gram(+) strain was used.     

Production and characterization of rabbit polyclonal sera against Shiga toxins Stx1 and Stx2 for detection of Shiga toxin-producing Escherichia coli

STEC has emerged as an important group of enteric pathogens worldwide. In this study, rabbit polyclonal Stx1 and Stx2 antisera were raised and employed in the standardization of immunoassays for STEC detection. Using their respective antisera, the limit of detection of the toxin was 35.0 pg for Stx1 and 5.4 pg for Stx2. By immunoblotting, these antisera recognized both toxin subunits. Cross-reactivity was observed in the A subunit, but only Stx2 antiserum was able to neutralize the cytotoxicity of both toxins in the Vero cell assay. Six stx-harboring E. coli isolates were analyzed for their virulence traits. They belonged to different serotypes, including the O48:H7, described for the first time in Brazil. Only three strains harbored eae, and the e-hly gene and hemolytic activity was detected in five strains. Three isolates showed new stx2 variants (stx(2v-ha) and stx(2vb-hb)). The ELISA assay detected all six isolates, including one VCA-negative isolate, while the immunodot assay failed to detect one isolate, which was VCA-positive. In contrast, the colony-immunoblot assay detected only one VCA-positive isolate. Our results demonstrate that among the immunoassays developed in this study, the immunodot, and particularly the ELISA, appear as perspective for STEC detection in developing countries.     

Antiproliferative effects of two amides, piperine and piplartine, from Piper species

The present work evaluated the cytotoxicity of piplartine {5,6-dihydro-1-[1-oxo-3-(3,4,5-trimethoxyphenyl)-trans-2-propenyl]-2(1H)pyridinone} and piperine {1-[5-(1,3)-benzodioxol-5-yl)-1-oxo-2,4-pentadienyl]piperidine}, components obtained from Piper species. The substances were tested for their cytotoxicity on the brine shrimp lethality assay, sea urchin eggs development, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay using tumor cell lines and lytic activity on mouse erythrocytes. Piperine showed higher toxicity in brine shrimp (DL50 = 2.8 +/- 0.3 microg/ml) than piplartine (DL50 = 32.3 +/- 3.4 microg/ml). Both piplartine and piperine inhibited the sea urchin eggs development during all phases examined, first and third cleavage and blastulae, but in this assay piplartine was more potent than piperine. In the MTT assay, piplartine was the most active with IC50 values in the range of 0.7 to 1.7 microg/ml. None of the tested substances induced hemolysis of mouse erythrocytes, suggesting that the cytotoxicity of piplartine and piperine was not related to membrane damage.     

Chloride channel 7 (CLCN7) gene mutations in intermediate autosomal recessive osteopetrosis

Osteopetrosis is a heterogeneous group of inherited disorders that includes a malignant autosomal recessive form, an intermediate autosomal recessive form and autosomal dominant forms of the disease. Most malignant osteopetroses have been ascribed to mutations in the OC116 gene encoding the human a3 subunit of vacuolar H(+)-ATPase. Few cases of autosomal recessive malignant osteopetrosis have been ascribed to mutations in the chloride channel 7 gene (CLCN7), which accounts for all autosomal dominant type II cases reported to date. Up until now, however, nothing has been known regarding the molecular basis of the intermediate form of osteopetrosis (IARO). Our study of two Portuguese IARO families shows that homozygosity for ClCN7 mutations also accounts for this form of osteopetrosis. The two patients presented with spontaneous fractures in the first years of life and generalised increase of bone density. Direct sequencing of the ClCN7 gene in both patients revealed homozygosity for two mutations (G203D and P470Q). We conclude therefore that ClCN7 mutations not only account for some dominant and malignant forms but also for intermediate forms of osteopetrosis.     

Protease and phospholipase inhibition protect Veneza zonata (Hemiptera Coreidae) against septicemia caused by parasite trypanosomatid 563DT

Veneza zonata (Hemiptera Coreidae) is an insect which causes losses in several crops, and it is also an important vector of lower trypanosomatids. V. zonata specimens were collected on rural properties in Londrina, state of Paraná, Southern Brazil. Inoculation of Leptomonas 563DT into V. zonata hemocoel caused insect death within approximately 24 h, with large bacterial proliferation into their hemocoels. Some bacteria which were found in the digestive tract of those insects, such as Escherichia coli, Providencia rettgeri, and Kluyveria ascorbata, were also found in their hemolymph, which suggests that trypanosomatid crossing into hemocoel caused mechanical lesions in the digestive tract that allowed intestinal bacteria to infect the hemolymph, thereby leading to lethal septicemia. In this study we analysed proteolytic activities from the 563DT Leptomonas strain, which is pathogenic for V. zonata, aiming at evaluating the potential use of this Leptomonas strain for the biocontrol of the insect. The proteolytic action was evaluated on cells and on culture supernatants of trypanosomatids. We also evaluated the gelatinolytic activities, the action over natural and synthetic substrates for aminopeptidases, and the action of protease inhibitors during all trypanosomatid growth stages. A significant reduction in the number of insect deaths was observed when Leptomonas 563DT were incubated with inhibitors of proteases and phospholipases before being inoculated into the insects, which suggests that those enzymes are involved in the pathogenic mechanism.     

Cytotoxic activity of pisosterol, a triterpene isolated from Pisolithus tinctorius (Mich.: Pers.) Coker & Couch, 1928

Pisolithus tinctorius (Basidiomycete) is an ectomicorrhizal fungus found in the roots and soil surrounding of many species of eucalyptus and pine trees. The present work verified the cytotoxic potential of pisosterol, a triterpene isolated from P. tinctorius collected in the Northeast region of Brazil, on three different animal cell models: mouse erythrocytes, sea urchin embryos and tumor cells. Pisosterol lacked activity on mouse erythrocytes as well as on the development of sea urchin eggs, but strongly inhibited the growth of all seven tumor cell lines tested, especially the leukemia and melanoma cells (IC50 of 1.55, 1.84 and 1.65 microg/ ml for CEM, HL-60 and B16, respectively). The results found for pisosterol were compared with those of doxorubicin and etoposide.     

Validation of reference genes for RT-qPCR studies in <Emphasis Type="Italic">Stevia rebaudiana</Emphasis> in response to elicitor agents

Stevia rebaudiana is an important source of natural steviol glycosides and is of increasing interest in various fields of study. Therefore, understanding the molecular processes regulating its metabolism is of great importance. In this study, the stability of seven reference genes (18S ribosomal RNA, Actin, Aquaporin, Calmodulin, Eukaryote elongation factor 1-α, Malate dehydrogenase, and Ubiquitin) under the effect of three stress-related elicitors (methyl jasmonate, salicylic acid, and spermidine) was evaluated in stevia plants. We used RefFinder software, which makes use of the four main currently available algorithms for reference gene selection: geNorm, NormFinder, BestKeeper, and the Comparative ?Ct method. The results indicated that Ubiquitin and Actin can be used as reference genes under all tested experimental conditions. The genes, 18S ribosomal RNA, traditionally used as a reference gene, along with Calmodulin, showed the lowest stability. The expression of Deoxyxylulose-5-phosphate synthase and Kaurenoic acid hydroxylase genes was used to confirm the validated reference genes, showing that inadequacy of the reference gene may lead to erroneous results. This is the first study on the stability of reference genes in Stevia rebaudiana plants, and is of great relevance for further analysis of the gene expression of the steviol glycoside biosynthetic pathway.     

Comparison of automated and conventional microbiological examination of donated human cardiac tissue in heart valve banking

Most tissue banks use the conventional method; however, the automated method has advantages over the conventional method. The aim of this study was to compare the conventional and automated methods of culture in human cardiac tissue using an artificial contamination model. Myocardial samples were contaminated with sequential concentration (10⁴ to 10^?1 CFU/mL) with Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus and Candida albicans. Cultures were obtained from solution were the fragment was immersed and minced tissue, before and after the routine decellularization solution, with automated and conventional culture methods. Automated and conventional methods were compared and a p value ≤?0.05 was considered significant. Staphylococcus aureus presented a significantly higher growth in the automated method, as well as faster than the conventional (p?<?0.05). The positivity for growth in the automated method was higher in concentrated inoculum (>?10² CFU/mL) (p?<?0.05). The growth in the automated method was significantly faster than conventional when inoculum concentration was above 10³ CFU/mL. The automated culture method is faster than conventional method with a higher positivity in a contaminated model of myocardial and transport solution used in tissue banks.