Effector responses of bovine blood neutrophils against Escherichia coli: Role of NOD1/NF-κB signalling pathway.

Neutrophils use a broad array of pattern recognition receptors to sense and respond to invading pathogens and are important in the early control of acute bacterial infections. Nucleotide-binding oligomerizing domain-1 (NOD1) is a cytoplasmic receptor involved in recognizing bacterial peptidoglycan. Reduced neutrophil NOD1 expression has been reported in periparturient dairy cows. The aim of this study was to investigate the role of NOD1 signalling in the early responses of bovine neutrophils to bacterial infections. Blood neutrophils from healthy heifers were preincubated for 2h with ML130, a selective inhibitor of NOD1-dependent nuclear factor-κB (NF-κB) activation. Thereafter, cells were cultured with live Escherichia coli for additional 30 min or subjected to Boyden chamber cell migration assay with E. coli in the lower chamber. Results showed that ML130 inhibited E. coli-induced NF-κB nuclear translocation. There was an indication, although not significant, that ML130 down-regulated gene expression of proinflammatory cytokines interleukin (IL)-1ß and tumour necrosis factor (TNF)-α, chemokines IL-8 and C-X-C motif ligand 2 (CXCL2), and adhesion molecule CD62L, in E. coli-challenged neutrophils. Flow cytometry-based Annexin V staining revealed a considerable increase in neutrophil survival upon E. coli infection, an effect that was attenuated in the presence of ML130. Additionally, inhibition of NOD1/NF-κB signalling resulted in reduced migration of neutrophils to E. coli, and impaired phagocytosis, intracellular bacterial killing and reactive oxygen species production by neutrophils. These results indicate that NOD1/NF-κB pathway plays a crucial role in modulating neutrophil responses that are important for early control of infections. Approaches aiming at restoring neutrophil NOD1 function could be beneficial for prevention or treatment of coliform mastitis.

[Study on method of paeonia radix alba producing and concocting integration processing].

OBJECTIVE: To study the origin pre-treating and processing integration techniques of Paeonia Radix Alba. METHOD: Different processing integration techniques were adopted and compared with traditional processing techniques to determine drying rate, aqueous extracts and peoniflori content. RESULT: Half-dry slices baked at 100 degrees C for 20 min and steamed at 100 degrees C for 10 min had the highest peoniflori contents. Half-dry slices baked at 100 degrees C for 20 min had the highest content of aqueous extracts. Products processed with conventional method and sulfur-fumigation had the lowest content of aqueous extracts. CONCLUSION: The origin processing integration techniques of Paeonia Radix Alba lose less active ingredients than conventional processing methods.

[Cloning and function identification of gene 'admA' and up-stream regulatory sequence related to antagonistic activity of Enterobacter cloacae B8].

To reveal the antagonistic mechanism of B8 strain to Xanthomonas oryzae pv. oryzae, transposon tagging method and chromosome walking were deployed to clone antagonistic related fragments around Tn5 insertion site in the mutant strain B8B. The function of up-stream regulatory sequence of gene 'admA' involved in the antagonistic activity was further identified by gene knocking out technique. An antagonistic related left fragment of Tn5 insertion site, 2 608 bp in length, was obtained by tagging with Kan resistance gene of Tn5. A 2 354 bp right fragment of Tn5 insertion site was amplified with 2 rounds of chromosome walking. The length of the B contig around the Tn5 insertion site was 4 611 bp, containing 7 open reading frames (ORFs). Bioinformatic analysis revealed that these ORFs corresponded to the partial coding regions of glyceraldehyde-3-phosphate dehydrogenase, two LysR family transcriptional regulators, hypothetical protein VSWAT3-20465 of Vibrionales and admA, admB, and partial sequence of admC gene of Pantoea agglomerans biosynthetic gene cluster, respectively. Tn5 was inserted in the up-stream of 200 bp or 894 bp of the sequence corresponding to anrP ORF or admA gene on B8B, respectively. The B-1 and B-2 mutants that lost antagonistic activity were selected by homeologuous recombination technology in association with knocking out plasmid pMB-BG. These results suggested that the transcription and expression of anrP gene might be disrupted as a result of the knocking out of up-stream regulatory sequence by Tn5 in B8B strain, further causing biosythesis regulation of the antagonistic related gene cluster. Thus, the antagonistic related genes in B8 strain is a gene family similar as andrimid biosynthetic gene cluster, and the upstream regulatory region appears to be critical for the antibiotics biosynthesis.

[Construction and transfection experiment of a goose circovirus infectious clone].

A pair of primers with BamH I restriction site were designed to amplify the complete genome of goose circovirus. Two copies of the genome were ligated in tandem and cloned into pGEM-T Easy vector to construct an infectious clone named as pGEMT-2GoCV. The pGEMT-2GoCV linearized with EcoR I was transfected to negative embryos and gosling with Lipfectamine. PCR detection verified the proliferation of GoCV in geese. Some sera of the embryo transfected group were detected to be positive at 2 and 4 weeks after hatching and one bursa was detected to be positive at 4 weeks. Some sera of the gosling transfected group were also detected to be positive at 2 weeks after transfection. Furthermore, the mark in the PCR products were identified by BamH I digestion and the GoCV in positive tissue and sera were quantitated by Real-time PCR. The results showed that the virus load in positive bursa was 1.57 x 10(6) copies/mg, the virus load in positive sera were 3.52 x 10(4)-5.92 x 10(5) copies/microL. In conclusion, the infectious DNA clone constructed with two copies of full-length GoCV genome in tandem can transfect embryo and gosling and propagate the marked goose circovirus.

[Study on the initial processing of Corydalis yanhusuo].

OBJECTIVE: To establish initial processing technology of Corydalis yanhusuo. METHODS: Investigated the effect of the factors such as slice method, dry method, drying temperature on the content of water-extract, ethanol-extract, effective component in Corydalis yanhusuo pieces. Compared the quality with that of the traditional initial processing samples. RESULTS: The best initial process method was: cut fresh Corydalis yanhusuo into 4 - 5 mm thick slices, dry at 70 - 80 degrees C or microwave dry. CONCLUSION: The study provides theoretical base for modifying the initial processing.

[Factors influence volatile oil content in Ocimun basilicum introduced from Xinjiang province to south-east China].

OBJECTIVE: To obtain the information on ecological adaptation of Ocimun basilicum introduced from Xinjiang to Hangzhou and study the effect of different harvesting times, drying methods, and different organs of Ocimun basilicum on Volatile oil content METHODS: Extraction was undertaken according to The Pharmacopoeia of China, 2010 edition. RESULTS: Sun-drying was the most efficient way to obtain Volatile oil compared with other methods. The largest biomass was harvested at 3rd, September. Furthermore, Volatile oil was found to accumulate mostly in the flowers and little in the stems. CONCLUSION: Ocimun basilicum can readily inhabit in Hangzhou and its economic value can be significant improved by growing two seasons per year. Only harvest leaves and flowers can significantly reduce the cost for transport and also increase oil extract rate of Volatile oil.

[Cloning and analysis of the complete genomes of pigeon circovirus from Zhejiang Province].

Pigeon circovrius (PiCV) is a member of circovirus, which is usually regarded as an immunosuppression agent. There were reports that pigeons infected by PiCV showed symptoms of lethargy, weight loss, vomiting, diarrhea, respiratory distress, etc. In this study, we established a PCR method for the detection of PiCV DNA. Samples from 5 different farms in Zhejiang Province were examined and samples from a farm in Hangzhou were positive. Furthermore, the genomic segments of 2 strains of PiCV were amplified, cloned and sequenced using designed primers and the complete genomes of the strains were then assembled and named as PiCV-zj1 and PiCV-zj2, respectively. The sequences were deposited in GenBank under the GenBank Accession number of DQ090945 and DQ090944, respectively. Sequence Analysis had shown that the complete genomes of 2 strains of PiCV from Zhejiang Province had 2 039 nucleotides totally in length and common characters of circovirus such as a stem-loop structure and conserved motifs for Rep protein, which were supposed to be related to the replication of the virus. Pairwise comparisons showed that the nucleotide sequence of the genome of PiCV strains from Zhejiang Province had 86%-89.1% identities to that of 11 published PiCV strains, and that the amino acid identities of the replication-associated protein (Rep) and capsid protein (Cap) displayed 92.1%-94.7% and 76.6%-81.4%, respectively. A phylogenic tree was built using PHYLIP with bootstrap support for 1 000 replicates. The result showed that 10 strains from Europe and America formed one big branch and the others from Zhejiang Province and Australia formed the other two, respectively. This was the first report on the detection and full genome sequencing of PiCV in China.

[Study on the influence on Rhizoma Corydalis by different harvest time and processing methods].

OBJECTIVE: To choose the best harvest time and initial processing method of Rhizoma Corydalis. METHODS: Taking the content of tetrahydropalmatine and dry rates as major indices, different samples from different harvest time and different processing methods were investigated by HPLC. The C18 column (250 mm x 4.6 mm, 5 microm) was used with mobile phase of methanol-0.1% phosphoric acid solution (pH = 6.0, adjusted with triethylamine) (58: 42). The mobile phase flow rate was 1.0 mL/min. The detection wavelength was 280 nm. RESULTS: The contents of tetrahydropalmatine were similar although the harvest time was different. The dry rate on May 21 was higher than the others. The content of tetrahydropalmatine was the highest by steaming and reached 0.11%. CONCLUSION: The best harvest time for Rhizoma Corydalis is around 10 days after the above-ground plant wilted, and the best initial processing method for Rhizoma Corydalis is steaming.

[Study on correlation between Atractylodes macrocephala viability and germination rate].

OBJECTIVE: To screen the optimized methods for detection seed viability and germination rate determination of Atractylodes macrocephala, and determine the relationship between seed viability and germination rate. METHOD: There were four methods, which including 2,3,5-triphynel tetrazolilum chloride (TTC) staining, red ink staining, BTB staining and Nongjia method, to evaluate the 12 A. macrocephala local varieties'seed viability and measure their germination rate. RESULT: Seed viability of A. macrocephala using TTC staining ranked the first compared to that of other three methods. Seed viability was significantly related with germination rate using TTC method. Their correlation coefficient reached 0.915 and regression equation was also found out between seed viability (X) and germination rate (Y), which was Y = -0.083 4 + 0.995 4X. CONCLUSION: TTC staining was the optimal method to determine A. macrocephala seed vitality. Furthermore, seed viability was significant related with germination rate of A. macrocephala.

[Mutagenic effects of gamma-rays on Coix lacryma-jobi var. ma-yuen].

OBJECTIVE: To study the mutagenic effect of gamma-rays on Coix lacryma-jobi var. ma-yuen. METHOD: Physiological and mutagenic effects of gamma-rays on C. lacryma-jobi var. ma-yuen dormant seeds were studied. The germination percentage, seeding survival, seeding height and root length of M1 plants and the frequency of chlorophyll mutation in M2 generation were selected as criteria. RESULT: The gamma-rays showed obvious inhibitory action to the seedling growth, and a strong ability in inducing the chlorophyll mutation. CONCLUSION: The gamma-rays is one kind of C. lacryma-jobi var. ma-yuen effective mutagen. The appropriate dose of gamma-rays is 450 Gy for C. lacryma-jobi var. ma-yuen dormant seeds.

[Study on cutting propagation of Tripterygium wilfordii].

The experiment of cutting propagation of Tripterygium wilfordii Hook. f. was carried out. It showed a better result when stems were instantly planted after being moistened or soaked by 500 - 1000 ppm IBA or NAA. A remarkably higher survival rate was achieved while plasticfilm and shade were accompanied. September and October or rainy June are prime moments for operation. It' s recommendable to choose those newly corking twigs for cuttage.

Identification of anrF gene, a homology of admM of andrimid biosynthetic gene cluster related to the antagonistic activity of Enterobacter cloacae B8.

AIM: To identify the gene (s) related to the antagonistic activity of Enterobacter cloacae B8 and to elucidate its antagonistic mechanism. METHODS: Transposon-mediated mutagenesis and tagging method and cassette PCR-based chromosomal walking method were adopted to isolate the mutant strain(s) of B8 that lost the antagonistic activity and to clone DNA fragments around Tn5 insertion site. Sequence compiling and open reading frame (ORF) finding were done with DNAStar program and homologous sequence and conserved domain searches were performed with BlastN or BlastP programs at www.ncbi.nlm.nih.gov. To verify the gene involved in the antagonistic activity, complementation of a full-length clone of the anrF gene to the mutant B8F strain was used. RESULTS: A 3 321 bp contig around the Tn5 insertion site was obtained and an ORF of 2 634 bp in length designated as anrF gene encoding for a 877 aa polyketide synthase-like protein was identified. It had a homology of 83% at the nucleotide level and 79% ID/87% SIM at the protein level, to the admM gene of Pantoea agglomerans andrimid biosynthetic gene cluster (AY192157). The Tn5 was inserted at 2 420 bp of the gene corresponding to the COG3319 (the thioesterase domain of type I polyketide synthase) coding region on B8F. The antagonistic activity against Xanthomonas oryzae pv. oryzae was resumed with complementation of the full-length anrF gene to the mutant B8F. CONCLUSION: The anrF gene obtained is related to the antagonistic activity of B8, and the antagonistic substances produced by B8 are andrimid and/or its analogs.

[Cloning and analysis of the complete genome of a Goose circovirus from Yongkang Zhejiang].

To investigate the mechanism of avian influenza outbreak wildly in water fowls, the co-pathogens, especially that caused immuno-depression were studied. A pair of degenerated primers, which amplified a fragment of 552bp in length, was designed and synthesized based on the published goose and other Circovirus sequences. The specific PCR product was amplified from the goose sample of Yongkang avian influenza case of Zhejiang Province. The fragment was then sequenced and the result showed the existence of the GoCV. The opposite part of the genome was further amplified using inverse primers designed based on the 552bp sequence obtained and the 1821bp full length genomic sequence of GoCV-yk01 was compiled using Seqman program of DNAStar. Sequence analysis showed that the GoCV-yk01 possessed common features of circovirus included potential replication associated stem-loop structure and the Rep protein motifs. Homology analysis showed that the sequence of GoCV-yk01 had 91% approximately 93% similarity to that of Taiwan and Germany strains. Phylogenetic analysis with ClustalW, however, showed that the GoCV-yk01 was on a different branch away from Taiwan and Germany strains. Circovirus usually causes immuno-depression and results in secondary infection, as it infects rapid growing cells as lymphocyte. It is speculated that the infection of GoCV may be a part of the reason of the avian influenza outbreak of the Yongkang case. The GoCV-yk01 is the first GoCV strain reported in mainland of China.

[Construction and verification of the effectiveness of pMBL: a cloning vector of exported proteins encoding genes].

The beta-lactamase was used as the reporter of expression and transmembrane secretion in this paper. A fragment of Amp resistance gene encoding the mature part of beta-lactamase (delta P delta SP Amp, i.e. without promoter and signal peptide coding sequences) was amplified from pUC18 vector. The upstream primer has BglII, BclI, BamHI in three reading frame respectively, in order to in frame fuse and express target genes together with the downstream reporter in finally constructed vector. Meanwhile, pET-28 was digested with the restriction enzymes BglII and Bst1107 I. The 2.8 kb fragment with replication origin, Kan resistance gene and MCS was recovered, filled, self-ligated and resulted in a plasmid pKan-B. The Bgl II site on pKan-B was then filled and the plasmid pKan was obtained. The delta P delta SP Amp gene, which was first cloned into pGEM-T-EASY vector, was inserted into pKan between EcoR I and XbaI sites. A plasmid pMBL-E was selected, with which the bacteria host could grew on Kan plate but not on plate with both Amp and Kan. An EcoRI site beside HindIII on the plasmid pMBL-E was then filled, and the plasmid pMBL, a cloning vector of the exported proteins encoding genes was finally obtained. Both results of the restriction enzyme digestion and sequencing demonstrated the correctness of the construction. The Tet resistance gene, a transmemebrane protein encoding gene, was applied to verify the effectiveness of the reporter in the vector. Cut with EcoRI and BamHI, a 375 bp fragment including promoter and 96 animo acids coding sequence (including signal peptide) of Tet was obtained from pBR322 vector. The fragment was then ligated to the vector pMBL which had been cut with both enzymes of EcoRI and BglI, or EcoRI and BclI, or EcoRI and BamHI (as 0, +1, +2 respectively of the beta-lactamase gene reading frame). Kan and Amp double resistant colonies only grew with the EcoRI and BglII combination (0 position). Restriction enzyme digestion and sequencing results of the recombinant plasmid showed that Tet resistance gene, which promoted the expression and transmembrane secretion of downstream beta-lactamase, was inserted in a correct reading frame into the vector. Thus, the results verified the effectiveness of the constructed vector pMBL, which may be used effectively to clone genes encoding exported proteins with promoters and signal peptide sequences.

The origin and genetic diversity of Chinese native chicken breeds.

The first 539 bases of mitochondrial DNA D-loop region of six Chinese native chicken breeds (Gallus gallus domesticus) were sequenced and compared to those of the red junglefowl (Gallus gallus), the gray junglefowl (Gallus sonneratii), the green junglefowl (Gallus varius) and Lafayette's junglefowl (Gallus lafayettei) reported in GenBank, and the phylogenetic trees for the chickens were constructed based on the D-loop sequences. The results showed that the four species of the genus Gallus had great differences among each other the G. g. domesticus was closest to the red junglefowl in Thailand and its adjacent regions, suggesting the Chinese domestic fowl probably originatedfrom the red junglefowl in these regions. The two subspecies of Thailand, G. g. gallus and G. g. spadiceus, should belong to one subspecies because of their resemblance. In the case of native breeds, there existed a great difference between the egg breeds and general purpose breeds, which suggested different maternal origins of the two types.