Augusto Parente

Seminal RNase: a unique member of the ribonuclease superfamily

The RNase found in bull semen, although a member of the mammalian superfamily of ribonucleases, possesses some unusual properties. Besides its unique structure and enzymic properties, it displays antispermatogenic, antitumor and immunosuppressive activities. Seminal RNase belongs to an interesting group of RNases, the RISBASES (RIbonucleases with Special, i.e. non catalytic, Biological Actions) other members of which include angiogenin, selectively neurotoxic RNases, a lectin and the self-incompatibility factors from a flowering plant.

New ribosome-inactivating proteins with polynucleotide:adenosine glycosidase and antiviral activities from Basella rubra L. and Bougainvillea spectabilis Willd.

Abstract. New single-chain (type 1) ribosome-inactivating proteins (RIPs) were isolated from the seeds of Basella rubra L. (two proteins) and from the leaves of Bougainvillea spectabilis Willd. (one protein). These RIPs inhibit protein synthesis both in a cell-free system, with an IC50 (concentration causing 50% inhibition) in the 10−10 M range, and by various cell lines, with IC50s in the 10−8–10−6 M range. All three RIPs released adenine not only from rat liver ribosomes but also from Escherichia coli rRNA, polyadenylic acid, herring sperm DNA, and artichoke mottled crinkle virus (AMCV) genomic RNA, thus being polynucleotide:adenosine glycosidases. The proteins from Basella rubra had toxicity to mice similar to that of most type 1 RIPs (Barbieri et al., 1993, Biochim Biophys Acta 1154: 237–282) with an LD50 (concentration that is 50% lethal) ≤ 8 mg · kg−1 body weight, whilst the RIP from Bougainvillea spectabilis had an LD50 >32 mg · kg−1. The N-terminal sequence of the two RIPs from Basella rubra had 80–93% identity, whereas it differed from the sequence of the RIP from Bougainvillea spectabilis. When tested with antibodies against various RIPs, the RIPs from Basella gave some cross-reactivity with sera against dianthin 32, and weak cross-reactivity with momordin I and momorcochin-S, whilst the RIP from Bougainvillea did not cross-react with any antiserum tested. An RIP from Basella rubra and one from Bougainvillea spectabilis were tested for antiviral activity, and both inhibited infection of Nicotiana benthamiana by AMCV.

Dimeric structure of seminal ribonuclease

RNAase BSl., the major component of ribonuclease activity in bull seminal plasma, is a basic protein with a molecular weight of 29,000 [ 11. Its catalytic properties are very similar to those of bovine pancreatic RNAase A [2] , except for a lower kcat and the ability to degrade also double-stranded RNA under conditions in which RNAase A is only slightly active [3]. We wish to report on the subunit structure of this enzyme, the first instance, to our knowledge, of a dimeric ribonuclease. This conclusion rests on several lines of evidence: i) peptide mapping of the protein; ii) quantitation of end groups; iii) estimation of the subunit molecular weight by electrophoresis on polyacrylamide gels in sodium dodecylsulphate; iv) estimation of the number of covalently linked species obtained by amidination of the protein with a crosslinking reagent. The peptide map of RNAase BS-1 was obtained by tryptic digestion of the protein oxidized with performic acid [4]. A total of 19 f 2 spots was obtained in several experiments (fig. la). From the ammo acid composition (28 lysine and 8 arginine residues per molecule), one would expect a number of about 37 peptides for a protein consisting of a single polypeptide chain. The observed value can therefore only be explained if one assumes that the protein is made up of two identical, or very similar, subunits. On the other hand, the possibility of RNAase BS1 being a dimer of RNAase A is ruled out by a comparison of the peptide maps of the two proteins (fig. la and b). Several peptides appear to be different, and this is in

Purification and partial characterization of single-chain ribosome-inactivating proteins from the seeds of Phytolacca dioica L.

Three ribosome-inactivating proteins (RIPs) similar to those already known (Stirpe et al. (1992) Bio/Technology 10, 405-412) were purified from the seeds of Phytolacca dioica. These proteins, called Phytolacca dioica RIPs (PD-S1, PD-S2 and PD-S3 RIPs), are glycoproteins, with M(r) approx. 30,000, inhibit protein synthesis by a rabbit reticulocyte lysate and phenylalanine polymerization by isolated ribosomes, and depurinate rat liver rRNA in an apparently identical manner as the A-chain of ricin and other RIPs (Endo et al. (1987) J. Biol. Chem. 262, 5908-5912). Part of the purified rat liver ribosomes appeared resistant to the action of PD-S RIPs. The most abundant protein, PD-S2 RIP, gave a weak or nil cross-reaction with sera against various other RIPs, including a pokeweed antiviral protein from the roots of Phytolacca americana. PD-S2 RIP was linked to a monoclonal antibody (Ber-H2) against the CD30 human lymphocyte antigen and the resulting immunotoxin was selectively toxic to the CD30 + Hodgkins lymphoma-derived L540 cell line.

Isolation and primary structure determination of a metallothionein from Paracentrotus lividus (Echinodermata, Echinoidea)☆

A low-molecular-mass zinc-binding protein was purified from the eggs of the sea urchin Paracentrotus lividus using procedures that included gel-permeation and anion-exchange chromatography followed by HPLC. The primary structure of this protein was derived from the sequences of peptide fragments obtained by digestion with trypsin and thermolysin. The reconstructed sequence showed the presence of 20 cysteinyl residues, thus resembling that of a metallothionein. The Paracentrotus protein was most similar to the metallothionein of Strongylocentrotus purpuratus, another member of the order of Echinoida, living along the coast of the Pacific Ocean. However, the presence of non-conservative amino acid substitution, together with a deletion of two residues in the Strongylocentrotus metallothionein, make the similarity scores of the two sea urchin proteins lower than that of metallothioneins from vertebrates of the same order. In addition, the present data show that sea urchin metallothioneins display no homology with metallothioneins of any other species.

Nutritional Traits of Bean (Phaseolus vulgaris) Seeds from Plants Chronically Exposed to Ozone Pollution

The effect of chronic exposure to ozone pollution on nutritional traits of bean ( Phaseolus vulgaris L. cv. Borlotto Nano Lingua di Fuoco) seeds from plants grown in filtered and nonfiltered open-top chambers (OTCs) has been investigated. Results showed that, among seed macronutrients, ozone significantly raised total lipids, crude proteins, and dietary fiber and slightly decreased total free amino acid content, although with a significant reduction of asparagine, lysine, valine, methionine, and glycine, compensated by a conspicuous augmentation of ornithine and tryptophan. Phytosterol analysis showed a marked increase of beta-sitosterol, stigmasterol, and campesterol in seeds collected from nonfiltered OTCs. With regard to secondary metabolites, ozone exposure induced a slight increase of total polyphenol content, although causing a significant reduction of some flavonols (aglycone kaempferol and its 3-glucoside derivative) and hydroxycinnamates (caffeic, p-coumaric, and sinapic acids). Total anthocyanins decreased significantly, too. Nevertheless, ozone-exposed seeds showed higher antioxidant activity, with higher Trolox equivalent antioxidant capacity (TEAC) values than those measured in seeds collected from filtered air.

Myoglobin as marker in meat adulteration: A UPLC method for determining the presence of pork meat in raw beef burger

The identification of meat animal species used in raw burgers is very important with respect to economic and religious considerations. Therefore, international supervisory bodies have implemented procedures to control the employed meat species. In this paper we propose myoglobin as a powerful molecular marker to evaluate the presence of non-declared meat addition in raw beef burgers by using ultra-performance liquid chromatography (UPLC) for the separation and identification of edible animal species (beef, chicken, horse, ostrich, pig and water buffalo). Meat samples were pre-treated with sodium nitrite to transform oxymyoglobin and deoxymyoglobin to the more stable metmyoglobin. The developed method was validated, preparing mixtures with different percentages of pork and beef minced meat. The obtained results show that using myoglobin as marker, 5% (25 mg/500 mg) of pork or beef meat can be detected in premixed minced meat samples.

Isolation and characterization of four type-1 ribosome-inactivating proteins, with polynucleotide:adenosine glycosidase activity, from leaves of Phytolacca dioica L.

Abstract. Four type-1 (single-chain) ribosome-inactivating proteins (RIPs), with isoelectric points between 9.5 and 9.7, were isolated from leaves of Phytolacca dioica L. The purification procedure furnished the four proteins with an overall yield of about 16% and separated them from a protein of 29 407 ± 2 Da, as determined by electrospray mass spectrometry, whose N-terminal amino acid sequence differed from that of pokeweed (Phytolacca americana L.) leaf chitinase (PLC-B) by only one amino acid (R17I). The four RIPs (PD-L1 to PD-L4) inhibited protein synthesis by a rabbit reticulocyte lysate with 50% inhibition at the picomolar level, and produced the β-fragment, diagnostic of the specific enzymatic action of RIPs, on yeast ribosomes. Comparison of their N-terminal sequences, up to residue 45, showed that PD-L1 is identical to PD-L2 [designated the isoleucine (Ile) form from the N-terminal residue] and PD-L3 is identical to PD-L4 [designated the valine (Val) form from the N-terminal residue] and that there are 35 identical residues between the two forms. Furthermore, the Val form presents the same number of identical residues as PD-S2, an RIP isolated from the seeds of the same plant. With the exception of PD-L4, the purified RIPs gave a positive reaction when stained for sugars on SDS-PAGE gels and, when analyzed by electrospray mass spectrometry, had Mr values of 32 715 ± 1 (PD-L1), 31 542 ± 1 (PD-L2), 30 356 ± 1 (PD-L3) and 29 185 ± 1 Da (PD-L4). The 1171 kDa difference in Mr, within the same RIP form, could be due to glycosylation. Like leaf saporins and many other RIPs, the four RIPs released several adenines from poly(A), herring sperm DNA and rRNA 16S + 23S, thus acting as polynucleotide:adenosine glycosidases. This property was less pronounced in PD-L1 and PD-L3 than in PD-L2 and PD-L4, respectively. The proteins PD-L1 and PD-L4 showed 3.7% reactivity with the antiserum anti-dianthin 32 and no reactivity with antisera to PAP-R saporin-S6, momordin I and even PD-S2, an RIP isolated from the seeds of the same plant. Protein PD-L4 showed 12.5% cross-reactivity with anti-PD-L1, while the opposite cross-reactivity was 100%.

Improved procedure for protein binder analysis in mural painting by LC-ESI/Q-q-TOF mass spectrometry: detection of different milk species by casein proteotypic peptides

Diagnostic techniques applied to the field of cultural heritage represent a very important aspect of scientific investigation. Recently, proteomic approaches based on mass spectrometry coupled with traditional spectroscopic methods have been used for painting analysis, generating promising results for binder’s protein identification. In the present work, an improved procedure based on LC-ESI/Q-q-TOF tandem mass spectrometry for the identification of protein binders has been developed for the molecular characterization of samples from an early-twentieth-century mural painting from the St. Dimitar Cathedral in Vidin, Bulgaria. The proteomic investigation has led to the identification of both egg white and egg yolk proteins, according to traditional old recipes for tempera paintings. In addition, beyond the egg components, the presence of caseins was also revealed, thus suggesting the use of milk as binding medium, fixative or stabilising agent. Furthermore, for the first time, the capability to discriminate the milk origin on the basis of alpha casein proteotypic peptides is reported, that are diagnostic for a given species, thus opening interesting perspectives in art and archaeological fields.

Structural characterization and comparative modeling of PD-Ls 1–3, type 1 ribosome-inactivating proteins from summer leaves of Phytolacca dioica L.

The amino acid sequence and glycan structure of PD-L1, PD-L2 and PD-L3, type 1 ribosome-inactivating proteins isolated from Phytolacca dioica L. leaves, were determined using a combined approach based on peptide mapping, Edman degradation and ESI-Q-TOF MS in precursor ion discovery mode. The comparative analysis of the 261 amino acid residue sequences showed that PD-L1 and PD-L2 have identical primary structure, as it is the case of PD-L3 and PD-L4. Furthermore, the primary structure of PD-Ls 1-2 and PD-Ls 3-4 have 81.6% identity (85.1% similarity). The ESI-Q-TOF MS analysis confirmed that PD-Ls 1-3 were glycosylated at different sites. In particular, PD-L1 contained three glycidic chains with the well known paucidomannosidic structure (Man)(3) (GlcNAc)(2) (Fuc)(1) (Xyl)(1) linked to Asn10, Asn43 and Asn255. PD-L2 was glycosylated at Asn10 and Asn43, and PD-L3 was glycosylated only at Asn10. PD-L4 was confirmed to be not glycosylated. Despite an overall high structural similarity, the comparative modeling of PD-L1, PD-L2, PD-L3 and PD-L4 has shown potential influences of the glycidic chains on their adenine polynucleotide glycosylase activity on different substrates.