Effect of LED Lights on Secondary Metabolites and Antioxidant Activities in Red Pakchoi Baby Leaves.

Pakchoi (Brassica rapa subsp. chinensis) is one of the most widely consumed vegetables in Asian countries, and it is high in secondary metabolites. The availability, quantity, and quality of light play a critical role in the growth and development of plants. In this study, we investigated the effect of LEDs (light-emitting diodes; white, blue, red, and red + blue) on anthocyanin, glucosinolates, and phenolic levels in red pakchoi baby leaves. On the 24th day after sowing (DAS), red baby pakchoi leaves were harvested, and shoot length, root length, and fresh weight were measured. Among the different LED treatments, there was no significant difference in shoot length, whereas the highest root length was achieved in the red + blue LED treatment (23.8 cm). The fresh weight also showed a significant difference among the different LED treatments. In total, 12 phenolic and 7 glucosinolate individual compounds were identified using high-performance liquid chromatography (HPLC) analysis. The highest total glucosinolate (2937 µg/g dry wt) and phenolic (1589 µg/g dry wt) contents were achieved in baby leaves exposed to red + blue light. Similarly, the highest contents of total anthocyanins (1726 µg/g dry wt), flavonoids (4920 µg/g dry wt), and phenolics (5900 µg/g dry wt) were achieved in the red + blue treatment. Plants exposed to red + blue LED light showed the highest accumulation of anthocyanin, glucosinolates, and phenolic compounds. For antioxidant activity, DPPH (2,2-diphenyl-1-picrylhydrazylradical) free radical scavenging, ABTS (2,2-azinobis (3-ethylbenzothiazoline)-6-sulfonic acid) radical scavenging, and reducing power assays were performed, and the antioxidant activity of red pakchoi baby leaves grown under red + blue LED light was found to be the best. The metabolic profiling of the identified metabolites revealed distinct separation based on the secondary metabolites. This research will be helpful for farmers to choose the best LED light combination to increase the secondary metabolic content in pakchoi plants.

Comparative Analysis of Primary and Secondary Metabolites in Different In Vitro Tissues of Narcissus tazetta var. chinensis.

Narcissus tazetta var. chinensis is a perennial monocot plant that is well known for its pharmaceutical and ornamental uses. This study aimed to understand the changes in the primary and secondary metabolites in different in vitro tissues of N. tazetta (callus, adventitious root, and shoot) using high-performance liquid chromatography and gas chromatography time-of-flight mass spectrometry. In addition, to optimize the most efficient in vitro culture methods for primary and secondary metabolite production, N. tazetta bulbs were used as explants and cultivated in Murashige and Skoog (MS) medium containing different hormones at various concentrations. In addition, the present study found suitable hormonal concentrations for callus, adventitious root, and shoot induction and analyzed the primary and secondary metabolites. The MS medium supplemented with 1.0 mg L-1 dicamba, 3.0 mg L-1 indole-3-butyric acid (IBA), and 3.0 mg L-1 6-benzylaminopurine (BAP) was the most efficient media for callus, adventitious root, and shoot induction in N. tazetta. The tissue induced in this medium was subjected to primary (amines, amino acids, organic acids, sugars, and sugar alcohols) and secondary metabolite (galantamine and phenolic acids) analysis. The shoots and roots showed the highest amounts of metabolites. This study showed that bulb in vitro culture can be an efficient micropropagation method for N. tazetta and the production of primary and secondary metabolites, offering implications for the mass production of primary and secondary metabolite compounds from N. tazetta tissues generated in vitro.

Metabolite profiles and biological activities of different phenotypes of Chinese cabbage (Brassica rapa ssp. Pekinensis).

Chinese cabbage is considered as one of the most important cruciferous vegetables in South Korea because of its use in salads, kimchi, and Korean cuisine. Secondary metabolites were quantified in three Chinese cabbage varieties: 65065, interspecific hybrid of Chinese cabbage × red cabbage exhibiting a deep purple color; 85772, interspecific hybrid of Chinese cabbage × red mustard exhibiting a reddish-purple color; and a typical Chinese green cabbage cultivar "CR Carotene" (Brassica rapa subsp. pekinensis cv. CR Carotene). A total of 54 metabolites (2 amines, 2 sugar alcohols, 2 sugar phosphates, 6 carbohydrates, 18 amino acids, 13 organic acids, 8 phenolic compounds, and 3 carotenoids) were detected in 85772. Of them, 52 metabolites excluding ß-carotene and 9-cis-ß-carotene, and 51 metabolites excluding leucine, ß-carotene, and 9-cis-ß-carotene, were detected in 65065 and CR Carotene, respectively. Amino acid content was the highest in 85772, followed by 65065 and CR Carotene. The cultivars 65065 and 85772 contained high levels of phenolic compounds and total anthocyanins. Cyanidin-, pelargonidin-, and petunidin-type anthocyanins were detected in 65065 and 85772. However, delphinidin-type anthocyanins which typically impart a deep purple color were identified only in the deep purple phenotype 65065. Furthermore, the total anthocyanin content was the highest in 85772 (4.38 ± 0.65 mg g -1 dry weight) followed by that in 65065 (3.72 ± 0.52 mg g-1 dry weight). Antibacterial and antioxidant analyses revealed remarkable antibacterial effects of the purple cultivars against pathogens Vibrio parahaemolyticus (KCTC 2471), Bacillus cereus (KCTC 3624), Pseudomonas aeruginosa (KCCM 11803), Staphylococcus aureus (KCTC 3881), Chryseobacterium gleum (KCTC 2094), and Proteus mirabilis (KCTC 2510)] and methicillin-resistant pathogenic strains of Pseudomonas aeruginosa (0826, 0225, 0254, 1113, 1378, 1731, p01827, and p01828) compared with the antibacterial effects of CR Carotene. Furthermore, 65065 and 85772 exhibited significantly higher antioxidant activity than that of the CR Carotene. Therefore, the novel purple Chinese cabbages (65065 and 85772), derived from interspecific hybridization, are potentially favorable alternatives to the typical green Chinese cabbage, given the higher content of amino acids, phenolic compounds, anthocyanins, and carotenoids, as well as an increased ability to scavenge free radicals and inhibit pathogen growth.

LED Lights Influenced Phytochemical Contents and Biological Activities in Kale (Brassica oleracea L. var. acephala) Microgreens.

Light-emitting diodes (LEDs) are regarded as an effective artificial light source for producing sprouts, microgreens, and baby leaves. Thus, this study aimed to investigate the influence of different LED lights (white, red, and blue) on the biosynthesis of secondary metabolites (glucosinolates, carotenoids, and phenolics) and the biological effects on kale microgreens. Microgreens irradiated with white LEDs showed higher levels of carotenoids, including lutein, 13-cis-ß-carotene, α-carotene, ß-carotene, and 9-cis-ß-carotene, than those irradiated with red or blue LEDs. These findings were consistent with higher expression levels of carotenoid biosynthetic genes (BoPDS and BoZDS) in white-irradiated kale microgreens. Similarly, microgreens irradiated with white and blue LEDs showed slightly higher levels of glucosinolates, including glucoiberin, progoitrin, sinigrin, and glucobrassicanapin, than those irradiated with red LEDs. These results agree with the high expression levels of BoMYB28-2, BoMYB28-3, and BoMYB29 in white- and blue-irradiated kale microgreens. In contrast, kale microgreens irradiated with blue LEDs contained higher levels of phenolic compounds (gallic acid, catechin, ferulic acid, sinapic acid, and quercetin). According to the total phenolic content (TPC) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) inhibition assays, the extracts of kale microgreens irradiated with blue LEDs had slightly higher antioxidant activities, and the DPPH inhibition percentage had a positive correlation with TPC in the microgreens. Furthermore, the extracts of kale microgreens irradiated with blue LEDs exhibited stronger antibacterial properties against normal pathogens and multidrug-resistant pathogens than those irradiated with white and red LEDs. These results indicate that white-LED lights are suitable for carotenoid production, whereas blue-LED lights are efficient in increasing the accumulation of phenolics and their biological activities in kale microgreens.

Tat-CIAPIN1 Prevents Pancreatic ß-Cell Death in hIAPP-Induced RINm5F Cells and T2DM Animal Model.

It is well known that the cytokine-induced apoptosis inhibitor 1 (CIAPIN1) protein plays an important role in biological progresses as an anti-apoptotic protein. Human islet amyloid peptide (hIAPP), known as amylin, is caused to pancreatic ß-cell death in type 2 diabetes mellitus (T2DM). However, the function of CIAPIN1 protein on T2DM is not yet well studied. Therefore, we investigated the effects of CIAPIN1 protein on a hIAPP-induced RINm5F cell and T2DM animal model induced by a high-fat diet (HFD) and streptozotocin (STZ). The Tat-CIAPIN1 protein reduced the activation of mitogen-activated protein kinase (MAPK) and regulated the apoptosis-related protein expression levels including COX-2, iNOS, Bcl-2, Bax, and Caspase-3 in hIAPP-induced RINm5F cells. In a T2DM mice model, the Tat-CIAPIN1 protein ameliorated the pathological changes of pancreatic ß-cells and reduced the fasting blood glucose, body weight and hemoglobin Alc (HbAlc) levels. In conclusion, the Tat-CIAPIN1 protein showed protective effects against T2DM by protection of ß-cells via inhibition of hIAPP toxicity and by regulation of a MAPK signal pathway, suggesting CIAPIN1 protein can be a therapeutic protein drug candidate by beneficial regulation of T2DM.

Protective effects of cell permeable Tat-PIM2 protein on oxidative stress induced dopaminergic neuronal cell death.

Background: Oxidative stress is considered as one of the main causes of Parkinson's disease (PD), however the exact etiology of PD is still unknown. Although it is known that Proviral Integration Moloney-2 (PIM2) promotes cell survival by its ability to inhibit formation of reactive oxygen species (ROS) in the brain, the precise functional role of PIM2 in PD has not been fully studied yet. Objective: We investigated the protective effect of PIM2 against apoptosis of dopaminergic neuronal cells caused by oxidative stress-induced ROS damage by using the cell permeable Tat-PIM2 fusion protein in vitro and in vivo. Methods: Transduction of Tat-PIM2 into SH-SY5Y cells and apoptotic signaling pathways were determined by Western blot analysis. Intracellular ROS production and DNA damage was confirmed by DCF-DA and TUNEL staining. Cell viability was determined by MTT assay. PD animal model was induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and protective effects were examined using immunohistochemistry. Results: Transduced Tat-PIM2 inhibited the apoptotic caspase signaling and reduced the production of ROS induced by 1-methyl-4-phenylpyridinium (MPP+) in SH-SY5Y cells. Furthermore, we confirmed that Tat-PIM2 transduced into the substantia nigra (SN) region through the blood-brain barrier and this protein protected the Tyrosine hydroxylase-positive cells by observation of immunohistostaining. Tat-PIM2 also regulated antioxidant biomolecules such as SOD1, catalase, 4-HNE, and 8-OHdG which reduce the formation of ROS in the MPTP-induced PD mouse model. Conclusion: These results indicated that Tat-PIM2 markedly inhibited the loss of dopaminergic neurons by reducing ROS damage, suggesting that Tat-PIM2 might be a suitable therapeutic agent for PD.

Tat-RAN attenuates brain ischemic injury in hippocampal HT-22 cells and ischemia animal model.

Oxidative stress plays a key role in the pathogenesis of neuronal injury, including ischemia. Ras-related nuclear protein (RAN), a member of the Ras superfamily, involves in a variety of biological roles, such as cell division, proliferation, and signal transduction. Although RAN reveals antioxidant effect, its precise neuroprotective mechanisms are still unclear. Therefore, we investigated the effects of RAN on HT-22 cell which were exposed to H2O2-induced oxidative stress and ischemia animal model by using the cell permeable Tat-RAN fusion protein. We showed that Tat-RAN transduced into HT-22 cells, and markedly inhibited cell death, DNA fragmentation, and reactive oxygen species (ROS) generation under oxidative stress. This fusion protein also controlled cellular signaling pathways, including mitogen-activated protein kinases (MAPKs), NF-κB, and apoptosis (Caspase-3, p53, Bax and Bcl-2). In the cerebral forebrain ischemia animal model, Tat-RAN significantly inhibited both neuronal cell death, and astrocyte and microglia activation. These results indicate that RAN significantly protects against hippocampal neuronal cell death, suggesting Tat-RAN will help to develop the therapies for neuronal brain diseases including ischemic injury.

The Effect of Light and Dark Treatment on the Production of Rosmarinic Acid and Biological Activities in Perilla frutescens Microgreens.

This study aimed to investigate the effect of light [a long-day photoperiod (16 h light/8 h dark cycle)] and dark treatment on the production of rosmarinic acid in P. frutescens microgreens and to determine its antioxidant and antibacterial activities. Microgreens of P. frutescens were grown under light and dark conditions and harvested after 10, 15, 20, and 25 days of each treatment. Although dry weight values of microgreens gradually increased from 10 to 25 days of both treatments, the microgreens grown under light treatment possessed slightly higher levels of dry weight than those grown in the dark. Rosmarinic acid and total phenolic content (TPC) were also analyzed using high-performance liquid chromatography (HPLC) and Folin-Ciocalteu assay. The accumulation patterns of rosmarinic acid and TPC gradually increased and decreased, respectively, in P. frutescens microgreens grown in continuous darkness. The highest accumulation was observed in microgreens grown for 20 days. However, rosmarinic acid and TPC values were not significantly different in microgreens grown under light conditions. According to the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical inhibition assay, the extracts of P. frutescens microgreens were confirmed to be strong antioxidants, and their ability to scavenge DPPH radicals was positively correlated with the total phenolic content in the microgreens after 10, 15, 20, and 25 days of both treatments. Considering the relatively higher values of dry weight, rosmarinic acid, TPC, and DPPH assay, P. frutescens microgreens after 20 days of darkness and 20 days of light treatment, respectively, were selected for screening antibacterial activity using nine pathogens. Both microgreen extracts showed strong antibacterial activity against pathogens. In particular, the extracts of microgreens grown for 20 days under light treatment showed higher antimicrobial effects. Therefore, the light treatments for 20 days, as well as the darkness treatment for 20 days, were the best conditions for P. frutescens microgreen production because of their high levels of dry weight, phenolics, and biological activities.

Tat-GSTpi Inhibits Dopaminergic Cells against MPP+-Induced Cellular Damage via the Reduction of Oxidative Stress and MAPK Activation.

Glutathione S-transferase pi (GSTpi) is a member of the GST family and plays many critical roles in cellular processes, including anti-oxidative and signal transduction. However, the role of anti-oxidant enzyme GSTpi against dopaminergic neuronal cell death has not been fully investigated. In the present study, we investigated the roles of cell permeable Tat-GSTpi fusion protein in a SH-SY5Y cell and a Parkinson's disease (PD) mouse model. In the 1-methyl-4-phenylpyridinium (MPP+)-exposed cells, Tat-GSTpi protein decreased DNA damage and reactive oxygen species (ROS) generation. Furthermore, this fusion protein increased cell viability by regulating MAPKs, Bcl-2, and Bax signaling. In addition, Tat-GSTpi protein delivered into the substantia nigra (SN) of mice brains protected dopaminergic neuronal cell death in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD animal model. Our results indicate that the Tat-GSTpi protein inhibited cell death from MPP+- and MPTP-induced damage, suggesting that it plays a protective role during the loss of dopaminergic neurons in PD and that it could help to identify the mechanism responsible for neurodegenerative diseases, including PD.

Effects of Carbohydrates on Rosmarinic Acid Production and In Vitro Antimicrobial Activities in Hairy Root Cultures of Agastache rugosa.

Agastache rugosa (popularly known as Korean mint) belongs to the Lamiaceae family and comprises 22 species of perennial aromatic medicinal species native to East Asian countries, such as Korea, Taiwan, Japan, and China. A. rugosa contains many phenolic compounds that exhibit pharmacological and physiological activities, including antioxidant, anticancer, antiviral, antifungal, and antibacterial activities. The highest concentrations of rosmarinic acid and its isomers have been reported in the roots of A. rugosa. In this in vitro study, hairy roots of A. rugosa were obtained and the carbohydrates (sorbitol, mannitol, glucose, maltose, galactose, mannose, and sucrose) were evaluated to determine those that were optimal for rosmarinic acid production and hairy root growth. Antioxidant and antibacterial activities of extracts of A. rugosa were also assessed. The best carbon source for A. rugosa hairy root cultures was sucrose, considering biomass productivity (0.460 ± 0.034 mg/30 mL), rosmarinic acid production (7.656 ± 0.407 mg/g dry weight), and total phenolic content (12.714 ± 0.202 mg/g gallic acid equivalent). Antioxidant and antimicrobial activities were displayed by A. rugosa hairy roots cultured in liquid medium supplemented with 100 mM sucrose. Twenty-five bacterial strains, including multidrug-resistant bacteria and one pathogenic yeast strain, were used for antimicrobial screening of A. rugosa hairy roots. The hairy root extracts displayed antibacterial activity against Micrococcus luteus (KCTC 3063) and Bacillus cereus (KCTC 3624). The inhibition of these bacteria was greater using A. rugosa hairy roots with the highest levels of phenolic compounds cultured in the presence of sucrose, compared to hairy roots with the lowest levels of phenolic compounds cultured in the presence of fructose. Considering hairy root biomass, phenolic compound production, and antibacterial activity, sucrose is the best carbon source for A. rugosa hairy root cultures.

Protective Effects of PEP-1-GSTA2 Protein in Hippocampal Neuronal Cell Damage Induced by Oxidative Stress.

Glutathione S-transferase alpha 2 (GSTA2), a member of the glutathione S-transferase family, plays the role of cellular detoxification against oxidative stress. Although oxidative stress is related to ischemic injury, the role of GSTA2 against ischemia has not been elucidated. Thus, we studied whether GSTA2 prevents ischemic injury by using the PEP-1-GSTA2 protein which has a cell-permeable protein transduction domain. We revealed that cell-permeable PEP-1-GSTA2 transduced into HT-22 cells and markedly protected cell death via the inhibition of reactive oxygen species (ROS) production and DNA damage induced by oxidative stress. Additionally, transduced PEP-1-GSTA2 promoted mitogen-activated protein kinase (MAPK), and nuclear factor-kappaB (NF-κB) activation. Furthermore, PEP-1-GSTA2 regulated Bcl-2, Bax, cleaved Caspase-3 and -9 expression protein levels. An in vivo ischemic animal model, PEP-1-GSTA2, markedly prevented the loss of hippocampal neurons and reduced the activation of microglia and astrocytes. These findings indicate that PEP-1-GSTA2 suppresses hippocampal cell death by regulating the MAPK and apoptotic signaling pathways. Therefore, we suggest that PEP-1-GSTA2 will help to develop the therapies for oxidative-stress-induced ischemic injury.

Protective effect of GK2 fused BLVRA protein against oxidative stress-induced dopaminergic neuronal cell damage.

It is well known that oxidative stress is highly associated with Parkinson's disease (PD), and biliverdin reductase A (BLVRA) is known to have antioxidant properties against oxidative stress. In this study, we developed a novel N-acetylgalactosamine kinase (GK2) protein transduction domain (PTD) derived from adenosine A2A and fused with BLVRA to determine whether the GK2-BLVRA fusion protein could protect dopaminergic neuronal cells (SH-SY5Y) from oxidative stress in vitro and in vivo using a PD animal model. GK2-BLVRA was transduced into various cells, including SH-SY5Y cells, without cytotoxic effects, and this fusion protein protected SH-SY5Y cells and reduced reactive oxygen species production and DNA damage after 1-methyl-4-phenylpyridinium (MPP+ ) exposure. GK2-BLVRA suppressed mitogen-activated protein kinase (MAPK) activation and modulated apoptosis-related protein (Bcl-2, Bax, cleaved Caspase-3 and -9) expression levels. In the PD animal model, GK2-BLVRA transduced into the substantia nigra crossed the blood-brain barrier and markedly reduced dopaminergic neuronal cell death in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced animals. These results indicate that our novel PTD GK-2 is useful for the transduction of protein, and GK2-BLVRA exhibits a beneficial effect against dopaminergic neuronal cell death in vitro and in vivo, suggesting that BLVRA can be used as a therapeutic agent for PD.

Tat-Thioredoxin-like protein 1 attenuates ischemic brain injury by regulation of MAPKs and apoptosis signaling.

Thioredoxin-like protein 1 (TXNL1), one of the thioredoxin superfamily known as redox-regulator, plays an essential in maintaining cell survival via various antioxidant and anti-apoptotic mechanisms. It is well known that relationship between ischemia and oxidative stress, however, the role of TXNL1 protein in ischemic damage has not been fully investigated. In the present study, we aimed to determine the protective role of TXNL1 against on ischemic injury in vitro and in vivo using cell permeable Tat-TXNL1 fusion protein. Transduced Tat-TXNL1 inhibited ROS production and cell death in H2O2-exposed hippocampal neuronal (HT-22) cells and modulated MAPKs and Akt activation, and pro-apoptotic protein expression levels in the cells. In an ischemia animal model, Tat-TXNL1 markedly decreased hippocampal neuronal cell death and the activation of astrocytes and microglia. These findings indicate that cell permeable Tat-TXNL1 protects against oxidative stress in vitro and in vivo ischemic animal model. Therefore, we suggest Tat-TXNL1 can be a potential therapeutic protein for ischemic injury. [BMB Reports 2023; 56(4): 234-239].

Effects of Chilling Treatment on Baicalin, Baicalein, and Wogonin Biosynthesis in Scutellaria baicalensis Plantlets.

When plants are exposed to stressful conditions, they modulate their nutrient balance by regulating their primary and secondary metabolisms to adapt. In this study, changes in primary and secondary metabolites elicited by chilling stress treatment and the effects of treatment duration were examined in roots of Scutellaria baicalensis (S. baicalensis) plantlets. The concentrations of most sugars (maltose, glucose, sucrose, and fructose) and of several amino acids (proline and GABA), which are crucial regarding plant defense mechanisms, increased with increasing duration of chilling stress. Furthermore, salicylic acid levels increased after two-day chilling treatments, which may enhance plant tolerance to cold temperatures. The concentrations of flavones (baicalin, baicalein, and wogonin) increased during chilling stress, and those of phenolic acids (ferulic acid and sinapic acid) increased after two-day chilling treatments. The concentrations of these flavones were positively correlated with sucrose levels which acted as energy sources.

Metabolic Analyses and Evaluation of Antioxidant Activity in Purple Kohlrabi Sprouts after Exposed to UVB Radiation.

Various metabolites act as plant defense molecules due to their antioxidant abilities. This study aimed to investigate the influence of UVB irradiation on the accumulation of metabolites, including primary metabolites (sugar, sugar alcohols, amino acids, organic acids, and an amine) and secondary metabolites (anthocyanins, fatty acids, and phenolic acids), and its synergistic antioxidant ability, in purple kohlrabi sprouts. Metabolite analyses revealed a total of 92 metabolites in the sprouts. Specifically, the levels of most amino acids increased after 24 h of UVB treatment, and then slightly decreased in the kohlrabi sprouts. The levels of most sugars and sugar alcohols increased after 24 h of UVB treatment and then decreased. The levels of TCA cycle intermediates and phenolic acids gradually increased during the UVB treatment. Furthermore, the levels of some fatty acids gradually increased during the UVB treatment, and the levels of the other fatty acids increased after 6 h of UVB treatment and then decreased. In particular, the levels of most anthocyanins, known to be strong antioxidants, gradually increased after 24 h of UVB treatment. In the in vitro ABTS scavenging assay, UVB-treated purple kohlrabi sprouts showed increased scavenging ability. This may be attributed to the increased accumulation of metabolites acting as antioxidants, in response to UVB treatment. This study confirmed that UVB irradiation induced the alteration of primary and secondary metabolism in the kohlrabi sprouts.

Gene Expression and Metabolic Analyses of Nontransgenic and AtPAP1 Transgenic Tobacco Infected with Potato Virus X (PVX).

Potato virus X (PVX), a species of the genus Potexvirus, is a plant pathogenic virus that causes severe symptoms such as mild mosaic, crinkling, necrosis, and mottling on leaves. The objectives of the present study were to investigate the effect of PVX virus infection on the metabolic system in nontransgenic and Arabidopsis thaliana production of anthocyanin pigment 1 (AtPAP1) transgenic tobacco using transcript expression analysis and metabolic profiling. Potato virus X inoculation increased the gene expression of phenylpropanoid and flavonoid biosynthesis and the production of chlorogenic acid, p-coumaric acid, benzoic acid, rutin, quercetin, and kaempferol in nontransgenic tobacco leaves. However, in the AtPAP1 transgenic tobacco leaves, PVX inoculation decreased the expression of AtPAP1 and phenylpropanoid and flavonoid biosynthesis genes, and the production of phenolics and anthocyanin also declined. In contrast, the levels of amino acids and tricarboxylic acid (TCA) cycle intermediates increased after infection in the AtPAP1 transgenic plant leaves. To date, these results have not been reported previously. We suggest that PVX infection decreases AtPAP1 expression, leading to the downregulation of phenylpropanoid and flavonoid biosynthesis in transgenic plants.

Tat-thioredoxin 1 reduces inflammation by inhibiting pro-inflammatory cytokines and modulating MAPK signaling.

Thioredoxin 1 (Trx1) serves a central role in redox homeostasis. It is involved in numerous other processes, including oxidative stress and apoptosis. However, to the best of our knowledge, the role of Trx1 in inflammation remains to be explored. The present study investigated the function and mechanism of cell permeable fused Tat-Trx1 protein in macrophages and a mouse model. Transduction levels of Tat-Trx1 were determined via western blotting. Cellular distribution of transduced Tat-Trx1 was determined by fluorescence microscopy. 2',7'-Dichlorofluorescein diacetate and TUNEL staining were performed to determine the production of reactive oxygen species and DNA fragmentation. Protein and gene expression were measured by western blotting and reverse transcription-quantitative PCR (RT-qPCR), respectively. Effects of skin inflammation were determined using hematoxylin and eosin staining, changes in ear weight and ear thickness, and RT-qPCR in ear edema animal models. Transduced Tat-Trx1 inhibited lipopolysaccharide-induced cytotoxicity and activation of NF-κB, MAPK and Akt. Additionally, Tat-Trx1 markedly reduced the production of inducible nitric oxide synthase, cyclooxygenase-2, IL-1ß, IL-6 and TNF-α in macrophages. In a 12-O-tetradecanoylphorbol-13-acetate-induced mouse model, Tat-Trx1 reduced inflammatory damage by inhibiting inflammatory mediator and cytokine production. Collectively, these results demonstrated that Tat-Trx1 could exert anti-inflammatory effects by inhibiting the production of pro-inflammatory mediators and cytokines and by modulating MAPK signaling. Therefore, Tat-Trx1 may be a useful therapeutic agent for diseases induced by inflammatory damage.

Molecular Characterization, Expression Analysis of Carotenoid, Xanthophyll, Apocarotenoid Pathway Genes, and Carotenoid and Xanthophyll Accumulation in Chelidonium majus L.

Chelidonium majus L. is a perennial herbaceous plant that has various medicinal properties. However, the genomic information about its carotenoid biosynthesis pathway (CBP), xanthophyll biosynthesis pathway (XBP), and apocarotenoid biosynthesis pathway (ABP) genes were limited. Thus, the CBP, XBP, and ABP genes of C. majus were identified and analyzed. Among the 15 carotenoid pathway genes identified, 11 full and 4 partial open reading frames were determined. Phylogenetic analysis of these gene sequences showed higher similarity with higher plants. Through 3D structural analysis and multiple alignments, several distinct conserved motifs were identified, including dinucleotide binding motif, carotene binding motif, and aspartate or glutamate residues. Quantitative RT-PCR showed that CBP, XBP, and ABP genes were expressed in a tissue-specific manner; the highest expression levels were achieved in flowers, followed by those in leaves, roots, and stems. The HPLC analysis of the different organs showed the presence of eight different carotenoids. The highest total carotenoid content was found in leaves, followed by that in flowers, stems, and roots. This study provides information on the molecular mechanisms involved in CBP, XBP, and ABP genes, which might help optimize the carotenoid production in C. majus. The results could also be a basis of further studies on the molecular genetics and functional analysis of CBP, XBP, and ABP genes.

Utilization of an Intracellular Calcium Mobilization Assay for the Screening of Transduced FK506-Binding Proteins.

FK506-binding proteins (FKBPs) belong to the immunophilin family and are linked to various disease states, including the inflammatory response. The inhibition of cytokine and chemokine expression in addition to positive effects of FKBPs on corneal inflammation in animal models suggests that they may be used for ophthalmic delivery in the treatment of dry eye disease. To pass the effective barriers protecting eye tissues, testing the transduction domains of FKBPs is essential. However, monitoring their transduction efficiencies is not a simple task. The quantitative measurement of FKBP interactions was performed using a cell model with a specific G protein-coupled receptor, as FKBPs had been known to act at the inositol 1,4,5-trisphosphate receptor (IP3R) leading to the inhibition of intracellular calcium mobilization. Because of its luminescence amplitude and stability, human urotensin II receptor was expressed in aequorin parental cells to measure the action of selected FKBPs. This luminescence-based functional assay platform exhibited a high signal-to-background ratio of more than 100 and a Z' factor at 0.6204. As expected, changes in the sequence of the transduction domain affected the function of the FKBPs. The intracellular calcium mobilization assay with selected FKBPs represented a robust and reliable platform to screen initial candidates. Although the precise nature of the control that FKBPs exert on the IP3R is uncertain, this approach can be used to develop innovative anti-inflammatory treatments for dry eye disease by optimizing protein transduction domain sequences.

PEP-1-GLRX1 Reduces Dopaminergic Neuronal Cell Loss by Modulating MAPK and Apoptosis Signaling in Parkinson's Disease.

Parkinson's disease (PD) is characterized mainly by the loss of dopaminergic neurons in the substantia nigra (SN) mediated via oxidative stress. Although glutaredoxin-1 (GLRX1) is known as one of the antioxidants involved in cell survival, the effects of GLRX1 on PD are still unclear. In this study, we investigated whether cell-permeable PEP-1-GLRX1 inhibits dopaminergic neuronal cell death induced by 1-methyl-4-phenylpyridinium (MPP+) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). We showed that PEP-1-GLRX1 protects cell death and DNA damage in MPP+-exposed SH-SY5Y cells via the inhibition of MAPK, Akt, and NF-κB activation and the regulation of apoptosis-related protein expression. Furthermore, we found that PEP-1-GLRX1 was delivered to the SN via the blood-brain barrier (BBB) and reduced the loss of dopaminergic neurons in the MPTP-induced PD model. These results indicate that PEP-1-GLRX1 markedly inhibited the loss of dopaminergic neurons in MPP+- and MPTP-induced cytotoxicity, suggesting that this fusion protein may represent a novel therapeutic agent against PD.