Hemi-synthesis of novel chiral benzodiazepine derivatives from Eucalyptus citriodora essential oil: 2D NMR experiments and differential scanning calorimetry study of diastereoisomers.

An efficient in situ condensation of citronellal, the main constituent of Eucalyptus citriodora essential oil (51%), with different amine derivatives of 2,3-diaminomaleonitrile and 3-[(2-aminoaryl)amino]dimedone has led to novel chiral benzodiazepine structures. All reactions were precipitated in ethanol and pure products were obtained in good yields (58-75%) without any purification. The synthesized benezodiazepines were characterized by spectroscopic techniques, namely 1H-NMR, 13C-NMR, 2D NMR and FTIR. Differential Scanning Calorimetry (DSC) and HPLC were used to confirm the formation diastereomeric mixtures of benzodiazepine derivatives.

Iron enrichment from hypoxic hypolimnion supports the blooming of Raphidiopsis raciborskii in a tropical reservoir.

Occurring worldwide, blooms of Raphidiopsis raciborskii threaten the use of water resources especially in tropical and subtropical waterbodies. Its high flexibility in the uses of light and macronutrients (C, N, P) frustrates any bloom prediction and control based on macronutrients regulation. To identify the critical factors promoting periodic blooms of R. raciborskii, the trends of meteorological, hydrodynamic, physical, and chemical variables (including macro- and micronutrients: N, P, Fe) were analyzed in a Chinese tropical large reservoir (Dashahe reservoir) over five years. It was hypothesized that Fe availability, mediated by the mixing pattern of the reservoir, played a crucial role in the periodic blooms of the cyanobacterium. To have a more complete understanding, the effects of Fe on growth of a local R. raciborskii strain were tested in a monoculture experiment. The biomass and relative abundance of R. raciborskii in the reservoir showed a clear seasonal trend, with relative abundance > 50% in summer/autumn (July to October). Three habitat types along a dominance gradient were identified in the reservoir and 17 variables were used to compare them. Statistical analysis and habitat comparison showed that temperature and stratification, dissolved Fe and N concentrations in the epilimnion, and dissolved Fe and oxygen concentrations in the hypolimnion were the critical factors driving the dynamics of R. raciborskii in the study reservoir. The habitat dominated by R. raciborskii was characterized by a relatively low availability of macro resources (Zeu/Zm < 1, SRP < 0.01 mg/L, DIN < 0.3 mg/L) and by a high Fe availability supplemented from hypoxic hypolimnion. The dependence of growth on Fe concentration increase was confirmed in culture where the maximum was reached at 0.689 mg Fe /L. Our results suggest that a high Fe bioavailability, also originating from the hypoxic hypolimnion, influences the dynamics R. raciborskii and favors the blooms of the species. As a consequence, Fe concentrations in the water column as well as oxygen measurements along the water column should be routinely included in the monitoring programs aimed at predicting and controlling R. raciborskii blooms.

Hemisynthesis of coumarin derivatives from Ammodaucus leucotrichus oil extract: organobase-catalyzed reaction, analytical study by diffusion-ordered spectroscopy NMR and differential scanning calorimetry.

This paper presents an in-depth chemical and analytical study of a natural substance extracted from Ammodaucus leucotrichus Coss. & Dur and its derivatives after hemisynthesis. The analysis was performed using Diffusion-Ordered Spectroscopy (NMR DOSY) and Differential Scanning Calorimetry (DSC) as general methods. The results show an interesting chemical reactivity towards coumarin-derived bisnucleophiles (4-hydroxycoumarin and triacetic acid lactone), and products obtained by hemisynthesis of pyrano[4,3-b]pyrane derivatives following Knoevenagel condensation and Michael's addition on this natural substance with the use of 4-pyrolidinopyridine organobase as catalyst.

[Intraspecific Variation in Growth and Alkaline Phosphatase Activity of Cylindrospermopsis raciborskii Strains in Response to Different Phosphorus Concentrations and Sources].

The cyanobacterial species C. raciborskii are ubiquitous in tropical regions, and its successful invasion into temperate zones has been partially attributed to its ability of survival in low P availability and the existence of multiple ecotypes. To explore the physiological response of different strains to phosphorus fluctuations, four strains of C. raciborskii isolated from the Zhenhai Reservoir were used to investigate their growth and alkaline phosphatase (ALP) activity at different inorganic phosphorus (Pi) concentrations (HP=7.13 mg ·L-1, MP=0.64 mg ·L-1, LP=0.03 mg ·L-1) and different phosphorus forms [dipotassium hydrogen phosphate (K2HPO4), sodium pyrophosphate (K4P2 O7), sodium polyphosphate (K5P3O10), D-glucose-6-phosphate (D-G-6-P), adenosine triphosphate (ATP), cyclic adenosine monophosphate (cAMP)]. Four C. raciborskii strains showed a similar growth response to phosphate changes: their biomass increased with an increase in Pi concentrations, while the ALP activity showed the opposite trend. The ALP activity of C. raciborskii N8 was significantly lower than that of other three strains, regardless of inorganic phosphorus concentrations, suggesting that this strain had a higher adaptability to phosphorus fluctuations. When cultured with different phosphorus forms, the biomass of C. raciborskii N8 and N9 in three dissolved inorganic phosphorus (DIP) compounds were significantly higher than those in three dissolved organic phosphorus (DOP) compounds, with the maximum and minimum specific growth rate in K2HPO4and ATP treatments, respectively. C. raciborskii preferred DIP although they can also utilize DOP to sustain its growth. Under the DOP conditions, the ALP activity of C. raciborskii N8 in the ATP treatment was significantly higher than that in the other two organic phosphorus compounds, while we did not observe similar results in C. raciborskii N9, indicating that strain N8 was more sensitive to DIP deficiency. Our results showed an intraspecific variation within C. raciborskii strains from the same reservoir. Compared with the other strains, strain N8 represented better adaptability to phosphorus fluctuations and DIP deficiency. Variations within C. raciborskii strains may make this species more adaptable to environmental changes and enhance its competitive advantage.

[Distribution and Factors Affecting Cylindrospermopsis raciborskii in Guangdong Reservoirs].

Cylindrospermopsis raciborskii originating from tropical and subtropical regions is potentially toxic and attracts much attention due to its extension to the global temperate zone in recent years. Based on historical data of 20 reservoirs with different trophic levels (dry season, wet season, and transitional season of 2010), this study focuses on the analysis of the occurrence and distribution of C. raciborskii in the Guangdong Province. Based on the results, C. raciborskii was found in 19 of the 20 reservoirs and its biomass ranges from 0.0001-39.740 mg·L-1 and accounts for 0.02%-97.07% of the total phytoplankton biomass. Both a notable spatial and seasonal distribution of C. raciborskii were observed. Its occurrence is higher in the western coastal area (77.78%) than in the Zhujiang Delta (66.67%) and northern coastal area (33.33%) and is relatively lower in the dry season (40%) compared with the rainy season (70%) and transition season (85%). The trophic level has a significant effect on the presence of C. raciborskii, which is notably higher in eutrophic reservoirs (81.48%) than in mesotrophic reservoirs (66.67%) and oligotrophic reservoirs (33.33%). The redundancy analysis shows that C. raciborskii biomass is positively correlated with total nitrogen (TN) and the trophic state index (TSI) and negatively correlated with dissolved inorganic nitrogen (DIN), soluble reactive phosphorus (SRP), and the secchi depth (SD). Thus, C. raciborskii in Guangdong reservoirs may be promoted by environmental factors such as high nitrogen contents, low phosphorus concentration, and transparency.

Inhibitory effect of Biejiajian pills on HepG2 cell xenograft growth and expression of β-catenin and Tbx3 in nude mice / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the molecular mechanism by which Biejiajian pills inhibit hepatocellular carcinoma in a nude mouse model bearing HepG2 cell xenograft.</p><p><b>METHODS</b>The inhibitory effect of Biejiajian pills on the growth of HepG2 cell xenograft in nude mice was observed. Immunohistochemical method was used to examine proliferating cell nuclear antigen (PCNA) expression in HepG2 cell xenograft, and TUNEL method was employed to detect the cell apoptosis; the expression levels of β-catenin and Tbx3 were measured by Western blotting.</p><p><b>RESULTS</b>Biejiajian pills significantly suppressed the growth of HepG2 cell xenograft in nude mice. The tumor-bearing mice treated with a high and a moderate dose of Biejiajian pills showed significantly increased apoptosis rate of the tumor cells [(22.9±1.220)% and (14.7±0.50)%, respectively] compared with the control group [(5.5±0.90)%, P<0.05]. Treatment with Biejiajian pills significantly decreased the expressions of PNCA, β-catenin, and Tbx3 in the cell xenograft (P<0.05).</p><p><b>CONCLUSIONS</b>Biejiajian pills can inhibit the growth of HepG2 cell xenograft in nude mice and promote tumor cell apoptosis possibly by inhibiting PNCA expression and the Wnt/β-catenin signaling pathway.</p>

Dual-labeled time-resolved immunofluorometric assay for the determination of IgM antibodies to rubella virus and cytomegalovirus in human serum.

OBJECTIVES: This study established a novel time-resolved fluorescence immunoassay (TRFIA) that allows the simultaneous determination of rubella virus (RV) IgM and cytomegalovirus (CMV) IgM in human serum. DESIGN AND METHODS: Lanthanum elements labeled antibody and streptavidin-biotin system were used in the "capture sandwich" format simultaneously. RESULTS: The working range of TRFIA for RV IgM was 2-80 AU/mL and for CMV IgM was 5-400 AU/mL. Intra- and inter-assay coefficient of variation (CV) for RV IgM and CMV IgM were both less than 10% and recoveries were from 90% to 110%. No significant statistical difference in sensitivity or specificity was observed between dual-TRFIA and commercial chemiluminescent immunoassays (CLIA) in serum samples. CONCLUSION: The novel dual-TRFIA for RV IgM and CMV IgM detection might have valuable clinical application, with satisfactory sensitivity, specificity and accuracy.

Hydrocamptothecin promotes the mRNA expression of Wnt signaling inhibitor DKK-1 / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effects of hydrocamptothecin on the expression of Wnt signaling pathway inhibitor DKK-1 in tumor cells.</p><p><b>METHODS</b>Human HepG2, Hep3B, LoVo and U251 cells were treated with the antitumor drug Hydrocamptothecin. DKK-1 mRNA expression in the cells was detected with RT-PCR, and beta-catenin expression was measured by fluorescence-activated cell sorting (FACS).</p><p><b>RESULTS</b>DKK mRNA in Hep3B, HepG2, LoVo and U251 cells was significantly increased after hydrocamptothecin treatment for 24 h, and the percentage of beta-catenin-positive cells and fluorescence intensity for beta-catenin expression was lowered in the cells after the treatment.</p><p><b>CONCLUSION</b>Hydrocamptothecin promotes mRNA expression of Wnt signaling pathway inhibitor DKK-1 in Hep3B, HepG2, LoVo and U251 cells.</p>

[One step time-resolved fluorescence immunoassay for direct estimation of microcystins].

A direct-competitive time-resolved fluorescence immunoassay (TRFIA) for microcystin detection was established, which was based on europium labeled MCLR-BSA conjugant and microtiter plates coated with anti-mouse IgG. The optimal dilution of europium labeled MCLR-BSA conjugant is 1/50 and most appropriate titration of anti-microcystin-LR (MCLR) monoclonal antibodies is 100 ng/mL. The standard curve under the optimal conditions shows that the quantitative range is from approximately 0.05 ng/mL to 10 ng/mL. The detection limit of the method is 0.02 ng/mL for MCLR, recoveries for microcystin (> 94%) in quantitative range is satisfactory.

[Usage of flocculation in emergent control of algal bloom in drinking water supplying reservoir].

An Anabaena circinalis bloom appeared in a reservoir for supplying drinking water in the south of China, in April 2006. Phytoplankton scums gathered and floated on the surface of the whole reservoir especially on the area of water intake, and the cell density of phytoplankton, cyanobacteria and Anabaena circinalis was as high as 7.3 x 10(7), 7.2 x 10(7), 4.1 x 10(7) cells x L(-1) respectively. To maintain drinking water supplying, an emergency program was initiated to control the cyanobacterial bloom. The zone immediately adjacent to the water intake was divided into two small zones by fishing nets and waterproof curtains to modify the water flow. Iron-based flocculants were then applied to control the algal bloom. As a result, the density of the phytoplankton decreased greatly, and at the first day the cell densities of phytoplankton, cyanobacterial, Anabaena circinalis decreased to 5.3 x 10(6), 4.7 x 10(6), 2 x 10(6) cells x L(-1) respectively, and the removal of them reached up to 93%, 94%, 95% respectively. The average of phytoplankton cell density was 1.2 x 10(7) cells x L(-1) and a highest density was 2.0 x 10(7) cells x L(-1) during the treatment from 22 to 30 April, while Chlorophyta and Bacillariophyta slightly increased. These encouraging results suggest that the flocculants used are efficient at removing Cyanobacteria.

Structural and functional characterization of microcystin detoxification-related liver genes in a phytoplanktivorous fish, Nile tilapia (Oreochromis niloticus).

Liver genes related to phase I and phase II detoxification, as well as inhibition of reactive oxygen species (ROS) production, were cloned, and their response to microcystin-LR (MC-LR) and lipopolysaccharide (LPS) exposure via intraperitoneal injection, was determined in a phytoplanktivorous fish, Nile tilapia (Oreochromis niloticus). The cloned full-length cDNA of tilapia soluble glutathione S-transferase (sGST) was classified as alpha-class GST based on their amino acid sequence identity with other species. The tilapia sGST clone was 861 bp in length, and contained a 25 bp 5'-UTR, a 167 bp 3'-UTR and an open reading frame of 669 bp, encoding a polypeptide of 222 amino acids. Using genome walker method, a 366 bp 5'-flanking sequence of tilapia sGST gene was further obtained, and the possible regulatory elements were identified. Partial cDNA sequences of glutathione peroxidase (GPX) and uncoupling protein 2 (UCP2) were also obtained by PCR using degenerate primers from tilapia liver. To study the transcriptional response of liver genes to microcystin treatment, tilapia were respectively exposed to a single 50 microg kg(-1) body weight (bwt) dose of pure MC-LR, a single 2 mg kg(-1) bwt dose of LPS and a co-exposure MC-LR and LPS (50 microg kg(-1) bwt+2 mg kg(-1) bwt), and were then sacrificed at 24 h post-exposure. Using beta-actin as external control, a significant increase (about 80%) in sGST mRNA expression was found in response to the MC-LR exposure after 24 h (P < 0.05), indicating the importance of sGST in microcystin detoxification. A slight decrease of sGST mRNA expression was observed in the liver of tilapia, exposed to LPS and MC-LR+LPS. It seems that the LPS response element (LPSRE), identified in the promoter region of tilapia sGST gene, may be functional at a rather low level. In contrast, the levels of cytochrome P450 1A (CYP1A) mRNA expression were found to keep unchanged to either MC-LR, or LPS, or MC-LR+LPS treatment, indicating that unlike the phase II enzyme (sGST), the phase I enzyme (CYP1A) might not play an important role in the detoxification process of microcystins. Although not significant, the mRNA expression level of GPX tended to increase in the liver of tilapia exposed to both MC-LR and LPS (P > 0.05). In addition, a significant increase in UCP2 mRNA expression was observed in the liver of tilapia exposed to LPS (P < 0.05), as well as an obvious but not significant increase in MC-LR exposure group. We suggest that phase II detoxification enzyme, instead of phase I detoxification enzyme, might be responsible for the strong tolerance of the phytoplanktivorous fish to microcystins, and hepatocyte proteins coping with oxidative stress (GPX and UCP2), might also have some auxiliary effect. In addition, the rather low and insignificant response of tilapia sGST gene to the inhibitory effect of LPS exposure, might possibly be critical to the phytoplanktivorous fish to utilize toxic blue-green algae.

Molecular cloning and characterization of alpha-class glutathione S-transferase gene from the liver of silver carp, bighead carp, and other major Chinese freshwater fishes.

Two full-length cDNAs encoding glutathione S-transferase (GST) were cloned and sequenced from the hepatopancreas of planktivorous silver carp (Hypophthalmichthys molitrix) and bighead carp (Aristichthys nobilis). The silver carp and bighead carp GST cDNA were 920 and 978 bp in length, respectively, and both contained an open reading frame that encoding 223 amino acids. Partial GST cDNA sequences were also obtained from the liver of grass carp (Ctenopharyngodon idellus), crucian carp (Carassius auratu), mud carp (Cirrhinus molitorella), and tilapia (Oreochromis nilotica). All these GSTs could be classified as alpha-class GSTs on the basis of their amino acid sequence identity with other species. The three-dimensional structure of the silver carp GST was predicted using a computer program, and was found to fit the classical two-domain GST structure. Using the genome walker method, a 875-bp 5'-flanking region of the silver carp GST gene was obtained, and several lipopolysaccharide (LPS) response elements were identified in the promoter region of the phytoplanktivorous fish GST gene, indicating that the GST gene expression of this fish might be regulated by LPS, released from the toxic blue-green algae producing microcystins. To compare the constitutive expression level of the liver GST gene among the six freshwater fishes with completely different tolerance to microcystins, beta-actin was used as control and the ratio GST/beta-actin mRNA (%) was determined as 130.7 +/- 6.6 (grass carp), 103.1 +/- 8.9 (bighead carp), 92.6 +/- 15.0 (crucian carp), 72.3 +/- 7.8 (mud carp), 58.8 +/- 11.5 (silver carp), and 33.6 +/- 13.7 (tilapia). The constitutive expression level of the liver GST gene clearly shows that all the six freshwater fishes had a negative relationship with their tolerance to microcystins: high-resistant fishes (phytoplanktivorous silver carp and tilapia) had the lowest tolerance to microcystins and the high-sensitive fish (herbivorous grass carp) had the highest tolerance to microcystins. Taken together with the reciprocal relationship of constitutive and inducible liver GST expression level in some of the tested fish species to microcystin exposure, a molecular mechanism for different microcystin detoxification abilities of the warm freshwater fishes was discussed.

[Microcystin-LR induces apoptosis in L-02 cell line].

OBJECTIVE: To investigate the toxicological mechanism of microcystin-LR (MCLR) on L-02 cells. METHODS: L-02 cells was treated with MCLR at different concentrations and the subsequent changes such as cell proliferation (MTT assay), morphology, lactate dehydrogenase (LDH) leakage, apoptosis rate and apoptosis-related gene expression were examined. RESULTS: MTT assay showed that MCLR mildly inhibited the cell growth within the initial 24 h of treatment but enhanced the cell viability after that till 60 h in a time- and dose-dependent manner. LDH leakage underwent no marked changes in response to 48-hour MCLR treatment but increased upon prolonged treatment for 60 h, indicating the presence of oxidative damage. After a 48-h treatment with MCLR at 50 microg/ml, obvious apoptosis of L-02 cells occurred as manifested by cell rounding, detachment from the substrate, cell shrinkage and membrane blebbing. The apoptosis rates were rather low (between 22% and 29%) after treatment with MCLR at different concentrations for 36 h, and increased to as much as 80% after a 60-h treatment with 50 microg/ml MCLR. The expressions of p53 and bcl-2 increased in the cells after treatment with high-concentration MCLR, suggesting that MCLR up-regulated the expression levels of the two proteins. CONCLUSION: MCLR can induce apoptosis and up-regulate p53 and bcl-2 expressions in human normal liver cell line L-02.

Evaluation of a parasite lactate dehydrogenase-based colloid gold-immunochromatography assay for diagnosis of Plasmodium falciparum.

OBJECTIVE: To establish a colloid gold-immunochromatography assay (GICA) for detecting Plasmodium falciparum. METHODS: The monoclonal antibodies (mAbs) against lactate dehydrogenase of P. falciparum (LDHpf) were screened for preparation of GICA strips. With microscopic examination of the blood smears and PCR test as control, GICA was evaluated for its sensitivity, specificity and stability in the diagnosis of malaria in the outpatient clinics in China. RESULTS: Four hybridoma cell lines against LDHpf were prepared. Enzyme-linked immunosorbent assay demonstrated that the 4 mAbs reacted only with P. falciparum, but not with protein of normal human red cell, P. vivax, Toxoplasma gondii, or Schistosoma japonicam. All the 4 mAbs recognized a 33 kD protein designated as LDHpf as shown by Western blot analysis. Compared with the microscopic examination of blood smears and PCR test, GICA had the sensitivities of 88.37% and 86.67% and the specificities of 95.65% and 97.78%, respectively. Concordance between microscopic examination and GICA for P. falciparum infection was 91.55%. CONCLUSION: GICA established in this study is simple, rapid, sensitive and specific for detecting P. falciparum, and is potentially useful in developing reagent kits for clinical use.

[Acute toxicity of microcystin-LR in BALB/c mice].

OBJECTIVE: To investigate acute toxicity of microcystin-LR in BALB/c mice. METHODS: BALB/c mice were subjected to intraperitoneal injection of microcystin-LR at the half lethal dose (LD50) and 1/2 LD50, and the organ weight indices and various biochemical indices were determined 30 min and 4 h after the treatment, respectively. RESULTS: Significant increase in liver or kidney weight index occurred in the treated mice, indicating hemorrhagic conditions of the two organs. A slight increase in serum levels of aspartate amino transferase and alanine aminotransferase was observed along with an obvious decrease in serum urea levels, suggesting liver damage. The increase of serum uric acid also indicated the presence of kidney damage. CONCLUSION: The kidney may be another target of microcystins besides the liver.

An ELISA-like time-resolved fluorescence immunoassay for microcystin detection.

BACKGROUND: A time-resolved fluorescence immunoassay (TRFIA), based on anti-microcystin-LR (MCLR) monoclonal antibodies (MAbs) and europium-labeled antimouse IgG conjugate, was first developed for microcystin detection. METHODS: Anti-MCLR MAbs were prepared by a standard method, and the attained MAbs showed a good cross reactivity with MCLR, MCRR and MCYR. The TRFIA was performed in an indirect competitive mode. The detection method of TRFIA was compared with indirect competitive enzyme-linked immunosorbent assay (ELISA) and high-performance liquid chromatography (HPLC). RESULTS: The TRFIA exhibited a typical sigmoidal response for MCLR at concentrations of 0.005-50 ng/ml, with a wide quantitative range between 0.01 and 10 ng/ml, indicating the broadest detective range and the most sensitive of all the methods for microcystins (MCs) detection. Additionally, the TRFIA maintained good reliability through its quantitative range, as evidenced by low coefficients of variation (1.6-12.2%). The toxin data of algal samples assayed from TRFIA were in the same range as those with ELISA and HPLC, implying that the method was reliable and practical for the detection of MCs. CONCLUSIONS: The TRFIA may offer a valuable alternative or a substitute for conventional ELISA for microcystin detection.

Study on the Preparation of Sulfadiazine Silver Collagen Sheeting for Burn and the Release Rate of Sulfadiazine-silver / 中国药房

AIM:To prepare the sulfadiazine silver collagen sheeting for burn(SD-Ag sheeting) and determine the release rate of sulfadiazine silver METHODS:To prepare the SD-Ag sheeting by cross-linking method and to determine the release rate by uniform design RESULTS:The release rate of SD-Ag from SD-Ag sheeting was (22 38?0 036)% CONCLUSION:The preparation of SD-Ag sheeting by cross-linking method was convenient The determining method was accurate,rapid and simple for the quality control of the SD-Ag sheeting