Paola Lusini

Proteomic response to physiological fermentation stresses in a wild-type wine strain of Saccharomyces cerevisiae

We report a study on the adaptive response of a wild-type wine Saccharomyces cerevisiae strain, isolated from natural spontaneous grape must, to mild and progressive physiological stresses due to fermentation. We observed by two-dimensional electrophoresis how the yeast proteome changes during glucose exhaustion, before the cell enters its complete stationary phase. On the basis of their identification, the proteins representing the S. cerevisiae proteomic response to fermentation stresses were divided into three classes: repressed proteins, induced proteins and autoproteolysed proteins. In an overall view, the proteome adaptation of S. cerevisiae at the time of glucose exhaustion seems to be directed mainly against the effects of ethanol, causing both hyperosmolarity and oxidative responses. Stress-induced autoproteolysis is directed mainly towards specific isoforms of glycolytic enzymes. Through the use of a wild-type S. cerevisiae strain and PMSF, a specific inhibitor of vacuolar proteinase B, we could also distinguish the specific contributions of the vacuole and the proteasome to the autoproteolytic process.

Helicobacter pylori: immunoproteomics related to different pathologies

Helicobacter pylori is a Gram-negative bacterium that causes ulcer, atrophic gastritis, adenocarcinoma and mucosa-associated lymphoid tissue lymphoma. Moreover, an ongoing controversial role of this bacterium infection has been suggested in the etiopathogenesis of some extradigestive diseases. The humoral response to H. pylori during a natural infection can be used for diagnostic purposes and as a basis for vaccine development. Host–pathogen interactions may be investigated by means of immunoproteomics, which provides global information about relevant specific and nonspecific antigens, and thus might be suitable to identify novel vaccine candidates or serological markers of H. pylori infection as well as of different related diseases. In this review, we describe how several research groups used H. pylori proteomics combined with western blotting analysis, using sera from patients affected with different H. pylori-related pathologies, to investigate potential associations between host immune response and clinical outcomes of H. pylori infection, resulting in the rapid identification of novel, highly immunoreactive antigens.

Pathogenic Mechanisms of Helicobacter pylori: Production of Cytotoxin

Bacteria associated with mucosal infections of the digestive system generally produce toxins, especially when they cause inflammatory lesions. Illnesses due to thermotolerant campylobacters, to enterohemorrhagic Escherichia coli, and to Clostridium difficileare only some examples. It would be surprising if Helicobacter pylori (HP) did not produce any toxic substances. The difficulty consists in attributing a pathogenic meaning to the toxin, since the range is quite wide of clinical and histological presentation of gastroduodenal inflammatory diseases linked to the presence in the stomach of H. pylori organisms [1]. Johnson and Lior [2] firstly reported the production of heat-labile cytotoxin by 80.6% of 36 HP strains they tested. However, most of our knowledge of the cytotoxigenicity of HP is from Leunk et al. [3] whose work has inspired us in part. They found that about 55% of 201 HP strains isolated in four different parts of the world produced a substance which caused intracellular vacuolization in cells of several lines in vitro, not only in lines generally employed in toxigenicity tests, like Chinese hamster ovary (CHO) cells, Vero cells, and Y-1 cells, but also in human tumoral cells like HeLa, KATO III, and HEp-2, as well as in human embryonic intestinal cells which were the most responsive. They also inferred that the toxin was proteinaceous in nature being heat labile (destroyed at 70 °C for 30 min), protease sensitive, and ammonium-sulfate precipitable. Its molecular weight ought to be higher than 100 kDa since cytotoxic activity could be found only in the retentate of a concentrated broth culture filtrate (CBCF) passed through 100 kDa molecular weight limit ultrafiltration membrane.

Oxidative damage mediated by herbicides on yeast cells.

Agricultural herbicides are among the most commonly used pesticides worldwide, posing serious concerns for both humans, exposed to these chemicals through many routes, and the environment. To clarify the effects of three herbicides as commercial formulations (namely, Pointer, Silglif, and Proper Energy), parameters related to oxidative issues were investigated on an autochthonous wine yeast strain. It was demonstrated that herbicides were able to affect the enzymatic activities of catalase and superoxide dismutase, as well as to induce carbonylation and thiol oxidation as post-translational modifications of proteins. Saccharomyces cerevisiae is an optimal model system to study responses to xenobiotics and oxidative stress. Thus, the results obtained could further the understanding of mechanisms underlying the toxicity of herbicides.

Adenylate cyclase in roots of Ricinus communis; stimulation by GTP and Mn 2+

Abstract A ‘sedimentable’ adenylate cyclase has been found in the roots of Ricinus communis plantlets. The enzymatic activity is stimulated by MnCl 2 , NaF and GTP. The results obtained constitute the first report of a higher plant adenylate cyclase requiring GTP.

Inactivation of Helicobacter pylori cagA Gene Affects Motility

Background.  The cytotoxin‐associated protein CagA is a Helicobacter pylori immunodominant antigen whose gene resides in the cag pathogenicity island. Our purpose was to determine if the disruption or deletion of cagA gene could have an effect on the expression of other proteins at the proteome level. We analyzed two H. pylori strains, 328 and G27 wild‐type, bearing the cag pathogenicity island, and their respective isogenic cagA− mutants.

Post-genomics of Neisseria meningitidis: an update

Neisseria meningitidis infection represents a major life-threatening bacterial disease worldwide. Genomics has revolutionized every aspect of the field of microbiology. As a consequence of genome sequencing, the postgenomic era commenced 15 years ago. Comparative genomics, functional genomics and proteomics, as well as a combination of these techniques, will play important roles in providing vital information regarding bacterial biological complexity and pathogenic traits, and accelerate the development of therapeutic drugs and vaccines for combating infections. This review summarizes the current knowledge regarding different approaches aimed to shed light on meningococcal biology and pathogenesis, and to accelerate the development and characterization of vaccines against pathogenic meningococci.

Inhibition of cAMP Phosphodiesterase by Some Phototherapeutic Agents

The behaviour of cyclic-3′,5′-AMP phosphodiesterase has been studied in the presence of psoralen, 8-methoxy-psoralen (8-MOP), 4,5′,8-trimethylpsoralen (TMP) (usu ally used in PUVA therapy), 4.6,4′-trimethylangelicin (TMA) and khellin recently proposed for the same thera peutical use. TMP and TMA exhibit a significant inhibitory effect on cyclic AMP phosphodiesterase; a light inhibition is produced by khellin at rather high concentration.

Pertussis toxin reverses the inhibition of the adenylyl cyclase system by khellin in HeLa cells

Khellin, a furochromone structurally related to furocoumarins, is a phototherapeutic agent used against psoriasis and vitiligo. This paper reports that pretreatment of HeLa cells with pertussis toxin reverses both the inhibition of NaF-stimulated adenylyl cyclase activity and the stimulation of GTPase by khellin alone and plus UVA light, as previously reported. Our results demonstrate, for the first time, that khellin is able to elicit an intracellular response by Gi alpha protein.

Khellin, but not 8-methoxypsoralen, inhibits adenylyl cyclase system in HeLa cells

Until recently, the therapeutic effects of furocoumarins and furochromones plus UV-A light were thought to be due to their ability to form photoadducts with DNA in the cell nuclei; now it appears that membrane effector systems may be involved as targets. Here we show that in HeLa cells khellin at 1 and 5 microM final concentration, in combination with UV-A light, inhibits NaF-stimulated adenylyl cyclase activity and Pertussis Toxin (PT)-catalyzed ADP-ribosylation of alpha-subunits of inhibitory guanine nucleotide regulatory proteins (Gi) and increases GTPase activity. In the same experimental conditions, 8-methoxypsoralen (8-MOP), either alone or plus UV-A, does not affect adenylyl cyclase and GTPase activities. Our results suggest that in HeLa cells, through an interaction with a receptor and the mediation of Gi proteins, the adenylyl cyclase system is a target for khellin but not for 8-MOP.