Truffle thio-flavours reversibly inhibit truffle tyrosinase

Tyrosinase is an enzyme having two copper atoms at the reactive site occurring in prokaryotic and eukaryotic organisms. In animals tyrosinase is responsible for pigmentation, in plants for protection of injured tissues or, as in fungi, to harden cell walls. Some of us have previously shown that tyrosinase is involved in truffle development and differentiation. Here we present the purification, the molecular properties and the reversible inhibition of Tuber melanosporum tyrosinase by dimethyl-sulfide and bis[methylthio]methane, the main flavour compounds of black and whitish truffles. The MW(r) is 39000. L-3,4-dihydroxyphenylalanine and L-tyrosine stain corresponding bands as expected for a true tyrosinase. Phenylthiourea, diethyldithiocarbamate and mimosine inhibit L-tyrosine and L-3,4-dihydroxyphenylalanine oxidation.     

A Kluyveromyces lactis mutant in the essential gene KlLSM4 shows phenotypic markers of apoptosis

We report the study of Kluyveromyces lactis cells expressing a truncated form of KlLSM4, a gene ortholog to LSM4 of Saccharomyces cerevisiae which encodes an essential protein involved in both pre-mRNA splicing and mRNA decapping. We had previously demonstrated that the first 72 amino acids of the K. lactis Lsm4p (KlLsm4Deltap) can restore cell growth in both K. lactis and S. cerevisiae cells not expressing the endogenous protein. However, cells showed a remarkable loss of viability in stationary phase. Here we report that cells expressing KlLsm4Deltap presented clear apoptotic markers such as chromatin condensation, DNA fragmentation, accumulation of reactive oxygen species, and showed increased sensitivity to different drugs. RNA analysis revealed that pre-mRNA splicing was almost normal while mRNA degradation was significantly delayed, pointing to this as the possible step responsible for the observed phenotypes.     

A specific enzymatic high-performance liquid chromatography method to determine N-methyl-D-aspartic acid in biological tissues

Recently we demonstrated that N-methyl-D-aspartic acid (NMDA) is present as an endogenous compound in the nervous tissues and endocrine glands of the rat where it plays a role in the regulation of the luteinizing hormone, growth hormone, and prolactin (FASEB J. 14 (2000) 699; Endocrinology 141 (2000) 3861). Based on the prediction that NMDA could have future importance in neuroendocrinology, we have devised an improved method for the specific and routine determination of NMDA in biological tissue. This method is based on the detection by HPLC of methylamine (CH(3)NH(2)) which comes from the oxidation of NMDA by D-aspartate oxidase, an enzyme which specifically oxidizes NMDA, yielding CH(3)NH(2) as one of the oxidative products of the reaction. The sensitivity of the method permits the accurate determination of NMDA in the supernatant of a tissue homogenate at levels of about 5-10 picomol/assay. However, for those tissues in which the concentration of NMDA is less than 1nmol/g, the sample must be further purified by treatment with o-phthaldialdehyde in order to separate the NMDA from the other amino acids and amino compounds and then concentrated and analyzed by HPLC. Using this method we have conducted a comparative study in order to measure the amount of NMDA in neuroendocrine and other tissues of various animal phyla from mollusks to mammals.     

Direct probing of RNA structure and RNA-protein interactions in purified HeLa cell's and yeast spliceosomal U4/U6.U5 tri-snRNP particles

The U4/U6.U5 tri-snRNP is a key component of spliceosomes. By using chemical reagents and RNases, we performed the first extensive experimental analysis of the structure and accessibility of U4 and U6 snRNAs in tri-snRNPs. These were purified from HeLa cell nuclear extract and Saccharomyces cerevisiae cellular extract. U5 accessibility was also investigated. For both species, data demonstrate the formation of the U4/U6 Y-shaped structure. In the human tri-snRNP and U4/U6 snRNP, U6 forms the long range interaction, that was previously proposed to be responsible for dissociation of the deproteinized U4/U6 duplex. In both yeast and human tri-snRNPs, U5 is more protected than U4 and U6, suggesting that the U5 snRNP-specific protein complex and other components of the tri-snRNP wrapped the 5' stem-loop of U5. Loop I of U5 is partially accessible, and chemical modifications of loop I were identical in yeast and human tri-snRNPs. This reflects a strong conservation of the interactions of proteins with the functional loop I. Only some parts of the U4/U6 Y-shaped motif (the 5' stem-loop of U4 and helix II) are protected. Due to difference of protein composition of yeast and human tri-snRNP, the U6 segment linking the 5' stem-loop to the Y-shaped structure and the U4 central single-stranded segment are more accessible in the yeast than in the human tri-snRNP, especially, the phylogenetically conserved ACAGAG sequence of U6. Data are discussed taking into account knowledge on RNA and protein components of yeast and human snRNPs and their involvement in splicesome assembly.     

Peach (Prunus persica) fruit ripening: aminoethoxyvinylglycine (AVG) and exogenous polyamines affect ethylene emission and flesh firmness

The effect of various concentrations of aminoethoxyvinylglycine (AVG; 0.32 and 1.28 m M ), an ethylene biosynthesis inhibitor, and of the polyamines putrescine (10 m M ), spermidine (0.1, 1 and 5 m M ) and spermine (2 m M ) on peach ( Prunus persica L. Batsch cv. Redhaven) fruit ripening was evaluated under field conditions. Treatments were performed 19 (polyamines) and 8 (AVG) days before harvest. Fruit growth (diameter, fresh and dry weight), flesh firmness, soluble solids content and ethylene emission were determined on treated and untreated (controls) fruits. Moreover, endogenous polyamine content and S-adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.21) activity were determined to check for a possible competition between polyamines and ethylene for their common precursor S-adenosylmethionine (SAM). Both treatments strongly inhibited ethylene emission and delayed flesh softening. On a biochemical level, AVG and exogenous polyamines both reduced the free-to-conjugate ratio of endogenous polyamines, and transiently altered SAMDC activity. The possible use of these compounds to control fruit ripening is discussed also in the light of their rejuvenating effect on peach fruits.     

A contribution to breast cancer cell proteomics: detection of new sequences

Ductal infiltrating carcinoma (DIC) of the breast is the most common and potentially aggressive form of cancer. Knowledge of proteomic profiles, attained both in vivo and in vitro, is fundamental to acquire as much information as possible on the proteins expressed in these pathologic conditions. We used the breast cancer cell line 8701-BC, established from a primary DIC, with the aim of contributing to the databases on mammary cancer cells, which in turn will be very useful for the identification of differentially expressed proteins in normal and neoplastic cells. Within an analysis window comprising about 1750 discernible spots, we have at present catalogued 84 protein spots. The proteins for which an identity was assigned were identified essentially using gel comparison, N-terminal (Nt) microseqencing and immune detection. Among the protein spots Nt-microsequenced, sixteen corresponded to known proteins, four resulted as modified, relative to matching sequences deposited on databases, and seven were unknown. These modified or novel sequences are thus of potential interest to the knowledge of breast cancer proteomics and its applications.     

Plasma carotenoid and malondialdehyde levels in ischemic stroke patients: relationship to early outcome

An association between ischemic stroke and increased oxidative stress has been suggested from animal studies. However, there is a lack of evidence with respect to this association in humans. Here, the time course of plasma levels of six carotenoids, which are lipophilic micronutrients with antioxidant properties, as well as of malondialdehyde (MDA), a marker of lipid peroxidation, was followed in ischemic stroke patients. Plasma levels of lutein, zeaxanthin, beta-cryptoxanthin, lycopene, alpha- and beta-carotene, as well as MDA were measured by high-performance liquid chromatography in 28 subjects (19 men and nine women aged 76.9+/-8.7 years) with an acute ischemic stroke of recent onset (<24h) on admission, after 6 and 24 h, and on days 3, 5, and 7. Carotenoid and MDA levels in patients on admission were compared with those of age- and sex-matched controls. Plasma levels of lutein, lycopene, alpha- and beta-carotene were significantly lower and levels of MDA were significantly higher in patients in comparison with controls. Significantly higher levels of MDA and lower levels of lutein were found in patients with a poor early-outcome (functional decline) after ischemic stroke as compared to patients who remained functionally stable. These findings suggest that the majority of plasma carotenoids are lowered immediately after an ischemic stroke, perhaps as a result of increased oxidative stress, as indicated by a concomitant rise in MDA concentrations. Among the carotenoids, only lutein plasma changes are associated with a poor early-outcome.