The Jinggangmycin-induced Mthl2 gene regulates the development and stress resistance in Nilaparvata lugens Stål (Hemiptera: Delphacidae).

Methuselah (Mth) belongs to the GPCR family B, which regulates various biological processes and stress responses. The previous transcriptome data showed jinggangmycin (JGM)-induced Mthl2 expression. However, its detailed functional role remained unclear in brown planthopper, Nilaparvata lugens Stål. In adult N. lugens, the Mthl2 gene showed dominant expressions, notably in ovaries and fat body tissues. The 3rd instar nymphs treated with JGM increased starvation, oxidative stress, and high temperature (34 °C) tolerance of the adults. On the contrary, under dsMthl2 treatment, completely opposite phenotypes were observed. The lipid synthesis genes (DGAT1and PNPLA3) of both females and males treated with JGM in the nymphal stage were observed with high expressions, while the lipolysis of the Lipase 3 gene was observed with low expressions. The JGM increased triglyceride (TG) content, fat body droplet size, and the number of fat body droplets. The same treatment also increased the Glutathione S-transferase (GST), catalase (CAT), and superoxide dismutase (SOD) activities. An increase in the heat shock protein (HSP70 and HSP90) expression levels was also observed under JGM treatment but not dsMthl2. The current study demonstrated the influential role of the Mthl genes, particularly the Mthl2 gene, in modulating the growth and development and stress-responsiveness in N. lugens. Thus, providing a platform for future applied research programs controlling N. lugens population in rice fields.

Assessment of the Purity of IMM-H014 and Its Related Substances for the Treatment of Metabolic-Associated Fatty Liver Disease Using Quantitative Nuclear Magnetic Resonance Spectroscopy.

An accurate, rapid, and selective quantitative nuclear magnetic resonance method was developed and validated to assess the purity of IMM-H014, a novel drug for the treatment of metabolic-associated fatty liver disease (MAFLD), and four related substances (impurities I, II, III, and IV). In this study, we obtained spectra of IMM--H014 and related substances in deuterated chloroform using dimethyl terephthalate (DMT) as the internal standard reference. Quantification was performed using the 1H resonance signals at δ 8.13 ppm for DMT and δ 6.5-7.5 ppm for IMM-H014 and its related substances. Several key experimental parameters were investigated and optimized, such as pulse angle and relaxation delay. Methodology validation was conducted based on the International Council for Harmonization guidelines and verified with satisfactory specificity, precision, linearity, accuracy, robustness, and stability. In addition, the calibration results of the samples were consistent with those obtained from the mass balance method. Thus, this research provides a reliable and practical protocol for purity analysis of IMM-H014 and its critical impurities and contributes to subsequent clinical quality control research.

Application of Band-Selective HSQC NMR in Species Discrimination and Adulteration Identification of Panax Linn.

The quality of Panax Linn products available in the market is threatened by adulteration with different Panax species, such as Panax quinquefolium (PQ), Panax ginseng (PG), and Panax notoginseng (PN). In this paper, we established a 2D band-selective heteronuclear single quantum coherence (bs-HSQC) NMR method to discriminate species and detect adulteration of Panax Linn. The method involves selective excitation of the anomeric carbon resonance region of saponins and non-uniform sampling (NUS) to obtain high-resolution spectra in less than 10 min. The combined strategy overcomes the signal overlap limitation in 1H NMR and the long acquisition time in traditional HSQC. The present results showed that twelve well-separated resonance peaks can be assigned in the bs-HSQC spectra, which are of high resolution, good repeatability, and precision. Notably, the identification accuracy of species was found to be 100% for all tests conducted in the present study. Furthermore, in combination with multivariate statistical methods, the proposed method can effectively determine the composition proportion of adulterants (from 10% to 90%). Based on the PLS-DA models, the identification accuracy was greater than 80% when composition proportion of adulterants was 10%. Thus, the proposed method may provide a fast, practical, and effective analysis technique for food quality control or authenticity identification.

The Phosphoserine Phosphatase Alters the Free Amino Acid Compositions and Fecundity in Cyrtorhinus lividipennis Reuter.

The mirid bug Cyrtorhinus lividipennis (Reuter) is an important predator that consumes eggs and young nymphs of the brown planthopper Nilaparvata lugens as a primary food source and thus becomes an important member of the rice ecosystem. We identified and characterized the ClPSP gene in C. lividipennis encoding the phosphoserine phosphatase enzyme. The ClPSP has an open reading frame (ORF) of 957 bp encoding a protein with a length of 294bp and it possesses a haloacid dehalogenase-like (HAD) hydrolase, phosphoserine phosphatase, eukaryotic-like (HAD_PSP_eu) conserved domain. Furthermore, the in silico analysis of the ClPSP gene unveiled its distinct characteristics and it serves as a key player in the modulation of amino acids. The ClPSP showed expression in all developmental stages, with higher expression observed in the ovary and fat body. Silencing the ClPSP by RNA interference (RNAi) significantly decreased PSP enzyme activity and expression compared to dsGFP at two days after emergence (2DAE). The dsPSP treatment altered free hemolymph amino acid compositions, resulting in a significant reduction of serine (Ser) and Arginine (Arg) proportions and a significant increase of Threonine (Thr), Cystine (Cys), and Tyrosine (Tyr) in the C. lividipennis female at 2 DAE. Additionally, a hindered total protein concentration in the ovary and fat body, and reduced vitellogenin (Vg) expression, body weight, and number of laid eggs, were also observed. The same treatment also prolonged the preoviposition period and hindered ovarian development. Our data, for the first time, demonstrated the influential role of the PSP gene in modulating the fecundity of C. lividipennis and provide a platform for future insect pest control programs using the PSP gene in modulating fecundity.

Intrinsic debromination potential of polybrominated diphenyl ethers in different sediment slurries.

The fate of BDE-153 (BDE = brominated diphenyl ethers) in different mangrove, fresh water pond, and marine subsurface sediments collected from Hong Kong SAR was investigated. Under anaerobic conditions, all sediments showed good intrinsic abilities to reductively debrominate BDE-153, producing debromination products ranging from hexa- to mono-BDEs in 90 days. The half-lives of BDE-153 in eight different sediments varied from 7.6 to 165 days, with higher debromination in mangrove than marine and fresh water pond sediments. All sediments exhibited the preference in removing the bromine in para, followed by meta, and the lowest in ortho positions; however, fresh water pond sediments had relatively higher fractions of meta (BDE-99) and ortho substitution (BDE-118) of the three penta-BDE products. Mai Po mangrove and fresh water pond subsurface sediments were also capable of debrominating BDE-47 in 90 days of anaerobic incubation with half-lives of 76.2 and 56.9 days, respectively; but not BDE-209. BDE-47, -153, and -209 in Mai Po surface sediment were not transformed under 30 day aerobic incubation. This study demonstrated that the microbial-mediated debromination of BDE-47 and -153 occurred in natural subsurface sediments under anaerobic conditions although the rates and pathways varied among the sediment types.

The superoxide dismutase (SOD) genes family mediates the response of Nilaparvata lugens to jinggangmycin and sugar.

Introduction: Brown planthopper (BPH), Nilaparvata lugens Stål (Hemiptera: Delphacidae), is a major rice pest causing significant damage to rice throughout the world. Intensive pesticide usage often causes resistance in these seasonal pests, mainly through the modulation of antioxidant machinery. The superoxide dismutase (SOD) gene family is known for regulating BPH response to pesticides. Methods: In the present study, we identified eight NlSOD genes from the NCBI using the BLASTP program. The bioinformatics analysis includes a phylogenetic tree, conserved domain, motifs, gene ontology (GO) analysis, Kyoto encyclopedia of genes and genomes (KEGG) pathways, and protein-protein interaction, highlighting the distinctive functional elements of NlSOD genes. Results and discussion: Additionally, the NlSOD genes showed expression in all developmental stages of BPH. Under three sugars (glucose, sucrose, and trehalose) treatment, the respective upregulation of NlSOD8, NlSOD6, and NlSOD2 was noted. The NlSOD1 induced significantly under jinggamycin (JGM) deduced its potential as a key regulator of BPH response to the pesticide. Our study has provided detailed knowledge of the NlSOD gene family in-silico analysis and the defensive response to insecticide and high sugar of BPH. We hope the results of this research will help to shed light on the resistance of BPH towards insecticide toxicity and high sugar and help to control it more efficiently.

Unravelling chilling-stress resistance mechanisms in endangered Mangrove plant Lumnitzera littorea (Jack) Voigt.

Lumnitzera littorea (Jack) Voigt is one of the most endangered mangrove species in China. Previous studies have showed the impact of chilling stress on L. littorea and the repsonses at physiological and biochemical levels, but few attentions have been paid at molecular level. In this study, we conducted genome-wide investigation of transcriptional and post-transcriptional dynamics in L. littorea in response to chilling stress (8 °C day/5 °C night). In the seedlings of L. littorea, chilling sensing and signal transducing, photosystem II regeneration and peroxidase-mediated reactive oxygen species (ROS) scavenging were substantially enhanced to combat the adverse impact induced by chilling exposure. We further revealed that alternative polyadenylation (APA) events participated in chilling stress-responsive processes, including energy metabolism and steroid biosynthesis. Furthermore, APA-mediated miRNA regulations downregulated the expression of the genes involved in fatty acid biosynthesis and elongation, and protein phosphorylation, reflecting the important role of post-transcriptional regulation in modulating chilling tolerance in L. littorea. Our findings present a molecular view to the adaptive characteristics of L. littorea and shed light on the conservation genomic approaches of endangered mangrove species.

The Homeodomain-Leucine Zipper Genes Family Regulates the Jinggangmycin Mediated Immune Response of Oryza sativa to Nilaparvata lugens, and Laodelphax striatellus.

The homeodomain-leucine zipper (HDZIP) is an important transcription factor family, instrumental not only in growth but in finetuning plant responses to environmental adversaries. Despite the plethora of literature available, the role of HDZIP genes under chewing and sucking insects remains elusive. Herein, we identified 40 OsHDZIP genes from the rice genome database. The evolutionary relationship, gene structure, conserved motifs, and chemical properties highlight the key aspects of OsHDZIP genes in rice. The OsHDZIP family is divided into a further four subfamilies (i.e., HDZIP I, HDZIP II, HDZIP III, and HDZIP IV). Moreover, the protein-protein interaction and Gene Ontology (GO) analysis showed that OsHDZIP genes regulate plant growth and response to various environmental stimuli. Various microRNA (miRNA) families targeted HDZIP III subfamily genes. The microarray data analysis showed that OsHDZIP was expressed in almost all tested tissues. Additionally, the differential expression patterns of the OsHDZIP genes were found under salinity stress and hormonal treatments, whereas under brown planthopper (BPH), striped stem borer (SSB), and rice leaf folder (RLF), only OsHDZIP3, OsHDZIP4, OsHDZIP40, OsHDZIP10, and OsHDZIP20 displayed expression. The qRT-PCR analysis further validated the expression of OsHDZIP20, OsHDZIP40, and OsHDZIP10 under BPH, small brown planthopper (SBPH) infestations, and jinggangmycin (JGM) spraying applications. Our results provide detailed knowledge of the OsHDZIP gene family resistance in rice plants and will facilitate the development of stress-resilient cultivars, particularly against chewing and sucking insect pests.

The Amino Acid-Mediated TOR Pathway Regulates Reproductive Potential and Population Growth in Cyrtorhinus lividipennis Reuter (Hemiptera: Miridae).

The predatory mirid bug, Cyrtorhinus lividipennis Reuter, feeds on brown planthopper (BPH) eggs that are deposited on rice and gramineous plants surrounding rice fields. The development and reproduction of C. lividipennis are inhibited by feeding on BPH eggs from gramineous species, and the underlining regulatory mechanism for this phenomenon is unclear. In the present study, HPLC-MS/MS analysis revealed that the concentrations of six amino acids (AAs:Ala, Arg, Ser, Lys, Thr, and Pro) were significantly higher in rice than in five gramineous species. When C. lividipennis fed on gramineous plants with BPH eggs, expression of several genes in the target of rapamycin (TOR) pathway (Rheb, TOR, and S6K) were significantly lower than that in the insects fed on rice plants with BPH eggs. Treatment of C. lividipennis females with rapamycin, dsRheb, dsTOR, or dsS6K caused a decrease in Rheb, TOR, and S6K expression, and these effects were partially rescued by the juvenile hormone (JH) analog, methoprene. Dietary dsTOR treatment significantly influenced a number of physiological parameters and resulted in impaired predatory capacity, fecundity, and population growth. This study indicates that these six AAs play an important role in the mediated-TOR pathway, which in turn regulates vitellogenin (Vg) synthesis, reproduction, and population growth in C. lividipennis.

CTL-Derived Granzyme B Participates in Hippocampal Neuronal Apoptosis Induced by Cardiac Arrest and Resuscitation in Rats.

Hippocampal neuronal apoptosis is a devastating consequence of cardiac arrest (CA) and subsequent cardiopulmonary resuscitation (CPR). In this study, we assessed the contribution of cytotoxic T lymphocyte (CTL)-derived toxic mediator granzyme B (Gra-b) to the hippocampal neuronal apoptosis following CA/CPR in rats. Rats that experienced CA/CPA presented with cytosomal shrinkage, dense cytoplasm, and intensive eosinophilic staining in the CA1 region of dorsal hippocampus. CA/CPR rats also exhibited inability in spatial navigation and a local infiltration of peripheral CD8+ T cells into the hippocampus. The protein levels of Gra-b, cleaved Caspase-3, and cleaved PARP1 were significantly elevated in rats undergoing CA/CPR. Pretreatment with Gra-b inhibitor suppressed Gra-b release, attenuated hippocampal neuronal apoptosis, as well as improved cognitive impairment. Together, this study indicates that CTL-derived Gra-b is involved in the CA/CPR-induced neuronal apoptosis, and pharmacological manipulation of Gra-b may represent a novel avenue for the treatment of brain injury following CA/CPR.

Bioactivity, pharmacokinetics, and immunogenicity assays in preclinical and clinical trials for recombinant human endostatin.

AIM: To determine the in vitro and in vivo bioactivity of recombinant human endostatin (rhEndostatin) and to analyze its pharmacokinetics and immunogenicity in rhesus monkeys and patients. METHODS: The physical chemical characteristics of rhEndostatin were detected according to Pharmacopoeia of the People's Republic of China (2005 edition, part III). Its in vitro and in vivo bioactivities were assayed via proliferation-inhibition on human umbilical vein endothelial cells and their inhibitory effect on tumor-bearing mice models. Serum concentrations of rhEndostatin in monkeys and patients were determined by an enzyme immunoassay method. RESULTS: The corresponding specific in vitro activities of rhEndostatin obtained from the cell counting method, 3-(4,5-dimethylthiazol-2- yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and lactate dehydrogenase assay, respectively, were 6.4 x 10(7), 6.7 x 10(7), and 3.8 x 10(8) U/mg, and the in vivo antitumoral potency was 4.04 x 10(7) U/mg. In rhesus monkeys, there were no gender differences in all pharmacokinetic parameters. Serum anti-rhEndostatin immunoglobulin (Ig)G antibodies were generated quickly after intravenous (iv) administration and decreased rapidly when therapy was stopped. In phase I clinical trials, linearity in the pharmacokinetics of rhEndostatin was indicated by dose-proportionate increases in the area under the curve and the maximum serum concentration. Serum rhEndostatin reached a steady-state level after 7 d of successive administration with the average concentration at a steady state of 272.44+/-91.98 ng/mL. Neither IgG nor IgM antibodies against rhEndostatin were observed in patients. CONCLUSION: RhEndostatin exhibited a definite proliferation- inhibition effect on HUVEC, and significant antitumoral activity in mice. The immunoreactivity of rhesus monkeys to rhEndostatin is common, and rhEndostatin showed no immunogenicity in patients in this trial. The results provide a basis for further clinical trials.

Zinc adsorption on goethite as affected by glyphosate.

Cosorption of metals with herbicides on minerals affects their mobility and their environmental effect. Batch experiments were conducted to evaluate the interaction between Zn and glyphosate [N-(phosphonomethyl)glycine (GPS; H3L)] with regard to the effect of GPS on Zn adsorption on goethite. The herbicide GPS markedly affected Zn adsorption on goethite when they coexisted in a goethite suspension. When solution pH was not intentionally adjusted, addition of GPS decreased Zn adsorption on goethite, since the equilibrium solution pH was significantly decreased in the presence of GPS and correspondingly the negative surface charges of goethite decreased. Zinc adsorption on goethite in the presence and absence of GPS at different pH of the equilibrium solution was studied in order to know if pH was the only variable for Zn adsorption with coexisting GPS. At lower pH (pH<5), the presence of GPS increased the adsorption of Zn, because Zn adsorbed on the sites of goethite via GPS bridge. However, at higher pH (pH>5), the presence of GPS decreased the adsorption of Zn on goethite, because GPS reacted with solution Zn to form water-soluble complexes that had lower affinity to the goethite surface in comparison with Zn itself. Zeta potential of goethite significantly decreased after adsorption of GPS, suggesting a chemical bond occurred between GPS and the mineral. FTIRs also show that GPS adsorbs on goethite by coordinating through caboxylate group.

A quantitative method for measuring the antitumor potency of recombinant human endostatin in vivo.

Recombinant human endostatin (rhEndostatin) has been shown to inhibit tumor growth, but the variable antitumor activity of different rhEndostatin preparations has necessitated the development of an accurate, reproducible in vivo bioassay for evaluating the rhEndostatin activity. To assess the in vivo antitumor efficacy of rhEndostatin, H22 tumor-bearing mice received three doses of rhEndostatin and the potency of rhEndostatin preparations in inhibiting tumor growth was determined by ED(50)-potency assay and validated by dose-response parallel-line assay. There was a consistent and highly reproducible linear regression relationship between rhEndostatin dosage and tumor growth inhibition rate. The ED(50) values were determined from dose-response regression lines for seven rhEndostatin preparations with high reproducibility. On the basis of the current study, the potency of rhEndostatin preparations was assigned a value of 6.09 x 10(5) U/ampoule and a 95% confidence limit of 5.96 x 10(5)-6.22 x 10(5). We consider that this procedure can be served as a potential candidate pharmacopoeial method for potency measurement of different rhEndostatin preparations.

Serum levels of anti-sperm-associated antigen 9 antibody are elevated in patients with hepatocellular carcinoma.

At present, there is a high incidence of viral hepatitis and high mortality rates due to hepatocellular carcinoma (HCC) in China. In the current study, the quantification of antibodies against the cancer-testis antigen sperm-associated antigen 9 (SPAG9), alone and combined with α-fetoprotein (AFP), were evaluated as biomarkers for the diagnosis of HCC. The levels of anti-SPAG9 antibody and AFP were quantified in serum samples from patients with HCC and hepatitis or cirrhosis, as well as healthy volunteers. The results revealed that the serum levels of anti-SPAG9 immunoglobulin G antibody in patients with HCC were significantly higher compared with those in patients with hepatitis/cirrhosis and healthy controls. Using receiver operator characteristic curves, the area under the curve (AUC, 0.870) of SPAG9 as a diagnostic marker of HCC was significant [P<0.001; 95% confidence interval (CI), 0.793-0.947], whereas the AUC of AFP was 0.832 (P<0.001; 95% CI, 0.736-0.928). Serum anti-SPAG9 antibody levels exhibited significant potential for the differential diagnosis of HCC, with an AUC value of 0.729, (P=0.008; 95% CI, 0.559-0.899). Similarly, serum AFP levels exhibited significant value for the differential diagnosis of HCC, with an AUC value of 0.842 (P<0.001; 95% CI, 0.732-0.953). When combined with quantification of AFP, the diagnostic sensitivity and specificity of anti-SPAG9 levels were increased. In summary, the results suggested that anti-SPAG9 antibody is a potential early diagnostic marker of HCC.

The expression of DAMP proteins HSP70 and cancer-testis antigen SPAG9 in peripheral blood of patients with HCC and lung cancer.

There are different views of how the immune system participates in the reaction to cancer. Here, we evaluated expression of DAMP proteins HSP70 and cancer-testis antigen SPAG9 in patients with hepatocellular carcinoma (HCC) and lung cancer to explore tumor immunity. Our analysis showed that levels of HSP70 and SPAG9 antibody were significantly higher in the serum of lung cancer and HCC patients than in the serum of healthy subjects (P < 0.001), but there were no differences in levels of HSP70 antibody in patients and controls. Levels of serum SPAG9 antibody in newly diagnosed lung cancer patients were significantly higher than in treated lung cancer patients (P < 0.05), but there were no differences in levels of HSP70 or HSP70 antibody. Levels of serum HSP70 and SPAG9 antibody, but not HSP70 antibody, were also higher in hepatitis/cirrhosis patients than in healthy subjects (P = 0.005, P < 0.001). Levels of serum SPAG9 antibody were significantly higher in HCC patients than in hepatitis/cirrhosis patients, but there were no differences in HSP70 or HSP70 antibody levels. Finally, levels of serum HSP70 and SPAG9 antibody were significantly higher in HCC patients than in lung cancer patients (P < 0.05, P < 0.001). These results indicate that cancer-testis antigen SPAG9 induces a strong humoral immune response in cancer patients but HSP70 does not. These results show that SPAG9 has potential as a tumor-specific biomarker.

Effects of site-specific polyethylene glycol modification of recombinant human granulocyte colony-stimulating factor on its biologic activities.

BACKGROUND: Recombinant human granulocyte colony-stimulating factor (rhG-CSF) is a long-chain cytokine that is administered to stimulate the production of white blood cells (WBCs) to reduce the risk of serious infection in immunocompromized patients. However, to achieve sustained stimulation of WBC production, rhG-CSF must be administered frequently, thus limiting its clinical use. METHODS: We conjugated rhG-CSF with linear monomethoxy-polyethylene glycol (PEG) maleimide at amino acid residue Cys(17) to test our hypothesis that this could extend the in vivo half-life of rhG-CSF in blood. RESULTS: The mono-PEG rhG-CSF became more stable to pH, temperature, and enzyme degradation in vitro, and had granulopoietic activity that was superior to the unmodified form in vivo. The granulopoietic activity of PEG-G-CSF was 2.82-fold greater than that of unmodified G-CSF. CONCLUSIONS: These results indicate that the thiol-specific PEGylation remarkably prolonged the half-life of rhG-CSF and represents a novel strategy to address the more clinically acceptable therapeutic application of hemopoietic growth factor.

Serum levels of HSP70 and other DAMP proteins can aid in patient diagnosis after traumatic injury.

Danger-associated molecular patterns (DAMPs) are activated by endogenous signals that originate from stressed, injured, or necrotic cells, signifying "danger" to the host. In this study, we evaluated the expression of the DAMP heat shock protein 70 (HSP70) in trauma patients with and without secondary infections. Levels of glucose (GLU), procalcitonin (PCT), total cholesterol (T-Chol), and white blood cell (WBC) counts were also evaluated at three time stages after trauma. Our analysis showed that the levels of serum HSP70 in patients with minor, moderate, and severe injuries were significantly higher than in healthy patients at each time point post-injury (P < 0.01), and levels of serum HSP70 in the severe injury group were significantly higher than in the minor injury group at 1-6 h after trauma (P = 0.047). HSP70 was correlated with GLU and was negatively correlated with T-Chol in the period 1-6 h after injury (P = 0.008/0.032). WBC and GLU were elevated after trauma, with mutual positive correlation (P < 0.001). PCT levels increased later than WBC counts and GLU levels; these levels were correlated at the two later time periods, 24-48 h and 60-90 h (P = 0.008/0.041). PCT continued to rise in patients with secondary infection, but PCT dropped at the third time period in patients without secondary infection. In summary, our results suggest that danger and stress theory can be used to predict severity of trauma.

Cancer testis antigen SPAG9 is a promising marker for the diagnosis and treatment of lung cancer.

Cancer testis antigen sperm-associated antigen 9 (SPAG9) is highly expressed in many types of cancers. In the present study, to obtain a better understanding of the relevance of SPAG9 in cancer diagnosis and treatment, the expression of SPAG9 mRNA and protein in lung cancer specimens was evaluated by RT-PCR, western blotting and immunohistochemistry. ELISA was used to quantify the SPAG9 autoantibody in the peripheral blood of lung cancer patients. The results showed that the expression of SPAG9 mRNA and protein in the lung cancer tissues was significantly higher than that in the adjacent non-cancerous tissues (P<0.01). The level of the SPAG9 autoantibody in the serum of lung cancer patients was significantly higher than the level in the healthy controls (P<0.001), and the level of the SPAG9 autoantibody in the serum of untreated patients was significantly higher than that in treated patients (P=0.002). SPAG9 IgG antibody levels were significantly lower in treated adenocarcinoma and small cell lung cancer patients than these levels in the untreated patients (P=0.006, P=0.026, respectively), while no statistical difference was found between treated and untreated squamous cell carcinoma patients. Our results suggest that the SPAG9 antibody in serum is a promising marker for the diagnosis of lung cancer, and the level of the humoral immune response to this antigen appears to be related to the type of lung cancer.

The methods for determining the purity and in vitro or in vivo activity of recombinant human endostatin.

In order to establish the methods of high-performance liquid chromatography (HPLC) for determining the purity of recombinant human endostatin (rhEndostatin) and in vitro or in vivo activity of rhEndostatin, two columns were firstly used in HPLC analysis for determining the purity of rhEndostatin, including Waters Symmetry 300C4 (4.6 mm x 250 mm, 5 microm) and the Superdex75 HR 10/30. Cell lines, bovine capillary endothelial cells (BCEs) or human umbilical vein endothelial cells (HUVECs) expression human vascular endothelial growth factor (hVEGF) were used in method MTT or LDH as substrate, respectively. The bioactivity in vivo was assayed by the anti-tumor proliferation rate in H22 liver tumor-bearing mice. The results showed that the retention time of rhEndostatin sample was stable at 19.066 min or 11.506 min in reverse phase HPLC (RP-HPLC) or gel filtering HPLC (GF-HPLC). The stableness, repeat and recovery rates were over 99% in both methods and there was no statistical difference between these two methods (p > 0.05). In nonserum culture medium, rhEndostatin can sensitively and stably inhibit the proliferation of the HUVEC cells that were transfected with plasmid encoding hVEGF. LDH substrate methods is the most sensitive and stable method. The anti-tumor activity in H22 tumor-bearing mice was also highly repeatable and had an inhibition rate over 50% at 20 mg kg(-1) weight. As a conclusion, the RP-HPLC and GF-HPLC set up in this paper are highly repeatable, accurate and sensitive for detecting the purity of rhEndostatin. The bioactivity of rhEndostatin can be measured through detection the proliferation-inhibition on HUVECs transfectants with hVEGF in vitro or on H22 liver tumor in vivo.

Effect of a polybrominated diphenyl ether congener (BDE-47) on growth and antioxidative enzymes of two mangrove plant species, Kandelia obovata and Avicennia marina, in South China.

The effects of BDE-47 on the growth and antioxidative responses of the seedlings of Kandelia obovata (Ko) and Avicennia marina (Am) were compared in an 8-week hydroponic culture spiked with different levels of BDE-47, 0.1, 1, 5 and 10 mg l(-1). The two highest BDE-47 levels significantly suppressed the growth and increased the activities of three antioxidative enzymes, superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT), of Ko in week 1. However, SOD and POD activities at high levels of BDE-47 became lower than the control in week 8. On the contrary, growth of Am was not affected at all contamination levels, and the activities of three enzymes were enhanced by BDE-47 in weeks 1 and 4, but such stimulatory effect became insignificant in week 8. Avicennia was more tolerant to BDE-47 toxicity than Kandelia, as its antioxidative enzymes could better counter-balance the oxidative stress caused by BDE-47.