swnk plays an important role in the biosynthesis of swainsonine in Metarhizium anisopliae.

OBJECTIVE: Swainsonine (SW) is the principal toxic ingredient of locoweeds, and is produced by multiple fungi. A key enzyme in the SW synthesis pathway is a hybrid swnk/nrps. To analyze the role of swnk in the SW biosynthesis pathway of Metarhizium anisopliae. RESULTS: The concentration of SW and the swnk expression in M. anisopliae fermentation from 1st to 7th day were determined using LC-MS and RT-qPCR, respectively. M. anisopliae had the highest SW content and swnk expression on the 5th day of fermentation; Mutant strain (MT) were obtained by PEG-mediated homologous recombination (HR) which knocked out swnk in the wild-type (WT) strain. Complemented-type (CT) strain were obtained by transforming a modified PUC19 complementation vector containing the geneticin (G418) resistance gene and swnK. SW was not detected in the MT strain and reverted to its original level in the CT strain; A Psilent-1 plasmid with Benomyl (ben)-resistant that was used interfered with swnk of WT strain. The level of SW was markedly diminished in the RNAi strain. RNAi of swnk affects the formation of the cell wall in M. anisopliae. CONCLUSION: These results indicate that swnk plays a crucial role in the SW biosynthesis of M. anisopliae.

Swainsonine-induced vacuolar degeneration is regulated by mTOR-mediated autophagy in HT22 cells.

The indolizidine alkaloid, swainsonine (SW), is the main toxic component of locoweed, which can cause locoism in animals with characteristic neurological dysfunction. Pathological manifestations at cellular level include extensive vacuolar degeneration. Studies have shown that SW can induces autophagy, but the role and mechanism of autophagy in SW-induced vacuolar degeneration is unclear. In this study, we analyzed the role of autophagy in SW-induced cell injury in mouse hippocampal neurons cell line (HT22) using western blotting, qRT-PCR, transmission electron microscopy and immunofluorescence microscopy. The results showed that the expressions of LC3-II, ATG5, Beclin1 and p62 proteins and their mRNAs in HT22 cells were induced by SW treatment. The SW treatment increased the number of autophagosomes with enhanced fluorescence intensity of monodansylcadaverine (MDC) and LC3-II in a time-dose dependent manner. The results of lysosome staining showed that SW could increase the number of lysosomes, increase the intraluminal pH. Transmission electron microscopy results indicate that SW induced autophagosomes, and Baf A1 could effectively alleviate SW-induced vacuolar degeneration. At the molecular level, SW treatment inhibited the expression of p-PI3K, p-AKT, p-ERK, p-AMPK, p-mTOR, p-p70S6K and p-4EBP1 and promoted the expression of p53. Our results collectively suggest, PI3K/AKT/mTOR, ERK/mTOR and p53/mTOR signaling pathways are involved in the regulation of SW-induced autophagy in HT22 cells, while the AMPK/mTOR signaling pathway is not involved in this regulation. Inhibition of autophagic degradation can effectively alleviate SW-induced vacuolar degeneration.

Host-Species Variation and Environment Influence Endophyte Symbiosis and Mycotoxin Levels in Chinese Oxytropis Species.

Oxytropis plants are widely distributed in the grasslands in northern China. Some Oxytropis species have been reported to contain the mycotoxin swainsonine, an alkaloid which causes poisoning in livestock, referred to as locoism. Previous studies showed that endophytic fungi (Alternaria oxytropis) symbiotically associate with these Oxytropis species to produce swainsonine. However, the influence of variation within the Oxytropis genus on the fixation or loss of symbiosis and toxicity is poorly understood, as is the influence of environmental factors. Here we used a collection of 17 common Oxytropis species sampled in northern China to assess genetic diversity using genotyping by sequencing which was compared with the levels of the endophyte and swainsonine. Results showed that nine Oxytropis species have detectable A. oxytropis colonisation, and seven Oxytropis species contain sufficient swainsonine to be considered poisonous, whereas the rest may be non-toxic. Species variation rather than the genetic lineage was associated with the fixation or loss of endophyte and swainsonine production, which appears to have resulted from genetic drift. Genotype × Environment (G × E) effects were also found to influence endophyte and swainsonine levels amongst species of the Oxytropis genus. Our study will provide a better understanding about the evolutionary basis of A. oxytropis symbiosis and swainsonine biosynthesis in locoweeds.

CHOP Regulates Endoplasmic Reticulum Stress-Mediated Hepatoxicity Induced by Monocrotaline.

Monocrotaline (MCT), a pyrrolizidine alkaloid, is the major toxin in Crotalaria, which causes cell apoptosis in humans and animals. It has been reported that the liver is a vulnerable target of MCT. However, the exact molecular mechanism of the interaction between endoplasmic reticulum (ER) stress and liver injury induced by MCT is still unclear. In this study, the cytotoxicity of MCT on primary rat hepatocytes was analyzed by a CCK-8 assay and Annexin V-FITC/PI assay. Protein expression was detected by western blotting and immunofluorescence staining. As a result, MCT significantly decreased the cell viability and mediated the apoptosis of primary rat hepatocytes. Meanwhile, MCT could also induce ER stress in hepatocytes, indicated by the expression of ER stress-related proteins, including GRP78, p-IRE1α, ATF6, p-eIF2α, ATF4, and CHOP. Pretreatment with 4-PBA, an inhibitor of ER stress, or knockdown of CHOP by siRNA could partly enhance cell viability and relieve the apoptosis. Our findings indicate that ER stress is involved in the hepatotoxicity induced by MCT, and CHOP plays an important role in this process.

The PERK/eIF2α/ATF4/CHOP pathway plays a role in regulating monocrotaline-induced endoplasmic reticulum stress in rat liver.

Monocrotaline (MCT) belongs to the category of Pyrrolizdine Alkaloids (PAs), which is one of important hepatotoxic alkaloid in Crotalaria Lin. Apoptosis is one mechanism of toxic responses induced by MCT. However, the underlying mechanism of liver apoptosis caused by MCT through Endoplasmic reticulum (ER) stress continues to be incompletely understood. In this study, we describe the role of ER stress in MCT induced hepatotoxicity in rats. 24 male rats were randomly divided into 3 groups: normal saline group, 45 mg/kg MCT group and 90 mg/kg MCT group. After 48 h of saline/MCT administration, the livers were collected for analysis of ER stress-related proteins by Western blotting. The expression of GRP78, p-IRE1α, ATF6 and caspase-12 showed a dose-dependent increase. PERK/eIF2α/ATF4/CHOP pathway is one of the major ER stress pathways which is required for cell survival. Therefore, through analyzing the effects of MCT on this pathway, we found the protein levels of p-PERK, p-eIF2α, ATF4 and CHOP were increase obviously. All these results indicate that MCT induces ER stress in rat liver. The PERK/eIF2α/ATF4/CHOP pathway is involved in the regulation of MCT-induced ER stress in the liver of rat.

[Extraction of spectral difference characteristics of Stellera chamaejasme in Qilian County of Qinghai Province, Northwest China].

Stellera chamaejasme is one of the main poisonous weeds distributed in alpine meadow of Qinghai Province. Rapid spreading of S. chamaejasme has done serious harm to local animal husbandry and caused continuous grassland ecosystem degradation. This paper focused on the spectral differences between S. chamaejasme and herbage, taking the typical degraded alpine meadow dominated by S. chamaejasme in Qilian County of Haibei Region as the test site and using the spectral measurements acquired in the full-blossom period of S. chamaejasme from 2012 to 2014. The results showed that the spectral behavior of flowers of S. chamaejasme differed significantly from green background that included leaves of S. chamaejasme and herbage within 350-900 nm of VIS-NIR wavebands. The biggest reflectance difference between flowers of S. chamaejasme and green background was located in the red valley, followed by the blue valley. The reflectance of S. chamaejasme community increased with the rising of coverage, the biggest reflectance difference between S. chamaejasme and herbage communities lied in the near-infrared peak, and the best separability between S. chamaejasme communities with different coverage was also at the point. The difference of first derivative spectra between flowers of S. chamaejasme and green background located in amplitude of yellow edge was remarkable, followed by amplitude of blue edge, the same as differences between S. chamaejasme and herbage communities. Linear regression analysis between coverage of S. chamaejasme and spectral feature parameters showed best result for red valley (R2 = 0.94). Finally, the red valley, the blue valley and the near-infrared peak were proposed for discriminating S. chamaejasme from herbage in the full-blossom period of S. chamaejasme, and the combination of corresponding red, blue and near-infrared bands could be used to build sensitive indices for S. chamaejasme recognition.