Giacomino Randazzo

Identification of a β-lactoglobulin lactosylation site

Abstract Thermal treatment of milk leads to non-enzymatic glycosylation of proteins through Maillard reaction. Free NH2 groups of basic amino acids react with the reducing carbonyl group of lactose forming the so-called Amadori products. Electrospray mass spectrometry analysis shows that β-lactoglobulin (β-LG), the major whey protein, undergoes lactosylation under industrial thermal treatment. In order to investigate the specificity of reactive sites for lactose binding the analysis of trypsin hydrolysates of β-LG isolated from different industrial milks was performed. Results demonstrate that Lys-100 is a preferential lactosylation site of β-LG during industrial milk treatment. These results were confirmed by an analysis of the three-dimensional model of the protein which showed that Lys-100 had the highest solvent accessibility and proximity to another amino group making Lys-100 the best candidate to lactosylation. Lys-47, previously identified by other authors, showed a good proximity to another Lys residue, but an intermediate level of exposition to solvent.

Formation of coloured Maillard reaction products in a gluten- glucose model system

A pasta model system consisting of durum wheat gluten proteins and glucose was heated at diAerent times and temperatures under wet (6.6% water) and dry conditions in order to study conditions which promote the formation of Maillard compounds in pasta. Formation of coloured compounds was very slow up to 120C and increased seven-fold at 150C. Under wet conditions, the coloured material formed could be better extracted with polar solvents whereas, under dry conditions, more hydrophobic coloured compounds were extracted. Methanol extracts of the wet and dry gluten‐glucose mixtures were separated by HPLC gel filtration. Two high molecular weight peaks were collected. They showed diAerent UV-vis properties: the first peak was colourless and the second one was brown and was absent from extracts of gluten heated without glucose. Both peaks were dialysed through a 12-kDa membrane and analysed by C18 reverse phase HPLC with diode array detection before and after tryptic hydrolysis. Analysis of chromatograms revealed that coloured compounds were present only in peak 2 and were better detectable after proteolysis. It is concluded that, in the gluten-glucose system, coloured low molecular weight molecules became entrapped in the high molecular weight polymers formed by gluten proteins and that trypsin treatment of gluten favours the release of the coloured compounds. # 1999 Elsevier Science Ltd. All rights reserved.

1H and 13c nmr analysis of lycorine and α-dihydrolycorine☆

Abstract -The 1 H and 13 C NMR spectra of lycorine and its α-dihydro derivative have been studied. The employment of nuclear magnetic double resonance, nuclear Overhauser effect and acetylated derivatives, allows the assignment of all proton resonances. The assignments of the carbon shifts have been obtained by means of proton noise decoupled, single frequency off-resonance decoupled, single frequency selective decoupling, time dependence nuclear Overhauser effect and by comparison with reference compounds.

Cytochalasins: structure-activity relationships

Abstract Seven cytochalasins, 17- O -acetylcytochalasin A and two derivatives of cytochalasin B were assayed for Pseudomonas syringae , Bacillus megaterium , and for Geotrichum candidum . Their ability to inhibit the growth of tomato seedlings and their toxicity to brine shrimp (% larvae mortality were also investigated. Among the compounds assayed only cytochalasin A showed antibiotic and fungicidal activity. Toxicity to tomato seedlings was exhibited by both [14]- and [11]-macrocyclic cytochalasans, while the activity decreased in the derivatives acetylated on the 7-hydroxy group and markedly in cytochalasin E. In the brine shrimp bioassay, cytochalasin E was the most active mycotoxin, but generally, at low concentrations, the cytochalasins with the [11]-macrocyclic ring were more active than those with the [14]-macrocyclic ring; cytochalasin A appeared to be the most toxic.

A cytokinin from the culture filtrate of Pseudomonas syringae pv. savastanoi

Abstract The structure of a new cytokinin, isolated from the culture filtrate of Pseudomonas syringae pv. savastanoi, is assigned on the basis of spectroscopic data including its tetracetyl derivative and comparison with related adenine derivatives. It was identified as 6-(4-hydroxy-1,3-dimethylbut-trans-2-enylamino-9-β- D -ribofuranosyl)purine.

Proliferin, a new sesterterpene from Fusarium proliferatum

Abstract A new bicyclic sestertepene, named proliferin, has been characterized from Fusarium proliferatum . The pure compound exhibit toxic activity. The structure elucidation of the molecula was accomplished using data from 2D-NMR strategies. Proliferin was shown to have a novel ring skeleton and molecular formula C27H40O5.

Observation of Non-covalent Interactions Between Beauvericin and Oligonucleotides Using Electrospray Ionization Mass Spectrometry

Electrospray ionization mass spectrometry has been used to study the possible non-covalent interaction between oligonucleotides and beauvericin (B) mycotoxin. Beauvericin-oligonucleotide adduct formation was observed even at low mycotoxin concentration (25 pmol/microL). Adducts were found with different numbers of B ligands attached. The selectivity of binding of B ligands to two different oligonucleotides has been shown to be similar indicating that beauvericin does not have a strongly preferred base sequence or base site in the DNA. In a competitive complexation reaction, beauvericin forms specific adducts with oligonucleotide while another mycotoxin, nigeromicin, which causes apoptosis without fragmentation of DNA, does not.

Further experiments on structure-activity relationships among the lycorine alkaloids

Abstract Synthetic lycorine analogues, five Amaryllidaceae alkaloids and narciclasine, all structurally related to lycorine, were tested for their ability to inhibit ascorbic acid biosynthesis in vivo . The highest potency observed was displayed by narciclasine followed by compounds having an aromatic C-ring. Derivatives modified at C-1 and/or C-2 were inactive, while the compound with a double bond between these positions is a weak inhibitor. Also lutessine and its deacetyl derivative having an α-methoxyl group bonded to C-4 of the D-ring appeared completely inactive. These results confirm that the presence of an appropriately substituted C-ring is a necessary requirement for optimal ‘response-triggering’ contact between the lycorine derivatives and the specific receptor. Functional groups jutting out from the α-side of the molecule do not allow a good fit with the binding sites.

1H NMR conformational study of fusicoccin and related compounds: molecular conformation and biological activity

Abstract A 1 H NMR study of the phytotoxin fusicoccin (FC) and of a group of related compounds has been carried out at 500 MHz in chloroform- d solutions. Among the seven investigated compounds five were biologically active while two were not. The NMR spectra were completely analysed in terms of chemical shifts and coupling constants. A computer program, designed to provide all possible conformations compatible with the experimental values of a molecular set of vicinal coupling constants, was then employed to attain the structural conformations of the aglycones of all the compounds. The procedure afforded one conformation for each compound, except in one case wherein the experimental data are better interpreted in terms of a mixture of two interconverting conformers. The results of the present investigation seem to suggest that the occurrence of a given overall conformation of the carbotricyclic system whilst contributory to, is not sufficient to account for the biological activity of these compounds.

Seiricuprolide, a new phytotoxic macrolide from a strain of Seiridium cupressi infecting cypress☆

Abstract A strain of Seiridium cupressi, a fungus causing a canker disease of cypress (Cupressus sempervirens) in Greece, produces several phytotoxins in culture. Two of them were identified as seiridin and iso-seiridin, the butenolides previously isolated from another cypress pathogen, S. cardinale. A third phytotoxin, which is present in small amounts in the culture filtrates of S. cupressi and is not produced by S. cardinale, was identified as a new macrolide which we have called seiricuprolide. Its structure was established by spectroscopic analysis of the metabolite and of some key derivatives.