Molecular Analysis Uncovers the Mechanism of Fertility Restoration in Temperature-Sensitive Polima Cytoplasmic Male-Sterile Brassica napus.

Temperature-sensitive male sterility is a heritable agronomic trait affected by genotype-environment interactions. In rapeseed (Brassica napus), Polima (pol) temperature-sensitive cytoplasmic male sterility (TCMS) is commonly used for two-line breeding, as the fertility of pol TCMS lines can be partially restored at certain temperatures. However, little is known about the underlying molecular mechanism that controls fertility restoration. Therefore, we aimed to investigate the fertility conversion mechanism of the pol TCMS line at two different ambient temperatures (16 °C and 25 °C). Our results showed that the anthers developed and produced vigorous pollen at 16 °C but not at 25 °C. In addition, we identified a novel co-transcript of orf224-atp6 in the mitochondria that might lead to fertility conversion of the pol TCMS line. RNA-seq analysis showed that 1637 genes were significantly differentially expressed in the fertile flowers of 596-L when compared to the sterile flower of 1318 and 596-H. Detailed analysis revealed that differentially expressed genes were involved in temperature response, ROS accumulation, anther development, and mitochondrial function. Single-molecule long-read isoform sequencing combined with RNA sequencing revealed numerous genes produce alternative splicing transcripts at high temperatures. Here, we also found that alternative oxidase, type II NAD(P)H dehydrogenases, and transcription factor Hsfs might play a crucial role in male fertility under the low-temperature condition. RNA sequencing and bulked segregant analysis coupled with whole-genome sequencing identified the candidate genes involved in the post-transcriptional modification of orf224. Overall, our study described a putative mechanism of fertility restoration in a pol TCMS line controlled by ambient temperature that might help utilise TCMS in the two-line breeding of Brassica crops.

Antioxidant Artemisia princeps Extract Enhances the Expression of Filaggrin and Loricrin via the AHR/OVOL1 Pathway.

The Japanese mugwort, Artemisia princeps (yomogi in Japanese), has anti-inflammatory and antioxidant effects. Skin care products containing Artemisia princeps extract (APE) are known to improve dry skin symptoms in atopic dermatitis. Atopic dry skin is associated with a marked reduction of skin barrier proteins, such as filaggrin (FLG) and loricrin (LOR). Recently, aryl hydrocarbon receptor (AHR), and its downstream transcription factor OVO-like 1 (OVOL1), have been shown to regulate the gene expression of FLG and LOR. The focus of this paper is to evaluate the effects of APE on the AHR/OVOL1/FLG or LOR pathway since they have remained unknown to this point. We first demonstrated that non-cytotoxic concentrations of APE significantly upregulated antioxidant enzymes, NAD(P)H dehydrogenase quinone 1 and heme oxygenase 1, in human keratinocytes. Even at these low concentrations, APE induced nuclear translocation of AHR and significantly upregulated CYP1A1 (a specific target gene for AHR activation), FLG, and LOR expression. AHR knockdown downregulated OVOL1 expression. The APE-induced upregulation of FLG and LOR was canceled in keratinocytes with AHR or OVOL1 knockdown. In conclusion, antioxidant APE is a potent phytoextract that upregulates FLG and LOR expression in an AHR/OVOL1-dependent manner and this may underpin the barrier-repairing effects of APE in treating atopic dry skin.

Elevated exhaled nitric oxide in allergen-provoked asthma is associated with airway epithelial iNOS.

BACKGROUND: Fractional exhaled nitric oxide is elevated in allergen-provoked asthma. The cellular and molecular source of the elevated fractional exhaled nitric oxide is, however, uncertain. OBJECTIVE: To investigate whether fractional exhaled nitric oxide is associated with increased airway epithelial inducible nitric oxide synthase (iNOS) in allergen-provoked asthma. METHODS: Fractional exhaled nitric oxide was measured in healthy controls (n = 14) and allergic asthmatics (n = 12), before and after bronchial provocation to birch pollen out of season. Bronchoscopy was performed before and 24 hours after allergen provocation. Bronchial biopsies and brush biopsies were processed for nitric oxide synthase activity staining with nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d), iNOS immunostaining, or gene expression analysis of iNOS by real-time PCR. NADPH-d and iNOS staining were quantified using automated morphometric analysis. RESULTS: Fractional exhaled nitric oxide and expression of iNOS mRNA were significantly higher in un-provoked asthmatics, compared to healthy controls. Allergic asthmatics exhibited a significant elevation of fractional exhaled nitric oxide after allergen provocation, as well as an accumulation of airway eosinophils. Moreover, nitric oxide synthase activity and expression of iNOS was significantly increased in the bronchial epithelium of asthmatics following allergen provocation. Fractional exhaled nitric oxide correlated with eosinophils and iNOS expression. CONCLUSION: Higher fractional exhaled nitric oxide concentration among asthmatics is associated with elevated iNOS mRNA in the bronchial epithelium. Furthermore, our data demonstrates for the first time increased expression and activity of iNOS in the bronchial epithelium after allergen provocation, and thus provide a mechanistic explanation for elevated fractional exhaled nitric oxide in allergen-provoked asthma.

A redox-mediated modulation of stem bolting in transgenic Nicotiana sylvestris differentially expressing the external mitochondrial NADPH dehydrogenase.

Cytosolic NADPH can be directly oxidized by a calcium-dependent NADPH dehydrogenase, NDB1, present in the plant mitochondrial electron transport chain. However, little is known regarding the impact of modified cytosolic NADPH reduction levels on growth and metabolism. Nicotiana sylvestris plants overexpressing potato (Solanum tuberosum) NDB1 displayed early bolting, whereas sense suppression of the same gene led to delayed bolting, with consequential changes in flowering time. The phenotype was dependent on light irradiance but not linked to any change in biomass accumulation. Whereas the leaf NADPH/NADP(+) ratio was unaffected, the stem NADPH/NADP(+) ratio was altered following the genetic modification and strongly correlated with the bolting phenotype. Metabolic profiling of the stem showed that the NADP(H) change affected relatively few, albeit central, metabolites, including 2-oxoglutarate, glutamate, ascorbate, sugars, and hexose-phosphates. Consistent with the phenotype, the modified NDB1 level also affected the expression of putative floral meristem identity genes of the SQUAMOSA and LEAFY types. Further evidence for involvement of the NADPH redox in stem development was seen in the distinct decrease in the stem apex NADPH/NADP(+) ratio during bolting. Additionally, the potato NDB1 protein was specifically detected in mitochondria, and a survey of its abundance in major organs revealed that the highest levels are found in green stems. These results thus strongly suggest that NDB1 in the mitochondrial electron transport chain can, by modifying cell redox levels, specifically affect developmental processes.

Polymeric black tea polyphenols induce phase II enzymes via Nrf2 in mouse liver and lungs.

Among tea polyphenols, the anti-initiating properties of polymeric black tea polyphenols (PBPs), the most abundant polyphenols in black tea, are poorly elucidated. Hence, this study was undertaken to investigate the effects of PBP extract on the induction of phase II enzymes. PBP extract induced transcriptional up-regulation of phase II enzymes in liver and lungs by increasing Nrf2-mediated antioxidant-responsive element (ARE) binding. PBP extract did not alter Nrf2 or Keap1 at the transcriptional level but may have increased their levels by posttranslational modifications such as phosphorylation and decreased ubiquitination. PKC and PI3-kinase-mediated phosphorylation of Nrf2 seems to be critical for the release of Nrf2 from Keap1 and its subsequent nuclear translocation. mafK was found to be the heterodimeric partner of Nrf2 for binding to ARE sequences in liver upon PBP extract pretreatment. Differences in phosphorylation, activation of cellular kinases, and speculated heterodimeric binding partners of Nrf2 by PBP extract in hepatic and pulmonary tissues suggested the possibility of tissue-specific differences in the activation of Nrf2. Thus, we conclude that the pathway of PBP extract-induced ARE activity involves the activation of Nrf2 through phosphorylation by PKC and PI3-kinases in hepatic cells, which is critical for the increased stability of Nrf2 upon release from Keap1 and nuclear translocation, respectively.

Induction of cancer chemopreventive enzymes by coffee is mediated by transcription factor Nrf2. Evidence that the coffee-specific diterpenes cafestol and kahweol confer protection against acrolein.

Mice fed diets containing 3% or 6% coffee for 5 days had increased levels of mRNA for NAD(P)H:quinone oxidoreductase 1 (NQO1) and glutathione S-transferase class Alpha 1 (GSTA1) of between 4- and 20-fold in the liver and small intestine. Mice fed 6% coffee also had increased amounts of mRNA for UDP-glucuronosyl transferase 1A6 (UGT1A6) and the glutamate cysteine ligase catalytic (GCLC) subunit of between 3- and 10-fold in the small intestine. Up-regulation of these mRNAs was significantly greater in mice possessing Nrf2 (NF-E2 p45 subunit-related factor 2) than those lacking the transcription factor. Basal levels of mRNAs for NQO1, GSTA1, UGT1A6 and GCLC were lower in tissues from nrf2(-/-) mice than from nrf2(+/+) mice, but modest induction occurred in the mutant animals. Treatment of mouse embryonic fibroblasts (MEFs) from nrf2(+/+) mice with either coffee or the coffee-specific diterpenes cafestol and kahweol (C+K) increased NQO1 mRNA up to 9-fold. MEFs from nrf2(-/-) mice expressed less NQO1 mRNA than did wild-type MEFs, but NQO1 was induced modestly by coffee or C+K in the mutant fibroblasts. Transfection of MEFs with nqo1-luciferase reporter constructs showed that induction by C+K was mediated primarily by Nrf2 and required the presence of an antioxidant response element in the 5'-upstream region of the gene. Luciferase reporter activity did not increase following treatment of MEFs with 100 mumol/l furan, suggesting that this ring structure within C+K is insufficient for gene induction. Priming of nrf2(+/+) MEFs, but not nrf2(-/-) MEFs, with C+K conferred 2-fold resistance towards acrolein.

Induction of drug-metabolizing enzymes by garlic and allyl sulfide compounds via activation of constitutive androstane receptor and nuclear factor E2-related factor 2.

Garlic oil (GO) contains several linear sulfur compounds, including diallyl sulfide (DAS), diallyl disulfide (DADS), and diallyl trisulfide (DATS), that induce drug-metabolizing enzymes such as CYP2B and NAD(P)H quinone oxidoreductase 1 (NQO1). CYP2B and NQO1 are primarily regulated by constitutive androstane receptor (CAR) and nuclear factor E2-related factor 2 (Nrf2) transcription factors, respectively. The purpose of this study was to determine whether GO and its specific constituents induce these two enzymes via CAR and Nrf2 activation. Female Wistar-Kyoto (WKY) rats express little CAR protein and exhibit less induction of CYP2B1/2 than males. GO, DAS, and DADS, but not DATS, induced CYP2B1/2 mRNA levels to a greater extent in WKY males than in females, suggesting CAR activation. Conversely, DAS induced NQO1 levels equally in WKY males and females, indicating CAR-independent induction in rats. DAS, but not GO, DADS, or DATS, induced CYP2B10 mRNA levels 530-fold in wild-type (WT) mice, whereas this induction was attenuated in CAR(-/-) mice. DAS induced NQO1 in WT and CAR(-/-) mice equally, suggesting CAR-independent induction in mice. DAS induced NQO1 5-fold in WT mice, whereas induction was completely absent in Nrf2(-/-) mice, indicating DAS also activates Nrf2. DAS induction of CYP2B10 mRNA was independent of Nrf2 presence or absence. In in vivo transcription assays, DAS activated the human CYP2B6 promoter, and the antioxidant response element of the human NQO1 promoter, respectively. These studies indicate that GO constituents, particularly DAS, activate CAR and Nrf2 to induce drug-metabolizing enzymes.

Increased expression of hypothalamic NADPH-diaphorase neurons in mice with iron supplement.

Iron deficiency is known as the most important nutritional problem in the world. The loss of appetite is a common characteristic of iron deficiency. Iron-containing heme is required as a cofactor for nitric oxide synthase (NOS) which produces nitric oxide (NO). NOS in the central nervous system has been suggested to regulate food intake. Hence, we examined the expression of hypothalamic NOS at various levels of dietary iron. ICR mice (n = 30) were randomly divided into three groups based on the level of dietary iron and fed experimental diets for 4 weeks: the normal-iron diet group (7 mg/kg diet, n = 10), the low-iron diet group (21 mg/kg diet, n = 10) and the high-iron diet group (42 mg/kg diet, n = 10). Expression of NOS in the paraventricular nucleus (PVN) and lateral hypothalamic area (LHA) of hypothalamus was examined by histochemistry for nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-diaphorase). The high-iron diet mice showed significantly higher staining intensity of NADPH-diaphorase-positive neurons in the PVN and LHA than the normal- and low-iron diet mice.

Adrenomedullin administration immediately after myocardial infarction ameliorates progression of heart failure in rats.

BACKGROUND: Adrenomedullin (AM) is expressed in cardiac tissue, and plasma AM levels increase in patients with acute myocardial infarction (MI). This study was performed to determine whether AM administration immediately after acute MI inhibits progression of heart failure in rats. METHODS AND RESULTS: Rats were infused with 1.0 microg/h IP AM or saline over 7 days immediately after MI inducted by left coronary ligation and were examined 9 weeks after MI. Compared with the saline infusion, AM infusion significantly improved survival (59% versus 81%; P<0.05) and body weight gain (32%; P<0.01) and reduced heart weight (-28%; P<0.01), lung weight (-26%; P<0.01), left ventricular (LV) end-diastolic pressure (11.4+/-2.0 versus 4.0+/-0.6 mm Hg, mean+/- SEM; P<0.01), collagen volume fraction of noninfarcted LV (-39%; P<0.05), and plasma levels of endogenous rat AM (-38%; P<0.05) without affecting infarct size. To investigate the mechanism of AM actions, another series of MI rats infused with AM were killed on day 7. AM infusion had no effect on organ weights and hemodynamic parameters on day 7 of MI but significantly reduced urinary excretion of isoprostane (-61%; P<0.01) and noninfarcted LV mRNA levels of ACE (-31%; P<0.05) and p22-phox (-30%; P<0.05). CONCLUSIONS: AM administration during the early period of MI improved the survival and ameliorated progression of LV remodeling and heart failure. This beneficial effect was accompanied by reductions in oxidative stress and ACE mRNA expression in noninfarcted LV in the AM infusion period.

Thermal treatment attenuates neointimal thickening with enhanced expression of heat-shock protein 72 and suppression of oxidative stress.

BACKGROUND: The beneficial effects of thermal therapy have been reported in several cardiovascular diseases. However, it is unknown whether the thermal treatment has some beneficial roles against the development of atherosclerosis. METHODS AND RESULTS: The inflammatory arterial lesion was introduced by placement of a polyethylene cuff on femoral arteries of male Sprague-Dawley rats for 4 weeks. Thermal-treated group underwent daily bathing in 41 degrees C hot water for 15 minutes. Neointimal thickening along with immunohistochemical expression of heat-shock proteins (HSPs), monocyte chemoattractant protein-1 (MCP-1), and NADPH oxidase were compared with those of a thermally untreated (Control) group. Morphometric analysis demonstrated a significant suppression of neointimal thickening in thermal-treated group compared with the Control group (intimal/medial area ratios, 0.01+/-0.01 versus 0.31+/-0.04, P<0.01). Expression of MCP-1 and infiltration of ED-positive cells were enhanced in the adventitial layer of Control. More importantly, expression of HSP72 in media was enhanced by thermal treatment. Expression of p22-phox, the major membrane subunit of NADPH oxidase, and MCP-1 was augmented in cuff-injured adventitia of the Control but not the thermal-treated groups. CONCLUSIONS: Thermal treatment significantly attenuated infiltration of inflammatory cells in adventitia and suppressed neointimal thickening in cuff-injured arteries with the enhancement of HSP72 expression and suppression of oxidative stress.

p22phox is a critical component of the superoxide-generating NADH/NADPH oxidase system and regulates angiotensin II-induced hypertrophy in vascular smooth muscle cells.

Superoxide anion formation is vital to the microbicidal activity of phagocytes. Recently, however, there is accumulating evidence that it is also involved in cell growth in vascular smooth muscle cells (VSMCs). We have shown that the hypertrophic agent angiotensin II stimulates superoxide production by activating the membrane-bound NADH/NADPH oxidase and that inhibition of this oxidase attenuates vascular hypertrophy. However, the molecular identity of this oxidase in VSMCs is unknown. We have recently cloned the cytochrome b558 alpha-subunit, p22(phox) (one of the key electron transfer elements of the NADPH oxidase in phagocytes), from a rat VSMC cDNA library, but its role in VSMC oxidase activity remains unclarified. Here we report that the complete inhibition of p22(phox) mRNA expression by stable transfection of antisense p22(phox) cDNA into VSMCs results in a decrease in cytochrome b content, which is accompanied by a significant inhibition of angiotensin II-stimulated NADH/NADPH-dependent superoxide production, subsequent hydrogen peroxide production, and [3H]leucine incorporation. We provide the first evidence that p22(phox) is a critical component of superoxide-generating vascular NADH/NADPH oxidase and suggest a central role for this oxidase system in vascular hypertrophy.

Gene expression of brain nitric oxide synthase and soluble guanylyl cyclase in hypothalamus and medulla of two-kidney, one clip hypertensive rats.

Nitric oxide may act at autonomic sites in the brain to regulate arterial blood pressure. Our goal was to determine whether gene expressions of the brain isoform of nitric oxide synthase and of the beta subunit of soluble guanylyl cyclase, the target of nitric oxide, were altered in discrete autonomic brain regions after induction of hypertension in rats. The two-kidney, one clip model was used to induce hypertension, and measurements were made 3 and 6 weeks after the left renal artery was clipped. Only experimental rats with blood pressures elevated by at least 25 mm Hg were used. Total RNA was purified from microdissected tissue blocks containing hypothalamus, dorsal medulla, rostral ventrolateral medulla, and caudal ventrolateral medulla. Changes in nitric oxide synthase and guanylyl cyclase mRNA were semiquantified in each region by use of reverse transcription-polymerase chain reactions in which known concentrations of deletion mutants of the two genes were coamplified as internal standards. Compared with controls, significant decreases and increases in nitric oxide synthase mRNA were found in the hypothalamus (x 2.2) and caudal ventrolateral medulla (x 6.4), respectively, of hypertensive rats 3 weeks after clipping. These alterations were reversed in hypertensive rats at 6 weeks; levels increased (x 4.6) in the hypothalamus and decreased (x 5.5) in the caudal ventrolateral medulla. Changes in guanylyl cyclase expression paralleled those for nitric oxide synthase in some but not all areas at both time points.(ABSTRACT TRUNCATED AT 250 WORDS)

Cloning and expression of the gene encoding the porcine NADPH oxidase light-chain subunit (p22-phox).

In previous studies, we showed that interleukin-4 (IL-4) suppressed porcine (p) macrophage superoxide production and that the mechanism of suppression involved down-regulation of the superoxide-generating enzyme NADPH oxidase heavy-chain 91-kDa subunit mRNA (gp91-phox) expression. In order to examine the effect of IL-4 on expression of the gene encoding the porcine NADPH oxidase light-chain 22-kDa subunit (p22-phox), we cloned the p22-phox cDNA from a macrophage library. The p22-phox cDNA is 786 bp in length and contains a 576-bp open reading frame which predicts a primary translation product of 192 amino acids (aa). Comparison of the porcine and human 22-phox cDNAs showed a high degree of similarity between the two species in their nucleotide (85%) and deduced aa (83%) sequences. as well as in their hydropathy profiles. Notable features, including a high proline content and an iron-coordinating His94, are conserved in both the porcine and human 22-Phox. A single species of mRNA of about 1 kb was detected in macrophages. The mRNA levels remained unchanged in cells treated with lipopolysaccharide (LPS) or with IL-4 at various concentrations from 0-50 ng/ml. Prolonged treatment with LPS or IL-4 did not enhance the effect of these substances on p22-phox mRNA expression. The effect of IL-4 on p22-phox mRNA expression was also compared with another immunosuppressive cytokine, transforming growth factor-beta 1 (TGF beta 1). No change in mRNA expression was found in the cells with or without TGF beta 1 treatment. The results indicated that the heavy and light chains of NADPH oxidase are independently regulated by IL-4 in macrophages.