Quinolone action in Escherichia coli cells carrying gyrA and gyrB mutations

Abstract We have isolated spontaneous mutant strains of Escherichia coli KL16 showing different levels of nalidixic acid (NAL) resistance. From 40 independent mutants, 36 had gyrA and four had gyrB mutations. Most of the gyrA mutations (30/36) conferred high level NAL resistance. In contrast, the only gyrB mutation that conferred a relatively high level of NAL resistance also determined enhanced susceptibility to quinolones with a piperazinyl substituent at C7 position of the quinolone ring (amphoteric quinolones). This gyrB mutation (denoted gyrB1604 ), jointly with a gyrA mutation (denoted gyrA972 ) which confers a high level of quinolone resistance, were used to construct strain IC2476, carrying the two gyr mutant alleles. The susceptibility of this strain to amphoteric quinolones (pipemidic acid, norfloxacin and ciprofloxacin) was similar to that of the gyrA972 single mutant. This result indicates that the change in GyrA subunit which determines a high level of quinolone-resistance has the capacity to mask the hypersusceptibility to amphoteric quinolones promoted by the GyrB1604 mutant subunit. This capacity was further confirmed by studying the effects of ciprofloxacin (CFX) on gyrase inhibition in the gyrA972 gyrB1604 strain.     

Detection of Pseudomonas savastanoi pv. savastanoi in Olive Plants by Enrichment and PCR

The sequence of the gene iaaL of Pseudomonas savastanoi EW2009 was used to design primers for PCR amplification. The iaaL-derived primers directed the amplification of a 454-bp fragment from genomic DNA isolated from 70 strains of P. savastanoi, whereas genomic DNA from 93 non-P. savastanoi isolates did not yield this amplified product. A previous bacterial enrichment in the semiselective liquid medium PVF-1 improved the PCR sensitivity level, allowing detection of 10 to 100 CFU/ml of plant extract. P. savastanoi was detected by the developed enrichment-PCR method in knots from different varieties of inoculated and naturally infected olive trees. Moreover, P. savastanoi was detected in symptomless stem tissues from naturally infected olive plants. This enrichment-PCR method is more sensitive and less cumbersome than the conventional isolation methods for detection of P. savastanoi.     

Flavones,sesquiterpene lactones and glycosides isolated from Centaurea aspera var. Stenophylla

From the alcoholic extract of Centaurea aspera var. stenophylla benzoic acid, p-hydroxybenzoic acid, apigenin, 6-methoxyluteolin, 11,13-dehydromelitensin, melitensin, stenophyliolide, ethyl-7-O-apigenin-glucuronate and the glucosides of sitosterol and stigmasterol were isolated and characterized. Stenophyllolide was shown to be 9,15-dihydroxygermacra-1(10),4,11-trien-12,6-olide.     

Increased rat alveolar macrophage expression of functional iNOS induced by a Dirofilaria immitis immunoglobulin superfamily protein.

Dirofilaria immitis is a worldwide filarial nematode causing heartworm disease in dogs and cats. Several mosquito species, which are able to feed both on humans and animals, can transmit this parasite. Inflammatory progression of host tissues induced by parasites are mediated by several molecules, including nitric oxide (NO), which usually exerts deleterious effects on parasites and occasionally on the host. We analyze the in vitro effect of total D. immitis adult worm somatic antigens on na?ve rat alveolar macrophage NO production and further separation of parasite proteins to define specific D. immitis somatic molecules influencing host cell NO secretion. Additionally, we address the possible influence of Wolbachia spp. on the in vitro production of NO by macrophages. Our results demonstrate that D. immitis adult worm soluble antigens are able to specifically induce NO production from host macrophages. Furthermore, we demonstrated that this effect is due to nematode antigens rather than to defined components (LPS and metabolic molecules) derived from its endosymbiont, Wolbachia spp. In addition, we were able to isolate and identify one of the parasite specific components from the DiSo extract, denominated DiID35.3 and putatively belonging to the Immunoglobulin Superfamily Protein (ISP) group, triggering NO release from macrophages in a dose-dependent and specific manner.     

Modulation of yeast alkaline cation tolerance by Ypi1 requires calcineurin

Ypi1 was discovered as an essential protein able to act as a regulatory subunit of the Saccharomyces cerevisiae type 1 protein phosphatase Glc7 and play a key role in mitosis. We show here that partial depletion of Ypi1 causes lithium sensitivity and that high levels of this protein confer a lithium-tolerant phenotype to yeast cells. Remarkably, this phenotype was independent of the role of Ypi1 as a Glc7 regulatory subunit. Lithium tolerance in cells overexpressing Ypi1 was caused by a combination of increased efflux of lithium, mediated by augmented expression of the alkaline cation ATPase ENA1, and decreased lithium influx through the Trk1,2 high-affinity potassium transporters. Deletion of CNB1, encoding the regulatory subunit of the calcineurin phosphatase, blocked Ypi1-induced expression of ENA1, normalized Li(+) fluxes, and abolished the Li(+) hypertolerant phenotype of Ypi1-overexpressing cells. These results point to a complex role of Ypi1 on the regulation of cation homeostasis, largely mediated by the calcineurin phosphatase.     

Reproducibility and Consistency of Proteomic Experiments on Natural Populations of a Non-Model Aquatic Insect

Population proteomics has a great potential to address evolutionary and ecological questions, but its use in wild populations of non-model organisms is hampered by uncontrolled sources of variation. Here we compare the response to temperature extremes of two geographically distant populations of a diving beetle species (Agabus ramblae) using 2-D DIGE. After one week of acclimation in the laboratory under standard conditions, a third of the specimens of each population were placed at either 4 or 27°C for 12 h, with another third left as a control. We then compared the protein expression level of three replicated samples of 2–3 specimens for each treatment. Within each population, variation between replicated samples of the same treatment was always lower than variation between treatments, except for some control samples that retained a wider range of expression levels. The two populations had a similar response, without significant differences in the number of protein spots over- or under-expressed in the pairwise comparisons between treatments. We identified exemplary proteins among those differently expressed between treatments, which proved to be proteins known to be related to thermal response or stress. Overall, our results indicate that specimens collected in the wild are suitable for proteomic analyses, as the additional sources of variation were not enough to mask the consistency and reproducibility of the response to the temperature treatments.     

Physiological and molecular characterisation of Saccharomyces cerevisiae cachaça strains isolated from different geographic regions in Brazil

In this work, 74 Saccharomyces cerevisiae strains isolated from cachaça fermentation of six different geographic regions in Brazil were characterized by mitochondrial DNA restriction fragment length polymorphism (mtDNA-RFLP) and by their ability to grow on stress conditions occurring during the cachaça fermentation process. Cachaça S. cerevisiae strains showed high mtDNA-RFLP polymorphism with the occurrence of 32 different molecular patterns. The S. cerevisiae strains presenting prevalent mtDNA were able to grow better in the stress conditions than strains represented by infrequent patterns. The principal coordinate analysis on 17 stress conditions revealed that the major source of growth variation were high ethanol concentrations and low temperatures. These results indicate that the stress conditions occurring in the fermentation process influence the prevalence of the most adapted S. cerevisiae strains in each distillery. The physiological tests used in our study can be used as a criterion for rapidly selecting autochthonous yeast strains for further purposes such as the selection of fermentative starters of S. cerevisiae strains.     

Frozen Cord Blood Hematopoietic Stem Cells Differentiate into Higher Numbers of Functional Natural Killer Cells In Vitro than Mobilized Hematopoietic Stem Cells or Freshly Isolated Cord Blood Hematopoietic Stem Cells

Adoptive natural killer (NK) cell therapy relies on the acquisition of large numbers of NK cells that are cytotoxic but not exhausted. NK cell differentiation from hematopoietic stem cells (HSC) has become an alluring option for NK cell therapy, with umbilical cord blood (UCB) and mobilized peripheral blood (PBCD34⁺) being the most accessible HSC sources as collection procedures are less invasive. In this study we compared the capacity of frozen or freshly isolated UCB hematopoietic stem cells (CBCD34⁺) and frozen PBCD34⁺ to generate NK cells in vitro. By modifying a previously published protocol, we showed that frozen CBCD34⁺ cultures generated higher NK cell numbers without loss of function compared to fresh CBCD34⁺ cultures. NK cells generated from CBCD34⁺ and PBCD34⁺ expressed low levels of killer-cell immunoglobulin-like receptors but high levels of activating receptors and of the myeloid marker CD33. However, blocking studies showed that CD33 expression did not impact on the functions of the generated cells. CBCD34⁺-NK cells exhibited increased capacity to secrete IFN-γ and kill K562 in vitro and in vivo as compared to PBCD34⁺-NK cells. Moreover, K562 killing by the generated NK cells could be further enhanced by IL-12 stimulation. Our data indicate that the use of frozen CBCD34⁺ for the production of NK cells in vitro results in higher cell numbers than PBCD34⁺, without jeopardizing their functionality, rendering them suitable for NK cell immunotherapy. The results presented here provide an optimal strategy to generate NK cells in vitro for immunotherapy that exhibit enhanced effector function when compared to alternate sources of HSC.