Differences between predicted and observed sequences in Saccharomyces cerevisiae.

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.

[Nasal surfactant: biochemical and histological study]. / Surfattante nasale: studio biochimico ed istologico.

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.

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.

Investigation of the mutagenic activity in Salmonella typhimurium of the furochromone khellin, proposed as a therapeutic agent for skin diseases.

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.

Photosensitization of DNA of defined sequence by furochromones, khellin and visnagin.

The sequence specificity in the in vitro DNA photobinding of khellin and visnagin, two naturally occurring furochromones proposed for chemotherapy of vitiligo, was investigated by using DNA sequencing methodology. The 3'-5' exonuclease associated with the T4 DNA polymerase served as a tool for determining photoadducts distribution on DNA fragments of the lac I gene of Escherichia coli. The photoadduct distribution of psoralen is also studied for comparison. Upon UVA irradiation, visnagin mainly forms monoadducts with thymine and to a lower extent with cytosine. Alternating (A-T)n sequences are hot spots for visnagin photoaddition. This is a property shared with furocoumarins. TTT sites are also quite reactive to visnagin, as they are to methylated angelicins. In contrast, with psoralen derivatives, there is no preferential photobinding in 5'-TpA sites, and 5'-ApT sites react as well. Furthermore, many sites such as T in the GC context, and C in any context, react, although weakly. The visnagin photoadduct distribution resembles very much the photoadduct distribution of methylated angelicins as described by Miolo et al. The photoreaction of these two series of compounds is less sequence dependent than the photobinding of psoralen derivatives as described by Sage and Moustacchi and by Boyer et al. The sequence specificity in khellin-DNA photobinding is the same as for visnagin, even though it forms much fewer photoadducts. The absence of photo-oxidation of DNA after treatment with visnagin or khellin plus UVA suggests that furochromones do not present any photodynamic effect on DNA.

Solvent spin-labelling for investigating the interaction of biological ligands with macromolecules. A 1H paramagnetic relaxation study.

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.

Inhibitory effects of psoralens on rat liver cyclic AMP phosphodiesterase.

TMP (4,5',8-trimethylpsoralen) usually employed in PUVA therapy and TMA (4,6,4'-trimethylangelicin), phototherapeutic agent under clinical and biological investigation, show an inhibitory effect of competitive type on the low Km cyclic AMP phosphodiesterase present in rat liver. The two drugs exhibit Ki values of 135 and 320 microM, respectively.

Khellin, a naturally occurring furochromone, used for the photochemotherapy of skin diseases: mechanism of action.

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.

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) (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.

Occurrence of cyclic AMP and related enzymes during germination of Pinus pinea seeds.

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 properties and physiological regulation of NAD glycohydrolase.

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.

The mechanisms of interaction between furanochromones and DNA. A heteronuclear Overhauser effect study on the khellin-thymidine model 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.

High performance liquid chromatography analysis of the furanochromones khellin and visnagin in various organs of Ammi visnaga (L.) Lam. at different developmental stages.

The content of the furanochromones khellin and visnagin, in the organs of Ammi visnaga (L.) Lam. at different developmental stages, has been examined. Determinations have been performed by means of a high performance liquid chromatography (HPLC) technique which allows both the separation and the quantitative determination of these chromones. Unripe fruits are the richest in both chromones, but the collection of ripe dry fruits--as it occurs in Egyptian folk-medicine--seems more reasonable because they might not undergo degradation processes during desiccation and storage.

Active oxygen forms in photoreaction between DNA and furanochromones khellin and visnagin.

The two furanochromones khellin and visnagin react with DNA under irradiation by 365 nm light, forming photoadducts. Recently, the use of khellin as therapeutic agent for skin diseases has been proposed. It is well known that during the formation of photoadducts toxic active oxygen forms are produced. We studied therefore the behaviour of the two furanochromones as producers of 1O-2 and O-2. Our results indicate that visnagin is a strong generator of both superoxide radicals and singlet oxygen, while khellin does not exhibit strong production of OO-2, which is promptly quenched by superoxide dismutase.

[Bioavailability of pyridine compounds in seeds of Ricinus communis L. at various stages of germination]. / Biodisponibilita' dei composti piridinici nei semi di Ricinus communis L. a vari stadi di germinazione.

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.

High performance liquid chromatography separation of the intermediates of pyridine coenzymes metabolism in Ricinus communis L. seedlings.

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.