Antiviral activity of ribosome-inactivating proteins

Producción CientíficaRibosome-inactivating proteins (RIPs) are rRNA N-glycosylases from plants (EC 3.2.2.22) that inactivate ribosomes thus inhibiting protein synthesis. The antiviral properties of RIPs have been investigated for more than four decades. However, interest in these proteins is rising due to the emergence of infectious diseases caused by new viruses and the difficulty in treating viral infections. On the other hand, there is a growing need to control crop diseases without resorting to the use of phytosanitary products which are very harmful to the environment and in this respect, RIPs have been shown as a promising tool that can be used to obtain transgenic plants resistant to viruses. The way in which RIPs exert their antiviral effect continues to be the subject of intense research and several mechanisms of action have been proposed. The purpose of this review is to examine the research studies that deal with this matter, placing special emphasis on the most recent findings.Junta de Castilla y León - (Grant VA033G19

Spontaneous malignant glaucoma: Case report and review of the literature.

Malignant glaucoma usually occurs after anterior segment surgery (typically after glaucoma surgery). The aim of this article is to report a case of spontaneous malignant glaucoma (SpMG), which required phacovitrectomy for resolution and to review the cases of SpMG reported in modern literature. Only nine cases were identified. SpMG has no gender predilection and age at onset seems to be lower (mean age 47 years) than in secondary malignant glaucoma (SeMG). Nearly in half of the reported patients (4 out of 9) the condition had a bilateral presentation. The risk factors that have been identified for SeMG (nanophthalmos, shallow anterior chamber, iris plateau, zonular laxity) are underrepresented in SpMG.post-print418 K

Killing cancer cells by targeting the EGF receptor

Producción CientíficaIn this issue of Cancer Biology & Therapy, Bachran et al. analyze the effect of two chimeric toxins (SE and SA2E) consisting of the epidermal growth factor (EGF) and the plant protein toxin saporin towards an oral squamous cell carcinoma cell line (BHY) as well as on primary oral squamous cell carcinoma (OSCC) tumour cells

Elicitor-dependent expression of the ribosome-inactivating protein beetin is developmentally regulated

Producción CientíficaBE27 and BE29 are two forms of beetin, a virus-inducible type 1 ribosome-inactivating protein isolated from leaves of Beta vulgaris L. Western blot analysis revealed the presence of beetin forms in adult plants but not in germ or young plants, indicating that the expression of these proteins is developmentally regulated. While beetins are expressed only in adult plants, their transcripts are present through all stages of development. In addition, the treatment of B. vulgaris leaves with mediators of plant-acquired resistance such as salicylic acid and hydrogen peroxide promoted the expression of beetin by induction of its transcript, but only in adult plants. The plant expresses three mRNAs which differ only in their 3′ untranslated region. All these observations suggest a dual regulation of beetin expression, i.e. at the post-transcriptional and transcriptional levels. Additionally, total RNA isolated from leaves treated with hydrogen peroxide, which express high levels of active beetin, is not de-adenylated by endogenous beetin, nor in vitro by the addition of BE27, thus suggesting that sugar beet ribosomes are resistant to beetin.Junta de Castilla y Leon (grant VA0150A7

Primary sequence and 3D structure prediction of the plant toxin stenodactylin

Producción CientíficaStenodactylin is one of the most potent type 2 ribosome-inactivating proteins (RIPs); its high toxicity has been demonstrated in several models both in vitro and in vivo. Due to its peculiarities, stenodactylin could have several medical and biotechnological applications in neuroscience and cancer treatment. In this work, we report the complete amino acid sequence of stenodactylin and 3D structure prediction. The comparison between the primary sequence of stenodactylin and other RIPs allowed us to identify homologies/differences and the amino acids involved in RIP toxic activity. Stenodactylin RNA was isolated from plant caudex, reverse transcribed through PCR and the cDNA was amplificated and cloned into a plasmid vector and further analyzed by sequencing. Nucleotide sequence analysis showed that stenodactylin A and B chains contain 251 and 258 amino acids, respectively. The key amino acids of the active site described for ricin and most other RIPs are also conserved in the stenodactylin A chain. Stenodactylin amino acid sequence shows a high identity degree with volkensin (81.7% for A chain, 90.3% for B chain), whilst when compared with other type 2 RIPs the identity degree ranges from 27.7 to 33.0% for the A chain and from 42.1 to 47.7% for the B chain.Universidad de Bolonia y Pallotti Legacies for Cancer Research; Fundación CARISBO - (Project 2019.0539)Junta de Castilla y León - (Project VA033G19

Sequence, structure, and binding site analysis of Kirkiin in comparison with ricin and other type 2 RIPs

Producción CientíficaKirkiin is a new type 2 ribosome-inactivating protein (RIP) purified from the caudex of Adenia kirkii with a cytotoxicity compared to that of stenodactylin. The high toxicity of RIPs from Adenia genus plants makes them interesting tools for biotechnology and therapeutic applications, particularly in cancer therapy. The complete amino acid sequence and 3D structure prediction of kirkiin are here reported. Gene sequence analysis revealed that kirkiin is encoded by a 1572 bp open reading frame, corresponding to 524 amino acid residues, without introns. The amino acid sequence analysis showed a high degree of identity with other Adenia RIPs. The 3D structure of kirkiin preserves the overall folding of type 2 RIPs. The key amino acids of the active site, described for ricin and other RIPs, are also conserved in the kirkiin A chain. Sugar affinity studies and docking experiments revealed that both the 1α and 2γ sites of the kirkiin B chain exhibit binding activity toward lactose and D-galactose, being lower than ricin. The replacement of His246 in the kirkiin 2γ site instead of Tyr248 in ricin causes a different structure arrangement that could explain the lower sugar affinity of kirkiin with respect to ricin.Universidad de Bolonia y Pallotti Legacies for Cancer Research; Fundación CARISBO - (Project 2019.0539)Junta de Castilla y León - (Grant VA033G19

Structural and functional characterization of the cytotoxic protein ledodin, an atypical ribosome‐inactivating protein from shiitake mushroom (Lentinula edodes)

Producción CientíficaWe 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. However, 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.Junta de Castilla y León (Consejería de Sanidad - grants BIO39/VA39/14 and BIO/VA17/15)Junta de Castilla y León (Consejería de Educación - grant VA033G19)NUTRABEST PON I&C 2014–2020 Prog.n. F/200050/01-03/X4

A review of NAFO 3LMN roughhead grenadier (Macrourus berglax Lacepède, 1801) reproductive biology including the evaluation of maturity ogive estimates.

A collection of 4770 histological slides collected in NAFO divisions 3LMN in a depth range from 200 to 1500 m and covering a time period between 2001 and 2015, has been reviewed to obtain information on reproductive biology of Macrourus berglax. Especial attention has been paid in atresia because its potential impact on stock productivity. Spatial and temporal variability of size and age at maturity of female roughhead grenadier was analyzed in NAFO divisions 3LMN based on microscopic maturity staging and individual age readings. Spawning capable females are homogeneously distributed nearly year-round, but in scarce numbers what prevent to define a spawning season. This statement united to the high levels of atresia could indicate a reproductive migration. We observed a clear decrease in length at first maturity (L50) of females from 27.8 cm in the period 2005-2011 to 25.6 cm in the last four years. The age at first maturity, A50, varied between 13.1 and 15 years, and there is not an evident trend of change over the years.Postprint0,000

Altered zinc homeostasis in a primary cell culture model of the retinal pigment epithelium

The retinal pigment epithelium (RPE) is progressively degenerated during age-related macular degeneration (AMD), one of the leading causes of irreversible blindness, which clinical hallmark is the buildup of sub-RPE extracellular material. Clinical observations indicate that Zn dyshomeostasis can initiate detrimental intracellular events in the RPE. In this study, we used a primary human fetal RPE cell culture model producing sub-RPE deposits accumulation that recapitulates features of early AMD to study Zn homeostasis and metalloproteins changes. RPE cell derived samples were collected at 10, 21 and 59 days in culture and processed for RNA sequencing, elemental mass spectrometry and the abundance and cellular localization of specific proteins. RPE cells developed processes normal to RPE, including intercellular unions formation and expression of RPE proteins. Punctate deposition of apolipoprotein E, marker of sub-RPE material accumulation, was observed from 3 weeks with profusion after 2 months in culture. Zn cytoplasmic concentrations significantly decreased 0.2 times at 59 days, from 0.264 ± 0.119 ng·μg−1 at 10 days to 0.062 ± 0.043 ng·μg−1 at 59 days (p < 0.05). Conversely, increased levels of Cu (1.5-fold in cytoplasm, 5.0-fold in cell nuclei and membranes), Na (3.5-fold in cytoplasm, 14.0-fold in cell nuclei and membranes) and K (6.8-fold in cytoplasm) were detected after 59-days long culture. The Zn-regulating proteins metallothioneins showed significant changes in gene expression over time, with a potent down-regulation at RNA and protein level of the most abundant isoform in primary RPE cells, from 0.141 ± 0.016 ng·mL−1 at 10 days to 0.056 ± 0.023 ng·mL−1 at 59 days (0.4-fold change, p < 0.05). Zn influx and efflux transporters were also deregulated, along with an increase in oxidative stress and alterations in the expression of antioxidant enzymes, including superoxide dismutase, catalase and glutathione peroxidase. The RPE cell model producing early accumulation of extracellular deposits provided evidences on an altered Zn homeostasis, exacerbated by changes in cytosolic Zn-binding proteins and Zn transporters, along with variations in other metals and metalloproteins, suggesting a potential role of altered Zn homeostasis during AMD development

Skipped BSCL2 Transcript in Celia’s Encephalopathy (PELD): New Insights on Fatty Acids Involvement, Senescence and Adipogenesis

Objective PELD (Progressive Encephalopathy with or without Lipodystrophy or Celia’s Encephalopathy) is a fatal and rare neurodegenerative syndrome associated with the BSCL2 mutation c.985C>T, that results in an aberrant transcript without the exon 7 (Celia seipin). The aim of this study was to evaluate both the process of cellular senescence and the effect of unsaturated fatty acids on preadipocytes from a homozygous c.985C>T patient. Also, the role of aberrant seipin isoform on adipogenesis was studied in adipose-derived human mesenchymal stem cells. Material and methods Cellular senescence was evaluated using β-galactosidase staining of preadipocytes obtained from a homozygous c.985C>T patient. Moreover, these cells were cultured during 24 hours with Intralipid, a soybean oil-based commercial lipid emulsion. The expression of the different BSCL2 transcripts was measured by qPCR. Adipose-derived human mesenchymal stem cells were differentiated to a fat lineage using StemPRO adipogenesis kit, and the expression of BSCL2 transcripts and several adipogenesis-related genes was measured by qPCR. Results the treatment of preadipocytes with unsaturated fatty acids significantly reduced the expression of the BSCL2 transcript without exon 7 by 34 to 63%. On the other hand, at least in preadipocytes, this mutation does not disturb cellular senescence rate. Finally, during adipocyte differentiation of adipose-derived human mesenchymal stem cells, the expression of adipogenic genes (PPARG, LPIN1, and LPL) increased significantly over 14 days, and noteworthy is that the BSCL2 transcript without exon 7 was differentially expressed by 332 to 723% when compared to day 0, suggesting an underlying role in adipogenesis. Conclusions our results suggest that Celia seipin is probably playing an underestimated role in adipocyte maturation, but not in senescence, and its expression can be modified by exogenous factors as fatty acidsThis work was supported by the Instituto de Salud Carlos III and the European Regional Development Fund, FEDER (grants number PI10/02873 and PI13/00314, http://www.isciii.es/), ISCIII/PI13/00314/Cofinanciado FEDER and by the Consellería de Industria, Xunta de Galicia (grant number 10PXIB208013PR, http://www.xunta.es/). SRG was awarded a Research Fellowship, granted by the Asociación Española de Familiares y Afectados de Lipodistrofias (AELIP, http://www.aelip.org/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscriptS