RESUMEN
The global incidence of economically motivated meat adulteration represents a crucial issue for the food industry. Undeclared addition of cheaper or low-quality species to meat products of high commercial value has become a common practice that needs to be countered with specific measures. In this framework, myoglobin (Mb) is a sarcoplasmic haemoprotein, primarily responsible for meat colour and has been successfully used in meat fraud authentication. Mb is highly soluble in water, easily monitored at 409 nm and species-specific. Knowing that various analytical DNA-based and protein-based methods, as well as spectroscopic techniques have been developed over the years for the detection of meat fraud, the aim of the present review is to take stock of the situation regarding the possible use of Mb as a molecular biomarker for the easy and rapid detection of undeclared species in meat products, avoiding the need of sophisticated or expensive equipment and specialised operators.
RESUMEN
Ribosome-inactivating proteins (RIPs) are a group of proteins with rRNA N-glycosylase activity that irreversibly inhibit protein synthesis and consequently cause cell death. Recently, an RIP called ledodin has been found in shiitake; it is cytotoxic, strongly inhibits protein synthesis, and shows rRNA N-glycosylase activity. In this work, we isolated and characterized a 50 kDa cytotoxic protein from shiitake that we named edodin. Edodin inhibits protein synthesis in a mammalian cell-free system, but not in insect-, yeast-, and bacteria-derived systems. It exhibits rRNA N-glycosylase and DNA-nicking activities, which relate it to plant RIPs. It was also shown to be toxic to HeLa and COLO 320 cells. Its structure is not related to other RIPs found in plants, bacteria, or fungi, but, instead, it presents the characteristic structure of the fold type I of pyridoxal phosphate-dependent enzymes. Homologous sequences have been found in other fungi of the class Agaricomycetes; thus, edodin could be a new type of toxin present in many fungi, some of them edible, which makes them of great interest in health, both for their involvement in food safety and for their potential biomedical and biotechnological applications.
Asunto(s)
Ribosomas , Hongos Shiitake , Humanos , Ribosomas/efectos de los fármacos , Ribosomas/metabolismo , Hongos Shiitake/química , Células HeLa , Animales , Micotoxinas/toxicidad , Micotoxinas/química , Proteínas Inactivadoras de Ribosomas/química , Proteínas Inactivadoras de Ribosomas/farmacología , Proteínas Fúngicas/química , Proteínas Fúngicas/toxicidad , Proteínas Fúngicas/farmacología , Proteínas Fúngicas/metabolismo , Línea Celular TumoralRESUMEN
Ribosome inactivating proteins (RIPs) are specific N-ß-glycosylases that are well-characterized in plants. Their enzymatic action is to damage ribosomes, thereby blocking protein translation. Recently, several research groups have been working on the screening for these toxins in edible plants to facilitate the use of RIPs as biotechnological tools and biopesticides and to overcome public prejudice. Here, four novel monomeric (type 1) RIPs have been isolated from the seeds of Atriplex hortensis L. var. rubra, which is commonly known as edible red mountain spinach. These enzymes, named hortensins 1, 2, 4, and 5, are able to release the ß-fragment and, like many other RIPs, adenines from salmon sperm DNA, thus, acting as polynucleotide:adenosine glycosidases. Structurally, hortensins have a different molecular weight and are purified with different yields (hortensin 1, ~29.5 kDa, 0.28 mg per 100 g; hortensin 2, ~29 kDa, 0.29 mg per 100 g; hortensin 4, ~28.5 kDa, 0.71 mg per 100 g; and hortensin 5, ~30 kDa, 0.65 mg per 100 g); only hortensins 2 and 4 are glycosylated. Furthermore, the major isoforms (hortensins 4 and 5) are cytotoxic toward human continuous glioblastoma U87MG cell line. In addition, the morphological change in U87MG cells in the presence of these toxins is indicative of cell death triggered by the apoptotic pathway, as revealed by nuclear DNA fragmentation (TUNEL assay).
Asunto(s)
Atriplex , Proteínas Inactivadoras de Ribosomas Tipo 1 , Semillas , Humanos , Glioblastoma , Ribosomas , Proteínas de Plantas , Línea Celular TumoralRESUMEN
Sodin 5 is a type 1 ribosome-inactivating protein isolated from the seeds of Salsola soda L., an edible halophytic plant that is widespread in southern Europe, close to the coast. This plant, known as 'agretti', is under consideration as a new potential crop on saline soils. Considering a possible defence role of sodin 5 in the plant, we report here its antifungal activity against different halophilic and halotolerant fungi. Our results show that sodin 5 at a concentration of 40 µg/mL (1.4 µM) was able to inhibit the growth of the fungi Trimmatostromma salinum (35.3%), Candida parapsilosis (24.4%), Rhodotorula mucilaginosa (18.2%), Aspergillus flavus (12.2%), and Aureobasidium melanogenum (9.1%). The inhibition observed after 72 h was concentration-dependent. On the other hand, very slight growth inhibition was observed in the fungus Hortaea werneckii (4.2%), which commonly inhabits salterns. In addition, sodin 5 showed a cytotoxic effect on the Sf9 insect cell line, decreasing the survival of these cells to 63% at 1.0 µg/mL (34.5 nM). Structural analysis of sodin 5 revealed that its N-terminal amino acid residue is blocked. Using mass spectrometry, sodin 5 was identified as a homologous to type 1 polynucleotide:adenosine glycosylases, commonly known as ribosome-inactivating proteins from the Amaranthaceae family. Twenty-three percent of its primary structure was determined, including the catalytic site.
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Salsola , Saporinas/metabolismo , Salsola/metabolismo , Hongos/metabolismo , Antifúngicos/metabolismo , Semillas/química , Proteínas de Plantas/químicaRESUMEN
BACKGROUND: Ribosome inactivating proteins (RIPs) are N-glycosylases found in various plants that are able to specifically and irreversibly inhibit protein translation, thereby leading to cell death. Their cytotoxic properties have attracted attention in the medical field in the context of developing new anticancer therapies. Quinoin is a novel toxic enzyme obtained from quinoa seeds and classified as a type 1 RIP (Chenopodium quinoa Willd.). Recently, quinoin was found to be cytotoxic to normal fibroblasts and keratinocytes in vitro, as well as to several tumor cell lines. METHODS: The aim of this study was to evaluate the in vitro and in vivo genotoxicity of quinoin in a zebrafish model. We evaluated its ability to induce DNA fragmentation, genomic instability, and reactive oxygen species (ROS) generation by means of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) reaction, randomly amplified polymorphic DNA (RAPD) Polymerase Chain Reaction (PCR) technique, and dichlorofluorescine (DCF) assay, respectively. RESULTS: Quinoin was found to cause genomic damage in zebrafish, as shown by DNA fragmentation, polymorphic variations leading to genomic instability, and oxidative stress. Interestingly, longer quinoin treatment caused less damage than shorter treatments. CONCLUSIONS: This study demonstrated ROS-mediated genotoxicity of quinoin toward the zebrafish genome. The reduced damage observed after longer quinoin treatment could indicate the activation of detoxification mechanisms, activation of repair mechanisms, or the loss of protein activity due to enzymatic digestion. In order to clarify the genotoxic actions of quinoin, further investigations of the response pathways to DNA damage are needed. Overall, the ability of quinoin to cause breaks and instability in DNA, together with its clear cytotoxicity, make it an interesting candidate for the development of new drugs for cancer treatment.
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Chenopodium quinoa , Pez Cebra , Animales , Pez Cebra/genética , Pez Cebra/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Chenopodium quinoa/metabolismo , Técnica del ADN Polimorfo Amplificado Aleatorio , Saporinas/metabolismo , Daño del ADN , Semillas/genética , Semillas/metabolismo , Inestabilidad Genómica , ADN/metabolismoRESUMEN
Ribotoxin-like proteins (RL-Ps) are specific ribonucleases found in mushrooms that are able to cleave a single phosphodiester bond located in the sarcin-ricin loop (SRL) of the large rRNA. The cleaved SRL interacts differently with some ribosomal proteins (P-stalk). This action blocks protein synthesis because the damaged ribosomes are unable to interact with elongation factors. Here, the amino acid sequences of eryngitin 3 and 4, RL-Ps isolated from Pleurotus eryngii fruiting bodies, were determined to (i) obtain structural information on this specific ribonuclease family from edible mushrooms and (ii) explore the structural determinants which justify their different biological and antipathogenic activities. Indeed, eryngitin 3 exhibited higher toxicity with respect to eryngitin 4 against tumoral cell lines and model fungi. Structurally, eryngitin 3 and 4 consist of 132 amino acids, most of them identical and exhibiting a single free cysteinyl residue. The amino acidic differences between the two toxins are (i) an additional phenylalanyl residue at the N-terminus of eryngitin 3, not retrieved in eryngitin 4, and (ii) an additional arginyl residue at the C-terminus of eryngitin 4, not retrieved in eryngitin 3. The 3D models of eryngitins show slight differences at the N- and C-terminal regions. In particular, the positive electrostatic surface at the C-terminal of eryngitin 4 is due to the additional arginyl residue not retrieved in eryngitin 3. This additional positive charge could interfere with the binding to the SRL (substrate) or with some ribosomal proteins (P-stalk structure) during substrate recognition.
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Agaricales , Ascomicetos , Pleurotus , Ricina , Endorribonucleasas/metabolismo , Proteínas Fúngicas/metabolismo , Pleurotus/metabolismo , Ribonucleasas/química , Agaricales/química , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/análisis , Ricina/metabolismo , Ascomicetos/metabolismo , Cuerpos Fructíferos de los Hongos/químicaRESUMEN
Ageritin from poplar mushrooms is a specific endonuclease that hydrolyzes a single phosphodiester bond located in the sarcin-ricin loop (SRL) of the large rRNA, thereby blocking protein synthesis. Considering the possible biotechnological use of this enzyme, here we report its antifungal activity against virulent fungi affecting crops of economic interest. Our results show that ageritin (200 µg/plug; ~13.5 nmole) inhibits the growth of Botrytis cinerea (57%), Colletotrichum truncatum (42%), and Alternaria alternata (57%), when tested on potato dextrose agar plates. At the same time, no effect was observed against Trichoderma harzianum (a fungus promoting beneficial effects in plants). To verify whether the antifungal action of ageritin against B. cinerea and T. harzianum was due to ribosome damage, we tested ageritin in vitro on partially isolated B. cinerea and T. harzianum ribosomes. Interestingly, ageritin was able to release the Endo's fragment from both tested fungal ribosomes. We therefore decided to test the antifungal effect of ageritin on B. cinerea and T. harzianum using a different growth condition (liquid medium). Differently from the result in solid medium, ageritin can inhibit both B. cinerea and T. harzianum fungal growth in liquid medium in a concentration-dependent manner up to 35.7% and 38.7%, respectively, at the highest concentration tested (~200 µg/mL; 12 µM), and the analysis of RNA isolated from ageritin-treated cells revealed the presence of Endo's fragment, highlighting its ability to cross the fungal cell wall and reach the ribosomes. Overall, these data highlight that the efficacy of antifungal treatment to prevent or treat a potential fungal disease may depend not only on the fungal species but also on the conditions of toxin application.
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Agaricales , Antifúngicos , Antifúngicos/farmacología , Agaricales/metabolismo , Ribonucleasas/metabolismo , Hongos/metabolismoRESUMEN
We have purified ledodin, a cytotoxic 22-kDa protein from shiitake mushroom (Lentinula edodes) consisting of a 197 amino acid chain. Ledodin possessed N-glycosylase activity on the sarcin-ricin loop of mammalian 28S rRNA and inhibited protein synthesis. However, it was not active against insect, fungal, and bacterial ribosomes. In vitro and in silico studies suggested that ledodin exhibits a catalytic mechanism like that of DNA glycosylases and plant ribosome-inactivating proteins. Moreover, the sequence and structure of ledodin was not related to any protein of known function, although ledodin-homologous sequences were found in the genome of several species of fungi, some edible, belonging to different orders of the class Agaricomycetes. Therefore, ledodin could be the first of a new family of enzymes widely distributed among this class of basidiomycetes. The interest of these proteins lies both, in the fact that they can be a toxic agent of some edible mushrooms and in their application in medicine and biotechnology.
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Hongos Shiitake , Animales , Saporinas , Hongos Shiitake/genética , Hongos Shiitake/química , MamíferosRESUMEN
White button mushroom (Agaricus bisporus (J.E. Lange) Imbach) is one of the widely consumed edible mushrooms. Indeed, A. bisporus fruiting bodies are a rich source of nutrients and bioactive molecules. In addition, several enzymes with biotechnological applications are found in A. bisporus (e.g., enzymes for lignocellulose degradation). Here, a novel ribotoxin-like protein (RL-P) from the edible mushroom A. bisporus was purified and characterized. This RL-P, named bisporitin, is a monomeric protein (17-kDa) exhibiting specific ribonucleolytic activity by releasing the α-fragment (hallmark of RL-Ps) when incubated with rabbit ribosomes. In addition, bisporitin shows magnesium-dependent endonuclease activity and displays a similar far-UV CD spectrum as ageritin, the prototype of RL-Ps, isolated from Cyclocybe aegerita fruiting bodies. Interestingly, bisporitin is the first member of RL-Ps to have noticeably lower thermal stability (Tm = 48.59 ± 0.98 °C) compared to RL-Ps isolated in other mushrooms (Tm > 70 °C). Finally, this protein is only partially hydrolyzed in an in vitro digestive system and does not produce adverse growing effects on eukaryotic cell lines. This evidence paves the way for future investigations on possible bioactivities of this RL-P in the digestive system.
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Agaricus , Animales , Conejos , Ribosomas/metabolismoRESUMEN
Cetuximab is a monoclonal antibody blocking the epidermal growth factor receptor (EGFR) in metastatic colorectal cancer (mCRC). However, cetuximab treatment has no clinical benefits in patients affected by mCRC with KRAS mutation or in the presence of constitutive activation of signalling pathways acting downstream of the EGFR. The aim of this study was to improve cetuximab's therapeutic action by conjugating cetuximab with the type 1 ribosome inactivating protein (RIP) quinoin isolated from quinoa seeds. A chemical conjugation strategy based on the use of heterobifunctional reagent succinimidyl 3-(2-pyridyldithio)propionate (SPDP) was applied to obtain the antibody-type 1 RIP chimeric immunoconjugate. The immunotoxin was then purified by chromatographic technique, and its enzymatic action was evaluated compared to quinoin alone. Functional assays were performed to test the cytotoxic action of the quinoin cetuximab immunoconjugate against the cetuximab-resistant GEO-CR cells. The novel quinoin cetuximab immunoconjugate showed a significant dose-dependent cytotoxicity towards GEO-CR cells, achieving IC50 values of 27.7 nM (~5.0 µg/mL) at 72 h compared to cetuximab (IC50 = 176.7 nM) or quinoin (IC50 = 149.3 nM) alone assayed in equimolar amounts. These results support the therapeutic potential of quinoin cetuximab immunoconjugate for the EGFR targeted therapy, providing a promising candidate for further development towards clinical use in the treatment of cetuximab-resistant metastatic colorectal cancer.
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Antineoplásicos , Neoplasias del Colon , Neoplasias Colorrectales , Inmunotoxinas , Humanos , Anticuerpos Monoclonales Humanizados , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Cetuximab/farmacología , Cetuximab/genética , Cetuximab/uso terapéutico , Neoplasias del Colon/tratamiento farmacológico , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Receptores ErbB/metabolismo , Inmunotoxinas/uso terapéutico , Mutación , Saporinas/uso terapéutico , Resistencia a AntineoplásicosRESUMEN
Ageritin is a ribotoxin-like protein of biotechnological interest, belonging to a family of ribonucleases from edible mushrooms. Its enzymatic activity is explicated through the hydrolysis of a single phosphodiester bond, located in the sarcin/ricin loop of ribosomes. Unlike other ribotoxins, ageritin activity requires divalent cations (Zn2+). Here we investigated the conformational stability of ageritin in the pH range 4.0-7.4, using calorimetric and spectroscopic techniques. We observed a high protein thermal stability at all pHs with a denaturation temperature of 78 °C. At pH 5.0 we calculated a value of 36 kJ mol-1 for the unfolding Gibbs energy at 25 °C. We also analysed the thermodynamic and catalytic behaviour of S-pyridylethylated form, obtained by alkylating the single Cys18 residue, which is predicted to bind Zn2+. We show that this form possesses the same activity and structure of ageritin, but lower stability. In fact, the corresponding values of 52 °C and 14 kJ mol-1 were found. Conservation of activity is consistent with the location of alkylation site on the opposite site of the catalytic site cleft. Inasmuch as Cys18 is part of a structurally stabilizing zinc-binding site, disrupted by cysteine alkylation, our results point to an important role of metal ions in ageritin stability.
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Agaricales , Ribonucleasas , Ribonucleasas/química , Ribosomas/metabolismo , Agaricales/química , Genes Fúngicos , Desnaturalización Proteica , TermodinámicaRESUMEN
Ribosome-inactivating proteins (RIPs) are known as RNA N-glycosylases. They depurinate the major rRNA, damaging ribosomes and inhibiting protein synthesis. Here, new single-chain (type-1) RIPs named sodins were isolated from the seeds (five proteins), edible leaves (one protein) and roots (one protein) of Salsola soda L. Sodins are able to release Endo's fragment when incubated with rabbit and yeast ribosomes and inhibit protein synthesis in cell-free systems (IC50 = 4.83-79.31 pM). In addition, sodin 5, the major form isolated from seeds, as well as sodin eL and sodin R, isolated from edible leaves and roots, respectively, display polynucleotide:adenosine glycosylase activity and are cytotoxic towards the Hela and COLO 320 cell lines (IC50 = 0.41-1200 nM), inducing apoptosis. The further characterization of sodin 5 reveals that this enzyme shows a secondary structure similar to other type-1 RIPs and a higher melting temperature (Tm = 76.03 ± 0.30 °C) and is non-glycosylated, as other sodins are. Finally, we proved that sodin 5 possesses antifungal activity against Penicillium digitatum.
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Salsola , Secuencia de Aminoácidos , Animales , Células HeLa , Humanos , N-Glicosil Hidrolasas/química , Proteínas de Plantas/química , Conejos , Proteínas Inactivadoras de Ribosomas/metabolismo , Proteínas Inactivadoras de Ribosomas/farmacología , Proteínas Inactivadoras de Ribosomas Tipo 1 , Ribosomas/metabolismo , Salsola/metabolismoRESUMEN
'Asprinio' is a white dry wine characteristic for its acidity and aromatic flavour, known as emerging DOP wine in Southern Italy. Nevertheless, little information is available on the metabolomic profile of this wine. Thus, in this paper we evaluated the colourimetric parameters, 1H NMR profiles and free amino acids content of 'Asprinio' wines, bottled by two different wineries (hereafter 'Asprinio_A' and 'Asprinio_B') collected in 2019 and 2020, using 'Greco di Tufo' for comparison. The colourimetric parameters are similar for both 'Asprinio' wines and differ from 'Greco di Tufo' wines. On the other hand, both 1H NMR and free amino acid content profiles show different chemometric profiles among the three wines analysed, although the profiles are similar for both vintages. Moreover, the multivariate analyses carried out highlight differences between 'Asprinio_A' and 'Asprinio_B', which exbibit also different residual yeast and plant DNA. Overall, considering that the two-manufacturing wineries use 100% 'Asprinio' grape, the difference retrieved between the two 'Asprinio' wines could be explained by the different grapevine training systems: 'vite maritata' (training system inherited from Etruscans) for 'Asprinio_A' and 'guyot' for 'Asprinio_B'.
RESUMEN
Ribotoxin-like proteins (RL-Ps) represent a novel specific ribonuclease family found in edible mushrooms and are able to inhibit protein synthesis. Here, we report the characterization and cytotoxic effects of four novel RL-Ps, named eryngitins, isolated from fruiting bodies of the king oyster mushroom (Pleurotus eryngii). These proteins induced formation of α-fragment from rabbit ribosomes, characteristic of their enzymatic action. The two 15 kDa eryngitins (3 and 4) are considerably more thermostable than the 21 kDa ones (1 and 2), however their overall structural features, as determined by far-UV CD spectrometry, are similar. Complete in vitro digestibility by pepsin-trypsin, and lack of cytotoxicity towards human HUVEC cells suggest low toxicity of eryngitins, if ingested. However, eryngitins exhibit cytotoxic action against insect Sf9 cells, suggesting their possible use in biotechnological applications as bioinsecticides. This cytotoxicity was not enhanced in the presence of cytolytic protein complexes based on aegerolysin proteins from Pleurotus mushrooms.
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Agaricales , Antineoplásicos , Pleurotus , Agaricales/química , Animales , Antineoplásicos/farmacología , Humanos , Pleurotus/química , Conejos , Ribonucleasas/química , Ribonucleasas/metabolismo , Ribonucleasas/farmacologíaRESUMEN
rRNA N-glycosylases (EC 3.2.2.22) remove a specific adenine (A4324, rat 28S rRNA) in the sarcin ricin loop (SRL) involved into ribosome interaction with elongation factors, causing the inhibition of translation, for which they are known as plant 'ribosome inactivating proteins' (RIPs). However, protein synthesis inactivation could be the result of other enzymes, which often have rRNA as the target. In this scenario, Endo's assay is the most used method to detect the enzymes that are able to hydrolyze a phosphodiester bond or cleave a single N-glycosidic bond (rRNA N-glycosylases). Indeed, the detection of a diagnostic fragment from rRNA after enzymatic action, with or without acid aniline, allows one to discriminate between the N-glycosylases or hydrolases, which release the ß-fragment after acid aniline treatment or α-fragment without acid aniline treatment, respectively. This assay is of great importance in the mushroom kingdom, considering the presence of enzymes that are able to hydrolyze phosphodiester bonds (e.g., ribonucleases, ribotoxins and ribotoxin-like proteins) or to remove a specific adenine (rRNA N-glycosylases). Thus, here we used the ß-fragment experimentally detected by Endo's assay as a hallmark to revise the literature available on enzymes from mushrooms and other fungi, whose action consists of protein biosynthesis inhibition.
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Agaricales , Ricina , Adenina/metabolismo , Agaricales/metabolismo , Compuestos de Anilina , Animales , Proteínas de Plantas/metabolismo , Inhibidores de la Síntesis de la Proteína/farmacología , ARN Ribosómico/análisis , ARN Ribosómico/metabolismo , Ratas , Proteínas Inactivadoras de Ribosomas/metabolismo , Ribosomas/metabolismo , Ricina/metabolismoRESUMEN
Here, we report the characterization (purification, autoxidation rate, pseudoperoxidase activity) and amino acid sequence determination of S. scombrus (Atlantic mackerel) and S. colias (Tinker mackerel) mioglobins (Mbs), considering the increasing consumption of fresh and canned mackerel meat and Mb implication in meat storage (e.g.: browning and lipid oxidation). We found that Atlantic mackerel Mb has major autoxidation rate (0.204 ± 0.013 h-1) compared to Tinker mackerel Mb (0.140 ± 0.009 h-1), while the pseudoperoxidase activity is major for Tinker mackerel (Km: 87.71 ± 7.19 µM; kcat: 0.32 s-1) Mb with respect to Atlantic mackerel (Km: 96.08 ± 6.91 µM; kcat: 0.50 s-1). These functional differences are confirmed by primary structure determination, in which six amino acid substitutions are found, with the first N-terminal amino acid residue acetylated. Furthermore, we predicted by AphaFold 3D model both fish Mbs and used them to investigate the possible structural differences. In addition, phylogenetic analysis using Mb sequences from Scombridae family confirms that Atlantic and Tinker mackerels are two distinct species. Finally, an analytic qualitative RP-HPLC method to distinguish S. scombrus and S. colias specimens was developed considering the different retention times of the two mackerel apoMbs.
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Mioglobina , Perciformes , Animales , Carne , Perciformes/genética , Filogenia , Alimentos MarinosRESUMEN
Here, we propose Ageritin, the prototype of the ribotoxin-like protein family, as an adjuvant treatment to control the growth of NULU and ZAR, two primary human glioblastoma cell lines, which exhibit a pharmacoresistance phenotype. Ageritin is able to inhibit NULU and ZAR growth with an IC50 of 0.53 ± 0.29 µM and 0.42 ± 0.49 µM, respectively. In this study, Ageritin treatment highlighted a macroscopic genotoxic response through the formation of micronuclei, which represents the morphological manifestation of genomic chaos induced by this toxin. DNA damage was not associated with either the deregulation of DNA repair enzymes (i.e., ATM and DNA-PK), as demonstrated by quantitative PCR, or reactive oxygen species. Indeed, the pretreatment of the most responsive cell line ZAR with the ROS scavenger N-acetylcysteine (NAC) did not follow the reverse cytotoxic effect of Ageritin, suggesting that this protein is not involved in cellular oxidative stress. Vice versa, Ageritin pretreatment strongly enhanced the sensitivity to temozolomide (TMZ) and inhibited MGMT protein expression, restoring the sensitivity to temozolomide. Overall, Ageritin could be considered as a possible innovative glioblastoma treatment, directly damaging DNA and downregulating the MGMT DNA repair protein. Finally, we verified the proteolysis susceptibility of Ageritin using an in vitro digestion system, and considered the future perspective use of this toxin as a bioconjugate in biomedicine.
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Agaricales , Glioblastoma , Toxinas Biológicas , Antineoplásicos Alquilantes , Línea Celular Tumoral , Metilasas de Modificación del ADN , Resistencia a Antineoplásicos , Glioblastoma/tratamiento farmacológico , Humanos , Ribonucleasas , Temozolomida/farmacologíaRESUMEN
Cannabidiolic acid (CBDA) is the main precannabinoid in industrial hemp. It represents a common constituent of hemp seed oil, but mainly abundant in the aerial parts of the plant (including their processing waste). Thus, the optimization of fast and low-cost purification strategies is mandatory, as well as a deep investigation on its nutraceutical and cosmeceutical properties. To this purpose, CBDA content in hemp seed oil is evaluated, and its recovery from wasted leaves is favorably achieved. The cytotoxicity screening towards HaCaT cells, by means of MTT, SRB and LDH release assays, suggested it was not able to decrease cell viability or perturb cell integrity up to 10 µM concentration. Thus, the ability of CBDA to differentially modulate the release of proinflammatory cytokines and chemokines mediators has been evaluated, finding that CBDA decreased IFN-γ, CXCL8, CXCL10, CCL2, CCL4 and CCL5, mostly in a dose-dependent manner, with 10 µM tested concentration exerting the highest activity. These data, together with those from assessing antimicrobial activity against Gram(+) and Gram(-) bacteria and the antibiofilm formation, suggest that CBDA is able to counteract the inflammatory response, also preventing bacteria colonization.
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Cannabinoides , Cannabis , Cannabinoides/farmacología , Extractos VegetalesRESUMEN
Here, we report the current status of the bioactive peptides isolated and characterized from mushrooms during the last 20 years, considering 'peptide' a succession from to 2 to 100 amino acid residues. According to this accepted biochemical definition, we adopt ~10 kDa as the upper limit of molecular weight for a peptide. In light of this, a careful revision of data reported in the literature was carried out. The search revealed that in the works describing the characterization of bioactive peptides from mushrooms, not all the peptides have been correctly classified according to their molecular weight, considering that some fungal proteins (>10 kDa MW) have been improperly classified as 'peptides'. Moreover, the biological action of each of these peptides, the principles of their isolation as well as the source/mushroom species were summarized. Finally, this review highlighted that these peptides possess antihypertensive, antifungal, antibiotic and antimicrobial, anticancer, antiviral, antioxidant and ACE inhibitory properties.
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Agaricales/química , Proteínas Fúngicas/química , Peso MolecularRESUMEN
Cow, sheep and goat raw milk raised in Ailano and Valle Agricola territories ('Alto Casertano', Italy) were characterized (raw proteins, free and total amino acids content) to assess milk quality. Raw milk with the highest total protein content is sheep milk followed by goat and cow milk from both localities. Total amino acid content in cow, goat and sheep raw milk is 4.58, 4.81 and 6.62 g per 100 g, respectively, in which the most abundant amino acid is glutamic acid (~20.36 g per 100 g of proteins). Vice versa, the free amino acids content characteristic profiles are different for each species. In particular, the most abundant free amino acid in cow, sheep and goat raw milk is glutamic acid (9.07 mg per 100 g), tyrosine (4.72 mg per 100 g) and glycine (4.54 mg per 100 g), respectively. In addition, goat raw milk is a source of taurine (14.92 mg per 100 g), retrieved in low amount in cow (1.38 mg per 100 g) and sheep (2.10 mg per 100 g) raw milk. Overall, raw milk from 'Alto Casertano' show a high total protein content and are a good source of essential amino acids.