Stepwise changes in flavonoids in spores/pollen contributed to terrestrial adaptation of plants.

Protecting haploid pollen and spores against UV-B light and high temperature, 2 major stresses inherent to the terrestrial environment, is critical for plant reproduction and dispersal. Here, we show flavonoids play an indispensable role in this process. First, we identified the flavanone naringenin, which serves to defend against UV-B damage, in the sporopollenin wall of all vascular plants tested. Second, we found that flavonols are present in the spore/pollen protoplasm of all euphyllophyte plants tested and that these flavonols scavenge reactive oxygen species to protect against environmental stresses, particularly heat. Genetic and biochemical analyses showed that these flavonoids are sequentially synthesized in both the tapetum and microspores during pollen ontogeny in Arabidopsis (Arabidopsis thaliana). We show that stepwise increases in the complexity of flavonoids in spores/pollen during plant evolution mirror their progressive adaptation to terrestrial environments. The close relationship between flavonoid complexity and phylogeny and its strong association with pollen survival phenotypes suggest that flavonoids played a central role in the progression of plants from aquatic environments into progressively dry land habitats.

Gap junction is essential for the antidepressant effects of fluoxetine.

OBJECTIVES: Fluoxetine has been used as the first line for the therapy of depression. However, lack of therapeutic efficacy and time lag still limit the application of fluoxetine. Gap junction dysfunction is a potentially novel pathogenic mechanism for depression. To clarify the mechanism underlying these limitations, we investigated whether gap junction was related to the antidepressant effects of fluoxetine. METHODS AND KEY FINDINGS: After chronic unpredictable stress (CUS), animals showed decreases in gap junction intracellular communication (GJIC). Treatment with fluoxetine 10 mg/kg significantly improved GJIC and anhedonia of rats until six days. These results indicated that fluoxetine improved gap junction indirectly. Furthermore, to test the role of gap junction on antidepressant effects of fluoxetine, we blocked gap junction using carbenoxolone (CBX) infusion in the prefrontal cortex. CBX dampened fluoxetine-induced decrease in immobility time of mice in tail suspension test (TST). CONCLUSIONS: Our study suggested that gap junction dysfunction blocks antidepressant effects of fluoxetine, contributing to understanding the mechanism underlying the time lag of fluoxetine.

Development of the Middle Layer in the Anther of Arabidopsis.

The middle layer is an essential cell layer of the anther wall located between the endothecium and tapetum in Arabidopsis. Based on sectioning, the middle layer was found to be degraded at stage 7, which led to the separation of the tapetum from the anther wall. Here, we established techniques for live imaging of the anther. We created a marker line with fluorescent proteins expressed in all anther layers to study anther development. Several staining methods were used in the intact anthers to study anther cell morphology. We clarified the initiation, development, and degradation of the middle layer in Arabidopsis. This layer is initiated from both the inner and outer secondary parietal cells at stage 4, stopped cell division at stage 6, and finally degraded at stage 11. The neighboring cell layers, the epidermis, and endothecium continued cell division until stage 10, which led to a thin middle layer. The degradation of the tapetum cell wall at stage 7 lead to its isolation from the anther wall. This work presents fundamental information on the development of the middle layer, which facilitates the further investigation of anther development and plant fertility. These live imaging methods could be useful in future studies.

Phenylpropanoid Derivatives Are Essential Components of Sporopollenin in Vascular Plants.

The outer wall of pollen and spores, namely the exine, is composed of sporopollenin, which is highly resistant to chemical reagents and enzymes. In this study, we demonstrated that phenylpropanoid pathway derivatives are essential components of sporopollenin in seed plants. Spectral analyses showed that the autofluorescence of Lilium and Arabidopsis sporopollenin is similar to that of lignin. Thioacidolysis and NMR analyses of pollen from Lilium and Cryptomeria further revealed that the sporopollenin of seed plants contains phenylpropanoid derivatives, including p-hydroxybenzoate (p-BA), p-coumarate (p-CA), ferulate (FA), and lignin guaiacyl (G) units. The phenylpropanoid pathway is expressed in the tapetum in Arabidopsis, consistent with the fact that the sporopollenin precursor originates from the tapetum. Further germination and comet assays showed that this pathway plays an important role in protection of pollen against UV radiation. In the pteridophyte plant species Ophioglossum vulgatum and Lycopodium clavata, phenylpropanoid derivatives including p-BA and p-CA were also detected, but G units were not. Taken together, our results indicate that phenylpropanoid derivatives are essential for sporopollenin synthesis in vascular plants. In addition, sporopollenin autofluorescence spectra of bryophytes, such as Physcomitrella and Haplocladium, exhibit distinct characteristics compared with those of vascular plants, indicating the diversity of sporopollenin among land plants.

The protective effect of ginsenoside Rg1 on depression may benefit from the gap junction function in hippocampal astrocytes.

Studies have shown that the ginsenoside Rg1 can improve depressive symptoms in vitro and in vivo. However, the efficacy of Rg1on the hippocampal astrocyte gap junctions in depression are unclear. We mainly aimed to explore the relationship between Rg1, hippocampal astrocyte gap junctions and depression. Using primary cultured astrocytes, corticosterone (CORT) was used to induce stress. CORT (100 µM) significantly reduced the survival rate in astrocytes, and this effect was prevented by additional Rg1 administration. Interestingly, the gap junction blocker carbenoxolone (CBX) was able to revert this Rg1 effect. In in vivo models, one group was exposed to chronic unpredictable stress (CUS) for 47 days, while another group was bilaterally injected with CBX (100 µM) into the hippocampal CA1 region. Rats treated with Rg1 (20 mg/kg) showed an improvement in the sucrose preference and the forced swimming test in both models, indicating an antidepressive activity of Rg1. The levels of astrocyte gap junction connexin 43 (Cx43) were detected by immunofluorescence (IF) and western blotting. The levels of glial fibrillary acidic protein (GFAP) were detected by IF. The gap junctions in the hippocampal CA1 area were evaluated using dye transfer and electron microscopy. The reduction in Cx43 expression, the decrease in the Cx43 to GFAP ratio, the shorter dye diffusion distance, and the abnormal ultrastructure of gap junctions in rats exposed to CUS were markedly alleviated by concomitant Rg1 treatment. Taken together, the ginsenoside Rg1 could improve depression-like behavior in rats induced by astrocyte gap junction dysfunction in the hippocampus.

Connexin 43: A novel ginsenoside Rg1-sensitive target in a rat model of depression.

Our previous studies have shown that ginsenoside Rg1 (Rg1) exerts antidepressant-like effects in animal models of depression, accompanied by an improvement of astrocytic gap junction functions. However, whether connexin 43 (Cx43), the major connexin forming gap junctions between astrocytes, is the key regulator of Rg1-induced antidepressant-like effects is still unknown. In this study, we examine in vitro and in vivo the involvement of Cx43 in the antidepressant effects of Rg1. Corticosterone was used to establish an in vitro rat model of depression. Treatment with Rg1 1 h prior to corticosterone significantly improved the cell viability of astrocytes, which was significantly inhibited by carbenoxolone, a widely used gap junction inhibitor. Moreover, Rg1 treatment significantly ameliorated antidepressant-sensitive behaviours induced by infusion of carbenoxolone or Gap26, a selective inhibitor of Cx43, into the prefrontal cortex of the animals. Rg1 treatment increased the expression of Cx43 compared with Gap26 group. According to these results, the antidepressant-like effects of Rg1 were mainly mediated by Cx43-formed gap junctions.

IMM-H004 reduced okadaic acid-induced neurotoxicity by inhibiting Tau pathology in vitro and in vivo.

This study aimed to explore effects and mechanisms of 004 (IMM-H004), a novel coumarin derivative, in OKA (okadaic acid)-induced AD (Alzheimer's disease)-like model. In vitro, MTT, LDH, and Annexin V/FITC flow cytometry assay were used to test cell survival. In vivo, OKA microinjection was conducted to simulate AD-like neuropathology. Morris water maze and Nissl staining were used to detect spatial memory function and neuronal damage respectively. Western blot and immunohistochemistry were used to study the mechanisms of 004 in Tau pathology. The results showed that 004 reduced cell death and increased survival in PC12 cells, and decreased neuronal injury in the hippocampus in rats. 004 improved learning and memory functions in OKA-treated rats. The mechanistic studies indicated that 004 inhibited phosphorylation of Tau protein by down-regulating the activity of protein kinases CDK5 and GSK3ß and increasing PP2A activity. Overall, 004 improved spatial memory impairments and neuron cells injury induced by OKA; on the other hand, 004 inhibited Tau hyperphosphorylation by regulating CDK5, GSK3ß and PP2A.

Glucocorticoid receptor activation induces decrease of hippocampal astrocyte number in rats.

RATIONALE: The decrease of astrocyte number and hypothalamic-pituitary-adrenal (HPA) axis overactivity are observed in individuals with major depressive disorder. Elevated levels of glucocorticoids induced by hyperactivation of the HPA axis may result in glucocorticoid receptor (GR) activation. However, it is unclear whether there is a direct link between GR activation and the decrease of astrocyte number. METHODS: Animals were exposed to chronic unpredictable stress (CUS) for 28 days and treated with continuous subcutaneous injections of vehicle or corticosterone (CORT; 40 mg/kg/day) for 21 days. We then administered mifepristone on day 21 after CUS and on day 18 after the CORT treatment. We observed behavioral deficits in the sucrose preference test, open field test, and forced swim test. Protein expression was analyzed using immunofluorescence (IF) and western blot (WB). RESULTS: Animals exposed to CUS exhibited behavioral deficits in tests measuring anhedonia, anxiety, and despair state. They also had decreases in glial fibrillary acidic protein (GFAP) expression and numbers of GFAP-positive cells in the hippocampus. The behavioral and cellular alterations induced by CUS were reversed by subchronic treatment with the GR antagonist mifepristone. We also found that the subcutaneous injection of glucocorticoids may induce depression-like behavior and reduce GFAP protein expression in rats, which was similarly reversed by mifepristone. CONCLUSIONS: These findings provide experimental evidence that GR activation due to elevated CORT levels induces the decrease of hippocampal astrocyte number in rats.

Corticosterone impairs gap junctions in the prefrontal cortical and hippocampal astrocytes via different mechanisms.

Increasing evidence has implicated astrocyte pathology in the etiopathology of major depressive disorder (MDD). In particular, dysfunction of gap junctions in astrocytes is a potential target for MDD treatment. However, the mechanism underlying stress-induced dysfunction of gap junctions is still unknown. We therefore studied the mechanism of stress-induced dysfunction of gap junctions in prefrontal cortical and hippocampal astrocytes. Corticosterone (CORT) was used to induce stress conditions; CORT damaged the function of gap junctions, which resulted from less distribution of connexin43 (Cx43) on membranes and the enhanced phosphorylation of Cx43 at S368. Moreover, CORT downregulated the biosynthesis of Cx43 but increased the degradation of Cx43. Interestingly, both autophagy and the proteasome system were involved in the degradation of Cx43 in prefrontal cortical astrocytes, but only the proteasome system was involved in the degradation of Cx43 in hippocampal astrocytes. CORT significantly induced the formation of annular gap junction vesicles in prefrontal cortical astrocytes; however, Cx43 mainly presented as small dots in the hippocampal astrocytes. Furthermore, CORT increased N-Cadherin expression and the interactions of Cx43 with ZO-1/drebrin in prefrontal cortical astrocytes, but these interactions were oppositely modulated in hippocampal astrocytes. In conclusion, this study clarified the alternations of the Cx43 life cycle in the prefrontal cortical and hippocampal astrocytes exposed to CORT, which may contribute to our understanding of the mechanisms underlying stress-induced dysfunction of gap junctions.

Ginsenoside Rg1 alleviates corticosterone-induced dysfunction of gap junctions in astrocytes.

ETHNOPHARMACOLOGICAL RELEVANCE: Ginsenoside Rg1 (Rg1), one of the major bioactive ingredients of Panax ginseng C. A. Mey, has neuroprotective effects in animal models of depression, but the mechanism underlying these effects is still largely unknown AIM OF THE STUDY: Gap junction intercellular communication (GJIC) dysfunction is a potentially novel pathogenic mechanism for depression. Thus, we investigated that whether antidepressant-like effects of Rg1 were related to GJIC. MATERIALS AND METHODS: Primary rat prefrontal cortical and hippocampal astrocytes cultures were treated with 50µM CORT for 24h to induce gap junction damage. Rg1 (0.1, 1, or 10µM) or fluoxetine (1µM) was added 1h prior to CORT treatment. A scrape loading and dye transfer assay was performed to identify the functional capacity of gap junctions. Western blot was used to detect the expression and phosphorylation of connexin43 (Cx43), the major component of gap junctions. RESULTS: Treatment of primary astrocytes with CORT for 24h inhibited GJIC, decreased total Cx43 expression, and increased the phosphorylation of Cx43 at serine368 in a dose-dependent manner. Pre-treatment with 1µM and 10µM Rg1 significantly improved GJIC in CORT-treated astrocytes from the prefrontal cortex and hippocampus, respectively, and this was accompanied by upregulation of Cx43 expression and downregulation of Cx43 phosphorylation. CONCLUSION: These findings provide the first evidence indicating that Rg1 can alleviate CORT-induced gap junction dysfunction, which may have clinical significance in the treatment of depression.

Ginsenoside Rg1-induced antidepressant effects involve the protection of astrocyte gap junctions within the prefrontal cortex.

Ginsenoside Rg1 (Rg1) exhibits antidepressant-like activity by increasing neurogenesis and dendritic spine density without discernible side effects. However, the molecular mechanisms underlying Rg1 antidepressant activity remain poorly understood. As the dysfunction of gap junctions between astrocytes in the prefrontal cortex (PFC) is implicated in major depression disorder, the aim of this study was to investigate the effects of Rg1 on astrocyte gap junctions in the PFC. Rats exposed to chronic unpredictable stress (CUS) were administered Rg1 (5, 10, and 20mg/kg) for 28days and analyzed for depressive symptoms using the sucrose preference and forced swimming tests. Functional and morphological changes of gap junction channels in the PFC were evaluated using dye transfer and electron microscopy, respectively. The expression of connexin 43 (Cx43) was analyzed by western blotting. Rg1 markedly alleviated depression-like behavior in rats. Long-term Rg1 treatment of CUS-exposed rats also significantly prevented the decrease in dye diffusion and improved the ultrastructure of astrocyte gap junctions in the PFC, indicating beneficial effects on the functional activity of gap junction channels in the brain. In addition, Rg1 upregulated Cx43 expression in the PFC reduced by CUS exposure, which significantly correlated with its antidepressant-like effects. The results demonstrate that Rg1-induced antidepressant effects are might be mediated, in part, by protecting astrocyte gap junctions within the prefrontal cortex.

Bibenzyl compound 20c protects against endoplasmic reticulum stress in tunicamycin-treated PC12 cells in vitro.

AIM: Accumulation of α-synuclein (α-syn) in the brain is a characteristic of Parkinson's disease (PD). In this study, we investigated whether treatment with tunicamycin, an endoplasmic reticulum (ER) stress inducer, led to the accumulation of α-syn in PC12 cells, and where α-syn protein was accumulated, and finally, whether bibenzyl compound 20c, a novel compound isolated from Gastrodia elata (Tian ma), could alleviate the accumulation of α-syn and ER stress activation in tunicamycin-treated PC12 cells. METHODS: PC12 cells were treated with tunicamycin for different time (6 h, 12 h, 24 h, 48 h). Cell viability was determined by a MTT assay. Subcellular fractions of ER and mitochondria were extracted with the Tissue Endoplasmic reticulum Isolation Kit. The levels of α-syn protein and ER-stress-associated downstream chaperones were detected using Western blots and immunofluorescence. RESULTS: Treatment of PC12 cells with tunicamycin (0.5-10 µg/mL) dose-dependently increased the accumulation of α-syn monomer (19 kDa) and oligomer (55 kDa), and decreased the cell viability. Accumulation of the two forms of α-syn was observed in both the ER and mitochondria with increasing treatment time. Co-treatment with 20c (10-5 mol/L) significantly increased the viability of tunicamycin-treated cells, reduced the level of α-syn protein and suppressed ER stress activation in the cells, evidenced by the reductions in phosphorylation of eIF2α and expression of spliced ATF6 and XBP1. CONCLUSION: Tunicamycin treatment caused accumulation of α-syn monomer and oligomer in PC12 cells. Bibenzyl compound 20c reduces the accumulation of α-syn and inhibits the activation of ER stress, which protected PC12 cells against the toxicity induced by tunicamycin.

[Bioindicating function of sulfur in Haplocladium under heavy metals pollution by SRXRF and XANES].

Haplocladium was cultivated in a special prepared nutrient medium containing different concentrations of Pb, Fe and Cr in laboratory. The sulfur content in moss was measured by synchrotron radiation X-ray fluorescence (SRXRF), and the percentage of various oxidation states of sulfur was analyzed by X-ray absorption near-edge structure (XANES) spectrum. The results show that the sulfur absorption increases under exposure to heavy metal ions of Pb and Fe, but it decreases under exposure to 400 mg/L Pb and 200 mg/L Fe. When Haplocladium was cultivated for 15 days, under the stress of 100 mg/L Pb, the relative content of low oxidation states sulfur increases from 17.8% to 23.6% and the sulfate sulfur decreases from 56.3% to 51.2%. Under the stress of 400 mg/L Pb, the relative content of low oxidation state sulfur increases from 17.8% to 24.8%, and the sulfate sulfur decreases from 56.3% to 48.4%. Under heavy metal exposure, the total relative content of low oxidation states sulfur such as cystine, cysteine, methionine and glutathione increases, and the relative content of sulfate sulfur apparently decreases. All these results indicate that the changing characteristics of sulfur content and oxidation states percentage in sulfur assimilation process under heavy metal exposure can be used as a bioindicator of heavy metal pollution.

[The application of infrared spectra and clustering analysis in the discrimination of geographical origin of Flos Chrysanthemi].

OBJECTIVE: To identify the origin of Flos Chrysanthemi. METHODS: FTIR (Fourier transform infrared spectroscopy) and SIMCA (sofe independent modeling of class analogy). RESULTS: The correctness of every classification was higher than 60%. CONCLUSION: This approach was proved to be a reliable and practicable method for the discrimination of geographical origin of Chrysanthemum.