Metabolic fingerprinting analysis of oil palm reveals a set of differentially expressed metabolites in fatal yellowing symptomatic and non-symptomatic plants

Introduction

Oil palm (E. guineensis), the most consumed vegetable oil in the world, is affected by fatal yellowing (FY), a condition that can lead to the plant’s death. Although studies have been performed since the 1980s, including investigations of biotic and abiotic factors, FY’s cause remains unknown and efforts in researches are still necessary.

Objectives

This work aims to investigate the metabolic expression in plants affected by FY using an untargeted metabolomics approach.

Method

Metabolic fingerprinting analysis of oil palm leaves was performed using ultra high liquid chromatography–electrospray ionization–mass spectrometry (UHPLC–ESI–MS). Chemometric analysis, using principal component analysis (PCA) and partial least square discriminant analysis (PLS-DA), was applied to data analysis. Metabolites identification was performed by high resolution mass spectrometry (HRMS), MS/MS experiments and comparison with databases and literature.

Results

Metabolomics analysis based on MS detected more than 50 metabolites in oil palm leaf samples. PCA and PLS-DS analysis provided group segregation and classification of symptomatic and non-symptomatic FY samples, with a great external validation of the results. Nine differentially expressed metabolites were identified as glycerophosphorylcholine, arginine, asparagine, apigenin 6,8-di-C-hexose, tyramine, chlorophyllide, 1,2-dihexanoyl-sn-glycero-3-phosphoethanolamine, proline and malvidin 3-glucoside-5-(6″-malonylglucoside). Metabolic pathways and biological importance of those metabolites were assigned.

Conclusion

Nine metabolites were detected in a higher concentration in non-symptomatic FY plants. Seven are related to stress factors i.e. plant defense and nutrient absorption, which can be affected by the metabolic depression of these compounds. Two of those metabolites (glycerophosphorylcholine and 1,2-dihexanoyl-sn-glycero-3-phosphoethanolamine) are presented as potential biomarkers, since they have no known direct relation to plant stress.

     

Fast protocol for the production of Histoplasma capsulatum antigens for antibody detection in the immunodiagnosis of histoplasmosis

Background

Current methods for the production of Histoplasma capsulatum antigens are problematic in terms of standardization, specificity, stability, repeatability and reproducibility.

Partial Purification and Characterization of Red Chlorophyll Catabolite Reductase,a Stroma Protein Involved in Chlorophyll Breakdown

Red chlorophyll (Chl) catabolite (RCC) reductase, which catalyzes the reaction of an intermediary Chl catabolite (RCC) in the two-step cleavage reaction of pheophorbide (Pheide) a into primary fluorescent catabolites (pFCCs) during Chl breakdown, was characterized and partially purified. RCC reductase activity was present at all stages of barley leaf development and even in roots. The highest specific activity was found in senescent leaves, which were used to purify RCC reductase 1000-fold. Among the remaining three proteins, RCC reductase activity was most likely associated with a 55-kD protein. RCC reductase exhibited saturation kinetics for RCC, with an apparent Michaelis constant of 0.6 mM. The reaction depended on reduced ferredoxin and was sensitive to oxygen. Assays of purified RCC reductase with chemically synthesized RCC as a substrate yielded three different FCCs, two of which could be identified as the stereoisomeric pFCCs from canola (Brassica napus) (pFCC-1) and sweet pepper (Capsicum annuum) (pFCC-2), respectively. In the coupled reaction with Pheide a oxidase and RCC reductase, either pFCC-1 or pFCC-2 was produced, depending on the plant species employed as a source of RCC reductase. Data from 18 species suggest that the stereospecific action of RCC reductase is uniform within a plant family.     

Oxytocin stimulates in vitro angiogenesis via a Pyk-2/Src-dependent mechanism

We previously reported that the hypothalamic hormone oxytocin (OT), best known for its uterotonic activity, also stimulates migration and invasion in human umbilical vein endothelial cells (HUVECs), thus suggesting a possible role for the peptide in the regulation of angiogenesis. We identified the Gq coupling of OT receptors (OTRs) and phospholipase C (PLC) as the main effectors of OT's action in HUVECs. Moreover, the pro-migratory effect of OT required the OTR-induced activation of the phosphatidylinositol-3-kinase (PI-3-K)/AKT/endothelial nitric oxide synthase (eNOS) pathway. To better characterize the proposed pro-angiogenic effect of OT in HUVECs, we have now utilized a three-dimensional (3-D) in vitro angiogenesis assay, and demonstrated that OT stimulates the outgrowth of capillary-like structures from HUVEC spheroids to an extent comparable to that of vascular endothelial growth factor (VEGF). This OT effect was abolished by inhibitors of PLC, PI-3-K and Src kinase. It was also found that OT phosphorylates proline-rich tyrosine kinase-2 (Pyk-2) and Src kinase in a PLC- and calcium-dependent manner. Furthermore, knockdown of Pyk-2 expression by RNA interference markedly impaired Src phosphorylation, migration and endothelial cell sprouting induced by OT. In conclusion, by using a pharmacological and genetic approach, the OT pro-angiogenic action and the cascade of intracellular signals responsible for it were defined by showing for the first time that OT, by interacting with its Gq-coupled receptor, induces HUVEC capillary outgrowth via Pyk-2 phosphorylation, which activates Src which in turn activates the PI-3-K/AKT pathway.     

Topical Formulations Containing Pimenta pseudocaryophyllus Extract: In Vitro Antioxidant Activity and In Vivo Efficacy Against UV-B-Induced Oxidative Stress

Pimenta pseudocaryophyllus is a Brazilian native plant that presents high concentrations of flavonoids and other polyphenolic compounds. Herein, we evaluated: (1) the chemical properties of P. pseudocaryophyllus ethanolic extract (PPE), (2) the in vitro antioxidant activity (AA) of PPE and of two different topical formulations (F1 and F2) containing PPE, (3) physico-chemical and functional stability, (4) in vitro release of PPE, and (5) in vivo capacity of formulations to prevent UV-B irradiation-induced skin damage. Results show that the polyphenol and flavonoid contents in PPE were 199.33 and 28.32 mg/g, respectively, and HPLC results show the presence of eugenol, tannic acid, and rutin. Evaluation of the in vitro AA of PPE demonstrated a dose-dependent effect and an IC₅₀ of 4.75 μg/mL in 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 3.0 μg/mL in 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assays. The ferric-reducing antioxidant power (FRAP assay) was 0.046 μmol/L trolox equivalent/μg/mL of extract. Among the AA, only the capacity to scavenge DPPH radical of PPE was maintained in F1 and F2. In addition, both formulations satisfactorily released the extract. The evaluation of the functional stability of F1 and F2 did not demonstrate loss of activity by storage at room temperature and at 4°C/6 months. In irradiated mice, treatment with F1 and F2 added with PPE significantly increased the capacity to scavenge ABTS radical and the FRAP of skin compared to vehicle-treated mice. In conclusion, the present results suggest that formulations containing PPE may be a topical source of antioxidant compounds to decrease oxidative damages of the skin.     

Human umbilical endothelial cells (HUVECs) have a sex: characterisation of the phenotype of male and female cells

Background

Human umbilical endothelial cells (HUVECs) are widely used to study the endothelial physiology and pathology that might be involved in sex and gender differences detected at the cardiovascular level. This study evaluated whether HUVECs are sexually dimorphic in their morphological, proliferative and migratory properties and in the gene and protein expression of oestrogen and androgen receptors and nitric oxide synthase 3 (NOS3). Moreover, because autophagy is influenced by sex, its degree was analysed in male and female HUVECs (MHUVECs and FHUVECs).

Methods

Umbilical cords from healthy, normal weight male and female neonates born to healthy non-obese and non-smoking women were studied. HUVEC morphology was analysed by electron microscopy, and their function was investigated by proliferation, viability, wound healing and chemotaxis assays. Gene and protein expression for oestrogen and androgen receptors and for NOS3 were evaluated by real-time PCR and Western blotting, respectively, and the expression of the primary molecules involved in autophagy regulation [protein kinase B (Akt), mammalian target of rapamycin (mTOR), beclin-1 and microtubule-associated protein 1 light chain 3 (LC3)] were detected by Western blotting.

Results

Cell proliferation, migration NOS3 mRNA and protein expression were significantly higher in FHUVECs than in MHUVECs. Conversely, beclin-1 and the LC3-II/LC3-I ratio were higher in MHUVECs than in FHUVECs, indicating that male cells are more autophagic than female cells. The expression of oestrogen and androgen receptor genes and proteins, the protein expression of Akt and mTOR and cellular size and shape were not influenced by sex. Body weights of male and female neonates were not significantly different, but the weight of male babies positively correlated with the weight of the mother, suggesting that the mother’s weight may exert a different influence on male and female babies.

Conclusions

The results indicate that sex differences exist in prenatal life and are parameter-specific, suggesting that HUVECs of both sexes should be used as an in vitro model to increase the quality and the translational value of research. The sex differences observed in HUVECs could be relevant in explaining the diseases of adulthood because endothelial dysfunction has a crucial role in the pathogenesis of cardiovascular diseases, diabetes mellitus, neurodegeneration and immune disease.

     

PDGF-induced receptor phosphorylation and phosphoinositide hydrolysis are unaffected by protein kinase C activation in mouse Swiss 3T3 and human skin fibroblasts

Short (1-10 min) pretreatment of intact cells with activators of protein kinase C (e.g. phorbol-12 myristate, 13-acetate, PMA) affects the activity of a variety of surface receptors (for growth factors, hormones and neurotransmitters), with inhibition of transmembrane signal generation. In two types of fibroblasts we demonstrate that the PDGF receptor is unaffected by PMA. Exposure to PMA at concentrations up to 100 nM for 10 min failed to inhibit either one of the agonist-induced, receptor-coupled responses of PDGF: the autophosphorylation of receptor molecules at tyrosine residues, and the hydrolysis of membrane polyphosphoinositides. In contrast, the EGF receptor autophosphorylation (in A 431 cells) and the bombesin-induced phosphoinositide hydrolysis were readily inhibited by PMA. Feed-back inhibition of surface receptors by protein kinase C-mediated phosphorylation is therefore not general, and cannot be the only process responsible for the attenuation of receptor-mediated responses in eukaryotic cells.     

Rapid construction of large synthetic genes: total chemical synthesis of two different versions of the bovine prochymosin gene

We have tested several different synthesis designs and assembly methodologies to develop an improved gene synthesis strategy which enables significantly longer nucleotide sequences to be easily constructed. This strategy, based in part upon our ability to synthesize high-quality extended-length oligodeoxynucleotides (over 100-mer in length), together with the use of chemical 5'-phosphorylation, and simplified low-melting-temperature agarose gel purification methods, combines ease, speed and high overall efficiency. We show that it is now feasible to synthesize routinely even long genes (at least 1-2 kb). To demonstrate this capability we have chemically synthesized and assembled two different versions of the gene encoding the bovine enzyme prochymosin (prorennin). One gene is essentially the natural bovine prochymosin gene sequence. In the second gene the codons have been optimized with regard to the codon bias of highly expressed yeast genes. Each synthetic gene was in excess of 1100 bp, yet they were assembled from only 13 or 14 pairs of complementary oligodeoxynucleotides (oligos), the average lengths of which were 87 and 82 bp, respectively. The 'mutation' rate was low enough to assess that more than 75% of all such oligo pairs (160-170 total nt) were error-free.     

The testicular cycle of Gambusia affinis holbrooki (Teleostei: Poeciliidae)

Fifteen male mosquito fish ( Gambusia affinis holbrooki ) were collected in 1989 on the 15th of each month to perform a quantitative histologic study of the annual testicular cycle including a calculation of the gonadosomatic index, testicular volume, and the total volume per testis occupied by each germ cell type. The cycle comprises two periods: spermatogenesis and quiescence. The spermatogenic period begins in April with the development of primary spermatogonia into secondary spermatogonia, spermatocytes and round spermatids. In May, the first spermatogenic wave is completed and the testicular volume begins to increase up to June when the maximum testicular volume and gonadosomatic index are reached. Germ cell proliferation with successive spermatogenetic waves continues until August. In September germ cell proliferation ceases and neither secondary spermatogonia nor spermatocytes are observed. However, spermiogenesis continues until October. In November, spermiogenesis has stopped and the testis enters the quiescent period up to April. During this period only primary spermatogonia and spermatozoa are present in the testis. In addition, a few spermatids whose spermiogenesis was arrested in November are observed. Testicular release of spermatozoa is continuous during the entire spermatogenesis period. The spermatozoa formed at the end of this period (September-October) remain in the testis during the quiescent period and are released at the beginning of the next spermatogenesis period in April. Developed Leydig cells appear all year long in the testicular interstitium, mainly around both efferent ducts and the testicular tubule sections showing S₄ spermatids.