Arginine catabolism by sourdough lactic acid bacteria: purification and characterization of the arginine deiminase pathway enzymes from Lactobacillus sanfranciscensis CB1

The cytoplasmic extracts of 70 strains of the most frequently isolated sourdough lactic acid bacteria were screened initially for arginine deiminase (ADI), ornithine transcarbamoylase (OTC), and carbamate kinase (CK) activities, which comprise the ADI (or arginine dihydrolase) pathway. Only obligately heterofermentative strains such as Lactobacillus sanfranciscensis CB1; Lactobacillus brevis AM1, AM8, and 10A; Lactobacillus hilgardii 51B; and Lactobacillus fructivorans DD3 and DA106 showed all three enzyme activities. Lactobacillus plantarum B14 did not show CK activity. L. sanfranciscensis CB1 showed the highest activities, and the three enzymes were purified from this microorganism to homogeneity by several chromatographic steps. ADI, OTC, and CK had apparent molecular masses of ca. 46, 39, and 37 kDa, respectively, and the pIs were in the range of 5.07 to 5.2. The OTCs, CKs, and especially ADIs were well adapted to pH (acidic, pH 3.5 to 4.5) and temperature (30 to 37 degrees C) conditions which are usually found during sourdough fermentation. Internal peptide sequences of the three enzymes had the highest level of homology with ADI, OTC, and CK of Lactobacillus sakei. L. sanfranciscensis CB1 expressed the ADI pathway either on MAM broth containing 17 mM arginine or during sourdough fermentation with 1 to 43 mM added arginine. Two-dimensional electrophoresis showed that ADI, OTC, and CK were induced by factors of ca. 10, 4, and 2 in the whole-cell extract of cells grown in MAM broth containing 17 mM arginine compared to cells cultivated without arginine. Arginine catabolism in L. sanfranciscensis CB1 depended on the presence of a carbon source and arginine; glucose at up to ca. 54 mM did not exert an inhibitory effect, and the pH was not relevant for induction. The pH of sourdoughs fermented by L. sanfranciscensis CB1 was dependent on the amount of arginine added to the dough. A low supply of arginine (6 mM) during sourdough fermentation by L. sanfranciscensis CB1 enhanced cell growth, cell survival during storage at 7 degrees C, and tolerance to acid environmental stress and favored the production of ornithine, which is an important precursor of crust aroma compounds.     

Emotion Processing in Parkinson’s Disease: A Three-Level Study on Recognition,Representation, and Regulation

Background

Parkinson’s disease (PD) is characterised by well-known motor symptoms, whereas the presence of cognitive non-motor symptoms, such as emotional disturbances, is still underestimated. One of the major problems in studying emotion deficits in PD is an atomising approach that does not take into account different levels of emotion elaboration. Our study addressed the question of whether people with PD exhibit difficulties in one or more specific dimensions of emotion processing, investigating three different levels of analyses, that is, recognition, representation, and regulation.

Methodology

Thirty-two consecutive medicated patients with PD and 25 healthy controls were enrolled in the study. Participants performed a three-level analysis assessment of emotional processing using quantitative standardised emotional tasks: the Ekman 60-Faces for emotion recognition, the full 36-item version of the Reading the Mind in the Eyes (RME) for emotion representation, and the 20-item Toronto Alexithymia Scale (TAS-20) for emotion regulation.

Principal Findings

Regarding emotion recognition, patients obtained significantly worse scores than controls in the total score of Ekman 60-Faces but not in any other basic emotions. For emotion representation, patients obtained significantly worse scores than controls in the RME experimental score but no in the RME gender control task. Finally, on emotion regulation, PD and controls did not perform differently at TAS-20 and no specific differences were found on TAS-20 subscales. The PD impairments on emotion recognition and representation do not correlate with dopamine therapy, disease severity, or with the duration of illness. These results are independent from other cognitive processes, such as global cognitive status and executive function, or from psychiatric status, such as depression, anxiety or apathy.

Conclusions

These results may contribute to better understanding of the emotional problems that are often seen in patients with PD and the measures used to test these problems, in particular on the use of different versions of the RME task.     

Detection of oligoclonal T lymphocytes in lymph nodes draining from advanced non-small-cell lung cancer

Despite the combined use of surgery and chemoradiotherapy, the poor prognosis of advanced non-smallcell lung cancer (NSCLC) requires the definition of new therapeutic approaches. The presence of T lymphocytes, with peculiar phenotypic, functional and molecular characteristics within the tumour, suggested the possible use of these cells, expanded in vitro, in protocols of adoptive immunotherapy. We have described how a population of oligoclonal T lymphocytes, derived from advanced NSCLC, can be expanded in vitro and has the capability of lysing autologous cancer cells. What is more important, we observed that patients with advanced NSCLC, treated with TIL expanded in vitro and recombinant interleukin-2, seemed to have a disease-free period longer than that of patients treated with conventional chemoradiotherapy. in an attempt to find new sources of specific lymphocytes for immunotherapy, we describe the analysis of the phenotypic, functional and molecular characteristics of T lymphocytes, derived from lymph nodes draining advanced NSCLC. In this paper we show that these cells, have restriction patterns of T cell receptor chain similar to those detectable in the population of infiltrating T lymphocytes. This finding suggests that T cells derived from draining lymph nodes of advanced NSCLC have peculiar characteristics and can be a suitable source of effector cells for protocols of adoptive immunotherapy in lung cancer treatment.     

Innovative metal oxide-based substrates for DNA microarrays

In the present report, we propose a novel approach to synthesize DNA microarrays that employs immobilization of the nucleic acid molecules onto zinc and iron oxide surfaces through their phosphate backbone. Oxide films were prepared by the sol–gel technique and the resulting surfaces were characterized especially with respect to surface chemistry and morphological features by both X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). ZnO films annealed at T ? 300 °C show the most promising surface features to be employed for DNA microarray preparation, i.e. high density of binding sites (hydroxyl groups), smooth and homogeneous surfaces, high optical transmittance in the visible spectral range suitable for detection using fluorescence, and easy handling during preparation procedures. The analysis of nucleic acid retention on the oxide layers was performed by the scanning of dye-labelled DNA previously printed on the substrate using the DNA microarray robotic arm. Clearly visible spots with regular shape were revealed above the background noise indicating that anchoring of the DNA on the treated surface is efficient and does not interfere with hybridization processes. The use of suitably engineered zinc oxide film makes the immobilization strategy ideal for facile, efficient, and cost-effective manufacturing of DNA microarrays.     

α-synuclein levels affect autophagosome numbers in vivo and modulate Huntington disease pathology

Huntington and Parkinson diseases (HD and PD) are two major neurodegenerative disorders pathologically characterized by the accumulation of the aggregate-prone proteins mutant huntingtin (in HD) and α-synuclein (in PD). Mutant huntingtin is an autophagy substrate and autophagy modulators affect HD pathology both in vitro and in vivo. In vitro, α-synuclein levels are able to modulate autophagy: α-synuclein overexpression inhibits autophagy, whereas downregulation promotes autophagy. Here, we review our recent studies showing that α-synuclein levels modulate mutant huntingtin toxicity in mouse models. This phenotypic modification is accompanied by the in vivo modulation of autophagosome numbers in mouse brains from both control and HD mice expressing different levels of α-synuclein.     

Diaminobutyric acid: A tool for discriminating between carrier-mediated and non-carrier-mediated release of GABA from synaptosomes?

The carrier-mediated transport of GABA in rat brain synaptosomes was strongly and permanently inhibited byl-2,4-diaminobutyric acid (DAB). In order to discriminate between carrier-mediated and non-carrier-mediated release of [³H]GABA, synaptosomes prelabeled with 0.5 M [³H]GABA in the presence of 100 M DAB, or with 0.2 M [³H]GABA without DAB, were superfused in conditions stimulating the release of [³H]GABA. Only the release elicited by unlabeled GABA or DAB (by homo- and heteroexchange, respectively) was strongly inhibited in DAB-pretreated synaptosomes. The spontaneous release and the release induced by 56 mM KCl in the presence of CaCl₂, by the ionophore A23187, by ouabain, by lack of K⁺, or by purified black widow spider toxin were unaffected or only barely decreased in DAB-treated synaptosomes, and therefore do not seem to be mediated by the DAB-blocked GABA carrier.     

A convenient synthesis of lubeluzole and its enantiomer: Evaluation as chemosensitizing agents on human ovarian adenocarcinoma and lung carcinoma cells

Lubeluzole, a neuroprotective anti-ischemic drug, and its enantiomer were prepared following a convenient procedure based on hydrolytic kinetic resolution. The ee values were >99% and 96%, respectively, as assessed by HPLC analysis. The chemosensitizing effects of both enantiomers were evaluated in combination with either doxorubicin (human ovarian adenocarcinoma A2780 cells) or paclitaxel (human lung carcinoma A549 cells) by the MTT assay. At the lowest concentrations used, lubeluzole showed an overall and remarkable tendency to synergize with both anticancer drugs. In ovarian cancer cells a clear prevalence of antagonistic effect was observed for the R-enantiomer. The synergistic effects of lubeluzole for both drugs were observed over a wide concentration window (0.005–5 μM), the lowest limit being at least 40 times lower than human plasma concentrations previously reported as causing serious side effects.