Effects of dietary Ginkgo biloba leaf extract on growth performance, immunity and environmental stress tolerance of Penaeus vannamei.

Ginkgo biloba leaf extract (GBE) has been extensively used in the treatment of diseases due to its anti-inflammatory, antioxidant, and immunomodulatory effects. In aquaculture, GBE is widely used as a feed additive, which is important to enhance the immunity of aquatic animals. The current study evaluated the effects of adding GBE to the diet of Penaeus vannamei (P. vannamei) under intensive aquaculture. The GBE0 (control group), GBE1, GBE2, and GBE4 groups were fed a commercial feed supplemented with 0.0, 1.0, 2.0, and 4.0 g/kg GBE for 21 days, respectively. The results showed that dietary GBE could alleviate hepatopancreas tissue damage and improve the survival rate of shrimp, and dietary 2 g/kg GBE could significantly increase the total hemocyte count (THC), the hemocyanin content, the antioxidant gene's expression, and the activity of their encoded enzymes in P. vannamei. Furthermore, transcriptome data revealed that immunity-related genes were upregulated in the GBE2 group compared with the GBE0 group after 21 days of culture. Drug metabolism-cytochrome P450, sphingolipid metabolism, linoleic acid metabolism, glycerolipid metabolism, fat digestion and protein digestion and absorption pathways were significantly enriched, according to KEGG results. Surprisingly, all of the above KEGG-enriched pathways were significantly upregulated. These findings demonstrated that supplementing P. vannamei with 2 g/kg GBE improved its environmental adaptability by improving immunity, lipid metabolism, and detoxification. In this study, a comprehensive evaluation of the effects of dietary GBE on the intensive aquaculture of P. vannamei was conducted to provide a reference for the healthy culture of P. vannamei.

Health Education and Nursing Needs of Breast Cancer Patients With Totally Implantable Venous Access Ports at Different Stages.

Context: The totally implantable venous access port (TIVAP) is an intravenous-infusion device, with a lower complication rate than other such devices. If patients fail to maintain the catheter, however, complications can still occur. Patients' needs may vary by the period of the port's use. Objective: The study intended to explore the differences in the needs of breast-cancer (BC) patients with TIVAPs for health education and nursing care at different periods of the port's use and to determine the kinds of targeted health education that can improve patients' quality of life. Design: The research team designed a questionnaire that the participants completed. Setting: The study took place at the Breast Center at the Fourth Hospital of Hebei Medical University in Shijiazhuang, China. Participants: Participants were 442 BC patients at the hospital between March and June 2020, who had TIVAPs at different stages. Groups: The study included three groups: (1) the preoperative group-participants in the preoperative period prior to the TIVAP implantation after they had signed a consent; (2) the chemotherapy group-participants in the chemotherapy period during the TIVAP's use for chemotherapy-agent transfusion, and (3) the maintenance group-participants in the maintenance period during which the TIVAD was in place but wasn't being used. Outcome Measures: The research team analyzed the results from the questionnaires, categorizing them as: (1) methods of knowledge acquisition, (2) methods of distribution of knowledge, (3) needs of participants in the different groups, and (4) distribution of symptoms among the groups. Results: Compared to other methods, the nursing staff was the main source that participants used to access the TIVAP-related information at different periods: preoperative group (79.6%), chemotherapy group (90.7%), and maintenance group (90.2%).The differences between the periods were statistically significant (P = .00). A traditional mode of education-the medical staff's explanations-was the most common in all groups: preoperative group (79.6%), chemotherapy group (83.3%), and (3) maintenance group (80.7%). Patients wanted new modes of receiving information: talks, a poster, and a medical system. TIVAP patients paid different amounts of attention to educational contents at the different stages (χ2 = 29.816, P = .00). Conclusions: BC patients' needs for health education and nursing vary at different stages when using TIVAPs. Nurses are the main source of knowledge about TIVAP in different periods for BC patients, and the nurses should obtain multidisciplinary health knowledge to enhance the benefits of the education for patients. The current education for patient is traditional, and hospitals need to implement new modes of education such as medical systems and network platforms, lectures, and posters for health education.

Andrographis paniculata improves growth and non-specific immunity of shrimp Litopenaeus vannamei, and protects it from Vibrio alginolyticus by reducing oxidative stress and apoptosis.

Andrographis paniculata (AP) is a traditional medicinal plant with many pharmacological activities, including anti-inflammatory, antimicrobial, immunity stimulation and so on. Several studies have reported that AP plays a strong role in promoting the immune system of aquatic animals to resist several pathogens. In the present study, we investigate the effects of a diet containing AP on the immune responses, growth, and the resistance to Vibrio alginolyticus (V. alginolyticus) in Litopenaeus vannamei (L. vannamei). Four diets were formulated by adding AP at the dosage of 0% (Control), 0.25%, 0.5%, and 1% in the basal diet, respectively. Each diet was randomly fed to one group with three replicates of shrimps in a 28-day feeding trial. The results showed that dietary AP improved the growth performance and non-specific immune function of shrimps. To investigate the effect of AP on disease resistance of L. vannamei, shrimps fed with diet containing AP were challenged with V. alginolyticus. Compared with the control group, the shrimps fed diet containing AP showed significantly higher survival. Furthermore, the hepatopancreas injury in the shrimp fed with AP was less than control group at 6 h after V. alginolyticus infection. However, no difference was observed in the degree of hepatopancreas injury between AP groups and control group at 12 h and 24 h after V. alginolyticus infection. Based on this result, the samples at 6 h after V. alginolyticus infection was selected for subsequent detection. Reactive oxygen species (ROS) accumulation in hemocytes and O2- production in hepatopancreas caused by V. alginolyticus infection was significantly reduced after feeding a diet containing 0.25% and 0.5% AP (p < 0.05). In addition, we found that feeding AP significantly up-regulated the expression of pro-apoptotic genes (Bax, Caspase 3, p53) and down-regulated the expression of anti-apoptotic genes (Bcl-2) in hepatopancreas after V. alginolyticus infection. In conclusion, AP promote the growth and immunity of L. vannamei, and protects shrimps against V. alginolyticus by regulating the oxidative damage and apoptosis. These results provide useful information regarding the effects of AP extracts as a shrimp feed additive for sustainable shrimp culture.

Nemo-like kinase (NLK) gene regulates apoptosis via the p53 signaling pathway in Litopenaeus vannamei under low-temperature stress.

The Nemo-like kinase (NLK) is an important serine/threonine-protein kinase in many signaling pathways. However, its function in crustaceans, such as shrimps, is still poorly understood and needs to be further explored. In the present study, the full-length cDNA of NLK from Litopenaeus vannamei (LvNLK) was cloned. The full-length LvNLK cDNA has 2497 bp, including an open reading frame (ORF) of 1524 bp encoding a protein with 507 amino acids and a predicted molecular mass of 56.1 kDa. Phylogenetic analysis revealed that LvNLK shared high similarities with NLK from other known species. Low-temperature stress markedly upregulated the expression of LvNLK. Its overexpression in hemocytes suppressed the expression of BCL2-associated X (Bax) and tumor protein P53 (p53) in vitro. Meanwhile, the BCL2 apoptosis regulator (Bcl-2), MDM2 proto-oncogene (MDM2), and Yin Yang 1 (YY1) were upregulated. Moreover, LvNLK silencing in vivo increased the susceptibility of shrimps to low-temperature stress. The generation of ROS and the rate of hemocyte apoptosis also increased when LvNLK was silenced. Additionally, qPCR results indicated that LvNLK might participate in apoptosis via the p53 signaling pathway in vitro and in vivo. These results suggested that LvNLK is indispensable for the environmental adaptation of L. vannamei. Our current findings also demonstrated that NLK is evolutionarily conserved in crustaceans and provided insights into the environmental adaptation of invertebrates.

Ciliary Type III Adenylyl Cyclase in the VMH Is Crucial for High-Fat Diet-Induced Obesity Mediated by Autophagy.

Neuronal primary cilia are crucial for body weight maintenance. Type III adenylyl cyclase (AC3) is abundantly enriched in neuronal cilia, and mice with global AC3 ablation are obese. However, whether AC3 regulates body weight through its ciliary expression and the mechanism underlying this potential regulation are not clear. In this study, humanized AC3 knock-in mice that are resistant to high-fat diet (HFD)-induced obesity are generated, and increases in the number and length of cilia in the ventromedial hypothalamus (VMH) are shown. It is demonstrated that mice with specifically knocked down ciliary AC3 expression in the VMH show pronounced HFD-induced obesity. In addition, in vitro and in vivo analyses of the VMH show that ciliary AC3 regulates autophagy by binding an autophagy-related gene, gamma-aminobutyric acid A receptor-associated protein (GABARAP). Mice with GABARAP knockdown in the VMH exhibit exacerbated HFD-induced obesity. Overall, the findings may reveal a potential mechanism by which ciliary AC3 expression regulates body weight in the mouse VMH.

PCAT1 induced by transcription factor YY1 promotes cholangiocarcinoma proliferation, migration and invasion by sponging miR-216a-3p to up-regulate oncogene BCL3.

This study was designed to illustrate the function and role of PCAT1 in CCA. The relative expression was confirmed by RT-qPCR and western blot. The biological function of PCAT1 was evaluated by CCK8, EdU, colony formation, wound healing, transwell, and subcutaneous tumor formation assays. Protein levels of EMT markers were measured by western blot. The binding relationship was predicted by JASPAR and starBase. The binding of YY1 to PCAT1 promoter was assessed by ChIP and luciferase reporter. The binding capacity between miR-216a-3p and PCAT1 as well as BCL3 was assessed by luciferase reporter and AGO2-RIP assays. In this study, we found that PCAT1 was up-regulated in CCA tissues and cells, and the PCAT1 overexpression was associated with poor prognosis. Moreover, PCAT1 was assessed as an independent risk factor of prognosis for CCA patients. Amplified PCAT1 was found to promote tumor proliferation, migration, invasion and EMT process, whereas PCAT1 knockdown inhibited these malignant phenotypes. Mechanistically, PCAT1 was predominantly localized in the cytoplasm and competitively bound miR-216a-3p to increase BCL3 expression. In addition, PCAT1 was activated by transcription factor YY1. This study revealed that PCAT1 acted as an oncogene in CCA, and the YY1/PCAT1/miR-216a-3p/BCL3 axis exhibited critical functions in CCA progression.

Tetrahydropalmatine protects against acute lung injury induced by limb ischemia/reperfusion through restoring PI3K/AKT/mTOR-mediated autophagy in rats.

BACKGROUND: Limb ischemia/reperfusion (I/R) is a common clinical process that frequently induces acute lung injury (ALI). Tetrahydropalmatine (THP) is a major bioactive constituent of various traditional Chinese medicine with protective effects on inflammation and oxidation. In this study, we aimed to investigate the possible protective effect of THP on ALI induced by limb I/R. METHODS: Rats were used to establish ALI through limb I/R. After administration of three doses of THP, the lung injury was evaluated by hematoxylin-eosin staining, tissue wet/dry weight ratio and ELISA examination of myeloperoxidase (MPO), malondialdehyde (MDA) and Super Oxide Dismutase (SOD). Additionally, PI3K/AKT/mTOR pathway and autophagy markers were determined by Western blot. To confirm the role of autophagy in the effect of THP on ALI, 3-methyladenine (3-MA), THP or THP + rapamycin (RAPA) was given to the model rats, and then evaluated the parameters above mentioned. RESULTS: The pulmonary histological lesions and wet/dry were significantly induced after limb I/R. Concurrently, I/R significantly increased MPO and MDA, and decreased SOD in lung tissues. These changes were reversed after THP treatment. Additionally, THP exerted inhibitory effect on the I/R-induced decrease of phosphorylation of PI3K/AKT/mTOR and increase of autophagy activity. The effects by THP on lung injury, PI3K/AKT/mTOR signaling and autophagy were also observed after treatment with 3-MA, an autophagy inhibitor, whereas were blocked by combinational treatment with RAPA, an autophagy inducer. CONCLUSION: Our data suggested that THP had significant protection against ALI and this might be achieved by autophagy inhibition through rescuing PI3K/AKT/mTOR activity.

The role of delta-1-pyrroline-5-carboxylate dehydrogenase (P5CDh) in the Pacific white shrimp (Litopenaeus vannamei) during biotic and abiotic stress.

Proline (Pro) metabolism is intimately associated with stress adaptation. The catabolism of Pro includes two dehydrogenation reactions catalyzed by proline dehydrogenase (ProDH) and Δ1-pyrroline-5-carboxylate dehydrogenase (P5CDh). P5CDh is a mitochondrial matrix NAD+-dependent dehydrogenase that is critical in preventing P5C-Pro intensive cycling and avoiding ROS production from electron run-off. Little is known about the roles of P5CDh in invertebrates, however. We cloned the P5CDh sequence in the Pacific white shrimp, Litopenaeus vannamei, and found that LvP5CDh is expressed predominantly in pleopod, hepatopancreas and gill. Subcellular localization analysis revealed that LvP5CDh protein was mainly found in the cytoplasm. In addition, overexpressing LvP5CDh in cells reduced ROS formation and inhibited apoptosis induced by LC50 Cd2+. Shrimp were exposed to various stress factors including infection with Vibrio alginolyticus, (½ LC50 and LC50) Cd2+, acid (pH 5.6) and alkali stress (pH 9.3). Both biotic and abiotic stress resulted in increased LvP5CDh expression and Pro accumulation; V. alginolyticus infection, pH 9.3 and LC50 Cd2+ stress apparently stimulated the Glu pathway of Pro synthesis, while pH 5.6 and ½ LC50 Cd2+ stress promoted the Orn pathway of Pro synthesis. Silencing of Lvp53 in shrimp attenuated LvP5CDh expression during Cd2+ stress, but had no effect on LvP5CDh mRNA levels if no Cd2+ stress was imposed. Our study contributes to the functional characterization of LvP5CDh in biotic and abiotic stress and reveals it to protect against ROS generation, damage to the cell, including the mitochondria, and apoptosis. Thus, LvP5CDh plays a critical role in immune defense and antioxidant responses.

Effects of a Dissostichus mawsoni-CaM recombinant proteins feed additive on the juvenile orange-spotted grouper (Epinephelus coioides) under the acute low temperature challenge.

The effects of Dissostichus mawsoni-Calmodulin (Dm-CaM) on growth performance, enzyme activities, respiratory burst, MDA level and immune-related gene expressions of the orange-spotted grouper (Epinephelus coioides) exposed to the acute low temperature stress were evaluated. The commercial diet supplemented with Dm-CaM protein was fed to the groupers for 6 weeks. No significant difference was observed in the specific growth rates, weight gains and survivals. After the feeding trial, the groupers were exposed to acute low temperature challenge. The groupers fed with Dm-CaM additive diet showed a significant decrease in the respiratory burst activity, while the blood cell number increased significantly at 25 °C by comparing with the control and additive control group. The enzymatic activity of SOD, ACP and ALP increased significantly in Dm-CaM additive group, while MDA level maintained stable with the lowest value. qRT-PCR analysis indicated that the up-regulated transcript expressions of CaM, C3, SOD2, LysC and HSPA4 were observed in Dm-CaM additive group. These results indicated that Dm-CaM additive diet may regulate the grouper immune response to the acute low temperature challenge.

Characterization and expression analysis of Calmodulin (CaM) in orange-spotted grouper (Epinephelus coioides) in response to Vibrio alginolyticus challenge.

Vibrio alginolyticus containing the highly toxic extracellular product is one of the most serious threats to grouper survival and its minimum lethal dose is approximately 500 CFU/g fish body weight in grouper. To study the toxic effects of V. alginolyticus on the immune system in teleost, Calmodulin (CaM), an important molecular indicator gene, was cloned from the orange-spotted grouper (Epinephelus coioides). The full-length Ec-CaM consisted of a 5'-UTR of 103 bp, an ORF of 450 bp and a 3'-UTR of 104 bp. The Ec-CaM gene encoded a protein of 149 amino acids with an estimated molecular mass of 16.4 kDa and a predicted isoelectric point of 3.93. The deduced amino acid sequence showed that Ec-CaM contained four highly conserved EF-hand domains known to be critical for the function of CaM. Ec-CaM was widely expressed and the highest expression level was observed in liver. Following V. alginolyticus challenge, a sharp increase level of respiratory burst activity and apoptosis ratio were observed. Further analyses of CaM expression and p53 expression in liver, kidney and spleen by qRT-PCR demonstrated that the up-regulated expression of CaM and p53 were observed in the vibrio challenge group. Western blotting analysis confirmed that the Ec-CaM protein was strongly induced in liver at 12 h post-injection, while a sharp increase of p53 protein expression was observed at 24 h post-injection. These results showed CaM expression serving as a potential molecular indicator may help to assess the toxicological effects of V. alginolyticus on the ROS generation and apoptotic process in grouper.

Effects of a recombinant complement component C3b functional fragment α2MR (α2-macroglobulin receptor) additive on the immune response of juvenile orange-spotted grouper (Epinephelus coioides) after the exposure to cold shock challenge.

The effects of Ec-α2MR (Epinephelus coiodes-α2-macroglobulin receptor) on growth performance, enzymatic activity, respiratory burst, MDA level, total antioxidant capacity, DPPH radical scavenging percentage and immune-related gene expressions of the juvenile orange-spotted grouper were evaluated. The commercial diet supplemented with α2MR additive was used to feed the orange-spotted grouper for six weeks. Although a slight increase was observed in the specific growth rate, survival rate and weight gain, no significance was observed among different group. After the feeding trial, the groupers were exposed to cold stress. Respiratory burst activity and MDA level decreased significantly in α2MR additive group by comparing with the control and additive control group, while a sharp increase of ACP activity, ALP activity, total antioxidant capacity and DPPH radial scavenging percentage was observed in α2MR additive group. qRT-PCR analyses confirmed that the up-regulated mRNA expressions of C3, TNF1, TNF2, IL-6, CTL, LysC, SOD1 and SOD2 were observed in α2MR additive group at 20 °C. These results showed that α2MR additive may moderate the immune response in grouper following cold shock challenge.

Molecular cloning, characterization and expression analysis of PPAR gamma in the orange-spotted grouper (Epinephelus coioides) after the Vibrio alginolyticus challenge.

PPAR gamma was a key nuclear receptor, playing an important role in the immune defense and the anti-inflammatory mechanism. In this study, the full-length PPAR gamma (EcPPAR gamma) was obtained, containing a 5'UTR of 133 bp, an ORF of 1602 bp and a 3'UTR of 26 bp besides the poly (A) tail. The EcPPAR gamma gene encoded a protein of 533 amino acids with an estimated molecular mass of 60.02 KDa and a predicted isoelectric point (pI) of 6.26. The deduced amino acid sequence showed that EcPPAR gamma consisted of the conserved residues and the domains known to be critical for the PPAR gamma function. The quantitative real-time PCR analysis revealed that EcPPAR gamma transcript was expressed in all the examined tissue, while the strong expression was observed in intestine, followed by the expression in liver, gill, spleen heart, kidney and muscle. Vibrio challenge could stimulate the inflammatory response in grouper and induce a sharp increase of pro-inflammatory cytokines expression, lipid peroxidation and DNA damage, while the up-regulation of vibrio-induced inflammation could also increase the non-specific immune defense. The groupers challenged with Vibrio alginolyticus showed a sharp increase of EcPPAR gamma transcript in immune tissues. Subcellular localization analysis revealed that EcPPAR gamma was distributed in the nucleus. Furthermore, overexpression of EcPPAR gamma could down-regulated the expression of IL1b, IL6, TNF1 and TNF2. In addition, the administration of PPAR gamma antagonist, GW9662, could up-regulate the expression of pro-inflammatory genes, including IL1b, IL6, TNF1 and TNF2. Together, these results indicated that EcPPAR gamma serving as a negative regulator of pro-inflammatory cytokines may play an important role in the immune defense against vibrio-induced inflammation in grouper.

One-step separation of four flavonoids from Herba Salviae Plbeiae by HSCCC.

High-speed countercurrent chromatography (HSCCC) was successfully applied to the isolation and purification of four flavonoids from Herba Salviae Plbeiae using stepwise and dual-mode elution with a pair of two-phase solvent systems. The systems composed of n-hexane-chloroform-methanol-water (0.5 : 4 : 3 : 2, v/v/v/v) and chloroform-methanol-water (4 : 3 : 2, v/v/v). Analytical HSCCC was used for the preliminary selection of a suitable system, and a linear scale-step procedure was performed at a preparative grade. Hispidulin (91 mg), nepetin (148 mg), homoplantaginin (405 mg) and nepetin-7-glucoside (192 mg) could be obtained from 1,500-mg crude sample in a one-step separation, with purities >95% as determined by high-performance liquid chromatography. Their chemical structures were identified by (1)H nuclear magnetic resonance (NMR) and (13)C NMR.

Functional analysis of a dietary recombinant fatty acid binding protein 10 (FABP10) on the Epinephelus coioides in response to acute low temperature challenge.

The effect of Ec-FABP10 (Epinephelus coiodes-FABP10) on growth performance, enzyme activity, respiratory burst, MDA level, ATP content, immune-related gene expression of juvenile orange-spotted grouper (E. coioides). The commercial diet supplemented with FABP10 protein was feed to orange-spotted grouper for six weeks. No significant difference was observed in the specific growth rates, while the survival rate in the FABP10 additive group was significantly higher. After the feeding trial, the groupers were exposed to acute low temperature challenge. The decreased level of respiratory burst activity was observed in the FABP10 additive group after the exposure to the acute low temperature stress, while the blood cell count increased significantly at 15 °C and a significant increase of ATP content was observed at 10 °C. Higher enzymatic activities of CAT and SOD were observed at 20 °C and 15 °C, respectively. Meanwhile, the lower level of MDA was observed after the exposure to acute low temperature challenge by comparing with the controls. Further transcript expression analyses of FABP10, SOD2, GPX4, HSPA4 and LIPC in liver by quantitative real-time PCR demonstrated that the up-regulated transcript expression of FABP10, SOD2, HSPA4 and LIPC was observed in FABP10 additive group at 15 °C, while the transcript expression of GPX4 increased significantly at 20 °C. Western blotting analysis confirmed that FABP10 protein expression strongly increased at 15 ± 0.5 °C in FABP10 additive group. These results showed that FABP10 additive diet could moderate the metabolic and immune abilities mainly via ROS pathway in the orange-spotted grouper.

A basic phenylalanine-rich oligo-peptide causes antibody cross-reactivity.

Glycolate oxidase (GO) and ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) are the two enzymes that serve key functions in the photorespiration and photosynthesis of plants. A 2 kDa highly basic phenylalanine-rich oligo-peptide (BOP) binds to the surface of acidic GO via ionic and hydrophobic interactions, forming the GO-BOP complex (GC). Previously, RubisCO was thought to exist as a single species composed of a large (rbc L, 54 kDa) and a small subunit (rbc S, 14 kDa). Here we show for the first time, using 2-DE, SDS-PAGE, immunoassays and amino acid determination, that BOP also interacts with RubisCO and that many RubisCO-BOP complexes (RCs), differing in pI, hydrophobicity and activity, coexist in green leaves. GCs, RCs and crude extract from green leaves analyzed by SDS-PAGE Western blotting showed that BOP exists either in subunit-BOP complexes (GO subunit-BOP, rbc L-BOP and rbc S-BOP etc.), with a wide variation in the number and the position of BOPs bound to each subunit molecular, or alone without a binding partner. When rbc L-BOP and rbc S-BOP were assayed by SDS-PAGE, BOP was dissociated from the subunit and it self-assembled to form 37 different BOP polymers (basic phenylalanine-rich protein) whose molecular weights (M(r)s) ranged from 34.0 to 91.6 kDa, indicating that the M(r) of BOP is about 2 kDa. Thus, the addition of BOP changes the M(r) of the subunit-BOP complexes so minimally that the rbc L and rbc S run at their predicted M(r)s on SDS-PAGE. In summary, the results described here demonstrate that the presence of BOP in complexes (both subunit-BOP complex and protein-BOP complex) can cause cross-reactivity of antibodies against different proteins.

Quick identification of apoptosis inducer from Isodon eriocalyx by a drug discovery platform composed of analytical high-speed counter-current chromatography and the fluorescence-based caspase-3 biosensor detection.

Analytical high-speed counter-current chromatography (HSCCC), a unique liquid-to-liquid separation technology, has an inherent capability to provide perfect fractionation for tracking active ingredients of medicinal herbs, in a quick, efficient, and high-recovery manner. A high throughput screening (HTS) method which utilizes a novel biosensor that selectively detects apoptosis based on the fluorescence resonance energy transfer (FRET) technique, was newly established and proved to be very sensitive in detecting apoptosis induced by various known anticancer drugs. The first combination of both advanced techniques formed an efficient platform for drug discovery and succeeded in quickly identifying the most potent apoptotic constituent of a Chinese herb namely Isodon eriocalyx. The system of n-hexane/ethyl acetate/methanol/water was used as the separation solvent. The solvent ratio was first set at 3:5:3:5 to check the water-soluble part of the crude extract, and then 1:1:1:1 was used to isolate the target compounds. The active fraction was tracked and purified continuously using HSCCC which was guided by the apoptosis detection at gradually decreased drug concentrations. As a result, the most potent apoptosis inducer in this herb was discovered by analytical HSCCC equipped with a 16 ml mini-coil column, using less than 50 ml diphase solvent, from about 50mg active fraction. It was identified as eriocalyxin B, a well-known antitumor natural product, by NMR analysis of the HSCCC purified fraction.

Effect of Oenanthe javanica flavone on human and duck hepatitis B virus infection.

AIM: To study the antiviral effect of Oenanthe javanica flavones (OjF) on human hepatoma HepG2.2.15 culture system and duck hepatitis B virus (DHBV) infection. METHODS: (1) After incubation for 24 h, the 2.2.15 cells were treated with different concentrations of OjF for 12 d. The cell alteration was observed by microscope. The presence of HBsAg and HBeAg were measured using the enzyme immunoassay kit after 2.2.15 cells were treated with OjF for 9 d. (2) Ducklings infected with DHBV intravenously were divided into 5 groups and treated with OjF, acyclovir (ACV), and normal saline respectively for 10 d. All the ducklings were bled before, during, and after treatments at different times, and serum levels of DHBV-DNA were detected by a dot-blot hybridization assay. RESULTS: (1) The 50% toxic concentration (TC50) of OjF was 2.28 g/L. The maximum nontoxic concentration (TC0) was 1.00 g/L. In nontoxic concentrations, OjF significantly inhibited HBsAg and HBeAg in 2.2.15 cells after 9 d of treatment (P<0.05, P<0.01). (2) The DHBV-DNA levels decreased significantly after the treatment with 0.50 and 1.00 g/kg of OjF (P<0.01). The inhibition of the peak of viremia was maximum at a dose of 1.00 g/kg and reached 54.3% on d 5 and 64.5% on d 10, respectively. CONCLUSION: The results demonstrate that OjF is a strong inhibitor of HBsAg and HBeAg secretion in 2.2.15 cells and DHBV-DNA levels in the HBV-infected duck model.

Effects of pH on survival, phosphorus concentration, adenylate energy charge and Na(+)-K(+) ATPase activities of Penaeus chinensis Osbeck juveniles.

Penaeus chinensis (Osbeck) juveniles were maintained for 14 days at pH 6.0, 7.0, 7.6, 8.0 and 8.5, respectively. The effects of pH on survival, phosphorus concentration, adenylate energy charge (AEC) and Na(+)-K(+) adenosine triphosphatase (ATPase) activities of prawns were investigated. The results showed that survival of P. chinensis was impaired at low and high pH levels. The total phosphate level and AEC in abdominal muscle increased with pH level in range of 6.0-7.6 reaching the maximum values at pH 7.6. Thereafter, the levels declined with increasing pH level in range of 7.6-8.5. The change of Na(+)-K(+) ATPase activity in gill of prawn was similar to that of total phosphorus content and AEC in muscle of prawn at different pH. The effect of pH on Na(+)-K(+) ATPase activity in the muscle was lower than on that in gill.