Proteomic analysis of apricot fruit during ripening

Ripening of climacteric fruits involves a complex network of biochemical and metabolic changes that make them palatable and rich in nutritional and health-beneficial compounds. Since fruit maturation has a profound impact on human nutrition, it has been recently the object of increasing research activity by holistic approaches, especially on model species. Here we report on the original proteomic characterization of ripening in apricot, a widely cultivated species of temperate zones appreciated for its taste and aromas, whose cultivation is yet hampered by specific limitations. Fruits of Prunus armeniaca cv. Vesuviana were harvested at three ripening stages and proteins extracted and resolved by 1D and 2D electrophoresis. Whole lanes from 1D gels were subjected to shot-gun analysis that identified 245 gene products, showing preliminary qualitative differences between maturation stages. In parallel, differential analysis of 2D proteomic maps highlighted 106 spots as differentially represented among variably ripen fruits. Most of these were further identified by means of MALDI-TOF-PMF and nanoLC–ESI–LIT–MS/MS as enzymes involved in main biochemical processes influencing metabolic/structural changes occurring during maturation, i.e. organic acids, carbohydrates and energy metabolism, ethylene biosynthesis, cell wall restructuring and stress response, or as protein species linkable to peculiar fruit organoleptic characteristics. In addition to originally present preliminary information on the main biochemical changes that characterize apricot ripening, this study also provides indications for future marker-assisted selection breeding programs aimed to ameliorate fruit quality

Article influence of the casein composite genotype on milk quality and coagulation properties in the endangered agerolese cattle breed

The aim of this study was the characterization of CSN1S1, CSN2 and CSN3 genetic variability in Agerolese cattle, and the investigation of the effect of casein composite genotypes (CSN1S1, CSN2 and CSN3) on quality and coagulation traits of the corresponding milk. To these purposes, blood and milk from 84 cows were sampled and analysed. Allele frequencies at CSN2 and CSN3 revealed no Hardy–Weinberg equilibrium in the population with a prevalence of allele A2 for CSN2 and allele B for CSN3. BBA1A2AB and BBA2A2AB composite genotypes were the most common in the population. BBA1A2AB showed a higher total solids and fat content (12.70 ± 0.16 and 3.93 ± 0.10, respectively), while BBA2A2BB showed the best coagulation properties (RCT 12.62 ± 0.81; k20 5.84 ± 0.37; a30 23.72 ± 1.10). Interestingly, the A2 allele of CSN2 was very widespread in the population; thus, it will be intriguing to verify if A2A2 Agerolese cattle milk and the derived cheese may have better nutraceutical characteristics

Identification of RNA-binding proteins that partner with Lin28a to regulate Dnmt3a expression

Lin28 is an evolutionary conserved RNA-binding protein that plays important roles during embryonic development and tumorigenesis. It regulates gene expression through two different post-transcriptional mechanisms. The first one is based on the regulation of miRNA biogenesis, in particular that of the let-7 family, whose expression is suppressed by Lin28. Thus, loss of Lin28 leads to the upregulation of mRNAs that are targets of let-7 species. The second mechanism is based on the direct interaction of Lin28 with a large number of mRNAs, which results in the regulation of their translation. This second mechanism remains poorly understood. To address this issue, we purified high molecular weight complexes containing Lin28a in mouse embryonic stem cells (ESCs). Numerous proteins, co-purified with Lin28a, were identified by proteomic procedures and tested for their possible role in Lin28a-dependent regulation of the mRNA encoding DNA methyltransferase 3a (Dnmt3a). The results show that Lin28a activity is dependent on many proteins, including three helicases and four RNA-binding proteins. The suppression of four of these proteins, namely Ddx3x, Hnrnph1, Hnrnpu or Syncrip, interferes with the binding of Lin28a to the Dnmt3a mRNA, thus suggesting that they are part of an oligomeric ribonucleoprotein complex that is necessary for Lin28a activity

Salt stress in olive tree shapes resident endophytic microbiota

Olea europaea L. is a glycophyte representing one of the most important plants in the Mediterranean area, both from an economic and agricultural point of view. Its adaptability to different environmental conditions enables its cultivation in numerous agricultural scenarios, even on marginal areas, characterized by soils unsuitable for other crops. Salt stress represents one current major threats to crop production, including olive tree. In order to overcome this constraint, several cultivars have been evaluated over the years using biochemical and physiological methods to select the most suitable ones for cultivation in harsh environments. Thus the development of novel methodologies have provided useful tools for evaluating the adaptive capacity of cultivars, among which the evaluation of the plant-microbiota ratio, which is important for the maintenance of plant homeostasis. In the present study, four olive tree cultivars (two traditional and two for intensive cultivation) were subjected to saline stress using two concentrations of salt, 100 mM and 200 mM. The effects of stress on diverse cultivars were assessed by using biochemical analyses (i.e., proline, carotenoid and chlorophyll content), showing a cultivar-dependent response. Additionally, the olive tree response to stress was correlated with the leaf endophytic bacterial community. Results of the metabarcoding analyses showed a significant shift in the resident microbiome for plants subjected to moderate salt stress, which did not occur under extreme salt-stress conditions. In the whole, these results showed that the integration of stress markers and endophytic community represents a suitable approach to evaluate the adaptation of cultivars to environmental stresses

Protoporphyrin IX enhancement by 5-aminolaevulinic acid peptide derivatives and the effect of RNA silencing on intracellular metabolism

Intracellular generation of the photosensitiser, protoporphyrin IX, from a series of dipeptide derivatives of the haem precursor, 5-aminolaevulinic acid (ALA), was investigated in transformed PAM212 murine keratinocytes, together with studies of their intracellular metabolism. Porphyrin production was substantially increased compared with equimolar ALA using N-acetyl terminated phenylalanyl, leucinyl and methionyl ALA methyl ester derivatives in the following order: Ac-L-phenylalanyl-ALA-Me, Ac-L-methionyl-ALA-Me and Ac-L-leucinyl-ALA-Me. The enhanced porphyrin production was in good correlation with improved photocytotoxicity, with no intrinsic dark toxicity apparent. However, phenylalanyl derivatives without the acetyl/acyl group at the N terminus induced significantly less porphyrin, and the replacement of the acetyl group by a benzyloxycarbonyl group resulted in no porphyrin production. Porphyrin production was reduced in the presence of class-specific protease inhibitors, namely serine protease inhibitors. Using siRNA knockdown of acylpeptide hydrolase (ACPH) protein expression, we showed the involvement of ACPH, a member of the prolyl oligopeptidase family of serine peptidases, in the hydrolytic cleavage of ALA from the peptide derivatives. In conclusion, ALA peptide derivatives are capable of delivering ALA efficiently to cells and enhancing porphyrin synthesis and photocytotoxicity; however, the N-terminus state, whether free or substituted, plays an important role in determining the biological efficacy of ALA peptide derivatives

SH3BGRL3 binds to myosin 1c in a calcium dependent manner and modulates migration in the MDA-MB-231 cell line

Background: The human SH3 domain Binding Glutamic acid Rich Like 3 (SH3BGRL3) gene is highly conserved in phylogeny and widely expressed in human tissues. However, its function is largely undetermined. The protein was found to be overexpressed in several tumors, and recent work suggested a possible relationship with EGFR family members. We aimed at further highlighting on these issues and investigated SH3BGRL3 molecular interactions and its role in cellular migration ability. Results: We first engineered the ErbB2-overexpressing SKBR3 cells to express exogenous SH3BGRL3, as well as wild type Myo1c or different deletion mutants. Confocal microscopy analysis indicated that SH3BGRL3 co-localized with Myo1c and ErbB2 at plasma membranes. However, co-immunoprecipitation assays and mass spectrometry demonstrated that SH3BGRL3 did not directly bind ErbB2, but specifically recognized Myo1c, on its IQ-bearing neck region. Importantly, the interaction with Myo1c was Ca2+-dependent. A role for SH3BGRL3 in cell migration was also assessed, as RNA interference of SH3BGRL3 in MDA-MB-231 cells, used as a classical migration model, remarkably impaired the migration ability of these cells. On the other side, its over-expression increased cell motility. Conclusion: The results of this study provide insights for the formulation of novel hypotheses on the putative role of SH3BGRL3 protein in the regulation of myosin-cytoskeleton dialog and in cell migration. It could be envisaged the SH3BGRL3-Myo1c interaction as a regulation mechanism for cytoskeleton dynamics. It is well known that, at low Ca2+ concentrations, the IQ domains of Myo1c are bound by calmodulin. Here we found that binding of Myo1c to SH3BGRL3 requires instead the presence of Ca2+. Thus, it could be hypothesized that Myo1c conformation may be modulated by Ca2+-driven mechanisms that involve alternative binding by calmodulin or SH3BGRL3, for the regulation of cytoskeletal activity

Ejection of damaged mitochondria and their removal by macrophages ensure efficient thermogenesis in brown adipose tissue

Recent findings have demonstrated that mitochondria can be transferred between cells to control metabolic homeostasis. Although the mitochondria of brown adipocytes comprise a large component of the cell volume and undergo reorganization to sustain thermogenesis, it remains unclear whether an intercellular mitochondrial transfer occurs in brown adipose tissue (BAT) and regulates adaptive thermogenesis. Herein, we demonstrated that thermogenically stressed brown adipocytes release extracellular vesicles (EVs) that contain oxidatively damaged mitochondrial parts to avoid failure of the thermogenic program. When re-uptaken by parental brown adipocytes, mitochondria-derived EVs reduced peroxisome proliferator-activated receptor-γ signaling and the levels of mitochondrial proteins, including UCP1. Their removal via the phagocytic activity of BAT-resident macrophages is instrumental in preserving BAT physiology. Depletion of macrophages in vivo causes the abnormal accumulation of extracellular mitochondrial vesicles in BAT, impairing the thermogenic response to cold exposure. These findings reveal a homeostatic role of tissue-resident macrophages in the mitochondrial quality control of BAT

Glomerular Autoimmune Multicomponents of Human Lupus Nephritis In Vivo (2): Planted Antigens.

Glomerular planted antigens (histones,DNA,andC1q) arepotential targets of autoimmunity in lupus nephritis (LN). However, the characterization of these antigens in human glomeruli in vivo remains inconsistent. We eluted glomerular autoantibodies recognizing planted antigens from laser-microdissected renal biopsy samples of 20 patientswith LN. Prevalent antibody isotypes were defined, levelswere determined, and glomerular colocalization was investigated. Renal and circulating antibodieswerematched, and serum levelswere compared in 104 patients with LN, 84 patients with SLE without LN, and 50 patients with rheumatoid arthritis (RA). Autoantibodies against podocyte antigens (antia-enolase/antiannexin AI) were also investigated. IgG2 autoantibodies against DNA, histones (H2A, H3, and H4), and C1q were detected in 50%, 55%, and 70% of biopsy samples, respectively. Anti-DNA IgG3 was the unique non-IgG2 anti-DNA deposit, and anti-C1q IgG4 was mainly detected in subepithelial membranous deposits. Anti-H3, anti-DNA, and anti-C1q IgG2 autoantibodies were also prevalent in LN serum, which also contained IgG3 against the antigen panel and anti-C1q IgG4. Serum and glomerular levels of autoantibodies were not strictly associated. High serum levels of all autoantibodies detected, including anti a-enolase and antiannexin AI, identified LN versus SLE and RA. Anti-H3 and antia-enolase IgG2 levels had the most remarkable increase in LN serum and represented a discriminating feature of LN in principal component analysis. The highest levels of these two autoantibodies were also associated with proteinuria.3.5 g/24 hours and creatinine>1.2 mg/dl. Our findings suggest that timely autoantibody characterization might allow outcome prediction and targeted therapies for patients with nephritis

Conserved Odorant-Binding Proteins from Aphids and Eavesdropping Predators

Background: The sesquiterpene (E)-ß-farnesene is the main component of the alarm pheromone system of various aphid species studied to date, including the English grain aphid, Sitobion avenae. Aphid natural enemies, such as the marmalade hoverfly Episyrphus balteatus and the multicolored Asian lady beetle Harmonia axyridis, eavesdrop on aphid chemical communication and utilize (E)-ß-farnesene as a kairomone to localize their immediate or offspring preys. These aphidpredator systems are important models to study how the olfactory systems of distant insect taxa process the same chemical signal. We postulated that odorant-binding proteins (OBPs), which are highly expressed in insect olfactory tissues and involved in the first step of odorant reception, have conserved regions involved in binding (E)-ß-farnesene. Methodology: We cloned OBP genes from the English grain aphid and two major predators of this aphid species. We then expressed these proteins and compare their binding affinities to the alarm pheromone/kairomone. By using a fluorescence reporter, we tested binding of (E)-ß-farnesene and other electrophysiologically and behaviorally active compounds, including a green leaf volatile attractant. Conclusion: We found that OBPs from disparate taxa of aphids and their predators are highly conserved proteins, with apparently no orthologue genes in other insect species. Properly folded, recombinant proteins from the English grain aphid, SaveOBP3, and the marmalade hoverfly, EbalOBP3, specifically bind (E)-ß-farnesene with apparent high affinity. For the firs