Function and regulation of Aurora/Ipllp kinase family in cell division

During mitosis,the parent cell distributes its genetic materials equally into two daughter cells through chromosome segregation,a complex movements orchestrated by mitotic kinases and its effector proteins.Faithful chromosome segregation and cytokinesis ensure that each daughter cell receives a full copy of genetic materials of parent cell.Defects in these processes can lead to aneuploidy or polyploidy.Aurora/Ipllp family, a class of conserved serine/threonine kinases,plays key roles in chromosome segregation and cytokinesis.This article highlights the function and regulation of Aurora/Ipllp family in mitosis and provides potential links between aberrant regulation of Aurora/Ipllp kinases and pathogenesis of human cancer.

Effects of tachyplesin on the regulation of cell cycle in human hepatocarcinoma SMMC-7721 cells

AIM: To investigate the effects of tachyplesin on the cell cycle regulation in human hepatcarcinoma cells.METHODS: Effects of tachyplesin on the cell cycle in human hepatocarcinoma SMMC-7721 cells were assayed with flow cytometry. The protein levels of p53, p16, cyclin D1 and CDK4 were assayed by immunocytochemistry. The mRNA levels of p21WAF1/CIP1 and c-myc genes were examined with in situ hybridization assay.RESULTS: After tachyplesin treatment, the cell cycle arrested at G0/G1 phase, the protein levels of mutant p53, cyclin D1 and CDK4 and the mRNA level of c-myc gene were decreased, whereas the levels of p16 protein and p21wWF1/CIP1 mRNA increased.CONCLUSION: Tachyplesin might arrest the cell at G0/G1 phase by upregulating the levels of p16 protein and p21WAF1/CIP1 mRNA and downregulating the levels of mutant p53, cyclin D1 and CDK4 proteins and c-myc mRNA, and induce the differentiation of human hepatocacinoma cells.

Effects of tachyplesin on proliferation and differentiation of human hepatocellular carcinoma SMMC-7721 cells

AIM: To investigate the antitumor activities of tachyplesin on human hepatocellula r carcinoma (HCC) cells.METHODS: Tachyplesin, isolated from acid extracts of Chinese horseshoe crab (Tachypleus tridentatus) hemocytes, was used to treat the human HCC cell line SMMC-7721. Effects of tachyplesin on the proliferation of SMMC-7721 cells were measured with trypan blue dye exclusion test and HE staining. The morphology and ultrastructure of the cells were examined by light microscopy and transmission electron microscopy, respectively. The activities of γ-glutamyltransferase (γ-GT) and tyrosine aminotransferase (TAT) were assayed with biochemical methods. The levels of alpha fetoprotein (α-FP), proliferating cell nuclear antigen (PCNA), p21wAF1/CIP1 and c-myc were examined by immunocytochemistry. RESULTS: After treatment with tachyplesin 3.0 mg/L, the proliferation of SMMC-7721 cells was inhibited significantly, with the cell growth inhibitory rate amounted to 55.57 % and the maximum cell mitotic index declined by 43.68 %. The morphology and ultrastructure underwent restorational alteration. The activity of γ-GT declined while TAT activity increased obviously, and the levels of α-FP and PCNA decreased. Moreover, the expression of p21WAF1/CIP1 protein was upregulated and that of c-myc protein was down-regulated. CONCLUSION: Tachyplesin could effectively inhibit the proliferation of hepatocarcinoma cells, reverse the malignant morphological and ultrastructural characteristics, alter the levels of enzymes and antigens, regulate the expression of differentiation-associated oncogene and tumor suppressor gene, and induce hepatocarcinama cell differentiation.

Cost-effective method of siRNA preparation and its application to inhibit hepatitis B virus replication in HepG2 cells

AIM: To find a cost-effective method of preparation of short interfering RNAs based on cloning, fermentation,digestion and purification (CFDP) and test its feasibility to inhibit hepatitis B virus replication in cell culture.METHODS: We constructed an expression vector containing T7 and tac promoter in a head-to-head orientation.cDNA fragment of interest was cloned into this vector between the opposing promoters. dsRNAs were expressed with this vector in Escherichia coli, and purified by affinity chromatography using CF 11 column. They were digested by RNase Ⅲ in a buffer containing manganese ions, then separated on 15% non-denaturing PAGE, and the siRNAs about 25 bp in length were recovered. siRNAs prepared with CFDP were co-transfected with target gene expression plasmid into human cell lines with lipofectamine 2000 to test their inhibition efficiency.RESULTS: siRNAs corresponding to part of the hepatitis3 virus polymerase gene (siHBVP) prepared by CFDP specifically and dramatically suppressed the virus protein expression. The HBsAg expression level was reduced to 10% that of the control by co-transfection of 60 nmol/L siHBVP in SMMC7721 cells. Dose-dependent effect on suppression of HBsAg and HBeAg expression was observed in HepG2 cells. The highest inhibition rate was kept at 70% during the six days after transfection of 7.5 nmol/L siHBVP.CONCLUSION: We show CFDP is a very promising method to prepare therapeutic agents in anti-virus applications.

Cloning and molecular characterization of a novel lectin gene from Pinellia ternata

The full-length cDNA of Pinellia ternata agglutinin (PTA) was cloned from inflorescences using RACE-PCR. Through comparative analysis of PTA gene (pta) and its deduced amino acid sequence with those of other Araceae species, pta was found to encode a precursor lectin with signal peptide and to have extensive homology with those of other Araceae species. PTA was a heterotetrameric mannose-binding lectin with three mannose-binding boxes like lectins from other Araceae and Amaryllidaceae species. Southern blot analysis of the genomic DNA revealed that pta belonged to a low-copy gene family. Northern blot analysis demonstrated that pta constitutively expressed in various plant tissues including root, leaf, stem and inflorescence. The pta cDNA sequence encoding for mature PTA protein was cloned into pET-32a plasmid and the resulting plasmid, pET-32a-PTA containing Trx-PTA fusion protein, was investigated for the expression in E. coli BL21. SDS-PAGE gel analysis showed that the Trx-PTA fusion protein was successfully expressed in E. coli BL21 when induced by IPTG. Artificial diet assay revealed that PTA fusion protein had significant levels of resistance against peach potato aphids when incorporated into artificial diet at 0.1% (w/v). The cloning of the pta gene will enable us to further test its effect in depth on aphids by transferring the gene into crop plants.

Induction of apoptosis by TPA and VP-16 is through translocation of TR3

AIM: To investigate the role of TR3 in induction of apoptosisin gastric cancer cells.METHODS: Human gastric cancer cell line, MGC80-3, wasused. Expression of TR3 mRNA and its protein was detectedby Northern blot and Westem blot. Localization of TR3protein was showed by immunofluorescence analysis underlaser-scanning confocal microscope. Apoptotic morphologywas observed by DAPI fluorescence staining, and apoptoticindex was counted among 1000 cells randomly. Stabletransfection assay was carried out by Lipofectamine.RESULTS: Treatment of MGC80-3 cells with TPA and VP-16resulted in apoptosis, accompanied by the repression ofBcl-2 protein in a time-dependent manner. At the sametime, TPA and VP-16 also up-regulated expression level ofTR3 mRNA in MGC80-3 cells that expressed TR3 mRNA.When antisense-TR3 expression vector was transfected intothe cells, expression of TR3 protein was repressed. In thiscase, TPA and VP-16 did not induce apoptosis. In addition,TPA and VP-16-induced apoptosis involved in translocationof TR3. In MGC80-3 cells, TR3 localized concentrative innucleus, after treatment of cells with TPA and VP-16, TR3translocated from nucleus to cytosol obviously. However,when this nuclear translocation was blocked by LMB,apoptosis was not occurred in MGC80-3 cells even in thepresence of TPA and VP-16.CONCLUSION: Induction of apoptosis by TPA and VP-16 isthrough induction of TR3 expression and translocation of TR3from nucleus to cytosol, which may be a novel signalpathway for TR3, and represent the new biological function ofTR3 to exert its effect on apoptosis in gastric cancer cells.

Codon 249 mutation in exon 7 of p53 gene in plasma DNA:maybe a new early diagnostic marker of hepatocellular carcinoma in Qidong risk area,China

AIM: One of the characteristics of hepatocellular carcinoma (HCC) in Qidong area is the selective mutation resulting in a serine substitution at codon 249 of the p53 gene (1,20),and it has been identified as a 'hotspot' mutation in heptocellular carcinomas occurring in populations exposed to aflatoxin and with high prevalence of hepatitis B virus carriers (2, 3, 9, 10, 16, 24). We evaluated in this paper whether this 'hotspot' mutation could be detected in cellfree DNA circulating in plasma of patients with hepatocellular carcinoma and cirrhosis in Qidong, China, and tried to illustrate the significance of the detection of this molecular biomarker.METHODS: We collected blood samples from 25hepatocellular carcinoma patients, 20 cirrhotic patients and 30 healthy controls in Qidong area. DNA was extracted and purified from 200 μl of plasma from each sample. The 249ser p53 mutation was detected by restriction digestion analysis and direct sequencing of exon-7 PCR products.RESULTS: We found in exon 7 of p53 gene G→T transversion at the third base of codon 249 resulting 249Arg→249ser mutation in 10/25 (40%) hepatocellular carcinoma cases,4/20 (20%) cirrhotics, and 2/30 (7 %) healthy controls.The adjusted odds ratio for having the mutation was 22.1(95 % CI, 3.2～91.7) for HCC cases compared to controls.CONCLUSION: These data show that the 249ser p53mutation in plasma is strongly associated with hepatocellular carcinoma in Qidong patients. We found this mutation was also detected, although it was at a much lower frequency,in plasma DNA of Qidong cirrhotics and healthy controls;We consider that these findings, together with the usual method of HCC diagnosis, will give more information in early diagnosis of HCC, and 249ser p53 mutation should be developed to a new early diagnostic marker for HCC.

Insulin expression in livers of diabetic mice mediated by hydrodynamics-based administration

AIM:Transfer and expression of insulin gene in vivo are an alternative strategy to improve glycemic control in type 1 diabetes. Hydrodynamics-based procedure has been proved to be very efficient to transfer naked DNA to mouse livers. The basal hepatic insulin production mediated by this rapid tail vein injection was studied to determine its effect on the resumption of glycemic control in type 1 diabetic mice.METHODS: Engineered insulin cDNA was inserted into plasmid vectors under a CMV promoter, and transferred into STZ induced diabetic mice by hydrodynamic procedure.Glucose levels, body weight of treated mice, insulin levels,immunohistology of the liver, and quantity of insulin mRNA in the liver were assayed to identify the improvement of hyperglycemic complication after plasmid administration.Sleeping Beauty, a transposon system, was also used to prolong the insulin expression in the liver.RESULTS: After plasmid administration, Plasma insulin was significantly increased in the diabetic mice and the livers were insulin-positive by immunostaining. At the same time the hyperglycemic complication was improved. The blood glucose levels of mice were reduced to normal. Glucose tolerance of the treated diabetic mice was improved. Body weight loss was also ameliorated. The rapid tail vein injection did not cause any fatal result.CONCLUSION: Our results suggested that insulin gene could be efficiently transferred into the livers of diabetic mice via rapid tail vein injection and it resulted in high level of insulin expression. The basal hepatic insulin production mediated by hydrodynamics-based administration improved the glycemic control in type 1 diabetes dramatically and ameliorated diabetic syndromes. Hydrodynamics-based administration offers a simple and efficient way in the study of gene therapy for type 1 diabetes.

Roles of main pro-and anti-angiogenic factors in tumor angiogenesis

Tumor growth without size restriction depends on vascular supply.The ability of tumor to induce new blood-vessel formation has been a major focus of cancer research over the past decade.It is now known that members of the vascular endothelial growth factor and angiopoietin families,mainly secreted by tumor cells, induce tumor angiogenesis,whereas other endogenous angiogenic inhibitors, including thrombospondin-1 and angiostatin, keep tumor in dormancy.Experimental and clinical evidence has suggested that the process of tumor metastasis depends on angiogenesis or lymphangiogenesis. This article summarizes the recent research progress for some basic pro- or anti-angiogenic factors in tumor angiogenesis.

Accumulation of heavy metals in four grasses grown on lead and zinc mine tailings

A field experiment was conducted to compare the growth and metal accumulation of Vetiveria zizanioides, Paspalum notatum, Cynodon dactylon and Imperata cylindraca var. major on the tailings, amended with 10 cm domestic refuse + complex NPK fertilizer(Treatment A), 10 cm domestic refuse(Treatment B) and complex NPK fertilizer(Treatment C) respectively, and without any amendment used as control(Treatment D). The results indicated that V. zizanioides was a typical heavy metal excluder, because the concentrations in shoots of the plants were the lowest among the four plants tested. The most of metal accumulated in V. zizanioides distributed in its root, and transportation of metal in this plant from root to shoot was restricted. Therefore, V. zizanioides was more suitable for phytostabilization of toxic mined lands than P. notatum and C. dactylon, which accumulated a relatively high level of metals in their shoots and roots. It was also found that I. cylindraca var. major accumulated lower amounts of Pb, Zn and Cu than C. dactylon and P. notatum, and could also be considered for phytostalilisaton of tailings. Although the metal(Pb, Zn and Cu) concentrations in shoots and roots of V. zizanioides were the lowest, the total amounts of heavy metals accumulated in shoots of V. zizanioides were the highest among the four tested plants due to the highest dry weight yield of it. The results indicated that V. zizanioides was the best choice among the four species used for phytoremediation(for both phytostabilization and phytoextraction) of metal contaminated soils.

Biomass,species composition and diversity of benthic diatoms in mangroves of the Houyu Bay,China

The biomass, species composition and diversity of benthic diatom assemblages in mud-flat soils in Kandelia candel (L.) Druce communities with and without vegetation were studied seasonally at the Houyu Bay in Fuding City, Fujian Province, China. A total of 103 taxa were identified (including varieties). Eighty-four taxa were found in the mud-flat with vegetation and 74 taxa in the mud-flat without vegetation, while the biomass was large in January and April and decreased from July to October. The most abundant species in the mud-flat with vegetation are Nitzschia cocconeiformis, Gyrosigma scalproides and N. fasciculata, compared with G. scalproides and N. obtusa var. scalpelliformis in the mud-flat without vegetation. High H' values at 2 sites during all seasons suggest that diatom assemblages in the sediments of the Houyu Bay represent an original environment. Multi-dimensional scaling of diatom assemblages from mud-flats with and without vegetation shows that a slight seasonal change and only a single association occur in the mangroves.

Integration of E.coli aroG-pheA tandem genes into Corynebacterium glutamicum tyrA locus and its effect on L-phenylalanine biosynthesis

AIM: To study the effect of integration of tandem aroG-pheA genes into the tyrA locus of Corynebacterium glutamicum (C. glutamicum) on the production of L-phenylalanine.METHODS: By nitrosoguanidine mutagenesis, five pfluorophenylalanine (FP)-resistant mutants of Cglutamicum FP were selected. The tyrA gene encoding prephenate dehydrogenase (PDH) of C.glutamicum was amplified by polymerase chain reaction (PCR) and cloned on the plasmid pPR. Kanamycin resistance gene (Km) and the PBF-aroGpheA-T (GA) fragment of pGA were inserted into tyrA gene to form targeting vectors pl-K and pTGAK, respectively. Then,they were transformed into C.glutamicum FP respectively by electroporation. Cultures were screened by a medium containing kanamycin and detected by PCR and phenotype analysis. The transformed strains were used for L-phenylalanine fermentation and enzyme assays.RESULTS: Engineering strains of C.glutamicum (Tyr-)were obtained. Compared with the original strain, the transformed strain C. glutamicum GAK was observed to have the highest elevation of L-phenylalanine production by a 1.71-fold,and 2.9-, 3.36-, and 3.0-fold in enzyme activities of chorismate mutase, prephenate dehydratase and 3-deoxy-Darabinoheptulosonate-7-phosphate synthase, respectively.CONCLUSION: Integration of tandem aroG-pheA genes into tyrA locus of C. glutamicum chromosome can disrupt tyrA gene and increase the yield of L-phenylalanine production.

Transfection of mEpo gene to intestinal epithelium in vivo mediated by oral delivery of chitosan-DNA nanoparticles

AIM: To prepare the chitosan-pmEpo nanoparticles and to study their ability for transcellular and paracellular transport across intestinal epithelia by oral administration. METHODS: ICR mice were fed with recombinant plasmid AAV-tetO-CMV-mEpo (containing mEpo gene) or pCMVβ(containing LacZ gene), whether it was wrapped by chitosan or no. Its size and shape were observed by transmission electron microscopy. Agarose gel electrophoresis was used to assess the efficiency of encapsulation and stability against nuclease digestion. Before and after oral treatmant, blood samples were collected by retro-orbital puncture, and hematocrits were used to show the physiological effect of mEpo. RESULTS: Chitosan was able to successfully wrap the plasmid and to protect it from DNase degradation. Transmission electron microscopy showed that freshly prepared particles were approximately 70-150 nm in size and fairly spherical.Three days after fed the chitosan-pCMVβ complex was fed,the mice were killed and most of the stomach and 30% of the small intestine were stained. Hematocrit was not modified in naive and ‘naked' mEpo-fed mice, a rapid increase of hematocrit was observed during the first 4 days of treatment in chitosan-mEpo-fed animals, reaching 60.9±1.2% (P<0.01),and sustained for a week. The second feed (6 days after the first feed) was still able to promote a second hematocrit increase in chitosan-mEpo-fed animals, reaching 65.9±1.4%(P<0.01), while the second hematocrit increase did not appearin the ‘naked' mEpo-second-fed mice. CONCLUSION: Oral chitosan-DNA nanoparticles can efficiently deliver genes to enterocytes, and may be used as a useful tool for gene transfer.

Characterization of a novel developmentally retarded mutant (drm1) associated with the autonomous flowering pathway in Arabidopsis

A developmentally retarded mutant (drm1) was identified from ethyl methanesulfonate (EMS)-mutagenized M2 seedsin Columbia (Col-0) genetic background. The drm1 flowers 109 d after sowing, with a whole life cycle of about 160 d.It also shows a pleiotropic phenotype, e.g., slow germination and lower germination rate, lower growth rate, curlingleaves and abnormal floral organs. The drm1 mutation was a single recessive nuclear mutation, which was mapped tothe bottom of chromosome 5 and located within a region of 20-30 kb around MXK3.1. There have been no mutantswith similar phenotypes reported in the literature, suggesting that DRM1 is a novel flowering promoting locus. Thefindings that the drm1 flowered lately under all photoperiod conditions and its late flowering phenotype was significantlyrestored by vernalization treatment suggest that the drm1 is a typical late flowering mutant and most likely associatedwith the autonomous flowering pathway. The conclusion was further confirmed by the revelation that the transcriptlevel of FLC was constantly upregulated in the drm1 at all the developmental phases examined, except for a very earlystage. Moreover, the transcript levels of two other important repressors, EMF and TFL1, were also upregulated in thedrm1, implying that the two repressors, along with FLC, seems to act in parallel pathways in the drm1 to regulateflowering as well as other aspects of floral development in a negatively additive way. This helps to explain why the drm1exhibits a much more severe late-flowering phenotype than most late-flowering mutants reported. It also implies that theDRM1 might act upstream of these repressors.

Regulation of swelling-activated chloride channels in embryonic chick heart cells

Swelling-activated Cl- currents, I(Cl,swell), were measured during hyposmotic shock in white Leghornembryonic chick heart cells using the whole-cell recording of patch-clamp technique. Genistein, an inhibitorof protein tyrosine kinase (PTK), suppressed I(Cl,swell). Under isosmotic condition phorbol 12-myristate 13-acetate (PMA), an activator of PKC, elicited the Cl- current similar to that in hyposmotic solution, whereashyposmotic shock did not elicit I(Cl,swell) in chelerythrine chloride(an inhibitor of PKC)-treated cells. Con-focal microscopy experiments using FITC-phalloidin as a fluorescent label of F-actin showed that the actinnetwork was moved from cortical region of the cell to the center after hyposmotic shock as compared withthe image under isosmotic condition. When the cells were treated with cytochalasin B (CB) or cytochalasinD (CD) under isosmotic condition the disruption of the F-actin integrity was observed, and I(Cl,swell) wasnot elicited. With combination treatment of CB with PMA, hyposmotic solution could not elicited I(Cl,swell).The results suggested that the role of PTK, probably receptor tyrosine kinase, for regulation of I(Cl,swell)appeared to be at upstream site related to the role of F-actin. Then PKC signal pathway was activatedsomehow and finally change in the polymerization state of cytoskeleton led to activate the swelling-activatedCl- channels. These results demonstrate clearly that PTK, PKC and F-actin are important factors for reg-ulation of I(Cl,swell), in embryonic chick heart cells as compared with often controversial results reported indifferent cell types.

Influence of water logging time on the growth of Kandelia candel seedlings

Influence of waterlogging time on the growth of Kandelia candel(L.) Druce seedlings grown for 70 d in the artificial- tidal tanks' simulated semidiurnal tide under greenhouse is studied. Sand and soil act as the substrate and artificial seawater with salinity of 15 is used in cultivation. Shorter waterlogging time(inundated for about 2 ~ 4 h) promotes the growth of K. candel seedlings, while longer time(inundated more than 8 h) or no waterlogging(0 h) inhibits their growth. The number and length of aerating roots increase with the increase of waterlogging time. Under existing conditions, the optimal waterlogging time for the growth of K. candel seedlings is about 2 ~ 4 h in every tide cycle. Compared with other treatments, the 2 h sanded treatments obtain the highest biomass of seedlings, have the lowest mass loss of hypocotyl and broaden the photosynthetic area by increasing the area per leaf after 70-d cultivation. And the soil treatments have the similar tendency. However, waterlogging for 8 h in every tide cycle is critical for normal development of seedlings.K. candel seedlings are highly tolerant to waterlogging and a proper waterlogging is beneficial to the growth of K. candel seedlings.

Bioconcentration of polycyclic aromatic hydrocarbons in roots of three mangrove species in Jiulong River Estuary

The polycyclic aromatic hydrocarbons(PAHs) concentrations were determined in the root of three mangrove species(Kandelia candel, Avicennia marina and Bruguiera gymnorrhiza) and their growing environment(sediment) in mangrove wetlands of Jiulong River Estuary, Fujian, China. The total PAHs(16 parent PAHs) in mangrove sediments ranged from 193.44 to 270.53 ng/g dw, with a mean value of 231.76±31.78 ng/g dw. Compared with other mangrove and coastal marine sediments, the PAHs concentrations of all the sampling areas in this study were at relatively lower level. The total PAHs(13 parent PAHs) values varied from 30.83 to 62.73 ng/g dw in mangrove roots. Benzo[a]pyrene(five-ring), fluoranthene(four-ring) and pyrene(four-ring) dominated in mangrove sediments. Based on ratios of phenathrene/anthracene, fluoranthene/pyrene and fluoranthene/pyrene + fluoranthene, the main possible sources of surface sediment PAHs were identified as grass, wood or coal combustion for mangrove wetlands of Jiulong River Estuary. Naphthalene(two-ring) and phenathrene(three-ring) were the most abundant compounds in mangrove roots. Sediment-to-vegetation bioconcentration factors(BCF SV s) were calculated and their relationships with PAHs' physico-chemical properties were investigated. The average BCF SV s of PAHs for three mangrove species roots were almost all under the level of 1 except for naphthalene. Good linear relationship between BCF SV values for mangrove roots and PAHs water solubility, octanol-water partitioning coefficients was derived in present study. The solubility and the octanol-water partition coefficient were proved to be good predictors for the accumulation of PAHs in mangrove roots, respectively.

Study on genetic diversity and resource conservation of amphioxus (Branchiostoma balcheri Gray) population

Amphioxus is the ancestor of vertebrates 5×108 a ago, it is a typical transitional sample of evolution from invertebrates to vertebrates. Inter simple sequence repeats (ISSRs) and random amplified polymorphic DNAs (RAPDs) technologies were applied to detect the genetic variation of 3 bulking samples and individually sampled populations in nowadays Xiamen sea areas (Xiekou, Nanxian and Huangcuo) where the amphioxuses are alive. For the bulking sampled populations, 5 ISSR and 10 RAPD primers generated 357 bands, of which 181 (50.7%) were polymorphic. Nei index and UPGMA statistical analysis indicated that amphioxuses in these 3 areas could be divided into 2 groups. The genetic distance between animals in Nanxian and Huangcuo areas was 0.07 and classified into 1 group, while the population in Xiekou belonged to another group because its genetic distances in Nanxian and Huangcuo were 0.12 and 0.14, respectively. The result was in accordance with the morphological comparison among animals from those areas. For individually sampled population, Shannon's index of genetic diversity was used to partition the diversity of the animals among these 3 sea areas, and the results showed that the indices in populations of Xiekou, Nanxian and Huangcuo, were 0.583, 0.482 and 0.374, respectively. The linear regression equation analysis for amphioxus' genetic diversity versus the environment factors revealed that granularity/sorting coefficient and water depth were the most important factors that affect amphioxus genetic diversity. On the basis of the results, the suggestions for amphioxus resource conservation in Xiamen sea areas are put forward.

Potent inhibition of angiogenesis and liver tumor growth by administration of an aerosol containing a transferrin-liposome-endostatin complex

AIM: To obtain an efficient delivery system for transportingendostatin gene to mouse liver tumor xenografts byadministration of aerosol.METHODS: Recombinant plasmid pcDNA3.0/endostatincontaining human endostatin gene together with signalpeptide from alkaline phosphatase were transferred intohuman umbilical vein endothelial cell (HUVEC) by transfenin(TF)-liposome-endostatin complex. Western blot was usedto detect the expression of human endostatin in transfectedHUVEC cells and its medium. After the tumor-bearing micewere administrated with TF-liposome-endostatin complex,the lung tissue was analyzed by immunohistochemicalmethod for expression of endostatin and the tumors weretreated with CD-31 antibody to detect the density ofmicrovesseles in tumor tissues. The inhibition of tumorgrowth was estimated by the weight of tumors from groupstreated with different dos es of TF-liposome-endostatincomplex. DNA fragmentation assay was used to detect theapoptosis of the cells from primary liver tumor.RESULTS: Western blot analysis and immunohistochemicalmethod confirmed the expression of endostatin proteininvitro and in vivo. After the tumor sections were treated withCD-31 antibody, the positive reaction cells appeared brownwhile the negative cells were colorless. The positively stainedarea of the TF-liposome-endostatin treated group wassignificantly smaller (P＜0.01, 645.8+55.2 μm2) than that ofthe control group (1325.4+198.5 μm2). The data showed asignificant inhibition of angiogenesis. After administrationof TF-liposome-endostatin, comparing with the control groupadministrated with TF-liposome-pcDNA3.0, liver tumorgrowth in the mice treated with 50, 250 and 500 mg DNA/kg was inhibited by 36.6 %, 40.8 %, and 72.8 %, respectively(P＜0.01). And a typical DNA fragmentation of apoptosis wasfound in the cells from tumor tissues of the mice treatedwith TF-liposome-endostatin but none in the control group.CONCLUSION: Endostatin gene could be efficientlytransported into the mice with TF-liposome-DNA deliverysystem by administration of aerosol. TF-liposome-mediatedendostatin gene therapy strongly inhibited angiogenesis andthe growth of mouse xenograft liver tumors. It also couldpromote the development of apoptosis of tumors withoutdirect influence on tumor cells.

Intermediate fertile Triticum aestivum (+) Agropyron elongatum somatic hybrids are generated by low doses of UV irradiation

We report the production and characterization of somatic hybrids between Triticum aestivum L. and Agropyron elongatum (Host) Nevishi (the synonym is Thinopyrum ponticum). Asymmetric protoplast fusion was performed between Agropyron elongatum protoplasts irradiated with a low UV dose and protoplasts of wheat taken from nonregenerable suspension cultures. More than 40 green plantlets were obtained from 15 regenerated clones and one of them produced seeds. The phenotypes of the hybrid plants and seeds were intermediate between wheat and Agropyron elongatum. All of the regenerated calli and plants were verified as intergeneric hybrids on the basis of morphological observation and analysis of isozyme,cytological,5SrDNA spacer sequences and random amplified polymorphic DNA (RAPD). RFLP analysis of the mitochondrial genome revealed evidence of random segregation and recombination of mtDNA.