Genomic analysis reveals the biotechnological ability of Enterococcus italicus to produce glutathione

Through the analysis of the recently available genome shotgun sequence of Enterococcus italicus DSM 15952^T type strain (Accession PRJNA61487, ID 61487), we found the presence of a gene encoding a bifunctional enzyme, termed γ-GCS-GS or GshF, involved in glutathione production and not influenced by feedback inhibition. The gshF gene exhibited high nucleotide and amino acid sequence similarity to other reported sequences from the Enterococcus genus and was constitutively expressed both in osmotic shock or in common cultural conditions. Several experimental studies concerning the culture medium, physiological stress, cell extract obtainment, and scaling-up showed that in selected conditions E. italicus was able to accumulate up to 250 μM of intracellular glutathione, which represented the main thiol group present into the cells. This is the first report regarding the production of glutathione by E. italicus, a species that could be used as a safe adjunct culture for glutathione-enriched dairy foods.     

Intestinal lymphoid tissue changes in the cortisone-treated Wistar rat

This paper continues the previous investigation of the Department on the lymphoid tissue of central and peripheral lymphoid organs under different experimental conditions. The morphological reactional modalities of the intestinal lymphoid tissue in the male Wistar rat were followed up under endocrine imbalance conditions following cortisone administration. Seven days after administration cortisone induced a hyperplasia of the intestinal lymphoid tissue in parallel with a depletion of the lymph node parenchyma and a hypercellularity of bone marrow. After a 6-week postcortisone interval, the lymphoid tissue showed changes corresponding to a cellular depletion in parallel with the restoration of the lymph node parenchyma and a normocellular bone marrow.     

Trace Elements and Metallothionein in Liver and Kidney of <Emphasis Type="Italic">Felis catus</Emphasis>

Trace metals such as Zn, Cu, and Fe are essential for life; differently, no biochemical function is known for Cd. Changes in dietary metal concentrations can cause deficiency or toxicity. Studies on trace elements in cat are lacking. This paper aimed to analyze Zn, Cu, Fe and Cd concentrations in liver and kidney of pathological domestic cat and to isolate metallothionein (MT) in these tissues. It was not possible to explore a possible correlation between metal concentrations and pathologies because the incidence for each of them was too low. Fe was the most abundant metal; in particular, the liver accumulates average Fe concentrations one order of magnitude higher than Zn and Cu, ranging from 66.75 and 1,444.23 μg/g. Significantly, higher levels of Fe were found in the liver of elder animals. Zn concentrations varied between 26.31 and 84.78 μg/g in the liver whereas in the kidney, ranged between 7.69 and 71.15 μg/g. Cu concentrations were between 2.37 and 112.91 μg/g in liver and between 2.12 and 9.85 μg/g in kidney. Cd was the least abundant metal with the exception of the kidney of the oldest cats where it reached a maximum of 13.71 μg/g. Gel-filtration metal distribution profiles from cytosolic extracts revealed the presence of Cd, Cu, Zn thioneins either in the liver or in the kidney. Because tissue samples were taken from pathological cats from different breed and age, care must be taken to use these data as a baseline profile of trace elements in healthy animals. Our results are indicative that for some specimens the feed levels of Fe and Cu could be higher than the optimal dietary intake and in few cats, there was also an exposure to Cd that was counteracted by MT biosynthesis.     

Mutagenic analysis of Thr-232 in rhodanese from Azotobacter vinelandii highlighted the differences of this prokaryotic enzyme from the known sulfurtransferases

Azotobacter vinelandii RhdA uses thiosulfate as the only sulfur donor in vitro, and this apparent selectivity seems to be a unique property among the characterized sulfurtransferases. To investigate the basis of substrate recognition in RhdA, we replaced Thr-232 with either Ala or Lys. Thr-232 was the target of this study since the corresponding Lys-249 in bovine rhodanese has been identified as necessary for catalytic sulfur transfer, and replacement of Lys-249 with Ala fully inactivates bovine rhodanese. Both T232K and T232A mutants of RhdA showed significant increase in thiosulfate-cyanide sulfurtransferase activity, and no detectable activity in the presence of 3-mercaptopyruvate as the sulfur donor substrate. Fluorescence measurements showed that wild-type and mutant RhdAs were overexpressed in the persulfurated form, thus conferring to this enzyme the potential of a persulfide sulfur donor compound. RhdA contains a unique sequence stretch around the catalytic cysteine, and the data here presented suggest a possible divergent physiological function of A. vinelandii sulfurtransferase.     

Regulation of locomotion and cell-cell contact area by the LFA-1 and ICAM-1 adhesion receptors.

We demonstrate complementary differences in the behavior of B lymphoblastoid cells adhering to LFA-1 or its counter-receptor ICAM-1. The interaction of B lymphoblastoid cells with glass-supported planar bilayers bearing LFA-1 or ICAM-1 was observed by time-lapse video microscopy, and the distribution of adhesion receptors on cells interacting with the planar bilayers was studied by immunofluorescence microscopy. B lymphoblasts formed a large contact area and crawled rapidly (up to 25 microns/min) on planar bilayers bearing ICAM-1. In contrast, these cells attached to planar bilayers bearing LFA-1 through a fixed point about which the cells actively pivoted, using a single stalk-like projection. Phorbol ester-stimulated lymphoblasts, which adhere more strongly to ICAM-1-bearing substrates than unstimulated lymphoblasts, were still capable of locomotion on ICAM-1. Phorbol ester stimulation of B lymphoblasts on planar bilayers bearing LFA-1 promoted a rapid conversion from "stalk" attachment to symmetrical spreading of the cell on the substrate. Cellular LFA-1 remained uniformly distributed on the cell surface during interaction with bilayers bearing purified ICAM-1 as determined by immunofluorescence. In contrast, ICAM-1 was concentrated in the stalk-like structure through which the unstimulated B lymphoblasts adhered to LFA-1 in planar bilayers, but ICAM-1 immunofluorescence became more uniformly distributed over the cell surface within minutes of phorbol ester addition. Neither LFA-1 or ICAM-1 colocalized with the prominent staining of filamentous actin in the ruffling membrane regions. Interaction through cell surface LFA-1 and ICAM-1, 2, or 3 promotes different cellular morphologies and behaviors, the correlation of which with previously observed patterns of lymphocyte interaction with different cell types is discussed.     

The proline-rich protein palladin is a binding partner for profilin

Palladin is an actin-associated protein that has been suggested to play critical roles in establishing cell morphology and maintaining cytoskeletal organization in a wide variety of cell types. Palladin has been shown previously to bind directly to three different actin-binding proteins vasodilator-stimulated phosphoprotein (VASP), alpha-actinin and ezrin, suggesting that it functions as an organizing unit that recruits actin-regulatory proteins to specific subcellular sites. Palladin contains sequences resembling a motif known to bind profilin. Here, we demonstrate that palladin is a binding partner for profilin, interacting with profilin via a poly proline-containing sequence in the amino-terminal half of palladin. Double-label immunofluorescence staining shows that palladin and profilin partially colocalize in actin-rich structures in cultured astrocytes. Our results suggest that palladin may play an important role in recruiting profilin to sites of actin dynamics.     

Palladin interacts with SH3 domains of SPIN90 and Src and is required for Src-induced cytoskeletal remodeling

Palladin and SPIN90 are widely expressed proteins, which participate in modulation of actin cytoskeleton by binding to a variety of scaffold and signaling molecules. Cytoskeletal reorganization can be induced by activation of signaling pathways, including the PDGF receptor and Src tyrosine kinase pathways. In this study we have analyzed the interplay between palladin, SPIN90 and Src and characterized the role of palladin and SPIN90 in PDGF and Src-induced cytoskeletal remodeling. We show that the SH3 domains of SPIN90 and Src directly bind palladin's poly-proline sequence and the interaction controls intracellular targeting of SPIN90. In PDGF-treated cells, palladin and SPIN90 co-localize in actin-rich membrane ruffles and lamellipodia. The effect of PDGF on the cytoskeleton is at least partly mediated by the Src kinase since PP2, a selective Src kinase family inhibitor, blocked PDGF-induced changes. Furthermore, expression of active Src kinase resulted in coordinated translocation of both palladin and SPIN90 to membrane protrusions. Knock-down of endogenous SPIN90 did not inhibit Src-induced cytoskeletal rearrangement, whereas knock-down of palladin resulted in cytoskeletal disorganization and inhibition of remodeling. Further studies showed that palladin is tyrosine phosphorylated in cells expressing active Src indicating bidirectional interplay between palladin and Src. These results may have implications in understanding the invasive and metastatic phenotype of neoplastic cells induced by Src.     

Effects of chronic treatment with sodium tetrachloroaurate(III) in mice and membrane models

Gold is a nonessential element with a variety of applications in medicine. A few gold(I) compounds are used in the clinics for treatment of rheumatoid arthritis and of discoid lupus. Some novel gold(III) compounds are under evaluation as anticancer agents. It is known that gold compounds generally produce toxic effects on the kidneys and characteristic lesions in the brain. However, information concerning the neurotoxicity of gold derivatives in humans as well as in experimental toxicology is rather scarce. For this reason we tried to shed some further light on this aspect of gold neurotoxicity by chronic treatment of mice with sodium tetrachloroaurate(III) in order to observe possible biophysical and morphological alterations that may occur in the brain. Chronic gold treatment resulted in a markedly decreased expression of metallothioneins and of glial fibrillary acidic protein in astrocytes of different brain areas. To examine its effects on cell membranes, interactions of sodium tetrachloroaurate(III) with molecular models were also evaluated. The models consisted in bilayers built-up of classes of phospholipids located in the outer and inner monolayers of biological membranes. Structural perturbation of cell membrane models was observed only at concentrations 10(5) times higher than those detected in the brains of animals after three months' treatment. These results show that toxic effects on animal brain upon treatment with sodium tetrachloroaurate develop with difficulty and may be observed only at high doses.