Experimental study on the therapeutic effect of different bleaching methods on white-spot lesions of the enamel using optical coherence tomography / 口腔医学

Objective@#To investigate the efficacy of different bleaching methods on white-spot lesions of the enamel using optical coherence tomography and to evaluate its feasibility for monitoring the therapeutic effects on white-spot lesions. @*Methods@#Forty-eight sound premolars extracted for orthodontic reasons were selected and cut for 4 mm×4 mm×2 mm enamel blocks in buccal surfaces of the crowns. The samples were covered with acid-resistant varnish (except for the buccal surfaces) and immersed in demineralization solution for 18 days to establish the white-spot lesion models of the enamels. Samples were randomly divided into four groups (n=12). Group A was given demineralization only. Specimens in Group B, C and D were treated with 40% hydrogen peroxide, resin infiltration and 40% hydrogen peroxide combined with resin infiltration, respectively. Eight samples in each group were randomly selected. OCT was applied to observe the optical changes of the enamel surface and according to the OCT scanning results, the demineralization depth of enamel samples in each group was calculated. Then, the enamel blocks were embedded in epoxy resins, except the buccal surfaces, and measured for the microhardness values of the enamel surface by a microindentation hardness tester. Four samples in each group were cut longitudinally, and the ultrastructural changes of enamel samples in each group were observed by scanning electron microscope. @* Results@#OCT showed that the light scattering characteristics of enamel surface changed in all groups, and the bright layer was formed, but the thickness of bright layer in Group C and D was significantly lower than that in Group A and B (P<0.05). The microhardness values (kg/mm2) of the samples in Group A-D were (214.99±31.70), (250.66±33.64), (312.42±18.01) and(286.53±26.65), respectively. The microhardness of enamel surfaces in Group C and D was significantly higher than that in Group A and B (P<0.05), and the ultrastructure of enamel surfaces in Group C and D were more flat and dense in SEM observation (P<0.05). @*Conclusion@#The methods of resin infiltration therapy or 40% hydrogen peroxide combined with resin infiltration could effectively improve white-spot lesions of the enamel and the non-invasive OCT can be used as a better evaluation method for the diagnosis and treatment of white-spot lesions of the enamel.

The genetic architecture of phenotypic diversity in the Betta fish (Betta splendens).

The Betta fish displays a remarkable variety of phenotypes selected during domestication. However, the genetic basis underlying these traits remains largely unexplored. Here, we report a high-quality genome assembly and resequencing of 727 individuals representing diverse morphotypes of the Betta fish. We show that current breeds have a complex domestication history with extensive introgression with wild species. Using a genome-wide association study, we identify the genetic basis of multiple traits, including coloration patterns, the "Dumbo" phenotype with pectoral fin outgrowth, extraordinary enlargement of body size that we map to a major locus on chromosome 8, the sex determination locus that we map to dmrt1, and the long-fin phenotype that maps to the locus containing kcnj15. We also identify a polygenic signal related to aggression, involving multiple neural system-related genes such as esyt2, apbb2, and pank2. Our study provides a resource for developing the Betta fish as a genetic model for morphological and behavioral research in vertebrates.

Cabbage defense response provoked by Trichoderma Th-LAAO.

To investigate the molecular mechanism of Trichoderma L-amino acid oxidase (Th-LAAO) in protecting and in promoting growth of cabbage infected with Botrytis cinerea, a three-way interaction system was established. Cabbage leaves treated with purified Th-LAAO significantly constrained damaged leaf area caused by B. cinerea infection. In response to Th-LAAO treatment, the expression levels of genes involved in photosynthesis, such as ribulose-1,5-bisphosphate carboxylase oxygenase, Rubisco activase, and ATP synthase increased 2.54, 2.18, and 1.41 folds, respectively. The transcription levels of sucrose transport protein 1 increased 7.6 fold. As to the expression of defense-related genes, the transcription level of ascorbate peroxidase increased 1.46 fold. On the contrary, pathogenesis-related protein 1, chitinase, ß-1,3 glucanase, and glutathione S-transferase decreased significantly. Overall, the results indicated that Th-LAAO may stimulate CO2 fixation and sucrose transport and elicit host defense responses in cabbage against B. cinerea, and this elicitation of defense response is likely to contribute to induced systemic resistance of host plant.

Expression of L-amino acid oxidase of Trichoderma harzianum in tobacco confers resistance to Sclerotinia sclerotiorum and Botrytis cinerea.

L-amino acid oxidase (ThLAAO) secreted by Trichoderma harzianum ETS323 is a flavoenzyme with antimicrobial characteristics. In this study, we transformed the ThLAAO gene into tobacco to elucidate whether ThLAAO can activate defense mechanisms and confer resistance against phytopathogens. Transgenic tobacco overexpressing ThLAAO showed enhanced resistance against Sclerotinia sclerotiorum and Botrytis cinerea and activated the expression of defense-related genes and the genes involved in salicylic acid, jasmonic acid, and ethylene biosynthesis accompanied by substantial accumulation of H2O2 in chloroplasts, cytosol around chloroplasts, and cell membranes of transgenic tobacco. Scavenge of H2O2 with ascorbic acid abolished disease resistance against B. cinerea infection and decreased the expression of defense-related genes. ThLAAO-FITC application on tobacco protoplast or overexpression of ThLAAO-GFP in tobacco revealed the localization of ThLAAO in chloroplasts. Chlorophyll a/b binding protein (CAB) was isolated through ThLAAO-ConA affinity chromatography. The pull down assay results confirmed ThLAAO-CAB binding. Application of ThLAAO-Cy5.5 on cabbage roots promptly translocated to the leaves. Treatment of ThLAAO on cabbage roots induces systemic resistance against B. cinerea. Overall, these results demonstrate that ThLAAO may target chloroplast and activate defense mechanisms via H2O2 signaling to confer resistance against S. sclerotiorum and B. cinerea.

Molecular characterization of an inhibitor of apoptosis protein (IAPs) in freshwater pearl mussel, Hyriopsis schlegelii.

The inhibitor of apoptosis proteins (IAPs) played important roles in inhibiting the apoptosis of tumor cells by regulating caspase activity in mammals. In this study, we first cloned the full-length cDNA sequence of IAPs gene (designated as Hs-IAPs) in Hyriopsis schlegelii. The Hs-IAPs gene contained an open reading frame of 1719 nucleotides, encoding a predicted protein of 572 amino acids. qRT-PCR assay indicated that the Hs-IAPs gene was ubiquitously expressed in different tissues, and the highest expression level was in gills. Furthermore, we purified and obtained the recombinant protein of Hs-IAPs which showed a molecular weight of 82.5 kDa. We used H2O2 stimulation experiment to explore the possible function of Hs-IAPs. The results showed that the percentage of viable cells significantly increased following the Hs-IAPs concentration. These indicated that the Hs-IAPs may play a role in anti-oxidation causing by H2O2, and its anti-oxidative may be crucial in the process of apoptosis.

Analysis of the structure and activity of the promoter regions of the metallothionein genes of the freshwater pearl mussel Hyriopsis schlegelii.

To investigate the regulation of metallothionein genes (HsMTs) of Hyriopsis schlegelii, 1,121-bp and 1,270-bp regions of the HsMT1 and HsMT2 promoters were cloned and analyzed, respectively. The two promoters shared partially conserved features and possessed distinct characteristics such as the number or position of metal response elements (MREs). Further analysis of the HsMT1 and HsMT2 promoters was performed by the reporter assay using the luciferase gene. Both promoters were activated by various metals, and presented different levels of metal ions inducibility in human hepatoblastoma cells. Deletion mutant assays demonstrated that both the longest promoter regions achieved the maximum inducibility, and the metal inducibility was dependent on the presence of the MRE in HsMT1 and the distal MRE in HsMT2. In addition, we cloned a putative metal responsive transcription factor (hereby designated as HsMTF-like) and studied its effect on HsMTs expression in human hepatoblastoma cells. An in vivo assay demonstrated that HsMTF-like activates basal HsMTs transcription level, and the MRE in the HsMTs promoter mediates this activation process. Moreover, this basal transcription level can be further boosted by zinc treatment. In conclusion, the regulation mechanism for MT activation in H. schlegelii should be evolutionarily conserved.

Gene-to-Gene Network Analysis of the Mediation of Plant Innate Immunity by the Eliciting Plant Response-Like 1 (Epl1) Elicitor of Trichoderma formosa.

A new clade, Trichoderma formosa, secretes eliciting plant response-like 1 (Epl1), a small peptide elicitor that stimulates plant immunity. Nicotiana benthamiana pretreated with Epl1 for 3 days developed immunity against Tomato mosaic virus (ToMV) infection. The transcriptome profiles of T. formosa and N. benthamiana were obtained by deep sequencing; the transcript of Epl1 is 736 nt in length and encodes a 12-kDa peptide. Identifying critical genes in Epl1-mediated immunity was challenging due to high similarity between the transcriptome expression profiles of Epl1-treated and ToMV-infected N. benthamiana samples. Therefore, an efficient bioinformatics data mining approach was used for high-throughput transcriptomic assays in this study. We integrated gene-to-gene network analysis into the ContigViews transcriptome database, and genes related to jasmonic acid and ethylene signaling, salicylic acid signaling, leucine-rich repeats, transcription factors, and histone variants were hubs in the gene-to-gene networks. In this study, the Epl1 of T. formosa triggers plant immunity against various pathogen infections. Moreover, we demonstrated that high-throughput data mining and gene-to-gene network analysis can be used to identify critical candidate genes for further studies on the mechanisms of plant immunity.

Effect of Pachybasin on General Toxicity and Developmental Toxicity in Vivo.

To document the safety of pachybasin, a secondary metabolite of Trichoderma harzianum, for use as a bioagricultural agent, it was subjected to general toxicological testing in mice and developmental toxicity in zebrafish. With either 5 or 20 mg kg-1 pachybasin i.p. injection, mice behavioral responses such as motor coordination, spontaneous locomotor activity, or nociceptive pain were not influenced. In long-term effect (daily injection for 14 days), the physiological, hematological, liver, and kidney functions were not altered either. Evidence for the developmental toxicity of pachybasin (10-100 µM) in 72-h exposure period was shown in zebrafish larvae, based on developmental retardation, impairment of chorion, and increase of mortality. In summary, there are no significant general toxicities presented in the pachybasin-treated adult male mice. However, the embryo-toxicity in aquatic biota should be taken into consideration during bioagricultural agent application.

Structural characteristics of gonadal development and hermaphroditic phenomenon in freshwater pearl mussel, Sinohyriopisis schlegelii (Hyriopisis schlegelii).

The period of gonads development was first studied from one to five years in the freshwater pearl mussel Hyriopisis schlegelii. It lasted for 36 months and was divided into three main stages: initiation of gonad formation, a stable growth phase, and a reproductive cell development phase. Each reproductive cycle consisted of five stages: proliferative stage (from late January to late February), growth stage (from late February to late March), maturation stage, spawning stage (from early April to late October) and recovery stage (from early November to late January). Interestingly, a hermaphroditic phenomenon was observed in this mussel for the first time, which appears during the development stage from 26 to 32 months. Male and female follicular tissues coexisted in hermaphrodite individuals with the male follicular tissue accounting for more than 90% of the whole gonad tissue. No hermaphroditic phenomenon was observed in matured gonad. We thus speculate that self-fertilization does not exist in H. schlegelii.

Comparative analysis of the complete mitochondrial genomes of three geographical topmouth culter (Culter alburnus) groups and implications for their phylogenetics.

Topmouth culter (C. alburnus) is an important commercial fish in China. We compared the nucleotide variations in the mtDNA genomes among three geographical groups of Culter alburnus: Liangzi Lake, Hubei Province (referred to as LZH); Taihu Lake, Jiangsu Province (TH); and Poyang Lake, Jiangxi Province (PYH). The similarity of whole mtDNA genomes ranged from 0.992 to 0.999. The similarity among 13 protein-coding genes, 2 rRNA genes, and the D-loop sequences was found to range from 0.982 to 0.996. This is useful data for future designing work for making specific molecular marker for distinguishing individuals of C. alburnus from the three geographical groups. An extended termination-associated sequence (ETAS) and several conserved blocks (CSB-F, CSB-E, CSB-D, CSB1, CSB2, and CSB3) were identified in the mtDNA control regions. A phylogenetic analysis shows a monophyletic relationship of the LZF-female and the LZF-male. However, the analysis also showed paraphyletic relationships for the other two geological groups. This result will be useful for the future breeding work of C. alburnus.

Molecular characterization and expression of metallothionein from freshwater pearl mussel, Hyriopsis schlegelii.

Two metallothionein genes (HsMT1 and HsMT2) were first identified and described from Hyriopsis schlegelii. The open reading frame of HsMT1 and HsMT2 were 216 and 222 bp, encoding a protein of 71 and 73 amino acid residues. The deduced amino acid sequences showed they contained parts of typical MT characteristics, apart from HsMT2 lacked Cys-Cys motifs. The phylogenetic tree showed HsMT1 shared a high similarity with that of other molluscs, but HsMT2 was split into a distinct group separated from known molluscan MTs. HsMT1 exhibited constitutive expression in all examined tissues and the highest expression occurred in hepatopancreas, however, nearly all HsMT2 was just detected in gonad. After Cd exposure, their mRNA levels presented similar expression patterns. The transgenic bacteria of HsMT1 showed higher tolerance than HsMT2 in Cd environment. It was implied that HsMT1 and HsMT2 were involved in metal response but HsMT2 might have other physiological functions.

Anticancer activity of Kalanchoe tubiflora extract against human lung cancer cells in vitro and in vivo.

Uncontrolled cell proliferation is a common feature of human cancer. Some of herbal extract or plant-derived medicine had been shown as an important source of effective anticancer agents. We previously reported that an n-BuOH-soluble fraction of Kalanchoe tubiflora has antiproliferative activity by inducing mitotic catastrophe. In this study, we showed that the H2 O-soluble fraction of Kalanchoe tubiflora (KT-W) caused cell cycle arrest, and senescence-inducing activities in A549 cells. We used 2 dimensional PAGE to analyze the protein expression levels after KT-W treatment, and identified that the energy metabolism-related proteins and senescence-related proteins were disturbed. In vivo experiments showed that the tumor growths in A549-xenografted nude mice were effectively inhibited by KT-W. Our findings implied that KT-W is a putative antitumor agent by inducing cell cycle arrest and senescence. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1663-1673, 2016.

Blockage of PD-L1/PD-1 pathway for enhancing the chemotherapeutic efficacy of cisplatin / 实用医学杂志

Objective To explore the anti-tumor effect and the influence of antitumor immunity of PD-L1/PD-1 blocked by PD-1 antibody combined with cisplatin. Methods Tumor models were established by injecting TC-1 cells into C57BL/6 mice, and the mice were divided into four groups (n = 4). The tumor growth curves and survival curves were drawn to observe the anti-tumor effect. The tumors were then removed; and the PD-L1 and CD8+ T cells were analyzed by immunohistochemical method. Results The anti-tumor effect was greater in the cisplatin group , PD-1 antibody group , and PD-1 antibody plus cisplatin group than in the control group (P < 0.05). Expression of PD-L1 in the tumor tissues was markedly increased in the cisplatin group and it was obviously decreased in the combination group (P < 0.05). CD8+ T cells decreased in the cisplatin group; and expression of CD8+ T cells was significantly increased the combination group (P < 0.05). Conclusion The anti-tumor effect and anti-tumor immunity of cisplatin are enhanced by blocking PD-L1/PD-1 pathway with PD-1 antibody.

De novo transcriptome sequencing to identify the sex-determination genes in Hyriopsis schlegelii.

This study presents the first analysis of expressed transcripts in the spermary and ovary of Hyriopsis schlegelii (H. schlegelii). A total of 132,055 unigenes were obtained and 31,781 of these genes were annotated. In addition, 19,511 upregulated and 25,911 downregulated unigenes were identified in the spermary. Ten sex-determination genes were selected and further analyzed by real-time PCR. In addition, mammalian genes reported to govern sex-determination pathways, including Sry, Dmrt1, Dmrt2, Sox9, GATA4, and WT1 in males and Wnt4, Rspo1, Foxl2, and ß-catenin in females, were also identified in H. schlegelii. These results suggest that H. schlegelii and mammals use similar gene regulatory mechanisms to control sex determination. Moreover, genes associated with dosage compensation mechanisms, such as Msl1, Msl2, and Msl3, and hermaphrodite phenotypes, such as Tra-1, Tra-2α, Tra-2ß, Fem1A, Fem1B, and Fem1C, were also identified in H. schlegelii. The identification of these genes indicates that diverse regulatory mechanisms regulate sexual polymorphism in H. schlegelii.

Transcriptional regulation of the iac locus from Acinetobacter baumannii by the phytohormone indole-3-acetic acid.

The iac locus is involved in indole-3-acetic acid (IAA) catabolism in Acinetobacter baumannii. Nine structural genes of iac are transcribed in the same direction, whereas iacR, which encodes a MarR-type transcriptional regulator, is transcribed in the opposite direction. The IacA protein, which is encoded by the second structural gene of the iac locus, is expressed in an IAA-dependent manner. Here, we characterized gene expression from this locus in wild type A. baumannii and in an iacR mutant; this revealed that the iacH promoter is negatively regulated by IacR. The transcriptional site of iacH was determined by using 5' rapid amplification of cDNA ends; one IacR-binding site was identified between positions -35 and +28 of the iacH promoter. Sequence analysis and an electrophoretic mobility shift assay indicated that recombinant IacR binds specifically to a sequence with dyad symmetry in the iacR-iacH overlapping promoters in the absence of IAA. In addition, a two-plasmid expression system in Escherichia coli showed that IAA probably serves as a ligand that binds to IacR and releases it from the iacH promoter, thereby allowing RNA polymerase to transcribe iac. Thus, iac is expressed in order to promote IAA degradation, whereas free IacR is required for iac repression. We conclude that IacR serves as a key regulator of IAA degradation in A. baumannii in the rhizosphere. These results provide new insights into the possible role of A. baumannii in the environment.

Effect of Paclitaxel on Expression of PD-L1 in Surface of Cervical Cancer TC-1 Cells / 医药导报

Objective To investigate effect of paclitaxel on expression of programmed death ligand-1 ( PD-L1 ) in the surface of cervical cancer TC-1 cells and its mechanism. Methods ①The cells were divided into two groups: paclitaxel group, paclitaxel combined with PKD blocker (G? 6976) group. There were 4 concentration gradient and 5 holes for each group, and each hole has its corresponding concentration of drugs. Influence of paclitaxel on TC-1 cell viability and effect of PKD blocker G? 6976 on IC50 value of paclitaxel were evaluated by MTT method.②The cells were divided into 0. 9% sodium chloride solution ( NS) group and paclitaxel group, There were 5 holes of each group. Effect of paclitaxel on PD-L1 expression on the surface of TC-1 cells were measured by immunohistochemistry.③The cells were divided into 4 groups:NS+DMSO group, G? 6976 group, paclitaxel group and paclitaxel+G? 6976 group. There were 5 holes for each group. Effect of paclitaxel and G? 6976 on PD-L1 expression on the surface of TC-1 cells were measured by immunohistochemistry. The expressions of PD-L1 on the surface of cells were measured by immunofluorescence treated with different drugs. Results The IC50 value of paclitaxel was 40 μg·mL-1 in paclitaxel group, and 38. 9 μg·mL-1 in paclitaxel combined with PKD blocker G? 6976 group, without significant difference between the two groups (P>0. 05). The expression of PD-L1 in the surface of TC-1 cells were significantly higher in paclitaxel group than in negative control group [(88. 48±13. 44)% vs. (39. 59±5. 99)%, P<0. 05]. The expression of PD-L1 in the surface of TC-1 cells was (79. 7%±4. 7)% after treatment with paclitaxel combined with PKD blocker G? 6976 for 24 h, and it was significantly lower than that in paclitaxel group [(96. 8±2. 5)%, P<0. 05]. Conclusion Paclitaxel promotes the expression of PD-L1 in the surface of TC-1 cells, which could be significantly inhibited by blocking PKD pathway. Paclitaxel may exert its effect through PKD pathway.

The role of Twist on cisplatin resistance of Hela cervical cancer cells in hypoxia microenvironment and its mechanism / 医学研究生学报

Objective Drug resistance is a major problem for the successful chemotherapeutic treatment and prognosis of cervi -cal cancer .The article investigated the role of Twist on cisplatin resistance of Hela cervical cancer cells in hypoxia microenvironment and its possible mechanism to provide an experimental basis to improve the therapeutic efficacy for cervical cancer . Methods Hela Cells were divided into two groups, normal oxygen group A (no CoCl2) and hypoxia group A (addition of 150μmol/L CoCl2 medium).6 hours later, different degrees(10-3, 10-4, 10-5, 10-6, 10-7 mol/L) of cisplatin (10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 mol/L) were added into two groups .24 hours later , MTT were added to measure IC 50 and growth inhibition ratio .CoCl2 were used to mimic hypoxia environ-ment.The working concentration and the treatment duration of Cispla-tin was optimized by MTT method.The expressions of Twist and MDR1 in normal oxygen group , cisplatin group , hypoxic group B and hypoxic cisplatin group B were determined by immunofluores-cence and western blot . Results The IC50 values were 10 -5.3 mol/L and 10 -4.5 mol/L of cisplatin respectively in normal oxygen group and hypoxia group, and there was significantly difference between them (P<0.05).The optimized working concentration and work time of cisplatin was 10 -5 mol/L and 24 h.Compare to the normal oxygen group (13.08 ±2.39, 29.57 ±12.80), the expres-sions of Twist (20.81 ±2.07, 24.25 ±4.51, 33.14 ±4.24) and MDR1 (35.26 ±8.41, 60.13 ±22.32, 76.00 ±9.96) was signifi-cantly higher than those in other three groups and which were the highest in hypoxic cisplatin group (P<0.05).There were significant positive correlations among them in hypoxic group and hypoxic cisplatin group (r =0.686,P <0.05;r =0.546,P <0.05). Conclusion The hypoxia microenvironment may be related to the cisplatin resistance of Hela cervical cancer cells .The possible mechanism is hypoxia activates Twist and upregulates MDR 1 expression , resulting in cisplatin resistance of Hela cells .

Molecular cloning, sequence analysis, and cadmium stress-rated expression changes of BTG1 in freshwater pearl mussel (Hyriopsis schlegelii).

The B cells translocation gene 1 (BTG1) is a member of the BTG/TOB family of anti-proliferative genes, which have recently emerged as important regulators of cell growth and differentiation among verteates. Here, for the first time we cloned the full-length cDNA sequence of Hyriopsis schlegelii (Hs-BTG1), an economically important freshwater shellfish and potential indicator of environmental heavy metal pollution, for the first time. Using rapid amplification of cDNA ends (RACE) together with splicing the EST sequence from a haemocyte cDNA liary, we found that Hs-BTG1 contains a 525 bp open reading frame (ORF) encoding a 174 amino-acid polypeptide, a 306 bp 5' untranslated region (5' UTR), and a 571 bp 3' UTR with a Poly(A) tail as well as a transcription termination signal (AATAAA). Homologue searching against GenBank revealed that Hs-BTG1 was closest to Crassostrea gigas BTG1, sharing 50.57% of protein identities. Hs-BTG1 also shares some typical features of the BTG/TOB family, possessing two well-conserved A and B boxes. Clustering analysis of Hs-BTG1 and other known BTGs showed that Hs-BTG1 was also closely related to BTG1 of C. gigas from the inverteate BTG1 clade. Function prediction via homology modeling showed that both Hs-BTG1 and C. gigas BTG1 share a similar three-dimensional structure with Homo sapiens BTG1. Tissue-specific expression analysis of the Hs-BTG1 via real-time PCR showed that the transcripts were constitutively expressed, with the highest levels in the hepatopancreas and gills, and the lowest in both haemocyte and muscle tissue. Expression levels of Hs-BTG1 in hepatopancreas (2.03-fold), mantle (2.07-fold), kidney (2.2-fold) and haemocyte (2.5-fold) were enhanced by cadmium (Cd²âº) stress, suggesting that Hs-BTG1 may have played a significant role in H. schlegelii adaptation to adverse environmental conditions.

Single nucleotide polymorphisms other than factor V Leiden are associated with coagulopathy and osteonecrosis of the femoral head in Chinese patients.

Single nucleotide polymorphisms (SNPs) of factor V Leiden have been associated with osteonecrosis of the femoral head (ONFH) in Caucasians but remains controversial in Asians. We used an SNP microarray to screen 55 loci of factor V gene in patients with ONFH of Chinese. Significantly different candidate SNPs at 14 loci were analyzed in 146 patients and 116 healthy controls using MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometry and gene sequencing. The factor V Leiden (rs6025) was not found in all participants. Six SNP loci (rs9332595, rs6020, rs9332647, rs3766110, rs10919186, and rs12040141) were confirmed with significant differences in patients but not in controls. The rs6020 G-to-A polymorphism was found in 88.9% of the patients. In addition, a high percentage (87.6%) of the patients had an abnormal coagulation profile that included hyperfibrinogen, elevated fibrinogen degradation products, elevated D-dimer, abnormal protein S, abnormal protein C, or a decrease in anti-thrombin III. Patients with the rs6020 G-to-A polymorphism (mutation) had a higher risk (odds ratio: 4.62; 95% confidence interval: 1.44-14.8) of having coagulation abnormalities than did those without the mutation (wild-type) (χ(2) p  =  0.006). Our findings suggested that the rs6020 polymorphism might be the genetic trait that accounts for the higher prevalence of ONFH in the Chinese population than in Westerners. Exposure to risk factors such as alcohol and steroids in patients with the rs6020 polymorphism causes coagulation abnormalities and, subsequently, thromboembolisms in the femoral head.

Characterization of a novel resistance-related deoxycytidine deaminase from Brassica oleracea var. capitata.

Brassica oleracea deoxycytidine deaminase (BoDCD), a deoxycytidine deaminase (DCD, EC 3.5.4.14) enzyme, is known to play an important role in the Trichoderma harzianum ETS 323 mediated resistance mechanism in young leaves of B. oleracea var. capitata during Rhizoctonia solani infection. BoDCD potentially neutralizes cytotoxic products of host lipoxygenase activity, and thereby BoDCD restricts the hypersensitivity-related programmed cell death induced in plants during the initial stages of infection. To determine the biochemical characteristics and to partially elucidate the designated functional properties of BoDCD, the enzyme was cloned into an Escherichia coli expression system, and its potential to neutralize the toxic analogues of 2'-deoxycytidine (dC) was examined. BoDCD transformants of E. coli cells were found to be resistant to 2'-deoxycytidine analogues at all of the concentrations tested. The BoDCD enzyme was also overexpressed as a histidine-tagged protein and purified using nickel chelating affinity chromatography. The molecular weight of BoDCD was determined to be 20.8 kDa as visualized by SDS-PAGE. The substrate specificity and other kinetic properties show that BoDCD is more active in neutralizing cytotoxic cytosine ß-d-arabinofuranoside than in deaminating 2'-deoxycytinde to 2'-deoxyuridine in nucleic acids or in metabolizing cytidine to uridine. The optimal temperature and pH of the enzyme were 27 °C and 7.5. The Km and Vmax values of BoDCD were, respectively, 91.3 µM and 1.475 mM for its natural substrate 2'-deoxycytidine and 63 µM and 2.072 mM for cytosine ß-d-arabinofuranoside. The phenomenon of neutralization of cytotoxic dC analogues by BoDCD is discussed in detail on the basis of enzyme biochemical properties.