Effect of fasting on hypogean (<Emphasis Type="Italic">Niphargus stygius</Emphasis>) and epigean (<Emphasis Type="Italic">Gammarus fossarum</Emphasis>) amphipods: a laboratory study

Two amphipods, the hypogean Niphargus stygius and epigean Gammarus fossarum, were analyzed for fatty acid (FA) composition, electron transport system (ETS) activity and respiration (R) during a laboratory fasting experiment. In agreement with ETS and R measurements (and the ETS/R ratio), the hypogean N. stygius utilized FA more slowly than the epigean G. fossarum. Inter-specific differences in the utilization of certain FA during fasting were also revealed. While N. stygius tended to preserve all of its FA during the experimental fasting period, G. fossarum showed a tendency to utilize MUFA (monounsaturated FA) and SAFA (saturated FA) and preferentially retain PUFA (polyunsaturated FA). The significant correlations between ETS activity and composition of specific FA during fasting can be linked to R. During the fasting, both ETS activity and respiration rate of G. fossarum decreased, however, ETS/R ratio increased. In contrast, N. stygius did not show significant changes in these parameters. This is the first report, which connects ETS activity with changes in concentrations of specific FA during fasting. Such evolutionary adaptations of hypogean species enables them to better survive chronically low and/or discontinuous food supplies compared to epigean species, which live in environments where food shortages are much less frequent.     

Efflux pumps expression and its association with porin down-regulation and β-lactamase production among <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> causing bloodstream infections in Brazil

Background  

Multi-drug efflux pumps have been increasingly recognized as a major component of resistance in P. aeruginosa. We have investigated the expression level of efflux systems among clinical isolates of P. aeruginosa, regardless of their antimicrobial susceptibility profile.     

Functional characterization of the translationally controlled tumor protein (<Emphasis Type="Italic">TCTP</Emphasis>) gene associated with growth and defense response in cabbage

The translationally controlled tumor protein (TCTP) is an important component of the target of rapamycin (TOR) signaling pathway, the major regulator of cell growth in animals and fungi. Despite its relevance, knowledge on plant TCTP homologs is still limited. In the present study, the full-length BoTCTP cDNA was isolated from a cabbage (Brassica oleracea L.) cDNA library. The BoTCTP cDNA encodes a polypeptide of 168 amino acids and shared the highly conserved GTPase binding surface in all of the species analyzed. Northern blotting analysis showed that BoTCTP was specifically expressed in the root and stem. Furthermore, the expression of BoTCTP could be obviously enhanced by stress stimuli, including high temperature and salt stresses, while no significant changes in the BoTCTP expression were observed under ABA stress. Functional analysis of BoTCTP was performed by the silencing of BoTCTP using RNA interference (RNAi) and the BoTCTP RNAi plants exhibited reduced vegetative growth rate and decreased tolerance of the cold, high temperature, and salt stresses. The reported results clearly suggest that the BoTCTP gene is involved in the regulation of both growth and stress response in cabbage.     

Quantitative real-time PCR and fluorescence in situ hybridization approaches for enumerating <Emphasis Type="Italic">Brevundimonas diminuta</Emphasis> in drinking water

Brevundimonas diminuta is a small Gram-negative bacterium used for validation of membranes and filters used in the pharmaceutical and drinking water treatment industries. Current assays are time consuming, nonselective, and may be subject to interference by competing indigenous microorganisms. The focus of this study is to develop rapid and specific enumeration methodologies for B. diminuta. Quantitative real-time polymerase chain reaction (qPCR) and fluorescence in situ hybridization (FISH) assays were developed based on the gyrB (1,166 bp) and rpoD (829 bp) gene sequences of B. diminuta ATCC 19146. Species-specific primers and probes were designed, and a 100–200 bp segment of each gene was targeted in the qPCR studies. For both the qPCR and FISH assays, an internal 25 bp sequence was selected for use as a TaqMan probe (labeled with 6-FAM and a Black Hole Quencher). Probe specificity studies, conducted against Gram-negative and Gram-positive reference strains as well as environmental strains, revealed high specificity of the primer/probe pairs to B. diminuta. Sensitivities of the qPCR reactions using purified genomic DNA from B. diminuta were determined to be 0.89 pg for rpoD and 8.9 pg for gyrB. The feasibility of using whole-cell B. diminuta suspensions directly with the rpoD qPCR protocol was also evaluated. The greatest sensitivity observed for B. diminuta was 1 × 10³ colony forming units (CFU) per mL when tryptic soy broth was used as the growth medium. When compared with direct microscopic enumeration using a 5′ 6-FAM FISH probe, traditional plating methods showed significant underestimation of B. diminuta concentration (P = 0.01) when this organism was cultivated in saline lactose broth. The results of this investigation demonstrate that qPCR and FISH are effective methods for rapid (<4 h) enumeration of B. diminuta and may be viable alternatives to plating when validating drinking water filtration systems.     

Early Changes of β-Catenins and Menins in Spinal Cord Dorsal Horn after Peripheral Nerve Injury

Injury to the peripheral nervous system can lead to spontaneous pain, hyperalgesia and allodynia. Previous studies have shown sprouting of Aβ-fibres into lamina II of the spinal cord dorsal horn after nerve injury and the formation of new synapses by these sprouts. β-Catenin and menin as synaptogenic factors are critically involved in synapse formation. However, the roles of β-catenin and menin in neuropathic pain are still unclear. Using Western blot analysis we investigated the changes of β-catenin and menin in the spinal dorsal horn after unilateral spared nerve injury (SNI). We demonstrated an increase in both β-catenin and menin protein levels in the ipsilateral spinal dorsal horn at days 1 and 3 following spared nerve injury (P < 0.05). These increases were associated with changes in paw withdrawal threshold to mechanical stimuli and weight bearing deficit suggestive of pain behavior and spontaneous ongoing pain respectively. However, the injury-associated increases in β-catenins and menins levels returned to control levels at day 14. In conclusion, these results indicate that peripheral nerve injury induces upregulation of β-catenins and menins in the dorsal horn of the spinal cord, which may contribute to the development of chronic neuropathic pain. Antagonists of these molecules may serve as new therapeutic agents.     

Differential activity of innate defense antimicrobial peptides against <Emphasis Type="Italic">Nocardia</Emphasis> species

Background  

Members of the genus Nocardia are ubiquitous environmental saprophytes capable to cause human pulmonary, disseminated and cutaneous nocardiosis or bovine mastitis. Innate immunity appears to play an important role in early defense against Nocardia species. To elucidate the contribution of antimicrobial peptides (AMPs) in innate defense against Nocardia, the activity of human α-defensins human neutrophil peptides (HNPs) 1-3, human β-defensin (hBD)-3 and cathelicidin LL-37 as well as bovine β-defensins lingual and tracheal antimicrobial peptides (LAP, TAP) and bovine neutrophil-derived indolicidin against four important Nocardia species was investigated.     

Flow Cytometry,HPLC-RP,and metAFLP Analyses to Assess Genetic Variability in Somatic Embryo-Derived Plantlets of <Emphasis Type="BoldItalic">Gentiana pannonica</Emphasis> Scop

Cytometric and molecular techniques were used to verify genetic uniformity among somatic embryo-derived plantlets of Gentiana pannonica Scop. Cytometric analysis of regenerants revealed absence of chromosomal changes and alterations in ploidy. However, reverse phase high pressure liquid chromatography detected higher levels of methylation in regenerated plants than those of control plants. These changes were further investigated using a quantitative molecular marker-based approach. This revealed that numerous tissue culture-induced variations, ∼3% (epi)mutations, were observed, including sequence variation and changes in methylation patterns. Moreover, complex patterns of variation, including combinations of genetic and epigenetic changes, were relatively high (ca. 9%). Overall, tissue culture-induced variation reached 16%; while, demethylation was lower than de novo methylation in heterozygotic material and similar in all regenerated plantlets.