A network pharmacology study with molecular docking to investigate the possibility of licorice against posttraumatic stress disorder.

Post traumatic stress disorder (PTSD) is a mental health condition that has a debilitating effect on a person's quality of life and leads to a high socioeconomic burden. Licorice has been demonstrated to have neuroprotective and antidepressant-like effects, but little is known about its effects for the treatment of PTSD. The present study aimed to explore the potential of licorice for PTSD therapy using a network pharmacology approach with molecular docking studies. The compounds of licorice were obtained from databases with screening by absorption, distribution, metabolism and excretion (ADME) evaluation. Genes associated with compounds or PTSD were obtained from public databases, and the genes overlapping between licorice compounds and PTSD were compared by Venn diagram. A network of medicine-ingredients-targets-disease was constructed, visualized, and analyzed using cytoscape software. Protein-protein interactions, gene ontology, pathway enrichment and molecular docking were performed to evaluate the effect of licorice for the treatment of PTSD. 69 potential compounds were screened after ADME evaluation. A total of 81 compound-related genes and 566 PTSD-related genes were identified in the databases with 27 overlapping genes. Licorice compounds (e.g., medicarpin, 7-methoxy-2-methyl isoflavone, shinpterocarpin, formononetin, licochalcone a) and target proteins (e.g., ESR1, PTGS2, NOS2, and ADRB2) with high degree in the network were involved in G protein-coupled receptor signaling pathways at the postsynaptic/synaptic membrane. Moreover, neuroactive ligand-receptor interactions, calcium signaling, cholinergic synapse, serotonergic synapse and adrenergic signaling in cardiomyocytes may play important roles in the treatment of PTSD by licorice. This study provides molecular evidence of the beneficial effects of licorice for the treatment of PTSD.

Attenuated acute salivary α-amylase responses to gustatory stimulation with citric acid in thin children.

Salivary α-amylase (sAA) is responsible for the 'pre-digestion' of starch in the oral cavity and accounts for up to 50 % of salivary protein in human saliva. An accumulating body of literature suggests that sAA is of nutritional importance; however, it is still not clear how sAA is related to individual's nutritional status. Although copy number variations (CNV) of the salivary amylase gene (AMY1) are associated with variation in sAA levels, a significant amount of sAA variation is not explained by AMY1 CNV. To measure sAA responses to gustatory stimulation with citric acid, we used sAA ratio (the ratio of stimulated sAA levels to those of resting sAA) and investigated acute sAA responses to citric acid in children with normal (Normal-BMI, n 22) and low (Low-BMI, n 21) BMI. The AMY1 gene copy number was determined by quantitative PCR. We, for the first time, demonstrated attenuated acute sAA responses (decreased sAA ratio) to gustatory stimulation in Low-BMI (thinness grade 3) children compared with the Normal-BMI children, which suggest that sAA responses to gustatory stimulation may be of nutritional importance. However, child's nutritional status was not directly related to their resting or stimulated sAA levels, and it was not associated with AMY1 gene copy number. Finally, AMY1 CNV might influence, but did not eventually determine, sAA levels in children.

[Effect of citric acid stimulation on salivary alpha-amylase, total protein, salivary flow rate and pH value in Pi deficiency children].

OBJECTIVE: To compare the effect of citric acid stimulation on salivary alpha-amylase (sAA), total protein (TP), salivary flow rate, and pH value between Pi deficiency (PD) children and healthy children, thereby providing evidence for Pi controlling saliva theory. METHODS: Twenty PD children were recruited, and 29 healthy children were also recruited at the same time. Saliva samples from all subjects were collected before and after citric acid stimulation. The sAA activity and amount, TP contents, salivary flow rate, and pH value were determined and compared. RESULTS: (1) Citric acid stimulation was able to significantly increase salivary flow rate, pH value, sAA activities, sAA specific activity and sAA amount (including glycosylated and non-glycosylated sAA amount) in healthy children (P<0.05), while it could markedly increase salivary flow rate, pH value, and glycosylated sAA levels in PD children (P<0.05); (2) Although there was no statistical difference in determined salivary indices between the two groups (P>0.05), salivary indices except salivary flow rate and glycosylated sAA levels decreased more in PD children. There was statistical difference in sAA activity ratio, sAA specific activity ratio, and the ratio of glycosylated sAA levels between PD children and healthy children (P<0.05). CONCLUSION: PD children had decreased response to citric acid stimulation.

[Molecular phylogeny analysis of full-length vacA and cagA genes from Helicobacter pylori].

All amino acid full-length sequences of VacA and CagA proteins from Helicobacter pylori strains including vacA and cagA genes were downloaded from GenBank. Molecular phylogenic trees of VacA and CagA were constructed by ClastalX 2.0 and MEGA 5.05 software to understand phylogenetic relationships of vacA and cagA genes, clinical infection effects, and genotype characteristics of different clustering groups. The results showed that the phylogenetic trees of VacA and CagA recapitulated the same three-clustering groups, i.e., East Asia group 1 and 2 and Western group, and all H. pylori strains had similar distribution. The strains of East Asia group 1 were significantly higher in patients with atrophic gastritis. Genotype vacA contained mainly s1c/m1b and s1a/m1b, while genotype cagA was mostly EPIYA-ABD. The strains of East Asia group 2 were higher in patients with duodenal ulcer. Genotype vacA was mainly s1c/m2 and s1a/m2, while genotype cagA was mostly EPIYA-AB'C. The strains of Western group were higher in patients with duodenal ulcer and chronic gastritis than with atrophic gastritis. Genotype vacA was mainly s1a/m1a and s1b/m1a, while genotype cagA was mostly EPIYA-AB/B'CC. All of these results illustrated that there might be inheritant relationship of coevolution between vacA and cagA genes; East Asia group 1 and 2 and Western group had different vacA and cagA sub-genotypes, which had close rela-tionship to its clinical infection effects. It might be necessary to deeply analyze vacA and cagA sub-genotypes in the re-search of H. pylori-related diseases.

[Study on gene differential expressions of substance and energy metabolism in chronic superficial gastritis patients of Pi deficiency syndrome and of pi-wei hygropyrexia syndrome].

OBJECTIVE: To analyze the metabolic levels of energy and substance in chronic superficial gastritis (CSG) patients of Pi deficiency syndrome (PDS) and of Pi-Wei hygropyrexia syndrome (PWHS), including lipid, protein, nucleic acid, carbohydrate, trace element, and energy metabolism, and to study the pathogenesis mechanism of PDS from substance and energy metabolisms. METHODS: Recruited were 8 CSG patients who visited at First Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine and Guangdong Provincial Hospital of Traditional Chinese Medicine from June 2004 to March 2005, including 4 patients of PDS and 4 of PWHS. Their gastric mucosae were used for experiments of DNA microarray. The dual-channel DNA microarray data were bioinformatically analyzed by BRB ArrayTools and IPA Software. RESULTS: Obtained were fifty-six differentially expressed genes involved in substance and energy metabolisms with the expression fold more than 2, including 11 genes up-regulated and 45 genes down-regulated. Of them, genes correlated to lipid metabolism included CRLS1, LRP11, FUT9, GPCPD1, PIGL, SULT1A4, B3GNT1, ST8SIA4, and ACADVL, mainly involved in the metabolic processes of fatty acid, cholesterol, phospholipids, and glycolipid. Genes correlated to protein metabolism included ASRGL1, AARSD1, EBNA1BP2, PUM2, MRPL52, C120RF65, PSMB8, PSME2, UBA7, RNF11, FBXO44, ZFYVE26, CHMP2A, SSR4, SNX4, RAB3B, RABL2A, GOLGA2, KDELR1, PHPT1, ACPP, PTPRF, CRKL, HDAC7, ADPRHL2, B3GNT1, ST8SIA4, DDOST, and FUT9, mainly involved in the biosynthesis processes of protein, ubiquitination, targeted transport and post-translation modification. Genes correlated to nucleic acid metabolism included DFFB, FLJ35220, TOP2A, SF3A3, CREB3, CRTC2, NR1D2, MED6, GTF2IRD1, C1ORF83, ZNF773, and ZMYND11, mainly involved in DNA replication and repair, transcription regulation. Genes correlated to carbohydrate metabolism included AGL, B3GNT1, FUT9, ST8SIA4, SULT1A4, DDOST, and PIGL, mainly involved in glucogen degradation and glycoconjugate biosynthesis. Genes correlated to trace element metabolism included COMMD1, SLC39A6, FTL, CHRFAM7A, SCGN, and S100A6, mainly involved in ion metabolisms of copper, zinc, ferri, and calcium. Genes correlated to energy metabolism included AK3 and COX7B, mainly involved in mitochondria structure and oxidative phosphorylation processes. CONCLUSION: The metabolic levels of energy and substance including lipid, protein, nucleic acid, carbohydrate, and trace element were obviously reduced in patients of PDS, which might be an important pathogenesis mechanism for its occurrence.

Astragalus Polysaccharides Alleviate Type 2 Diabetic Rats by Reversing the Expressions of Sweet Taste Receptors and Genes Related to Glycolipid Metabolism in Liver.

Sweet taste receptors (STRs) play an important role in glucose metabolism, and type 2 diabetic rats have abnormal expressions of STRs in multiple tissues. Astragalus polysaccharides (APS) has shown a significant therapeutic effect on type 2 diabetes mellitus (T2DM), but its mechanism needs to be further clarified. T2DM rat model was induced by intraperitoneal streptozotocin injection and treated with APS for 8 weeks. Daily indicators of experimental rats were observed, and expression levels of STRs and genes related to glycolipid metabolism were determined by real-time quantitative PCR and western blot. The present study revealed that APS alleviated the symptoms of T2DM rats, improved HOMA-IR and promoted insulin secretion. Gene expression analysis found that APS significantly increased the expressions of signaling molecules in STRs pathways, including taste receptor family 1 member 2 (T1R2), α-gustducin (Gα) and transient receptor potential cation channel subfamily member 5 (TRPM5), and reversed the expressions of genes related to glucolipid metabolism, including glucose transporters 2 and 4 (GLUT2 and GLUT4), pyruvate carboxylase (PC), fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC) in the liver of T2DM rats. However, APS had no influences on the expressions of genes, including glycogen synthase kinase-3 beta (GSK-3ß), pyruvate kinase (PK) and phosphoenolpyruvate carboxykinase (PEPCK) in the liver of T2DM rats. These results suggested that the physiological roles of STRs in the liver were involved with glucose transport and metabolism. APS alleviated T2DM rats by activating the STRs pathway, and promoted glucose transport and lipogenesis.

Decreased basal and stimulated salivary parameters by histopathological lesions and secretory dysfunction of parotid and submandibular glands in rats with type 2 diabetes.

Patients with type 2 diabetes mellitus (T2DM) present with dry mouth, polydipsia and taste impairment due to salivary secretion disorder. However, the underlying functional mechanism of T2DM remains unknown. The present study found that T2DM rats had significantly lower salivary flow rate and salivary alpha amylase activity, and attenuated salivary secretion responses to acid stimulation compared with control rats. Histopathological observation found that T2DM rats had inflammatory cell infiltration with increased expressions of IL-6 and TNF-α, oxidative stress, including decreased total superoxide dismutase activity and increased malondialdehyde content, and decreased expressions of ß1 adrenergic receptor, cholinergic receptor, aquaporin-5 and protein kinase A in salivary glands, in particular the parotid gland. These results indicated that parotid gland impairment was more severe compared with submandibular gland impairment. Reduced salivary secretion may be associated with histopathological lesions and decreased regulation in secretory pathways in salivary glands.

Serum microRNA profiling and bioinformatics analysis of patients with type 2 diabetes mellitus in a Chinese population.

Type 2 diabetes mellitus (T2DM) is characterized by islet ß-cell dysfunction and insulin resistance, which leads to an inability to maintain blood glucose homeostasis. Circulating microRNAs (miRNAs) have been suggested as novel biomarkers for T2DM prediction or disease progression. However, miRNAs and their roles in the pathogenesis of T2DM remain to be fully elucidated. In the present study, the serum miRNA expression profiles of T2DM patients in Chinese cohorts were examined. Total RNA was extracted from serum samples of 10 patients with T2DM and five healthy controls, and these was used in reverse-transcription­quantitative polymerase chain reaction analysis with the Exiqon PCR system of 384 serum/plasma miRNAs. A total of seven miRNAs were differentially expressed between the two groups (fold change >3 or <0.33; P<0.05). The serum expression levels of miR­455­5p, miR­454­3p, miR­144­3p and miR­96­5p were higher in patients with T2DM, compared with those of healthy subjects, however, the levels of miR­409­3p, miR­665 and miR­766­3p were lower. Hierarchical cluster analysis indicated that it was possible to separate patients with T2DM and control individuals into their own similar categories by these differential miRNAs. Target prediction showed that 97 T2DM candidate genes were potentially modulated by these seven miRNAs. Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed that 24 pathways were enriched for these genes, and the majority of these pathways were enriched for the targets of induced and repressed miRNAs, among which insulin, adipocytokine and T2DM pathways, and several cancer­associated pathways have been previously associated with T2DM. In conclusion, the present study demonstrated that serum miRNAs may be novel biomarkers for T2DM and provided novel insights into the pathogenesis of T2DM.

Serum MicroRNA Profiling and Bioinformatics of Patients with Spleen-Deficiency Syndrome.

To investigate serum microRNA (miRNA) profile and bioinformatics of patients with spleen-deficiency syndrome (SDS) and explore pathogenesis of SDS patients from miRNA levels, 10 patients with type 2 diabetes mellitus (T2DM), within which 5 patients were with SDS and the remaining were with blood stasis syndrome (BSS), and 5 healthy volunteers were recruited. Serum miRNA profiles of SDS patients were identified by quantitative PCR array. Target prediction and functional annotation for miRNAs were performed by miRSystem database. The present study identified 11 candidate serum miRNAs for SDS patients, and their targets were significantly enriched in 18 KEGG pathways and 7 GO molecular functions. Those enriched KEGG pathways included (1) metabolisms of carbohydrate, protein, amino acid, and fatty acid, (2) signaling pathways of insulin, ErbB, chemokine, calcium, and type II diabetes mellitus, (3) invasions of bacterium, Escherichia coli, and Shigella (Shigellosis), and (4) endocytosis and phagocytosis. Those enriched GO molecular functions were mainly involved in transcription regulation and regulation of metabolism. Our findings might elucidate the pathogenesis of SDS patients with disorders of substance metabolism and hypoimmunity from miRNA levels, as well as providing some miRNA biomarkers for clinical syndrome differentiation of SDS.

The roles of AMY1 copies and protein expression in human salivary α-amylase activity.

Salivary α-amylase (sAA) activity has been extensively investigated in nutrition and psychology. But few studies were performed to assess the role played by sAA gene (AMY1) copies and protein expression in basal and stimulus-induced sAA activity. The sAA activity, amount and AMY1 copy number were determined from 184 saliva samples pre- and post-citric acid stimulation. Our findings showed that citric acid could induce significant increase in sAA activity, total sAA amount, and glycosylated sAA amount, among which the glycosylated sAA amount had the largest response. The correlation analysis showed that AMY1 copy number, total sAA amount and AMY1 copy number×total sAA amount had significantly positive and successively increasing correlations with sAA activity in unstimulated and stimulated saliva, respectively, and furthermore, we observed higher correlations in unstimulated saliva when compared with the corresponding correlations in stimulated saliva. We also observed significant correlations between glycosylated sAA amount and sAA activity in unstimulated and stimulated saliva, respectively. Interestingly, the correlations were higher in stimulated saliva than in unstimulated saliva, and the correlations between glycosylated sAA amount and sAA activity were higher than that of between total sAA amount and sAA activity in stimulated saliva. Moreover, total sAA amount ratio and glycosylated sAA amount ratio showed significantly positive correlation with sAA activity ratio. AMY1 copy number had no correlation with sAA activity ratio. These findings suggested that AMY1 copy number and sAA amount played crucial roles in sAA activity; however, the roles were attenuated after stimulation due to fortified release of glycosylated sAA.

Age Differences of Salivary Alpha-Amylase Levels of Basal and Acute Responses to Citric Acid Stimulation Between Chinese Children and Adults.

It remains unclear how salivary alpha-amylase (sAA) levels respond to mechanical stimuli in different age groups. In addition, the role played by the sAA gene (AMY1) copy number and protein expression (glycosylated and non-glycosylated) in sAA activity has also been rarely reported. In this study, we analyzed saliva samples collected before and after citric acid stimulation from 47 child and 47 adult Chinese subjects. We observed that adults had higher sAA activity and sAA glycosylated levels (glycosylated sAA amount/total sAA amount) in basal and stimulated saliva when compared with children, while no differences were found in total or glycosylated sAA amount between them. Interestingly, adults showed attenuated sAA activity levels increase over those of children after stimulation. Correlation analysis showed that total sAA amount, glycosylated sAA amount, and AMY1 copy number × total sAA amount were all positively correlated with sAA activity before and after stimulation in both groups. Interestingly, correlation r between sAA levels (glycosylated sAA amount and total sAA amount) and sAA activity decreased after stimulation in children, while adults showed an increase in correlation r. In addition, the correlation r between AMY1 copy number × total sAA amount and sAA activity was higher than that between AMY1 copy number, total sAA amount, and sAA activity, respectively. Taken together, our results suggest that total sAA amount, glycosylated sAA amount, and the positive interaction between AMY1 copy number and total sAA amount are crucial in influencing sAA activity before and after stimulation in children and adults.

Parallel increase in secretory activity between N-glycosylated and nonglycosylated α-amylase without protein synthesis after short-term ß-adrenergic receptor activation in isolated rat parotid acinar cells.

OBJECTIVE: To investigate comparisons of the secretory activity between N-glycosylated and nonglycosylated α-amylase, and α-amylase synthetic activity, after ß-adrenergic receptor activation in rat parotid acinar cells in vitro. STUDY DESIGN: Rat parotid acinar cells were incubated in the presence or absence of (-)-isoproterenol. For ß-adrenergic blocking experiments, acinar cells were pretreated with (±)-propranolol prior to adding agonist. After the time indicated, the "released amylase" and "total amylase" were obtained. Western blotting was applied to identify and quantify the N-glycosylated and nonglycosylated α-amylase. Amylase activity was also measured. RESULTS: The potent ß-adrenergic agonist (-)-isoproterenol induced a dramatic increase (2-3-fold) of α-amylase secretion for 30 minutes (p < 0.05 vs. control), while the effect was completely abolished when cells were pretreated with (±)-propranolol for 15 minutes. Moreover, the N-glycosylated level of released and total amylase among groups was measured accordingly. Our data showed the N-glycosylated level ratios (released amylase/total amylase) did not differ among groups, which indicated that the N-glycosylated form of α-amylase was not secreted more easily than the nonglycosylated one after stimulation. Interestingly, the total amylase concentration remained unchanged after stimulation within 30 minutes, which might indicate no α-amylase synthesized within the time indicated. CONCLUSION: Our findings suggest a parallel increase in secretory activity between N-glycosylated and nonglycosylated α-amylase after ß-adrenergic receptor activation. There seems to be a dissociation of α-amylase synthesis from secretion within 30 minutes.

Gene expression profiling in gastric mucosa from Helicobacter pylori-infected and uninfected patients undergoing chronic superficial gastritis.

Helicobacter pylori infection reprograms host gene expression and influences various cellular processes, which have been investigated by cDNA microarray using in vitro culture cells and in vivo gastric biopsies from patients of the Chronic Abdominal Complaint. To further explore the effects of H. pylori infection on host gene expression, we have collected the gastric antral mucosa samples from 6 untreated patients with gastroscopic and pathologic confirmation of chronic superficial gastritis. Among them three patients were infected by H. pylori and the other three patients were not. These samples were analyzed by a microarray chip which contains 14,112 cloned cDNAs, and microarray data were analyzed via BRB ArrayTools software and Ingenuity Pathways Analysis (IPA) website. The results showed 34 genes of 38 differentially expressed genes regulated by H. pylori infection had been annotated. The annotated genes were involved in protein metabolism, inflammatory and immunological reaction, signal transduction, gene transcription, trace element metabolism, and so on. The 82% of these genes (28/34) were categorized in three molecular interaction networks involved in gene expression, cancer progress, antigen presentation and inflammatory response. The expression data of the array hybridization was confirmed by quantitative real-time PCR assays. Taken together, these data indicated that H. pylori infection could alter cellular gene expression processes, escape host defense mechanism, increase inflammatory and immune responses, activate NF-κB and Wnt/ß-catenin signaling pathway, disturb metal ion homeostasis, and induce carcinogenesis. All of these might help to explain H. pylori pathogenic mechanism and the gastroduodenal pathogenesis induced by H. pylori infection.

Research on differentially expressed genes related to substance and energy metabolism between healthy volunteers and splenasthenic syndrome patients with chronic superficial gastritis / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To analyze the metabolic states of the lipids, protein, carbohydrate, and nucleic acid for chronic superficial gastritis patients of splenasthenic syndrome (SS), and to explore the pathogenesis mechanism of SS based on substance and energy metabolisms.</p><p><b>METHODS</b>During June 2004 to March 2005, recruited were four chronic superficial gastritis patients of SS who visited at the First Hospital of Guangzhou University of Chinese Medicine and Guangdong Provincial Hospital of Traditional Chinese Medicine. Four healthy volunteers were recruited from Guangzhou University of Chinese Medicine. Their gastric mucosa was extracted to perform experiments of DNA microarray. The dual-channel DNA microarray data were mined and bioinformatics analyzed by BRB ArrayTools and IPA software.</p><p><b>RESULTS</b>Fifteen genes were involved in substance and energy metabolisms in 20 differentially expressed genes, accounting for 75%.Among these genes, one gene was up-regulated, 14 genes down-regulated, and 11 genes were enzyme gene. Differentially expressed genes related to lipid metabolism included ACAA2 and CYP20A1, manifested as fatty acid catabolism and cholesterol transformation. Genes related to protein metabolism included ALDH9A1, ASL, ASS1, PCY-OX1L, RPS28, UBE2D2, UBXN1, B3GNT1, GCNT1, and PPP1R3C, manifested as decreased amino acid metabolism that may affect the biologic processes such as autonomic nerve, urea cycle, etc., reduced protein synthesis, increased ubiquitination of fault fold proteins, and decreased post-translated modification of glycosylation and dephosphorylation. Genes related to carbohydrate metabolism included PPP1R3C, B3GNT1, and GCNT1, manifested as decreased glycogen and glycan syntheses. Genes related to nucleic acid metabolism included RMI1, SMARCD3, and PARP1, manifested as degraded DNA duplication and transcription, and increased DNA damage repair.</p><p><b>CONCLUSIONS</b>The metabolisms of the lipids, protein, carbohydrate, and nucleic acid in chronic superficial gastritis patients of SS obviously decreased, manifested mainly as down-regulated enzyme gene expression. We inferred that these might be one of the vital pathogenesis mechanisms for nutrition dysmetabolism of SS.</p>

[Dynamic change of ATPase activity on amyloplasts and protein bodies during the endosperm development in rice (Oryza sative L.)].

The ultracytochemical localizations of ATPase activity on amyloplasts and protein bodies were studied by using a lead precipitation technique at middle and late developmental stage of endosperm in rice (Oryza sativa L. cv. Minghui 63). The results showed that the inner and outer plasma membrane of amyloplasts, the web-like passages among starch grains and the amorphous substance surrounding the amyloplasts exhibited high ATPase activity. ATPase activity products were present on the membranes of protein body I and protein body II, and on the vacuoles and vesicle-like structures surrounding protein bodies. Active products of ATPase were also located on the plasma membrane and cell wall of starchy endosperm cells, on the cell wall, plasma membrane, nucleus and plasmodesma of aleurone and subaleurone layer cells. According to the distribution pattern of active products of ATPase, we infer that the web-like passage in amyloplast may be the transporting channel for nutrients flowing into the amyloplast. The ATPase on amyloplasts and protein bodies can supply the power for nutrients passing through the plasma membranes.

[The starchy endosperm denucleation by a process of programmed cell death during rice grain development].

The cellular ultrastructural morphology, grain filling ratio and activity of related enzymes during denucleation development stage in starchy endosperm cell of an Indica rice variety Zhongxian 8836 were observed and analyzed. The endosperm cellularization was completed at about the 3rd day after flowering (DAF). At 5 DAF a few endosperm cells initiated denucleation development. At 13 DAF almost all the starchy endosperm cells completed their denucleation. The nuclear degradation is the first stage of programmed cell death (PCD) in starchy endosperm. It occurred unsynchronously among different starchy endosperm cells. The nuclear degradation of starchy endosperm cell during denucleation development stage showed not only morphological features commonly observed in animal and plant PCD, but also some unique characteristics. Mitochondria degeneration was observed along with nuclear degradation, indicating there were interrelations between the two processes. Enzymes related to PCD, such as super-oxide dismutases (SOD) and catalase (CAT), as well as enzymes related to starch synthesis, such as ADP-glucose pyriphosphorylase (AGP), soluble starch synthase (SSS) and starch branching enzyme or Q-enzyme, showed very high activity during the denucleation development stage. Maximum grain filling rate and grain weight increase were also achieved in the denucleation developing stage of most starch endosperm cells. The denucleated cell remained alive in the developing endosperm, keeping its normal metabolisms, the synthesis and accumulation of starch and storage proteins. However, enzyme activities and grain filling rate were apparently dropped to a lower level after denucleation. The starchy endosperm cell finally completed its PCD process when it was completely filled with reserves. Evan's blue staining indicated that cell death occurred unsynchronously among the starchy endosperm cells with initiation points randomly distributed in the endosperm tissue.