Down-modulation of heat shock protein 70 and up-modulation of Caspase-3 during schisandrin B-induced apoptosis in human hepatoma SMMC-7721 cells

AIM: To investigate the effect of schisandrin B (Sch B) on proliferation and apoptosis of human hepatoma SMMC-7721 cells in vitro and regulation of Hsp70 and Caspases-3, 7, 9 expression by Sch B. METHODS: Human hepatoma cell line SMMC-7721 was cultured and treated with Sch B at various concentrations. Growth suppression was detected with MTT colorimetric assay. Cell apoptosis was confirmed by DNA ladder detection and flow cytometric analysis. The expression of Hsp70, Caspases-3, 7, 9 were analyzed by Western blot analysis. RESULTS: Sch B inhibited the growth of hepatoma SMMC-7721 cells in a dose-dependent manner, leading to a 50% decrease in cell number (LC50) value of 23.50 mg/L. Treatment with Sch B resulted in degradation of chromosomal DNA into small internucleosomal fragments, evidenced by the formation of a 180-200 bp DNA ladder on agarose gels. FCM analysis showed the peak areas of subdiploid at the increased concentration of Sch B. The results of Western bolt analysis showed that Hsp70 was down-regulated and Caspase-3 was up-regulated, while the activity of Caspases-7,-9 had no significant change. CONCLUSION: Sch B is able to inhibit the proliferation ofhuman hepatoma SMMC-7721 cells and induce apoptosis, which goes through Caspase-3-dependent and Caspase-9-independent pathway accompanied with the down-regulation of Hsp70 protein expression at an early event.

K^+channels inhibited by hydrogen peroxide mediate abscisic acid signaling in Vicia guard cells

A number of studies show that environmental stress conditions increase abscisic acid (ABA) and hydrogen peroxide (H2O2) levels in plant cells. Despite this central role of ABA in altering stomatal aperture by regulating guard cell ion transport, little is known concerning the relationship between ABA and H2O2 in signal transduction leading to stomatal movement. Epidermal strip bioassay illustrated that ABA- inhibited stomatal opening and ABA-induced stomatal closure were abolished partly by externally added catalase (CAT) or diphenylene iodonium (DPl), which are a H2O2 scavenger and a NADPH oxidase inhibitor respectively. In contrast, internally added CAT or DPI nearly completely or partly reversed ABA-induced closure in half-stoma. Consistent with these results, whole-cell patch-clamp analysis showed that intracellular application of CAT or DPI partly abolished ABA-inhibited inward K+ current across the plasma membrane of guard cells. H2O2 mimicked ABA to inhibit inward K+ current, an effect which was reversed by the addition of ascorbic acid (Vc) in patch clamping micropipettes. These results suggested that H2O2 mediated ABA-induced stomatal movement by targeting inward K+ channels at plasma membrane.

Reversal of the phenotype by K-ras^(val12) silencing mediated by adenovirus-delivered siRNA in human pancreatic cancer cell line Panc-1

AIM: To investigate the in vitro antitumor effect of adenovirus-mediated small interfering RNAs (siRNAs) on pancreatic cancer and the associated mechanism. METHODS: A 63-nucleotide (nt) oligonucleotide encoding K-rasval12 and specific siRNA were introduced into pSilencer 3.1-H1, then the H1-RNA promoter and siRNA coding insert were subcloned into pAdTrack to get plasmid pAdTrackH1-Avasval12. After homologous recombination in bacteria and transfections of such plasmids into a mammalian packaging cell line 293, siRNA expressing adenovirus Adh1-K-rasval12 was obtained. Stable suppression of K-rasval12 was detected by Northern blot and Western blot. Apoptosis in Panc-1 cells was detected by flow cytometry. RESULTS: We obtained adenovirus AdHl-K-rasval12 carrying the pSilencer 3.1-H1 cassette, which could mediate gene silencing. Through siRNA targeted K-rasval12, the oncogenic phenotype of cancer cells was reversed. Flow cytometry showed that apoptotic index of Panc-1 cells was significantly higher in the AdH1-K-rasval12-treatment group (18.70% at 72 h post-infection, 49.55% at 96 h post-infection) compared to the control groups (3.47%, 3.98% at 72 and 96 h post-infection of AdH1-empty, respectively; 4.21%, 3.78% at 72 and 96 h post-infection of AdHl-p53, respectively) (P<0.05). CONCLUSION: These results demonstrate that adenoviral vectors can be used to mediate RNA interference (RNAi) to induce persistent loss of functional phenotypes. In gene therapy, the selective down-regulation of only the mutant version of a gene allows for highly specific effects on tumor cells, while leaving the normal cells untouched. In addition, the apoptosis of pancreatic cancer cell line Panc-1 can be induced after AdH1-K-rasval12 infection. This kind of adenovirus based on RNAi might be a promising vector for cancer therapy.

Protective effects of transplanted and mobilized bone marrow stem cells on mice with severe acute pancreatitis

AIM: To evaluate the protective effects of transplanted and mobilized bone marrow stem cells (BMSCs) on mice with severe acute pancreatitis (SAP) and to probe into their possible mechanisms.METHODS: A mouse model of SAP induced by intraparitoneal injections of L-arginine was employed in the present study.Two hundred female Balb/c mice weighing 18-22 g were randomly assigned into 4 groups. Group A was the stem cell mobilized group treated by injection of granulocytecolony stimulating factor (G-CSF) into mice for 4 days at a dose of 40 μg@kg-1@d-1 before induction of SAP. Group B was the group of BMSCs transplantation, in which the mice were given the isolated BMSCs via the tail vein 4 days prior to induction of SAP. Group C served as the model control and only SAP was induced. The mice without induction of SAP in group D acted as the normal control. At the time of animal sacrifice at 24, 48 and 72 h after induction of SAP, blood samples were obtained and prepared to detect serum amylase, while the abdominal viscera were examined both grossly and microscopically for the observation of pathological changes.RESULTS: The mortality of mice in the model control, groups A and B was 34%, 8% and 10% respectively within 72 h after induction of SAP. The serum level of amylase in the model control was significantly increased at all time points after induction of SAP as compared with that of the normal control (P＜0.05-0.01). When the mice were pretreated with BMSCs' transplantation or G-CSF injection, their serum level of amylase was significantly reduced at 48 h and 72 h after induction of SAP in comparison with that of the model control (P＜0.05-0.01). In accordance with these observations,both gross and microscopic examinations revealed that the pathological changes of SAP in mice pretreated with BMSCs transplantation or G-CSF injection were considerably attenuated as compared with those in the model control at all observed time points.CONCLUSION: Both transplanted allogenic and mobilized autologous BMSCs can protect mouse pancreas from severe damage in the process of SAP.

Ability of Bacillus mucilaginosus GY03 Strain to Adsorb Chromium Ions

A research with Bacillus mucilaginosus cultured in nitrogen-free medium for forming a flocculant material to adsorb Cr+6 was conducted to determine the effects of different pH, volume, treatment time, and chromium (VI) concentrations on chromium (VI) adsorption by microbial flocculant (MBF), which was produced from the B. mucilaginosus GY03 strain. The results showed that MBF had outstanding flocculation on chromium (VI). Based on the results of a oneway experiment and actual wastewater treatment conditions, the optimum conditions, obtained by using orthogonal experiments, for chromium (VI) adsorption by MBF were: Cr6+ solution pH of 9, flocculant material volume of 15 mL, treatment time of 12 h and chromium ion concentration of 30 mg L-1. The results demonstrated that the MBF produced from GY03 could be used in the chromium-containing wastewater treatment. Meanwhile, after extraction and analysis of the MBF polysaccharides, it was found that MBF was mainly composed of glycoprotein. Analysis on constituents of monosaccharide showed that polysaccharides of B. mucilaginosus were composed of rhamnose, glucose etc. Thus, because it was applied over a wide range of pH, in small amounts and had a rapid flocculation speed the flocculant used in this experiment had a vast field of application potential.

Cloning and functional characterization of two cDNAs encoding NADPH-dependent 3-ketoacyl-CoA reductased from developing cotton fibers

Genes encoding enzymes involved in biosynthesis of very long chain fatty acids were significantly up-regulatedduring early cotton fiber development. Two cDNAs, GhKCR1 and GhKCR2 encoding putative cotton 3-ketoacyl-CoAreductases that catalyze the second step in fatty acid elongation, were isolated from developing cotton fibers. GhKCR1and 2 contain open reading frames of 963 bp and 924 bp encoding proteins of 320 and 307 amino acid residues,respectively. Quantatitive RT-PCR analysis showed that both these genes were highly preferentially expressed duringthe cotton fiber elongation period with much lower levels recovered from roots, stems and leaves. GhKCR1 and 2showed 30%-32% identity to Saccharomyces cerevisiae Ybr159p at the deduced amino acid level. These cotton cDNAswere cloned and expressed in yeast haploid ybr159w? mutant that was deficient in 3-ketoacyl-CoA reductase activity.Wild-type growth rate was restored in ybr159w? cells that expressed either GhKCR1 or 2. Further analysis showed thatGhKCR1 and 2 were co-sedimented within the membranous pellet fraction after high-speed centrifugation, similar to theyeast endoplasmic reticulum marker ScKar2p. Both GhKCR(s) showed NADPH-dependent 3-ketoacyl-CoA reductaseactivity in an in vitro assay system using palmitoyl-CoA and malonyl-CoA as substrates. Our results suggest thatGhKCR1 and 2 are functional orthologues of ScYbr159p.

Relationship between ATPase activity and conjugated polyamines in mito-chondrial membrane from wheat seedling roots under osmotic stress

The effects of osmotic stress on the ATPase activity, the contents of —SH group and conjugated polyamines in mitochondrial membrane from wheat seedling [Triticum aestivum L. cv. Yumai No.18(drought-tolerant) and cv. Yumai No.9(drought-sensitive)] roots were investigated. The results showed that ATPase activity and —SH group content decreased with polyethylene glycol(PEG) 6000(-0.55 MPa) treatment for 7 d, in concert with the decrease of the ratio of noncovalently conjugated spermidine(NCC-Spd)/noncovalently conjugated putrescine(NCC-Put) and increase of the covalently conjugated putrescine(CC-Put). Osmotic stress injury to Yangmai No.9 seedlings was alleviated greatly with 1 mmol/L exogenous spermidine(Spd), in concert with marked increases of the ratio of NCC-Spd/NCC-Put, —SH group contents and ATPase activity in mitochondrial membrane. Under osmotic stress, the concomitant treatment of Yumai No.18 seedlings with methylglyoxyl bis(guanylhydrazone) (MGBG), an inhibitor of S-adenosyl methionine decarboxylase(SAMDC), and phenanthrolin (o-Phen), an inhibitor of transglutaminase(TGase), caused a significant decrease of the ratio of NCC-Spd / NCC-Put, CC-Put contents, respectively, in concert with the marked decreases of ATPase activity, —SH group content and its tolerance to osmotic stress. All the results above suggested that osmotic stress tolerance of wheat seedlings was associated with the ATPase activity, the contents of —SH group, NCC-Spd and CC-Put in mitochondrial membrane.

In vitro cultivation and differentiation of fetal liver stem cells from mice

During embryonic development, pluripotent endoderm tissue in the developing foregut may adopt pancreatic fate or hepatic fate depending on the activation of key developmental regulators. Transdifferentiation occurs between hepato- cytes and pancreatic cells under specific conditions. Hepatocytes and pancreatic cells have the common endodermal progenitor cells. In this study we isolated hepatic stem/progenitor cells from embryonic day (ED) 12-14 Kun-Ming mice with fluorescence-activated cell sorting (FACS). The cells were cultured under specific conditions. The cultured cells deploy dithizone staining and immunocytochemical staining at the 15th, 30th and 40th day after isolation. The results indicated the presence of insulin-producing cells. When the insulin-producing cells were transplanted into alloxan- induced diabetic mice, the nonfasting blood glucose level was reduced. These results suggested that fetal liver stem/ progenitor cells could be converted into insulin-producing cells under specific culture conditions. Fetal liver stem/ progenitor cells could become the potential source of insulin-producing cells for successful cell transplantation therapy strategies of diabetes.

Aggregate formation of hepatitis B virus X protein affects cell cycle and apoptosis

AIM: To investigate whether the formation of aggregated HBx has a potential linking with its cellular responses.METHODS: Recombinant HBx was expressed in Escherichia coli and purified by Ni-NTA metal-affinity chromatography.Anti-HBx monoclonal antibody was developed for immunocytochemical detection. Bicistronic expression vector harboring full-length DNA of HBx was employed for transfection of human HepG2 ceils. Immunocytochemical staining was used to examine the intracellular HBx aggregates in cells. The effects of HBx aggregation on cell cycle and apoptosis were assessed by flow cytometry.RESULTS: Immunocytochemical staining revealed most of the HBx was formed intracellular aggregate in cytoplasm and frequently accumulated in large granules. Flow cytometry analysis showed that HepG2 cells transfected with vector harboring HBx significantly increased apoptosis and largely accumulated in the G0-G1 phase by maintenance in serum medium for 36 hours. Control cells without HBx aggregates in the presence of serum entered S phase and proliferated more rapidly at the same time.EGFP fluorescence in HBx expression cells was significantly decreased.CONCLUSION: Our observations show that cells with HBx aggregate undergo growth arrest and apoptosis, whereas control cells without HBx remain in growth and progression into S phase. Our data may provide helpful information to understand the biological effects of HBx aggregates on cells.

Shedding of TNFR1 in regenerative liver can be induced with TNF α and PMA

AIM: Liver regeneration is associated with apoptosis of hepatocytes, which is mediated via tumor necrosis factor receptor 1(TNFR1). The shedding of TNFR1 in liver regeneration and its mechanism to regulate this shedding were investigated.METHODS: The shedding of TNFR1 in liver regeneration and changes of TNF-α, PMA and plasma membrane purified from hepatocytes on this shedding process were measured with Western blot. Then, the relationship between TNFR1 shedding and apoptosis of hepatocytes induced by TNFα was studied by detecting apoptotic index.RESULTS: The shedding of TNFR1 began at 4 hours and terminated before 2 months after partial hepatectomy. In culture system, serum from rats at 36 h after partial hepatectomy could also promote this shedding process. With the stimulation of TNF α, PMA or purified plasma membrane from hepatocytes at 36 h after partial hepatectomy or from hepatocytes treated with TNF α for 2 h, membranous TNFR1 was also shed. With the stimulation of both TNF α and plasma membrane from hepatocytes affected with TNF α for 2 hor from hepatocytes at 36 h after partial hepatectomy, apoptotic index of hepatocytes decreased from 21% to 7.52 % and 8.45 %, respectively. PMA could also reduce apoptotic index to 13.67 %. This descent occurred in hepatocytes cultured in serum from rats at 36 h after partial hepatectomy too,but not in serum from rats at 2 months after partial hepatectomy and sham-operated rats.CONCLUSION: Shedding of TNFR1 may help reduce apoptosis of hepatocytes induced by TNFα. Membraneanchored metalloprotases could play a role in shedding membranous TNFR1. At the same time, PKC may take part in regulation of this shedding process.

Effects of salinity on activities of H^+-ATPase, H^+-PPase and membrane lipid composition in plasma membrane and tonoplast vesicles isolated from soybean(Glycine max L.) seedlings

The effects of NaCl stress on the H +-ATPase, H +-PPase activity and lipid composition of plasma membrane(PM) and tonoplast(TP) vesicles isolated from roots and leaves of two soybean cultivars(Glycine max L.) differing in salt tolerance(Wenfeng7, salt-tolerant; Union, salt-sensitive) were investigated. When Wenfeng7 was treated with 0.3%(W/V) NaCl for 3 d, the H +-ATPase activities in PM and TP from roots and leaves exhibited a reduction and an enhancement, respectively. The H +-PPase activity in TP from roots also increased. Similar effects were not observed in roots of Union. In addition, the increases of phospholipid content and ratios of phospholipid to galactolipid in PM and TP from roots and leaves of Wenfeng7 may also change membrane permeability and hence affect salt tolerance.

Effect of Amendments on Growth and Element Uptake of Pakchoi in a Cadmium, Zinc and Lead Contaminated Soil

A pot experiment was carried out to study the effects of two amendments, limeand calcium magnesium phosphate, on the growth and Cd, Pb, Zn, Cu, Mu, Fe, N, P and K uptake ofpakchoi (Brassica chinensis) in a Cd, Pb and Zn polluted acid soil in the southern part of China.The growth of pakchoi was apparently improved by lime and calcium magnesium phosphate application,the uptake of Cd, Pb, Cu and Zn by pakchoi was significantly depressed and the symptom caused byheavy metals pollution was eliminated. Meanwhile, the absorption of N, K and Mn was also inhibitedby these amendments. Soil pH was the main factor controlling the uptake of the heavy metals bypakchoi. This suggests that lime and calcium magnesium phosphate could be used as effectiveamendments for eliminating the toxicity of heavy metals to the vegetable and inhibiting theirabsorption by the crop.

Short-Term Influence of Herbicide Quinclorac on Enzyme Activities in Flooded Paddy Soils

The influence of quinclorac (3,7-dichloroquinoline-8-carboxylic acid) on enzyme activities in flooded paddy soils was assessed under laboratory conditions. The enzymes differed markedly in their response to quinclorac. Quinclorac inhibited proteinase, hydrogen peroxidase, phosphorylase, and urease activities.The higher the concentration of quinclorac applied, the more significant the inhibition to these observed activities with a longer time required to recover to the level of the control. However, soils supplemented with quinclorac were nonpersistent for proteinase, phosphorylase and urease as opposed to soils without quinclorac. Dehydrogenase activity was also sensitive to quinclorac. Three soil samples with concentrations of quinclorac higher than 1 μg g-1 soil declined to less than 20% of that in the control. However, the highest dehydrogenase activity (up to 3.28-fold) was detected in soils with 2 μg g-1 soil quinclorac on the 25th day after treatment. Quinclorac had a relatively mild effect on saccharase activity at the concentrations used in this experiment and a stimulatory one on soil respiration when added to soil at normal field concentrations.Nonetheless it was inhibited at higher concentrations in paddy soils. Quinclorac is still relatively safe to the soil ecosystem when applied at a normal concentration (0.67 μg g-1 dried soil) but may have some effects on soil enzymes at higher concentrations.

Effect of Nano-anatase TiO_2 on Spectral Characterization of PhotosystemⅡParticles from Spinach

The photosystem Ⅱ(PSⅡ) particles were purified by means of nano-anatase TiO_2 treatment of spinach and studied by spectroscopy. The results show that the electron transport and the oxygen-evolving rate of PSⅡ are accelerated after it has been treated with nano-anatase TiO_2; the UV-Vis absorption spectrum of PSⅡ particles is increased; the red shift of fluorescence emission peak of PSⅡ is 2 nm; the peak intensity is decreased; the PSⅡ signal Ⅱs of low temperature electron paramagnetic resonanace(EPR) spectrum is intensified under light, and the PSⅡ circular dichroism(CD) spectrum is similar to that of control. It is suggested that nano-anatase TiO_2 might bind to the PSⅡ reaction center complex and intensify the function of the PSⅡ electron donor, however, nano-anatase TiO_2 treatment does not change the configuration of the PSⅡ reaction center complex.

QTLs for Rice Leaf Chlorophyll Content Under Low N Stress

Quantitative trait loci (QTLs) for chlorophyll content of a rice leaf weremapped on to the molecular marker linkage map of a double-haploid (DH) population derived from across between two rice varieties 'IR64' and 'Azucena' in both nutrient solutionand soil cultureexperiments to detect rice nitrogen nutrition status under low N stress A chlorophyll meter was usedto measure the soil plant analysis development (SPAD) value of the topmost fully expanded leaf asthe index of chlorophyll content thatexpressed nitrogen status in rice plants. Totally 3 QTLs forSPAD values, two on chromosome 3 located at interval RG179-CDO337 and RG348-RZ329, respectively, andone on chromosome 10 at interval RZ500-RG134, were detected under stressed conditions of low N inthe soil and/or nutrient solution culture experiments. One QTL located at interval RG179-GDO337 onchromosome 3 associated with a relative change in SPAD value from a high N level to a low N level inthe soil culture experiment was also detected.Based on the different responses to low N stressbetween the two parents, it was supposed that the QTLs identified in this study associated withnitrogen efficiency in rice at low N levels might be useful in applying marker technology to ricebreeding programs.

Characterization of enhancer trap and gene trap harboring Ac／Ds transposon in transgenic rice

Insertion mutagenesis has become one of the most popular methods for gene functions analysis.Here we report a two-element Ac/Ds transposon system containing enhancer trap and gene trap for gene tagging in rice.The excision of Ds element was examined by PCR amplification.The excision frequency of Ds element varied from 0% to 40% among 20 F2 populations derived from 11 different Ds parents.Southern blot analysis revealed that more than 70% of excised Ds elements reinserted into rice genome and above 70% of the reinserted Ds elements were located at different positions of the chromosome in rice.The result of histochemical GUS analysis indicated that 28% of enhancer trap and 22% of gene trap tagging plants displayed GUS activity in leaves, roots,flowers or seeds.The GUS positive lines will be useful for identifying gene function in rice.

Aberrant expression and function of TCF4 in the proliferation of hepatocellular carcinoma cell line BEL-7402

Wnt signaling pathway is essential for development and tumorigenesis,however,this signaling pathway in the progress of hepatocellular carcinoma (HCC) remains unclear. In this paper,we studied the function of human T-cell transcription factor-4 (TCF4),a key factor of Wnt signaling pathway,on the proliferation of HCC cell line. We showed that the expression of TCF4 mRNA in HCC cell line BEL-7402 was higher than that in immortalized normal liver cell line L02. Blockage of Wnt pathway by △NTCF4,a dominant negative TCF4,could suppress BEL-7402 cells growth and decrease the expression of cyclin D1 and c-myc,two of target genes of Wnt pathway. On the other hand,stimulating Wnt pathway by introducing a degradation-resistant β-catenin S37A could increase BEL-7402 cells proliferation. But all the treatments had no effect on L02 cells. Our data indicated that TCF4 might be another key factor in Wnt pathway involved in HCC cells proliferation and TCF4 could be an effective therapeutic target for suppressing the growth of hepatocellular cancers.

Characterization and phylogenetic analysis of a phenanthrene-degrading strain isolated from oil-contaminated soil

Bacterium strain EVA17 was isolated from an oil-contaminated soil, and identified as Sphingomonas sp. based on analysis of 16S rDNA sequence,cellular fatty acid composition and physiological-chemical tests. The salicylate hydroxylase and catechol 2,3-dioxygenase(C23O) were detected in cell-free lysates, suggesting a pathway for phenanthrene catabolism via salicylate and catechol. Alignment showed that both of the C23O and GST genes of the strain EVA17 had high similarity with homologues of strains from genus Sphingomonas. The phylogenetic analysis based on 16S rDNA and C23O gene sequence indicated that EVA17 should be classified into genus Sphingomonas, although the two phylogenetic trees were slightly different from each other. The results of co-amplification and sequence determination indicated that GST gene should be located upstream of the C23O gene.

Mitotic cell death in BEL-7402 cells induced by enediyne antibiotic lidamycin is associated with centrosome overduplication

AIM: Mitotic cell death has been focused on in tumor therapy.However, the precise mechanisms underlying it remain unclear. We have reported previously that enediyne antibiotic lidamycin induces mitotic cell death at low concentrations in human epithelial tumor cells. The aim of this study was to investigate the possible link between centrosome dynamics and lidamycin-induced mitotic cell death in human hepatoma BEL-7402 cells.METHODS: Growth curve was established by Ml-l assay.Cell multinucleation was detected by staining with Hoechst 33342. Flow cytometry was used to analyze cell cycle.Aberrant centrosomes were detected by indirect immunofluorescence. Western blot and senescenceassociated β-galactosidase (SA-β-gal) staining were used to analyze protein expression and senescence-like phenotype,respectively.RESULTS: Exposure of BEL-7402 cells to a low concentration of lidamycin resulted in an increase in cells containing multiple centrosomes in association with the appearance of mitotic cell death and activation of SA-β-gal in some cells, accompanied by the changes of protein expression for the regulation of proliferation and apoptosis. The mitochondrial signaling pathway, one of the major apoptotic pathways, was not activated during mitotic cell death. The aberrant centrosomes contributed to the multipolar mitotic spindles formation, which might lead to an unbalanced division of chromosomes and mitotic cell death characterized by the manifestation of multi- or micronucleated giant cells.Cell cycle analysis revealed that the lidamycin treatment provoked the retardation at G2/M phase, which might be involved in the centrosome overduplication.CONCLUSION: Mitotic cell death and senescence can be induced by treatment of BEL-7402 cells with a low concentration of lidamycin. Centrosome dysregulation may play a critical role in mitotic failure and ultimate cell death following exposure to intermediate dose of lidamycin.

Freeliving marine nematodes as a pollution indicator of the Bohai Sea

A hierarchical diversity index—taxonomic distinctness index Δ +, which was first defined by Warwick and Clark in 1998, was employed to evaluate the pollution status of the Bohai Sea with freeliving marine nematodes. The result showed that the Bohai Bay and other coastal sampling sites might be affected by oil and gas production and other anthropogenic influences. In other words,anthropogenic disturbance was affecting this component of the benthos in these locations. And most offshore sampling sites in the middle of the Bohai Sea were clear and unpolluted.