Type II secretion system secretin PulD localizes in clusters in the Escherichia coli outer membrane

The cellular localization of a chimera formed by fusing a monomeric red fluorescent protein to the C terminus of the Klebsiella oxytoca type II secretion system outer membrane secretin PulD (PulD-mCherry) in Escherichia coli was determined in vivo by fluorescence microscopy. Like PulD, PulD-mCherry formed sodium dodecyl sulfate- and heat-resistant multimers and was functional in pullulanase secretion. Chromosome-encoded PulD-mCherry formed fluorescent foci on the periphery of the cell in the presence of high (plasmid-encoded) levels of its cognate chaperone, the pilotin PulS. Subcellular fractionation demonstrated that the chimera was located exclusively in the outer membrane under these circumstances. A similar localization pattern was observed by fluorescence microscopy of fixed cells treated with green fluorescent protein-tagged affitin, which binds with high affinity to an epitope in the N-terminal region of PulD. At lower levels of (chromosome-encoded) PulS, PulD-mCherry was less stable, was located mainly in the inner membrane, from which it could not be solubilized with urea, and did not induce the phage shock response, unlike PulD in the absence of PulS. The fluorescence pattern of PulD-mCherry under these conditions was similar to that observed when PulS levels were high. The complete absence of PulS caused the appearance of bright and almost exclusively polar fluorescent foci.     

Subjective assessment of palatability, digestibility and emotions in healthy volunteers after ingestion of an iced dessert: preliminary report

Dietary habits can be influenced by several factors such as emotional status and food palatability represented by food smell, taste, texture, appearance and temperature. The aim of this study is to assess the palatability and digestibility of a coffee-flavored iced dessert ingested at the end of a standardized meal and its impact on emotional status in a sample of 30 healthy female volunteers. Thirty healthy female volunteers, after ENT and psychological assessment, were asked to fill in a Psycho-Emotional Questionnaire to assess their basal emotional pattern before the consumption of an iced coffee-flavored dessert after a standard meal. After the meal they filled in an Organoleptic-Sensory questionnaire, a Dynamic Digeribility questionnaire and again a Psycho-Emotional Questionnaire. In our study, most of the subjects found the tested coffee-flavoured iced dessert pleasant according to the Organoleptic-Sensorial Questionnaire (OSQ), in terms of taste, aspect, texture and smell; moreover, in 29 subjects the Dynamic Digestibility Questionnaire (DDQ) resulted in a good digestive experience. By means of the Psycho-Emotional Questionnaire (PEQ), an improvement of feelings and mood, associated with good data of digestibility and palatability was recorded. Although this observation is not statistically significant, the results seem to show a positive correlation between pleasure in eating such a product and emotional status. These data are preliminary and need further investigations on a larger population, in order to confirm this association, also in a mixed population, comparing male and female eating behaviour.     

PTPD1 supports receptor stability and mitogenic signaling in bladder cancer cells

PTPD1, a cytosolic non-receptor protein-tyrosine phosphatase, stimulates the Src-EGF transduction pathway. Localization of PTPD1 at actin cytoskeleton and adhesion sites is required for cell scattering and migration. Here, we show that during EGF stimulation, PTPD1 is rapidly recruited to endocytic vesicles containing the EGF receptor. Endosomal localization of PTPD1 is mediated by interaction with KIF16B, an endosomal kinesin that modulates receptor recycling at the plasma membrane. Silencing of PTPD1 promotes degradation of EGF receptor and inhibits downstream ERK signaling. We also found that PTPD1 is markedly increased in bladder cancer tissue samples. PTPD1 levels positively correlated with the grading and invasiveness potential of these tumors. Transgenic expression of an inactive PTPD1 mutant or genetic knockdown of the endogenous PTPD1 severely inhibited both growth and motility of human bladder cancer cells. These findings identify PTPD1 as a novel component of the endocytic machinery that impacts on EGF receptor stability and on growth and motility of bladder cancer cells.     

Previously unrecognized vaccine candidates against group B meningococcus identified by DNA microarrays

We have used DNA microarrays to follow Neisseria meningitidis serogroup B (MenB) gene regulation during interaction with human epithelial cells. Host-cell contact induced changes in the expression of 347 genes, more than 30% of which encode proteins with unknown function. The upregulated genes included transporters of iron, chloride, amino acids, and sulfate, many virulence factors, and the entire pathway of sulfur-containing amino acids. Approximately 40% of the 189 upregulated genes coded for peripherally located proteins, suggesting that cell contact promoted a substantial reorganization of the cell membrane. This was confirmed by fluorescence activated cell sorting (FACS) analysis on adhering bacteria using mouse sera against twelve adhesion-induced proteins. Of the 12 adhesion-induced surface antigens, 5 were able to induce bactericidal antibodies in mice, demonstrating that microarray technology is a valid approach for identifying new vaccine candidates and nicely complements other genome mining strategies used for vaccine discovery.     

Caspase activation in etoposide-treated fibroblasts is correlated to ERK phosphorylation and both events are blocked by polyamine depletion

Activation of the extracellular signal-regulated kinases (ERKs) 1 and 2 is correlated to cell survival, but in some cases ERKs can act in signal transduction pathways leading to apoptosis. Treatment of mouse fibroblasts with 20 microM etoposide elicited a sustained phosphorylation of ERK 1/2, that increased until 24 h from the treatment in parallel with caspase activity. The inhibitor of ERK activation PD98059 abolished caspase activation, but caspase inhibition did not reduce ERK 1/2 phosphorylation, suggesting that ERK activation is placed upstream of caspases. Both ERK and caspase activation were blocked in cells depleted of polyamines by the ornithine decarboxylase inhibitor alpha-difluoromethylornithine (DFMO). In etoposide-treated cells, DFMO also abolished phosphorylation of c-Jun NH(2)-terminal kinases triggered by the drug. Polyamine replenishment with exogenous putrescine restored the ability of the cells to undergo caspase activation and ERK 1/2 phosphorylation in response to etoposide. Ornithine decarboxylase activity decreased after etoposide, indicating that DFMO exerts its effect by depleting cellular polyamines before induction of apoptosis. These results reveal a role for polyamines in the transduction of the death signal triggered by etoposide.     

Symptomatic infections by toscana virus in the Modena province in the triennium 1999-2001

We report nine cases of clinical manifestations by Toscana virus (TOSV) diagnosed at the Centre for the Diagnosis of Viral Diseases of the University of Modena and Reggio Emilia, in the inhabitants of Modena province (Emilia Romagna region), in the triennium 1999-2001. TOSV was involved in six cases of meningitis, two of meningoencephalitis and one case of febrile erythema without meningeal manifestations. Six TOSV-infected patients had stayed for a few days in a different area of the Tuscany region before the onset of clinical manifestations so, only three cases of infections by TOSV had an autochthonous origin.