Search

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.

A low-molecular weight chromium-containing fraction of the material resulting from dichromate reduction by bovine liver homogenate was investigated by NMR and ES-MS. The ES-MS spectrum showed a readily detectable peak at m/z = 786.1. The same molecular weight reasonably agreed with the relatively low diffusion coefficient measured by NMR-DOSY experiments on the main species observed in the (1)H NMR spectrum. At least two downfield shifted and broad paramagnetic signals were apparent in the (1)H NMR spectrum. Temperature dependence of chemical shift was exploited in order to estimate the diamagnetic shift of the signals in the diamagnetic region of the spectrum. 2D TOCSY, NOESY, COSY and (1)H-(3)C HMQC spectra revealed the presence of aromatic protons (which were assigned as His residues), Gly and some other short chain amino-acids. Combinations of the molecular masses of such components together with acetate (which is present in the solution) and chromium atoms allowed a tentative proposal of a model for the compound.

The products of hexavalent chromium [Cr(VI)] reduction by glutathione (GSH) alone or in the presence of equimolar quantities of aspartate (Asp) and/or glutamate (Glu) and a chromium-containing material extracted from bovine liver were studied by ultraviolet-visible spectrum (UV-vis) studies, electrospray mass spectrometry (ES-MS), electron paramagnetic resonance (EPR), and nuclear magnetic resonance (NMR). Reduction of chromate by GSH was followed by UV-vis and NMR, revealing the formation of a paramagnetic complex in which GSH acts as a ligand. ES-MS and EPR measurements provided unequivocal evidence of a dimeric Cr(V)(2)GSH(2) species in which the two metal ions are bridged by the Gamma-Glu carboxylate. The analysis of the (1)H and (13)C shifts experienced by GSH protons and the values of paramagnetic contributions to proton spin-lattice relaxation rates provided a set of constraints for structural determination. The same experiments were repeated in the presence of an equimolar concentration of Asp, revealing the formation of a dimeric Cr(V) paramagnetic complex in which the two metals are now bridged by Asp. Nuclear magnetic resonance dispersion profiles show that water is not displaced by Asp and that the correlation time of this complex is slowed by the increased complexity. When Glu is also included in the solution in equimolar concentration to GSH and Asp, data are consistent with the formation of many mono- and dinuclear species, with the three ligands competing with each other. Finally, the spectroscopic investigation of the chromium-containing material extracted from bovine liver revealed the presence of a complicate mixture of Cr(IV) or Cr(V) complexes, among which some Cr(V)-GSH species are present alone or with other ligands in the metal coordination sphere.

We recently studied the protein composition of a Saccharomyces cerevisiae wine yeast strain (K310) of enological interest. About 2,500 spots of 8-250 kDa observed molecular mass were resolved by two-dimensional gel electrophoresis. Experimental molecular masses and isoelectric points were calculated for most of them. Twenty-seven proteins were subjected to Edman microsequencing. N-terminal sequences of 12/27 proteins were determined, whereas internal sequences of 6/27 proteins were obtained following in situ proteolysis. Comparison between the experimental data and those reported in the SWISS-PROT database revealed some differences between genotypic and phenotypic sequences. These are indicative of the changes a protein can undergo with respect to the primary structure coded by the genomic DNA. Our results highlight the need to complement genomic analysis with detailed proteomics in order to refine the vast amount of information provided by DNA sequencing and to find an exact correlation between genome and proteome.

The existence of a Surface Tension Lower Substance (STLS) in the lung and the Eustachian tube was established a few decades ago. The histological and physiological characteristics of the epithelium of the whole respiratory tract, from the nasal cavities to rhinopharynx and bronchiolar segments, induced the Authors to search for such a similar substance also in nasal secretion. To this purpose nasal secretions were studied using High Pressure Liquid Chromatography (HPLC) to determine the presence of phospholipids. In addition, ultrastructural examination of the nasal mucosa was performed looking for lamellar body-like structures of 2nd degree pulmonary 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.

The photomutagenicity of the furochromone khellin was tested in Ames Salmonella strains using 8-methoxypsoralen (8-MOP) and 4,5', 8-trimethylpsoralen (TMP) as positive controls. When khellin was assayed with strain TA1537, mutation induction was not detectable; in the same strain, an equitoxic dose (52-56% level of survival) of TMP (used at a concentration 12-fold lower than khellin and with a UVA dose 83-fold lower than that used with khellin) yielded an increase in revertants/plate 3-fold above the spontaneous background. In strain TA102, khellin plus UVA treatment yielded a 2-fold increase in revertants/plate above the spontaneous background (79% survival). 8-MOP, however, used at a concentration 8-fold lower than khellin with a UVA dose 13-fold lower than khellin, yielded an increase in revertants/plate about 14-fold above background (66% survival) in the same strain. These data show that khellin has a weak photomutagenic potential and, along with the previously reported low photogenotoxic potential in eukaryotic cell systems, support the notion that khellin may be safer than bifunctional psoralens for clinical use.

The paramagnetic contributions to the spin-lattice relaxation rates of khellin protons, induced by the presence in an aqueous solution of TEMPO nitroxide, have been analyzed in the interaction of the furochromone with DNA. The relaxation data obtained at different temperatures, nitroxide and DNA concentrations indicate that the average solvent exposure of the furanic moiety of khellin is lower than that of the pyranic group. This feature suggests that the former is the main site of approach of khellin to DNA.

Khellin, a naturally occurring furochromone, used in the past as a coronary vasodilator, has recently been used in the photochemotherapeutic treatment of vitiligo and psoriasis. With the aim of elucidating its mechanism of action, the interactions both in ground and excited states between the drug and DNA were studied in vitro. Khellin forms in the dark a molecular complex with DNA. By subsequent irradiation (365 nm) the drug photoconjugates covalently with the macromolecule, although the rate of photobinding is rather low. The in vivo photobinding of khellin to the DNA of Ehrlich ascites tumor cells is also low. In photoaddition with the macromolecule the drug forms inter-strand cross-links, although again in small amounts. The furan side monoadduct between khellin and thymine, formed in the photoreaction between the drug and DNA, was isolated and characterized, and shows a cis-syn configuration.

The intermediates of metabolic cycle of pyridine compounds have been isolated in Ricinus communis seeds, at various stages of development, by reversed-phase HPLC with a linear gradient elution. NAm, NAc, QAc, NMN, des-NMN, des-NAD+, NAD+ and NADP+ were separated in less than 32 min. Pyridine compounds show typical behaviours during the considered periods (0-6th day). On the basis of the obtained results the metabolic availabilities of these vegetable tissues are discussed.

The metabolism of pyridine coenzymes is not yet well clarified in vegetable cells. We propose a HPLC method to separate the compounds involved in the pyridine nucleotides cycle in less than 32 min. The method has been applied to Ricinus communis seedlings. On the basis of the results obtained, the well known "de novo" and "salvage" pathways, demonstrated in other biological tissues for NAD biosynthesis, are likely to occur in this vegetable system.

The furanochromones khellin and visnagin have been characterised by 1H and 13C mono- and bidimensional NMR spectroscopies. Their strong affinity with DNA was experimentally confirmed by the complete disappearance of the furano-chromones' NMR signals upon additions of DNA. An intermolecular interaction between furanochromones and the thymidyl moieties of DNA, stabilized by the formation of a hydrogen bond between the thymidyl NH hydrogen and the C = O group of khellin or visnagin, is here proposed. This is suggested by the strong donor-acceptor behavior of these two molecular moieties, as pointed out by a selective 1H-13C Overhauser effect study of the khellin-thymidine model system.

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) (usually used in PUVA therapy), 4,6,4'-trimethylangelicin (TMA) and khellin recently proposed for the same therapeutical use. TMP and TMA exhibit a significant inhibitory effect on cyclic AMP phosphodiesterase; a light inhibition is produced by khellin at rather high concentration.

The occurrence of cAMP, adenylate cyclase and cAMP phosphodiesterase has been tested in Pinus pinea seed during germination. The study has been carried out on dormant and imbibed seeds, seedlings, endospermic residues, roots and cotyledons. cAMP has been detected by the protein binding method and its occurrence has been verified by HPLC detections. cAMP phosphodiesterase shows a very high activity at acidic pH, while being completely inactive at pH 7.4. At this pH value, well detectable levels of adenylate cyclase have been observed. Therefore, the classical pathway of synthesis and breakdown of cAMP, already accepted for animal and bacterial cells, seems to be operating in Pinus pinea plant too.

Kinetic studies on horse spleen NAD-glycohydrolase demonstrate that the hydrolysis of NAD+ is extensively inhibited by physiological concentrations of nicotinamide and NADP+. These compounds act as reversibly released product and competitive inhibitor respectively.