Genome-Wide Analysis of the RNase T2 Family and Identification of Interacting Proteins of Four ClS-RNase Genes in ‘XiangShui’ Lemon

S-RNase plays vital roles in the process of self-incompatibility (SI) in Rutaceae plants. Data have shown that the rejection phenomenon during self-pollination is due to the degradation of pollen tube RNA by S-RNase. The cytoskeleton microfilaments of pollen tubes are destroyed, and other components cannot extend downwards from the stigma and, ultimately, cannot reach the ovary to complete fertilisation. In this study, four S-RNase gene sequences were identified from the ‘XiangShui’ lemon genome and ubiquitome. Sequence analysis revealed that the conserved RNase T2 domains within S-RNases in ‘XiangShui’ lemon are the same as those within other species. Expression pattern analysis revealed that S₃-RNase and S₄-RNase are specifically expressed in the pistils, and spatiotemporal expression analysis showed that the S₃-RNase expression levels in the stigmas, styles and ovaries were significantly higher after self-pollination than after cross-pollination. Subcellular localisation analysis showed that the S₁-RNase, S₂-RNase, S₃-RNase and S₄-RNase were found to be expressed in the nucleus according to laser confocal microscopy. In addition, yeast two-hybrid (Y2H) assays showed that S₃-RNase interacted with F-box, Bifunctional fucokinase/fucose pyrophosphorylase (FKGP), aspartic proteinase A1, RRP46, pectinesterase/pectinesterase inhibitor 51 (PME51), phospholipid:diacylglycerol acyltransferase 1 (PDAT1), gibberellin receptor GID1B, GDT1-like protein 4, putative invertase inhibitor, tRNA ligase, PAP15, PAE8, TIM14-2, PGIP1 and p24beta2. Moreover, S₃-RNase interacted with TOPP4. Therefore, S₃-RNase may play an important role in the SI of ‘XiangShui’ lemon.     

Genome-Wide Analysis of Genes Involved in the GA Signal Transduction Pathway in ‘duli’ Pear (Pyrus betulifolia Bunge)

Gibberellic acid (GA) is an important phytohormone that regulates every aspect of plant growth and development. While elements involved in GA signaling have been identified and, hence, their functions have been well studied in model plants, such as Arabidopsis and rice, very little is known in pear. We, therefore, analyzed the genes related to GA signaling from the recently sequenced genome of the wildtype ‘duli’ pear (Pyrus betulifolia Bunge), a widely used rootstock for grafting in pear cultivation in China due to its vigorous growth and resistance to abiotic and biotic stress. In total, 15 genes were identified, including five GA receptors PbGID1s (GA-INSENSTIVE DWARF 1), six GA negative regulators, PbDELLAs, and four GA positive regulators, PbSLYs. Exogenous application of GA could promote the expression of PbGID1s but inhibit that of PbDELLAs and PbSLYs in tissue culture ‘duli’ pear seedlings. The expression profiles of these genes in field-grown trees under normal growth conditions, as well as in tissue-cultured seedlings treated with auxin (IAA), GA, paclobutrazol (PAC), abscisic acid (ABA), and sodium chloride (NaCl), were also studied, providing further evidence of the involvement of these genes in GA signaling in ‘duli’ pear plants. The preliminary results obtained in this report lay a good foundation for future research into GA signaling pathways in pear. Importantly, the identification and preliminary functional verification of these genes could guide molecular breeding in order to obtain the highly desired dwarf pear rootstocks for high-density plantation to aid easy orchard management and high yielding of pear fruits.     

Oxidative Stress-Induced Sirtuin1 Downregulation Correlates to HIF-1α, GLUT-1, and VEGF-A Upregulation in Th1 Autoimmune Hashimoto’s Thyroiditis

In Hashimoto’s thyroiditis (HT), oxidative stress (OS) is driven by Th1 cytokines’ response interfering with the normal function of thyrocytes. OS results from an imbalance between an excessive production of reactive oxygen species (ROS) and a lowering of antioxidant production. Moreover, OS has been shown to inhibit Sirtuin 1 (SIRT1), which is able to prevent hypoxia-inducible factor (HIF)-1α stabilization. The aims of this study were to determine the involvement of NADPH-oxidases (NOX), SIRT1, and HIF-1α in HT pathophysiology as well as the status of antioxidant proteins such as peroxiredoxin 1 (PRDX1), catalase, and superoxide dismutase 1 (SOD1). The protein expressions of NOX2, NOX4, antioxidant enzymes, SIRT1, and HIF-1α, as well as glucose transporter-1 (GLUT-1) and vascular endothelial growth factor A (VEGF-A), were analyzed by Western blot in primary cultures of human thyrocytes that were or were not incubated with Th1 cytokines. The same proteins were also analyzed by immunohistochemistry in thyroid samples from control and HT patients. In human thyrocytes incubated with Th1 cytokines, NOX4 expression was increased whereas antioxidants, such as PRDX1, catalase, and SOD1, were reduced. Th1 cytokines also induced a significant decrease of SIRT1 protein expression associated with an upregulation of HIF-1α, GLUT-1, and VEGF-A proteins. With the exception of PRDX1 and SOD1, similar results were obtained in HT thyroids. OS due to an increase of ROS produced by NOX4 and a loss of antioxidant defenses (PRDX1, catalase, SOD1) correlates to a reduction of SIRT1 and an upregulation of HIF 1α, GLUT-1, and VEGF-A. Our study placed SIRT1 as a key regulator of OS and we, therefore, believe it could be considered as a potential therapeutic target in HT.     

Effect of Overexpression of γ-Tocopherol Methyltransferase on α-Tocopherol and Fatty Acid Accumulation and Tolerance to Salt Stress during Seed Germination in Brassica napus L.

Rapeseed (Brassica napus L.) is an important oil crop and a major source of tocopherols, also known as vitamin E, in human nutrition. Enhancing the quality and composition of fatty acids (FAs) and tocopherols in seeds has long been a target for rapeseed breeding. The gene γ-Tocopherol methyltransferase (γ-TMT) encodes an enzyme catalysing the conversion of γ-tocopherol to α-tocopherol, which has the highest biological activity. However, the genetic basis of γ-TMT in B. napus seeds remains unclear. In the present study, BnaC02.TMT.a, one paralogue of Brassica napus γ-TMT, was isolated from the B. napus cultivar “Zhongshuang11” by nested PCR, and two homozygous transgenic overexpression lines were further characterised. Our results demonstrated that the overexpression of BnaC02.TMT.a mediated an increase in the α- and total tocopherol content in transgenic B. napus seeds. Interestingly, the FA composition was also altered in the transgenic plants; a reduction in the levels of oleic acid and an increase in the levels of linoleic acid and linolenic acid were observed. Consistently, BnaC02.TMT.a promoted the expression of BnFAD2 and BnFAD3, which are involved in the biosynthesis of polyunsaturated fatty acids during seed development. In addition, BnaC02.TMT.a enhanced the tolerance to salt stress by scavenging reactive oxygen species (ROS) during seed germination in B. napus. Our results suggest that BnaC02.TMT.a could affect the tocopherol content and FA composition and play a positive role in regulating the rapeseed response to salt stress by modulating the ROS scavenging system. This study broadens our understanding of the function of the Bnγ-TMT gene and provides a novel strategy for genetic engineering in rapeseed breeding.     

Sequence Analysis and Functional Verification of the Effects of Three Key Structural Genes,PdTHC2’GT,PdCHS and PdCHI,on the Isosalipurposide Synthesis Pathway in Paeonia delavayi var. lutea

Isosalipurposide (ISP) is the most important yellow pigment in tree peony. In ISP biosynthesis, CHS catalyzes 1-molecule coumaroyl-CoA and 3-molecule malonyl-CoA to form 2′,4′,6′,4-tetrahyroxychalcone (THC), and THC generates a stable ISP in the vacuole under the action of chalcone2′-glucosyltransferases (THC2′GT). In tree peony, the details of the THC2’GT gene have not yet been reported. In this study, the candidate THC2’GT gene (PdTHC2’GT) in Paeonia delavayi var. lutea was screened. At the same time, we selected the upstream CHS gene (PdCHS) and the competitive CHI gene (PdCHI) to study the biosynthesis pathway of ISP. We successfully cloned three genes and sequenced them; subcellular localization showed that the three genes were located in the nucleus and cytoplasm. The overexpression of PdTHC2’GT in tobacco caused the accumulation of ISP in tobacco petals, which indicated that PdTHC2’GT was the key structural gene in the synthesis of ISP. After the overexpression of PdCHS and PdCHI in tobacco, the accumulation of anthocyanins in tobacco petals increased to different degrees, showing the role of PdCHS and PdCHI in anthocyanin accumulation. The analysis of NtCHS and NtCHI of transgenic tobacco lines by qRT-PCR showed that the THC2’GT gene could increase the expression of CHS. THC2’GT and CHI were found to be competitive; hence, the overexpression of THC2’GT could lead to a decrease in CHI expression. The CHS gene and CHI gene could increase the expression of each other. In conclusion, we verified the key structural gene PdTHC2’GT and studied the operation of the genes in its upstream and competitive pathway, providing a new perspective for the biosynthesis of ISP and new candidate genes for the directional breeding of tree peony.     

A Pilot Study of Changes in the Level of Catecholamines and the Activity of α-2-Macroglobulin in the Tear Fluid of Patients with Parkinson’s Disease and Parkinsonian Mice

Development of differential and early (preclinical) diagnostics of Parkinson’s disease (PD) is among the priorities in neuroscience. We searched for changes in the level of catecholamines and α-2-macroglobulin activity in the tear fluid (TF) in PD patients at an early clinical stage. It was shown that TF in patients is characterized by an increased level of noradrenaline mainly on the ipsilateral side of pronounced motor symptoms (72%, p = 0.049), a decreased level of adrenaline on both sides (ipsilateral—53%, p = 0.004; contralateral—42%, p = 0.02), and an increased α-2-macroglobulin activity on both sides (ipsilateral—53%, p = 0.03; contralateral—56%, p = 0.037) compared to controls. These changes are considered as potential biomarkers for differential diagnosis. Similar changes in the TF were found in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice when modeling clinical and preclinical stages of PD. These data show the adequacy of models to the pathogenesis of PD along the selected metabolic pathways, and also suggest that the found TF changes can be considered as potential biomarkers for preclinical diagnosis of PD. In Parkinsonian mice, the level of catecholamines also changes in the lacrimal glands, which makes it possible to consider them as one of the sources of catecholamines in the TF.     

The Effect of Oxidized Dopamine on the Structure and Molecular Chaperone Function of the Small Heat-Shock Proteins, αB-Crystallin and Hsp27

Oxidation of the neurotransmitter, dopamine (DA), is a pathological hallmark of Parkinson’s disease (PD). Oxidized DA forms adducts with proteins which can alter their functionality. αB-crystallin and Hsp27 are intracellular, small heat-shock molecular chaperone proteins (sHsps) which form the first line of defense to prevent protein aggregation under conditions of cellular stress. In vitro, the effects of oxidized DA on the structure and function of αB-crystallin and Hsp27 were investigated. Oxidized DA promoted the cross-linking of αB-crystallin and Hsp27 to form well-defined dimer, trimer, tetramer, etc., species, as monitored by SDS-PAGE. Lysine residues were involved in the cross-links. The secondary structure of the sHsps was not altered significantly upon cross-linking with oxidized DA but their oligomeric size was increased. When modified with a molar equivalent of DA, sHsp chaperone functionality was largely retained in preventing both amorphous and amyloid fibrillar aggregation, including fibril formation of mutant (A53T) α-synuclein, a protein whose aggregation is associated with autosomal PD. In the main, higher levels of sHsp modification with DA led to a reduction in chaperone effectiveness. In vivo, DA is sequestered into acidic vesicles to prevent its oxidation and, intracellularly, oxidation is minimized by mM levels of the antioxidant, glutathione. In vitro, acidic pH and glutathione prevented the formation of oxidized DA-induced cross-linking of the sHsps. Oxidized DA-modified αB-crystallin and Hsp27 were not cytotoxic. In a cellular context, retention of significant chaperone functionality by mildly oxidized DA-modified sHsps would contribute to proteostasis by preventing protein aggregation (particularly of α-synuclein) that is associated with PD.     

The Influence of Interaction between Cadmium with 17β-Estradiol, 2-Methoxyestradiol and 16α-Hydroxyestrone on Viability and p-Glycoprotein in Ovarian Cancer Cell Line

Occupational and environmental exposure to xenoestrogens, a subgroup of endocrine disruptors (EDCs), can affect the endocrine system and increase the risk of cancer, primarily the hormone-dependent kind. This type of cancer includes ovarian cancer, which is the leading cause of death from gynecological tumors. The aim of this study was to assess the role of 17β-estradiol and its metabolites: 2-MeOE2, 16α-OHE1 in exposure to the metalloestrogen cadmium. The effect of interactions of cadmium with estrogens on the viability of cells in malignant ovarian cancer cells SKOV-3 was investigated, both in simultaneous action and in the pre-incubation model. There are no known interactions between estrogens and cadmium in ovarian cancer cells. Due to the frequent occurrence of multidrug resistance (MDR) in ovarian cancer, the effects of estrogens and cadmium on MDR in SKOV-3, measured as P-glycoprotein (P-gp), were assessed. An interaction study showed that E2 had an antagonistic effect on cadmium-induced cell damage, while 2-MeOE2 showed less of a protective effect in combination with CdCl₂ than E2. There were two types of interaction: toxic synergism and beneficial antagonism. E2 and cadmium increased P-gp expression in SKOV-3 cells, while 2-MeOE2 decreased P-gp expression to a potentially beneficial effect on MDR prevention. The obtained results constitute an interesting starting point for further research in the field of interactions between estrogens and xenoestrogens in ovarian cancer.     

Worsening of the Toxic Effects of (±)Cis-4,4′-DMAR Following Its Co-Administration with (±)Trans-4,4′-DMAR: Neuro-Behavioural,Physiological, Immunohistochemical and Metabolic Studies in Mice

4,4’-Dimethylaminorex (4,4’-DMAR) is a new synthetic stimulant, and only a little information has been made available so far regarding its pharmaco-toxicological effects. The aim of this study was to investigate the effects of the systemic administration of both the single (±)cis (0.1–60 mg/kg) and (±)trans (30 and 60 mg/kg) stereoisomers and their co-administration (e.g., (±)cis at 1, 10 or 60 mg/kg + (±)trans at 30 mg/kg) in mice. Moreover, we investigated the effect of 4,4′-DMAR on the expression of markers of oxidative/nitrosative stress (8-OHdG, iNOS, NT and NOX2), apoptosis (Smac/DIABLO and NF-κB), and heat shock proteins (HSP27, HSP70, HSP90) in the cerebral cortex. Our study demonstrated that the (±)cis stereoisomer dose-dependently induced psychomotor agitation, sweating, salivation, hyperthermia, stimulated aggression, convulsions and death. Conversely, the (±)trans stereoisomer was ineffective whilst the stereoisomers’ co-administration resulted in a worsening of the toxic (±)cis stereoisomer effects. This trend of responses was confirmed by immunohistochemical analysis on the cortex. Finally, we investigated the potentially toxic effects of stereoisomer co-administration by studying urinary excretion. The excretion study showed that the (±)trans stereoisomer reduced the metabolism of the (±)cis form and increased its amount in the urine, possibly reflecting its increased plasma levels and, therefore, the worsening of its toxicity.     

Impact of Two Neuronal Sigma-1 Receptor Modulators,PRE084 and DMT,on Neurogenesis and Neuroinflammation in an Aβ1–42-Injected,Wild-Type Mouse Model of AD

Alzheimer’s disease (AD) is the most common form of dementia characterized by cognitive dysfunctions. Pharmacological interventions to slow the progression of AD are intensively studied. A potential direction targets neuronal sigma-1 receptors (S1Rs). S1R ligands are recognized as promising therapeutic agents that may alleviate symptom severity of AD, possibly via preventing amyloid-β-(Aβ-) induced neurotoxicity on the endoplasmic reticulum stress-associated pathways. Furthermore, S1Rs may also modulate adult neurogenesis, and the impairment of this process is reported to be associated with AD. We aimed to investigate the effects of two S1R agonists, dimethyltryptamine (DMT) and PRE084, in an Aβ-induced in vivo mouse model characterizing neurogenic and anti-neuroinflammatory symptoms of AD, and the modulatory effects of S1R agonists were analyzed by immunohistochemical methods and western blotting. DMT, binding moderately to S1R but with high affinity to 5-HT receptors, negatively influenced neurogenesis, possibly as a result of activating both receptors differently. In contrast, the highly selective S1R agonist PRE084 stimulated hippocampal cell proliferation and differentiation. Regarding neuroinflammation, DMT and PRE084 significantly reduced Aβ_1–42-induced astrogliosis, but neither had remarkable effects on microglial activation. In summary, the highly selective S1R agonist PRE084 may be a promising therapeutic agent for AD. Further studies are required to clarify the multifaceted neurogenic and anti-neuroinflammatory roles of these agonists.     

Variability in Behavioral Phenotypes after Forced Swimming-Induced Stress in Rats Is Associated with Expression of the Glucocorticoid Receptor,Nurr1, and IL-1β in the Hippocampus

Individual differences in coping with stress may determine either a vulnerable or resilient phenotype. Therefore, it is important to better understand the biology underlying the behavioral phenotype. We assessed whether individual behavioral phenotype to acute stress is related with the hippocampal expression of glucocorticoid receptor (GR), Nurr1, interleukin-1 beta (IL-1β) or brain-derived neurotrophic factor (BDNF). Wistar male rats were exposed to forced swimming for 15 min and sacrificed at different times. Behavioral response was analyzed, and it was compared with the gene and protein expression of GR, Nurr1, IL-1β and BDNF in the hippocampus for each time point. Behavioral phenotyping showed a group with high immobility (vulnerable) while another had low immobility (resilient). No significant differences were found in the Nurr1, IL-1β and BDNF mRNA levels between resilient and vulnerable rats at different recovery times except for Nr3c1 (gene for GR). However, exposure to stress caused significantly higher levels of GR, Nurr1 and IL-1β proteins of vulnerable compared to resilient rats. This variability of behavioral phenotypes is associated with a differential molecular response to stress that involves GR, Nurr1, and IL-1β as mediators in coping with stress. This contributes to identifying biomarkers of susceptibility to stress.     

Cardioprotective Effects of PPARβ/δ Activation against Ischemia/Reperfusion Injury in Rat Heart Are Associated with ALDH2 Upregulation,Amelioration of Oxidative Stress and Preservation of Mitochondrial Energy Production

Accumulating evidence support the cardioprotective properties of the nuclear receptor peroxisome proliferator activated receptor β/δ (PPARβ/δ); however, the underlying mechanisms are not yet fully elucidated. The aim of the study was to further investigate the mechanisms underlying PPARβ/δ-mediated cardioprotection in the setting of myocardial ischemia/reperfusion (I/R). For this purpose, rats were treated with PPARβ/δ agonist GW0742 and/or antagonist GSK0660 in vivo and hearts were subjected to ex vivo global ischemia followed by reperfusion. PPARβ/δ activation improved left ventricular developed pressure recovery, reduced infarct size (IS) and incidence of reperfusion-induced ventricular arrhythmias while it also up-regulated superoxide dismutase 2, catalase and uncoupling protein 3 resulting in attenuation of oxidative stress as evidenced by the reduction in 4-hydroxy-2-nonenal protein adducts and protein carbonyl formation. PPARβ/δ activation also increased both mRNA expression and enzymatic activity of aldehyde dehydrogenase 2 (ALDH2); inhibition of ALDH2 abrogated the IS limiting effect of PPARβ/δ activation. Furthermore, upregulation of PGC-1α and isocitrate dehydrogenase 2 mRNA expression, increased citrate synthase activity as well as mitochondrial ATP content indicated improvement in mitochondrial content and energy production. These data provide new mechanistic insight into the cardioprotective properties of PPARβ/δ in I/R pointing to ALDH2 as a direct downstream target and suggesting that PPARβ/δ activation alleviates myocardial I/R injury through coordinated stimulation of the antioxidant defense of the heart and preservation of mitochondrial function.     

Effect of Lipopolysaccharide-Induced Inflammatory Challenge on β-Glucuronidase Activity and the Concentration of Quercetin and Its Metabolites in the Choroid Plexus,Blood Plasma and Cerebrospinal Fluid

Quercetin-3-glucuronide (Q3GA), the main phase II metabolite of quercetin (Q) in human plasma, is considered to be a more stable form of Q for transport with the bloodstream to tissues, where it can be potentially deconjugated by β-glucuronidase (β-Gluc) to Q aglycone, which easily enters the brain. This study evaluates the effect of lipopolysaccharide (LPS)-induced acute inflammation on β-Gluc gene expression in the choroid plexus (ChP) and its activity in blood plasma, ChP and cerebrospinal fluid (CSF), and the concentration of Q and its phase II metabolites in blood plasma and CSF. Studies were performed on saline- and LPS-treated adult ewes (n = 40) receiving Q3GA intravenously (n = 16) and on primary rat ChP epithelial cells and human ChP epithelial papilloma cells. We observed that acute inflammation stimulated β-Gluc activity in the ChP and blood plasma, but not in ChP epithelial cells and CSF, and did not affect Q and its phase II metabolite concentrations in plasma and CSF, except Q3GA, for which the plasma concentration was higher 30 min after administration (p < 0.05) in LPS- compared to saline-treated ewes. The lack of Q3GA deconjugation in the ChP observed under physiological and acute inflammatory conditions, however, does not exclude its possible role in the course of neurodegenerative diseases.     

The Effect of the BCO2 Genotype on the Expression of Genes Related to Carotenoid,Retinol, and α-Tocopherol Metabolism in Rabbits Fed a Diet with Aztec Marigold Flower Extract

This study investigated the effect of the BCO2 genotype and the addition of Aztec marigold flower extract to rabbit diets on the expression of BCO1, BCO2, LRAT, and TTPA genes in the liver. The levels of lutein, zeaxanthin, β-carotene, retinol, and α-tocopherol in the liver and blood serum of rabbits, as well as plasma biochemical parameters and serum antioxidant enzyme activities were also determined. Sixty male Termond White growing rabbits were divided into three groups based on their genotype at codon 248 of the BCO2 gene (ins/ins, ins/del and del/del); each group was divided into two subgroups: one subgroup received a standard diet, and the other subgroup was fed a diet supplemented with 6 g/kg of marigold flower extract. The obtained results indicate that the BCO2 genotype may affect the expression levels of BCO1 and BCO2 genes in rabbits. Moreover, the addition of marigold extract to the diet of BCO2 del/del rabbits may increase the expression level of the BCO2 gene. Finally, an increase in the amount of lutein in the diet of rabbits with the BCO2 del/del genotype contributes to its increased accumulation in the liver and blood of animals without compromising their health status or liver function.