Co-delivery of doxorubicin-dihydroartemisinin prodrug/TEPP-46 nano-liposomes for improving antitumor and decreasing cardiotoxicity in B16-F10 tumor-bearing mice.

In order to reduce the cardiotoxicity of doxorubicin (DOX) and improve its antitumor effect, dihydroartemisinin (DHA) and DOX prodrug (DOX-S-DHA) synthesized via a single sulfur bond was used with TEPP-46 to prepare nano-liposomes (DOX-S-DHA@TEPP-46 Lips). In which, TEPP-46 was expected to exert p53 bidirectional regulation to promote the synergistic antitumor effect of DOX and DHA while reducing cardiotoxicity. DOX-S-DHA@TEPP-46 Lips exhibited uniform particle size, good stability, and excellent redox-responsive activity. DOX-S-DHA@TEPP-46 Lips could significantly inhibit the proliferation of tumor cells, but had less cytotoxicity on normal cells. The presence of TEPP-46 increased the content of p53 protein, which further induced tumor cell apoptosis. DOX-S-DHA@TEPP-46 Lips had satisfactory long circulation to enhance the antitumor efficacy and reversed the cardiotoxicity of DOX in B16-F10 tumor-bearing mice. In conclusion, DOX-S-DHA@TEPP-46 Lips provides a new insight on creating sophisticated redox-sensitive nano-liposomes for cancer therapy as well as the decreased cardiotoxicity of DOX.

Asymmetric Synthesis of Spiropyrazolone-Fused Dihydrofuran-naphthoquinones Bearing Contiguous Stereocenters via a Michael Addition/Chlorination/Nucleophilic Substitution Sequence.

An enantioselective synthesis of spiropyrazolone-fused dihydrofuran-naphthoquinones is first demonstrated via a Michael addition/Chlorination/Nucleophilic substitution sequence. The reactions of 2-hydroxy-1,4-naphthoquinone and α,ß-unsaturated pyrazolones in the presence of the cinchona alkaloid derived hydrogen-bonding catalyst and NCS provide spiropyrazolone-fused 2,3-dihydronaphtho[2,3-b]furan-4,9-diones bearing contiguous stereocenters, of which one is the spiro quaternary stereocenter in high yields with exclusive diastereoselectivity and good to excellent enantioselectivities.

Novel multi-component crystals of berberine with improved pharmaceutical properties.

As an extremely popular natural product, berberine (BER) is mainly used for gastroenteritis and diarrhoea caused by bacteria. Research has also revealed the potent and extensive pharmacological properties of BER including its anti-arrhythmic, anti-tumour, anti-inflammatory and hypoglycemic activities and so on; therefore, BER is a promising drug for further development. However, its commercial form with hydrochloride exhibits poor stability and solubility, which are detrimental to its clinical therapeutic effects. For these purposes, the salt form was regulated via the reactive crystallization of 8-hydroxy-7,8-dihydroberberine (8H-HBER) with five pharmaceutically suitable organic acids including malonic acid (MA), L-tartaric acid (LTA), D-tartaric acid (DTA), DL-tartaric acid (DLTA) and citric acid (CA), resulting in the six novel solid forms 1BER-1LTA-1W, 1BER-1DTA-1W, 1BER-1DLTA and 2BER-2CA as well as two rare multi-stoichiometric solid forms 1BER-1MA and 1BER-2MA-2W. The preparation of the multi-stoichiometric products was greatly influenced by both the crystallization solvent type and the molar ratio of reactants. The structures of these multi-component solid forms were determined using single-crystal X-ray diffraction and further characterized by powder X-ray diffraction, thermal analysis and Fourier transform infrared spectroscopy. Stability experiments showed that all samples prepared had superior physical stability under high temperature and high humidity. Furthermore, dissolution experiments demonstrated that the maximum apparent solubilities (MAS) of all the products were significantly improved compared with the commercial form of BER in dilute hydrochloric solution (pH = 1.2). In particular, the MAS of 1BER-1MA in dilute hydrochloric solution is as high as 34 times that of the commercial form. In addition, it is preliminarily confirmed that the MAS of the samples prepared in pure water and dilute hydrochloric solution is primarily influenced by a combination of factors including the packing index, intermolecular interactions, affinity of the counter-ion to the solvent, the molar ratio of the drug to counter-ion in the product and the common ion effect. These novel solids are potential candidates for BER solid forms with improved oral dosage design and may prompt further development.

Docetaxel prodrug and hematoporphyrin co-assembled nanoparticles for anti-tumor combination of chemotherapy and photodynamic therapy.

To realize the synergistic anti-tumor effect of chemotherapy and photodynamic therapy, the mono sulfide-modified docetaxel (DTX) prodrugs (DSD) provided by our laboratory and hematoporphyrin (HP) were used to physically prepare co-assembled nanoparticles (DSD/HP NPs) by nano-precipitation. For the first time, this study showed its characteristics, in vitro anti-tumor activity, pharmacokinetic behavior in rats, in vivo distribution, and pharmacodynamic effects on 4T1 tumor-bearing Bal b/c mice. DSD/HP NPs optimized by single-factor and response surface optimization had several distinct characteristics. First, it had dark purple appearance with particle size of 105.16 ± 1.24 nm, PDI of 0.168 ± 0.15, entrapment efficiency and drug loading of DSD and HP in DSD/HP NPs of 96.27 ± 1.03% and 97.70 ± 0.20%, 69.22 ± 1.03% and 20.03 ± 3.12%, respectively. Second, it had good stability and could release DTX and HP slowly in the media of pH 7.4 PBS with 10 mM DTT (H2O2). Moreover, DSD/HP NPs along with NiR treatment significantly inhibited 4T1 cells proliferation, and induced more reactive oxygen species and cells apoptosis. In vivo pharmacokinetic and pharmacodynamic studies showed that DSD/HP NPs could prolong the drug circulation time in rats, increase drug distribution in tumor site, obviously inhibit tumor growth, and decrease the exposure of drug to normal tissues. Therefore, DSD/HP NPs as a promising co-assembled nano-drug delivery system could potentially improve the therapeutic efficiency of chemotherapeutic drug and achieve better anti-tumor effects due to the combination of chemotherapy and photodynamic therapy.

High expression of serine and arginine-rich splicing factor 9 (SRSF9) is associated with hepatocellular carcinoma progression and a poor prognosis.

BACKGROUND: Serine and arginine-rich splicing factor 9 (SRSF9) has been linked to the occurrence and progression of various cancers; however, its effects and mechanism of action hepatocellular carcinoma (HCC) have not been reported. In this study, we used a bioinformatics approach and in vitro assays to evaluate the expression of SRSF9 in HCC, its prognostic value, and its underlying regulatory mechanisms, including analyses of related pathways and the role of methylation. METHODS: Transcriptomic and DNA methylation data for 357 HCC cases and 50 paratumor tissues in The Cancer Genome Atlas database were obtained. Additionally, protein expression data for cell lines and tissue samples were obtained from the Human Protein Atlas. The CMap databased was used to predict candidate drugs targeting SRSF9. Various cell lines were used for in vitro validation. RESULTS: SRSF9 expression was significantly elevated in HCC and was negatively regulated by its methylation site cg06116271. The low expression of SRSF9 and hypermethylation of cg06116271 were both associated with a longer overall survival time. A correlation analysis revealed ten genes that were co-expressed with SRSF9; levels of CDK4, RAN, DENR, RNF34, and ANAPC5 were positively correlated and levels of RBP4, APOC1, MASP2, HP, and HPX were negatively correlated with SRSF9 expression. The knockdown of SRSF9 in vitro inhibited the proliferation and migration of HCC cells and significantly reduced the expression of proteins in the Wnt signaling pathway (DVL2 and ß-catenin) and cell cycle pathway (Cyclin D and Cyclin E). A CMap analysis identified two drugs, camptothecin and apigenin, able to target and inhibit the expression of SRSF9. CONCLUSIONS: This study expands our understanding of the molecular biological functions of SRSF9 and cg06116271 and provides candidate diagnostic and therapeutic targets for HCC.

Increased ATP2A1 Predicts Poor Prognosis in Patients With Colorectal Carcinoma.

Colorectal cancer is one of the most common malignant tumors in the digestive system. Traditional diagnosis and treatment methods have not significantly improved the overall survival of patients. In this study, we explored the value of ATP2A1 as a biomarker in predicting the prognosis of colorectal cancer patients. We used the TCGA database to reveal the relationship between ATP2A1 mRNA level and prognosis, methylation, and immune invasion in colorectal cancer. The results showed that the expression of ATP2A1 was increased in colorectal cancer. The overall survival of patients with high expression of ATP2A1 was significantly lower than patients with low expression of ATP2A1. Cox regression analysis showed that high expression of ATP2A1 was an independent risk factor for poor prognosis in colorectal cancer patients. In addition, we used three datasets to perform a meta-analysis, which further confirmed the reliability of the results. Furthermore, we revealed that ATP2A1 could significantly inhibit the proliferation of colorectal cancer cells by inhibiting the autophagy process and was associated with several immune cells, especially CD8 + T cells. Finally, four small molecule drugs with potential inhibition of ATP2A1 expression were found by CMap analysis. This study demonstrates for the first time that ATP2A1 is a potential pathogenic factor, which may play a significant role in colorectal cancer.

Construction of reduction-sensitive heterodimer prodrugs of doxorubicin and dihydroartemisinin self-assembled nanoparticles with antitumor activity.

Doxorubicin (DOX) is used as a first-line chemotherapeutic drug, whereas dihydroartemisinin (DHA) also shows a certain degree of antitumor activity. Disulfide bonds (-SS-) in prodrug molecules can be degraded in highly reducing environments. Thus, heterodimer prodrugs of DOX and DHA linked by a disulfide bond was designed and subsequently prepared as reduction-responsive self-assembled nanoparticles (DOX-SS-DHA NPs). In an in vitro release study, DOX-SS-DHA NPs exhibited reduction-responsive activity. Upon cellular evaluation, DOX-SS-DHA NPs were found to have better selectivity toward tumor cells and less cytotoxicity to normal cells. Compared to free DiR, DOX-SS-DHA NPs showed improved accumulation at the tumor site and even had a longer clearance half-life. More importantly, DOX-SS-DHA NPs possessed a much higher tumor inhibition efficacy than DOX-sol and MIX-sol in 4T1 tumor-bearing mice. Our results suggested the superior antitumor efficacy of DOX-SS-DHA NPs with less cytotoxicity.

Design of red blood cell membrane-cloaked dihydroartemisinin nanoparticles with enhanced antimalarial efficacy.

Targeting delivery and prolonging action duration of artemisinin drugs are effective strategies for improving antimalarial treatment outcomes. Here, dihydroartemisinin (DHA) loaded poly (lactic-co-glycolic acid) (PLGA) nanoparticles (PDNs) were prepared and further cloaked with red blood cell (RBC) membranes via electrostatic interactions to yield RBC membrane-cloaked PDNs (RPDNs). The prepared RPDNs displayed a notable "core-shell" structure, with a negative surface charge of -29.2 ± 4.19 mV, a relatively uniform size distribution (86.4 ± 2.54 nm, polydispersity index of 0.179 ± 0.011), an average encapsulation efficiency (70.1 ± 0.79%), and a 24-h sustained-release behavior in vitro. Compared with PDNs, RPDNs showed markedly decreased phagocytic activity by RAW 264.7 cells and had prolonged blood circulation duration. The Pearson correlation coefficient of RPDNs distribution in infected red blood cells (iRBCs) was 0.7173, suggesting that RPDNs could effectively target Plasmodium-iRBCs. In PyBy265-infected mice, RPDNs showed a higher inhibition ratio (88.39 ± 2.69%) than PDNs (83.13 ± 2.12%) or DHA (58.74 ± 3.78%), at the same dose of 8.8 µmol/kg. The ED90 of RPDNs (8.13 ± 0.18 µmol/kg) was substantially lower than that of PDNs (14.48 ± 0.23 µmol/kg) and DHA (17.67 ± 3.38 µmol/kg). Furthermore, no apparent abnormalities were detected in routine blood examination, liver function indexes, and pathological analysis of tissue sections of PyBy265-infected mice following RPDNs treatment. In conclusion, the prepared RPDNs exhibited enhanced antimalarial efficacy, prolonged circulation, targeted delivery to Plasmodium-iRBCs, and satisfactory biocompatibility.

Murine pharmacokinetics and antimalarial pharmacodynamics of dihydroartemisinin trimer self-assembled nanoparticles.

Currently, conjugation of artemisinin-derived dimers, trimers, and tetramers is a viable strategy for developing new effective antimalarial candidates. Furthermore, nanotechnology is an effective means to achieve intravenous administration of hydrophobic drugs. In this paper, an ester-linked dihydroartemisinin trimer (DHA3) was synthesized and further prepared as self-assembled nanoparticles (DHA3NPs) by a one-step nanoprecipitation method. The pharmacokinetics and antimalarial pharmacodynamics of DHA3NPs were studied in rats and mice infected with Plasmodium yoelii BY265 (PyBY265). DHA3NPs had a regular spherical shape with a uniform size distribution of 140.27 ± 3.59 nm, entrapment efficiency (EE) of 99.63 ± 0.17%, and drug loading efficiency (DL) of 79.62 ± 0.11%. The in vitro release characterization revealed that DHA3NPs were easily hydrolysed into DHA in an esterase environment. The pharmacokinetics study demonstrated that the area under the concentration-time curve (AUC0-t) of DHA in DHA3NPs group was 2070.52 ± 578.76 h×ng×mL-1, which was higher than that of DHA and artesunate (AS) control groups (AUC0-t values of 724.18 ± 94.32 and 448.40 ± 94.45 h×ng×mL-1, respectively) (P < 0.05). The antimalarial pharmacodynamics in vivo suggested that DHA3NPS (ED90 7.82 ± 1.16 µmol×(kg×day)-1) had a superior antimalarial effect compared with that of control groups (ED90 values of 14.68 ± 0.98 (DHA) and 14.34 ± 1.96 (AS) µmol×(kg×day)-1) (P < 0.05). In addition, DHA3NPS reduced the recurrence ratio and improved the cure ratio and survival time. In summary, DHA3NPs exhibited promising pharmacokinetic characteristics and antimalarial pharmacodynamics in vivo.

Squaramide-catalysed asymmetric Friedel-Crafts alkylation of naphthol and unsaturated pyrazolones.

The first method for highly efficient asymmetric Michael-type Friedel-Crafts alkylation of naphthol and unsaturated pyrazolones has been accomplished under mild reaction conditions. In the presence of the chiral squaramide catalyst, a wide range of substrates are tolerated in excellent yields (up to 99%) with reasonable enantioselectivities (up to 96% ee).

Choline and PEG dually modified artemether nano delivery system targeting intra-erythrocytic Plasmodium and its pharmacodynamics in vivo.

OBJECTIVE: The choline derivative (CD) and polyethylene-glycol (PEG) dually modified artemether (ARM) nanostructured lipid carriers (CD-PEG-ARM-NLC) have been designed to prolong the circulation of ARM in blood, as well as to develop targeting for new permeability pathways (NPPs) and erythrocyte choline carriers (ECCs) that are expressed on the Plasmodium-infected erythrocyte membrane. SIGNIFICANCE: The CD-PEG-ARM-NLC constructed in this study was found to be able to target endoerythrocytic Plasmodium by increasing the drug concentration and residence time in the infected erythrocytic microenvironment and minimizing toxicity and side effects. METHODS: CD-PEG-ARM-NLC was prepared using high-pressure homogenization followed by physicochemical characterization. The targeting ability of CD-PEG-NLC to infected erythrocytes probed by coumarin-6 was investigated by using fluorescence microscopy imaging. The SYBR Green I assay for parasite nucleic acid was adapted in order to assess the efficacy of inhibition against parasite growth in vitro. The antimalarial activity of ARM-loaded NLCs was evaluated by a Pearson four-day suppressive test in Pyy265BY-bearing mice. RESULTS: In vitro imaging indicated that the intracellular delivery of CD-PEG-ARM-NLC was efficiently taken up by the infected erythrocytes via ECCs and NPPs, which could be inhibited by addition of furosemide (an inhibitor of NPPs) and excessive choline (native substrate of ECCs). Moreover, in vitro and in vivo studies that evaluated antimalarial activity suggested that CD-PEG-ARM-NLC exhibited higher antimalarial activity in comparison to ARM-NLC and PEG-ARM-NLC. CONCLUSION: These findings suggested that choline and PEG dually modified NLC could be promising preparations for the production of hydrophobic antimalarial drugs, particularly for ARM.

In vivo and in vitro evaluation of dihydroartemisinin prodrug nanocomplexes as a nano-drug delivery system: characterization, pharmacokinetics and pharmacodynamics.

To develop new, more effective and lower toxicity antitumor dihydroartemisinin (DHA) nanocomplexes, a DHA prodrug synthesized in this study was used to prepare DHA prodrug self-assembled nanocomplexes (DHANPs) by molecular self-assembly technology. The optimization, pharmacokinetics and in vitro and in vivo antitumor efficiency of DHANPs were assessed. The results showed that the entrapment efficiency, drug loading, particle size and zeta potential of the optimized formulation were 92.37 ± 3.68%, 76.98 ± 3.07%, 145.9 ± 2.11 nm and -16.0 ± 0.52 mV, respectively. DHANPs had a uniform size distribution and good stability during storage. The release of DHA prodrugs from DHANPs was slow in a PBS solution (pH 7.4). The pharmacokinetic study indicated that DHANPs could significantly improve the blood concentration of DHA. DHANPs exhibited lower cytotoxicity to 4T1 cells. More importantly, DHANPs could increase the quality life of mice in comparison with that of the DHA solution in 4T1 tumor-bearing mice. In short, the optimized DHA prodrug nanocomplexes show good long-term stability during the experimental time, extend the life-cycle of DHA in rats and can act as a prospective nano-drug delivery system for future artemisinin-based anti-tumor drugs.

Solvates of acotiamide hydrochloride: characterization and phase transformation.

Five novel crystals of acotiamide hydrochloride (AH) with solvates dimethyl formide (DMF), dimethyl sulfoxide (DMSO), ethyl acetate (EA) and water (W) [1AH-1W-1DMF, 1AH-1DMSO-I, 1AH-1DMSO-II, 1AH-1W-1DMSO and 2AH-2DMSO-1EA] were characterized using single-crystal X-ray diffraction, powder X-ray diffraction, thermal analysis and Fourier transform infrared spectroscopy. The five crystals can be divided into four distinct structural types based on analysis of their similarities; 1AH-1W-1DMF and 1AH-1W-1DMSO are isostructural. The benzene rings in 1AH-1DMSO-I, 1AH-1DMSO-II and 2AH-2DMSO-1EA can rotate 180°, and the intramolecular hydrogen bond changes from an OH...C=O bond to an NH...OH bond relative to what is seen in 1AH-1W-1DMF and 1AH-1W-1DMSO. Phase transformation experiments show that a humidity-induced phase transformation is closely tied to moisture content.

Antimalarial activity and metabolism of dihydroartemisinin-derived dimer.

The dihydroartemisinin-derived dimer (DHA dimer) was synthesized, and its antimalarial activities were evaluated both in vitro and in vivo. The dimer IC50 value of 0.51 ± 0.12 nM in vitro was significantly lower than that of DHA at 1.81 ± 0.70 nM. The dimer ED50 values were 0.44 ± 0.03 and 0.18 ± 0.03 mg/(kg·day) in vivo for intragastric (i.g.) and intravenous (i.v.) groups, respectively, to Plasmodium yoelii rodent malaria. It also performed better relative to those of DHA which had ED50 values of 0.76 ± 0.03 mg/(kg·day) (i.g.) and 0.32 ± 0.03 mg/(kg·day) (i.v.). Moreover, the recrudescence rate, negative conversion rate, and cure rate of the dimer showed superior performance. Furthermore, the metabolites and major metabolic pathways of the dimer in rats were preliminarily investigated using the HPLC-HRMSn method. Twenty-seven metabolites, including DHA, 11 metabolites of DHA, and 15 other novel metabolites, were detected in rats after i.g. administration of dimer. The metabolic pathways of the 15 novel metabolites were inferred: deoxygenation, hydroxylation, and hydroxylation with dehydration.

Polymorphs and Versatile Solvates of 7-Hydroxyisoflavone.

7-hydroxyisoflavone has been crystallized, identified, and characterized as 2 solvent-free conformational polymorphs and 5 solvates, which differ from each other in the mode of packing and in molecular conformation. All the 7 crystal structures were previously unreported. The conformational polymorphs and solvates were compared by Hirshfeld surface and fingerprint plot analysis and were spectroscopically characterized by powder X-ray diffraction, differential scanning calorimetry, and thermal gravimetric analysis. Hydrogen bond played an important role in the formation of polymorphs. From this study, we can predict that more solvates could be cultivated in other polarity solvents such as isopropanol or 2-butanol at appropriate conditions.

Salt screening and characterization of ciprofloxacin.

With the aim of improving the solubility of ciprofloxacin, polybasic organic acids were utilized to react with ciprofloxacin in different stoichiometric proportions. The use of the solvent drop grinding (SDG) method, as well as the solvent evaporation method, resulted in the crystalline salts ciprofloxacin/fumaric acid (1:1, 2:1), ciprofloxacin/maleic acid (1:1) and ciprofloxacin/citric acid (2:1). The solubilities of these salts in pure water (pH 7.0) were determined using high-performance liquid chromatography (HPLC) at 310 K, with the salts showing considerably greater solubility than ciprofloxacin itself and, interestingly, ciprofloxacin/fumaric acid (2:1) being more soluble than ciprofloxacin/fumaric acid (1:1). Intrigued by this phenomenon, we undertook a comparison of the crystal structures of the salts: the three-dimensional sandwich-like structure observed in the 2:1 salt indicates that the preferred stacking may be a factor in increasing the solubility of ciprofloxacin.

Morphology and Microstructure of As-Synthesized Anodic TiO2 Nanotube Arrays.

The as-grown structure of electrochemically synthesized titania nanotube arrays is investigated by scanning electron microscope (SEM) in combination with transmission electron microscope (TEM) as well as X-ray diffraction (XRD). The analysis reveals a preferred growth direction of the nanotubes relative to the substrate surface and the well control on the nanotube arrays morphology. The crystal structure of the anatase phase is detected and exists in the tube walls without any thermal treatment, which makes it possible to realize the application of as-formed TiO2 nanotubes avoiding the degradation of the nanotube structures when sintering. In addition, a new growth, layered model of the anodic TiO2 nanotubes is presented to obtain further understanding of the growth mechanism.

[A study of chronic hepatitis B infection superinfected with hepatitis E infection].

OBJECTIVES: To compare the influence of hepatitis E virus (HEV) infection on the replication of hepatitis B virus (HBV) by observing clinical features, the relationship between the number of HBV DNA copies in serum, the degree of hepatic function impairments and prognosis of chronic hepatitis B patients with hepatitis E superinfection. METHODS: One hundred and fifteen chronic hepatitis B (CHB) patients and 115 CHB patients with hepatitis E (HE) superinfection were studied and compared. 74 liver tissue biopsy samples of the CHB and 51 of the CHB-HE sufferers were obtained. HBsAg, anti-HBs, HBeAg, anti-HBe, anti-HBc, anti-HBc-IgG, anti-HBc-IgM, HBV DNA and anti-HEVIgM were detected respectively by enzyme linked immunosorbent assay and polymerase chain reaction. RESULTS: The number of HBV DNA copies in sera in the CHB-HE group and the severity of the clinical features were significantly higher than those in the CHB group. The former group had a poorer prognosis with a 49.6% occurrence rate of severe hepatitis and a 25.2% death rate while in the latter group the corresponding rates were just 4.4% and 1.7%. CONCLUSION: Re-infection with HEV, in those CHB patients, severely damaged their hepatic functions and increased their mortality; the older the patient and the higher the level of HBV DNA, the poorer the prognosis.

[Isolation and characterization of a HCH degradation Sphingomanas sp. stain BHC-A].

An aerobic bacterium was isolated successfully from a long-term contaminated upland field, which was named BHC-A. The bacterium can utilize Hexachlorocycloexane as a sole carbon source and decompose this substance rapidly and completely. According to its physiological & biochemical characters and the homology analysis of its 16S rDNA sequence, this strain was identified as Sphingomonas sp. Stain BHC-A canmineralize not only alpha, gamma and delta-HCH rapidly, but also 5 mg/L beta-HCH in 12h completely. However, Sphingomonas paucimobilis UT26, a gamma-HCH-decomposing bacterium, can only degrade 12.6% of 5 mg/L alpha-HCH, but not beta-HCH and delta-HCH, even for more than 24h in the medium. The seedling of cucumber was selected as the model plant subject to the inhibition of HCH to the growth of cucurbitaceous plants. The results shows that the degradation of HCH isomers by BHC-A in soil is even steady and excellent, that BHC-A can eliminate the contamination of the soil and the harm to the plants.

[Cloning, expression of the gene of dehydrochlorination of BHC from enrichment and its degradation experiment].

A high effective enrichment that could degrade four isomers of BHC completely was got from the soil polluted by gamma-BHC, but the pure culture was not obtained yet. The 471 bp sequence of linN was amplified from the total DNA of enrichment by polymerase chain reaction (PCR). The nucleotide sequence analysis showed that the linN gene had high homology with reported linA up to 99%. Then the amplified fragment linN was cloned in the proper orientation into the site between Nde I and Hind III of pET-29alpha via restriction endonuclease Nde I and Hind III. The recombinant was transformed into its host E. coli strain BL21 and a recombinant protein of about 17 kD was highly expressed and showed high ability of degrading gamma-BHC after induced by IPTG. The expressed protein occupied about 30% of the total bacterial protein. The cell extracts also showed some ability of degradation of gamma-BHC. It offered basic theory for the isolation of pure culture and the construction of genetic engineering microorganisms.