Chemical composition analysis and transcriptomics reveal the R2R3-MYB genes and phenol oxidases regulating the melanin formation in black radish.

Melanins are dark-brown to black-colored biomacromolecules which have been thoroughly studied in animals and microorganisms. However, the biochemical and molecular basis of plant melanins are poorly understood. We first characterized melanin from the black radish (Raphanus sativus var. niger) 'HLB' through spectroscopic techniques. p-Coumaric acid was identified as the main precursor of radish melanin. Moreover, a joint analysis of transcriptome and coexpression network was performed for the two radish accessions with black and white cortexes, 'HLB' and '55'. A set of R2R3-type RsMYBs and enzyme-coding genes exhibited a coexpression pattern, and were strongly correlated with melanin formation in radish. Transient overexpression of two phenol oxidases RsLAC7 (laccase 7) or RsPOD22-1 (peroxidase 22-1) resulted in a deeper brown color around the infiltration sites and a significant increase in the total phenol content. Furthermore, co-injection of the transcriptional activator RsMYB48/RsMYB97 with RsLAC7 and/or RsPOD22-1, markedly increased the yield of black extracts. Spectroscopic analyses revealed that these extracts are similar to the melanin found in 'HLB'. Our findings advance the understanding of structural information and the transcriptional regulatory mechanism underlying melanin formation in radish.

Deciphering the Molecular Signatures Associated With Resistance to Botrytis cinerea in Strawberry Flower by Comparative and Dynamic Transcriptome Analysis.

Gray mold caused by Botrytis cinerea, which is considered to be the second most destructive necrotrophic fungus, leads to major economic losses in strawberry (Fragaria × ananassa) production. B. cinerea preferentially infects strawberry flowers and fruits, leading to flower blight and fruit rot. Compared with those of the fruit, the mechanisms of flower defense against B. cinerea remain largely unexplored. Therefore, in this study, we aimed to unveil the resistance mechanisms of strawberry flower through dynamic and comparative transcriptome analysis with resistant and susceptible strawberry cultivars. Our experimental data suggest that resistance to B. cinerea in the strawberry flower is probably regulated at the transcriptome level during the early stages of infection and strawberry flower has highly complex and dynamic regulatory networks controlling a multi-layered defense response to B. cinerea. First of all, the higher expression of disease-resistance genes but lower expression of cell wall degrading enzymes and peroxidases leads to higher resistance to B. cinerea in the resistant cultivar. Interestingly, CPKs, RBOHDs, CNGCs, and CMLs comprised a calcium signaling pathway especially play a crucial role in enhancing resistance by increasing their expression. Besides, six types of phytohormones forming a complex regulatory network mediated flower resistance, especially JA and auxin. Finally, the genes involved in the phenylpropanoid and amino acids biosynthesis pathways were gene sets specially expressed or different expression genes, both of them contribute to the flower resistance to B. cinerea. These data provide the foundation for a better understanding of strawberry gray mold, along with detailed genetic information and resistant materials to enable genetic improvement of strawberry plant resistance to gray mold.

Genetic Loci Conferring Reducing Sugar Accumulation and Conversion of Cold-Stored Potato Tubers Revealed by QTL Analysis in a Diploid Population.

Cold-induced sweetening (CIS) caused by reducing sugar (RS) accumulation during storage in low temperature in potato tubers is a critical factor influencing the quality of fried potato products. The reconditioning (REC) by arising storage temperature is a common measure to lower down RS. However, both CIS and REC are genotype-dependent and the genetic basis remains uncertain. In the present study, with a diploid potato population with broad genetic background, four reproducible QTL of CIS and two of REC were resolved on chromosomes 5, 6, and 7 of the CIS-sensitive parent and chromosomes 5 and 11 of the CIS-resistant parent, respectively, implying that these two traits may be genetically independent. This hypothesis was also supported by the colocalization of two functional genes, a starch synthesis gene AGPS2 mapped in QTL CIS_E_07-1 and a starch hydrolysis gene GWD colocated with QTL REC_B_05-1. The cumulative effects of identified QTL were proved to contribute largely and stably to CIS and REC and confirmed with a natural population composed of a range of cultivars and breeding lines. The present research identified reproducible QTL for CIS and REC of potato in diverse conditions and elucidated for the first time their cumulative genetic effects, which provides theoretical bases and applicable means for tuber quality improvement.

[Effects of aconitine poisoning and Shuang-huang-lian treatment on the expression of myocardial ryanodine receptor].

OBJECTIVE: To study the impact of aconitine and Shuang-huang-lian injection on the expression of myocardial ryanodine receptor (RyR2) and to discuss the anti-arrhythmic mechanisms of Shuanghuanglian against aconitine poisoning.
 METHODS: A total of 52 SD rats were randomly divided into 3 groups: aconitine group (group A, n=20), Shuang-huang-lian group (group B, n=20) and control group (group C, n=12). Aconitine at a dose of 0.3 mg/kg was firstly administrated to the rats of group A and group B through gavage. 30 minutes later, normal saline was delivered to rats in group A through intraperitoneal injection, while rats in group B received Shuang-huang-lian at a dose of 100 mg/kg through intraperitoneal injection. Group C accepted twice administration of normal saline by gavage and intraperitoneal injection. The disposals for each group were implemented for 10 consecutive days. The rats' electrocardiograms (ECG) were recorded on day 1, 3, 6 and 10. Immunofluorescent staining technique and real-time quantitative PCR technique was used to detect the protein and mRNA expression of myocardial RyR2.
 RESULTS: The arrhythmia scores of group B at different time were lower than group A and the difference was statistically significant (P<0.05 or P<0.01). The protein and mRNA expression of RyR2 in group A was slightly increased on the first day compared to group B and group C, with no statistical significance (P>0.05). On day 3, 6 and 10, the expression of RyR2 protein and mRNA in group A was dramatically increased compared to group B and group C (P<0.01). 
 CONCLUSION: Shuang-huang-lian injection can effectively prevent the arrhythmia caused by aconitine, which is related to suppressing the aconitine-induced RyR2 expression.

[Effects of Chaihu Shugan San on behavior and plasma levels of corticotropin releasing hormone and adrenocorticotropic hormone of rats with chronic mild unpredicted stress depression].

OBJECTIVE: To investigate the effects of Chaihu Shugan San (CHSGS), a compound traditional Chinese herbal medicine, on behavior and plasma levels of corticotropin releasing hormone (CRH) and adrenocorticotropic hormone (ACTH) of rats with chronic mild unpredicted stress depression. METHODS: Forty male Sprague-Dawley rats were randomly divided into 4 groups: normal control group, untreated group, fluoxetine group and CHSGS group. Except the normal control group, rats were singly housed and exposed to an unpredicted sequence of mild stressor for continuous 4 weeks to induce depression. Since the fifteenth day, rats were intragastrically administered with equal volume agents respectively for 2 weeks [normal saline for the normal control group and the untreated group, fluoxetine (1.8 mg/kg) for the fluoxetine group and CHSGS (5.9 g/kg) for the CHSGS group]. Behavioral scores of rats were detected by open-field test and sucrose preference test, and the plasma levels of CRH and ACTH in different groups were detected by radioimmunoassay. RESULTS: Compared with the normal control group, body weights of the rats in the untreated group were significantly decreased. Scores of crossing, rears and grooming in open-field test were reduced significantly. Pure water consumption in sucrose preference test was increased significantly. The levels of plasma CRH and ACTH were significantly increased. The depressive behaviors of the rats were improved significantly and the levels of plasma CRH and ACTH were obviously reduced in the CHSGS group. CONCLUSION: Chronic mild unpredicted mild stress can affect the neuroendocrine and behavior and cause depression in rats. CHSGS can regulate HPA hyperactivity of rats caused by chronic stress and has antidepressive effects.

[Clinical experience of successful management of a human case with A/H5N1 infection in Hunan Province].

OBJECTIVE: To study the clinical features and successful management of a survived human case with A/H5N1 infection. METHODS: The data of a confirmed case of human case with A/H5N1 infection in Hunan province were collected and analyzed. RESULTS: This patient with A/H5N1 infection in Hunan province was confirmed by reverse-transcription polymerase chain reaction (RT-PCR) for A/H5N1 in airway secretions. The main clinical presentations included fever, cough and dyspnea. An extensive pulmonary infiltration developed quickly, followed by multi-organ dysfunction. Early administration of oseltamivir, early protection of organ function and extensive support were effective for the cure of the disease in this case. CONCLUSION: Early administration of oseltamivir, early protection of organ function and adequate support therapy may be useful for the treatment of human A/H5N1 infection.

[Correlation between serum MMP-2 and MMP-9 in patients with acute myocardial infarction before and after PCI].

AIM: To investigate the correlation between serum MMP-2 and MMP-9 in patients with acute myocardial infarction (AMI) before and after PCI. METHODS: The enzymatic activity and protein expression level of serum MMP-2 and MMP-9 in 56 patients with AMI and 20 control subjects were detected by the SDS-PAGE enzymograph and Western blot before and 1-7 days after PCI. The correlation between MMP-2 and MMP-9 was also analyzed. RESULTS: The enzymatic activity and protein expression level of serum MMP-2 and MMP-9 in 56 patients with AMI were both significantly higher than those in the control subjects before and after PCI (P<0.01). The enzymatic activity and protein expression level of serum MMP-2 and MMP-9 in patients with AMI began to increase on the first day and were at peak on the third day (P<0.01). They began to decrease on the fifth day after PCI (P<0.05). The enzymatic activity of serum MMP-2 was correlated positively with that of MMP-9 (P<0.01). The protein expression level of serum MMP-2 was also correlated positively with that of MMP-9 (P<0.01). CONCLUSION: The serum MMP-2 and MMP-9 may be related to inflammatory injuries after myocardial ischemia and reperfusion. MMP-2 can be regarded as an important marker of inflammation as MMP-9 in the pathogenesis of AMI.

[Mechanism and protective effect of glossy ganoderma decoction on the activities of RNA polymerase in hepatocyte of rabbits with Amanita mushroom poisoning].

OBJECTIVE: To investigate the mechanism of glossy ganoderma decoction in Amanita mushroom poisoning. METHODS: Twenty male New Zealand white rabbits were randomly divided into 4 groups, including a normal control, a model poison group, and 2 treatment groups (different doses of glossy ganoderma decoction). The activities of hepatocyte RNA polymerase were measured by ultraviolet spectrophotometry and liver function were measured. RESULTS: The activities of hepatocyte RNA polymerase of the model group significantly decreased, and those of the 2 treatment groups were significantly higher than those of the model group. There was a dose-dependent manner between the 2 treatment groups ( all Ps<0.01), and the differences of liver function test including total bilirubin (TBIL), direct bilirubin (DB), total bile acid (TBA), and alanine aminotransferase (ALT) in the 4 groups were significant (P<0.01). CONCLUSION: Glossy ganoderma decoction may protect the liver from Amanita mushroom poisoning. Its mechanism may be related to the increase of the activities of hepatocyte RNA polymerase.

Clinical experience in treatment of Amanita mushroom poisoning with Glossy Ganoderma Decoction and routine Western medicines.

OBJECTIVE: To assess the effects of treatment of Amanita mushroom poisoning with Glossy anoderma Decoction (, GGD). METHODS: Twelve patients with acute Amanita mushroom poisoning received conventional treatment (penicillin and reduced glutathione) combined with oral administration of GGD (treated group), which was prepared out of 200 g Glossy ganoderma decocted in water to 600 mL, and 200 ml was given once, three times a day for 7 successive days; while conventional treatment alone was given to the other 11 patients assigned to the control group. The therapeutic efficacy and changes in serum levels of total bilirubin (TBil), bile acids (BA), alanine transaminase (ALT), and aspartate transaminase (AST) activities in the two groups were compared. RESULTS: The cured-markedly effective rate in the treated group was more significant than that in the control group (P<0.01). Elevation in TBil, BA, ALT, and AST activities were observed in both groups 3 days after poisoning, which progressively increased thereafter in the control group. In the treated group, they reached their peak on the 3rd day and then declined gradually. The differences between pre-treatment and post-treatment in both groups were obviously significant (P<0.01), so were the differences between the two groups at corresponding time points (P<0.01). CONCLUSION: GGD shows excellent clinical efficacy in the treatment of acute Amanita mushroom poisoning and can reduce mortality significantly.

[Clinical observation on treatment of Russula subnigricans poisoning patients by Ganoderma lucidum decoction].

OBJECTIVE: To observe the effect of Ganoderma lucidum decoction in treating Russula subnigricans poisoning (RSP) patients. METHODS: The 14 patients of RSP in the treated group were treated with GLD (GLD, one dose was prepared by 100 g of Ganoderma lucidum decocted with water to 600 ml), on the base of conventional treatment, and 11 patients received conventional therapy in the previous year were taken as control. The clinical efficacy and parameters in them were compared, including the urine N-acetyl-D-glucosaminidase (NAG, which reflects the injury of kidney), the red blood cell and protein in urine, the alanine transaminase (ALT, which reflects the injury of liver), and the aspartate aminotransferase (AST, which reflects the injury of heart). RESULTS: A better clinical cure-markedly improving rate was showed in the treated group as compared with the control group, P < 0.01. In the treated group, red blood cell in urine disappeared after 24 hrs treatment in the majority of patients, urinary protein reduced obviously and the other three parameters reached the peak at the 3rd day then lowered gradually. In the control group, all the parameters increased continuously. Comparison between the parameters at corresponding time in the two groups showed significant difference (P < 0.01), those in the treated group were markedly lower than those in the control group respectively. CONCLUSION: GLD has good effect in treating RSP, could obviously lower the fatat rate of RSP.

Multiple organ dysfunction syndrome due to ingestion of fish gall bladder.

OBJECTIVE: To investigate changes in renal function, urine N-acetyl-beta-D-glucosaminidase enzyme (N-AG),liver function, myocardial enzymes, the pathology of renal damage and the mechanism of acute renal failure (ARF) associated with fish gall bladder poisoning. METHODS: Eleven patients with acute fish gall bladder poisoning were consecutively admitted to our hospital from September 1997 to October 1999. Renal function, urine N-AG enzyme, liver function, and myocardial enzymes were assayed before and after treatment. One patient consented to a kidney biopsy and the pathology of renal damage was observed under light and electron microscopes. RESULTS: All patients had multiple organ dysfunction syndrome (MODS) and 11 patients suffered from ARF. Ten patients had liver dysfunction, ten patients had poisonous myocarditis, and 8 patients had gastrointestinal dysfunction. Renal function, urine N-AG enzyme, liver function, and myocardial enzymes were significantly improved after treatment compared with those of before treatment (P < 0.05). Kidney biopsy showed that the main damage site was the proximal renal tubule. All eleven patients recovered and were discharged from the hospital. CONCLUSIONS: Ingestion of fish gall bladder leads to kidney damage, as well as liver, heart and gastrointestinal tract injury. The mechanism of acute renal function failure is the serious tubular damage, confirming the location of kidney damage. Necrosis of the proximal tubules plays an important role in the development of ARF. Immediate hemodialysis is the most effective treatment.