ZEB2 Attenuates LPS-Induced Inflammation by the NF-κB Pathway in HK-2 Cells.

As a transcription factor, zinc finger E-box binding homeobox 2 (ZEB2) includes multiple functional domains which interact with kinds of transcriptional co-effectors. It has been reported that ZEB2 was involved in signal transduction and multiple cellular functions. However, the functional role of ZEB2 in inflammation is still obscure. The aim of the current study is to explore the function of ZEB2 in inflammation cytokine secretion and the role of the nuclear factor-κB (NF-κB) signaling pathway in lipopolysaccharide (LPS)-induced human proximal tubule cell line (HK-2) cells. Our result demonstrated that expression of ZEB2 was significantly downregulated and expression of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) was upregulated in response to LPS. Meanwhile, knockdown of ZEB2 by transfecting siRNA increased TNF-α and IL-6 secretion. Overexpression of ZEB2 resulted in a decrease of TNF-α and IL-6 secretion in HK-2 cells. Additionally, Western blot analysis indicated that ZEB2 suppressed the activation of the NF-κB signaling pathway via downregulating the levels of phosphorylated p65 and IκBα compared with LPS stimulation. Collectively, our data demonstrated that ZEB2 attenuated LPS-induced inflammation cytokine secretion possibly through suppressing the NF-κB signaling pathway.

Functional characterization of the promoter of carbonyl reductase 1 gene in porcine endometrial cells.

Prostaglandins (PGs) play a critical role in porcine reproduction, of which prostaglandin E2 (PGE2) and prostaglandin F2α (PGF2α) exert antiluteolytic and luteolysis actions, respectively. As a rate-limiting enzyme, carbonyl reductase 1 (CBR1) catalyzes the conversion of PGE2 to PGF2α. A high ratio of PGE2:PGF2α is beneficial to the establishment and maintenance of porcine pregnancy. PG is essential for the establishment of pregnancy which resembles the proinflammatory response and nuclear factor κB (NF-κB) is involved in the process. Bioinformatic analysis has shown that NF-κB is a possible factor bound to two cis-regulatory elements in CBR1 promoter. In this study, we cloned the 2997 bp (-2875/+122) of the promoter, and constructed six 5'-deleted dual-luciferase reporter recombinant vectors. In endometrial cells, the region of P2 (-1640/+7) exhibited the greatest transcriptional activity at driving luciferase expression, but not significantly different from that of P1 (-2089/+7). The activity of P1, P2, and P3 (-1019/+7) was highly significantly higher than that of others (P<0.01), suggesting that two positive regulatory elements were likely present in the regions of -1640/-1019 and -1019/-647. The results also showed that the -1640/-647 region was indispensable for the promoter. The results of chromatin immunoprecipitation (ChIP) demonstrated that the NF-κB subunit p65 binds to one site around -1545/-1531. Using four reference genes, we found that the over-expression of p65 enhanced the expression of CBR1 (P<0.05) in porcine endometrial epithelial cells, while knockdown of the p65 did not down-regulate the CBR1 expression. These results indicated that NF-κB (p65) could bind to the special element of CBR1 gene promoter in porcine endometrial epithelial cells in vitro. The binding site of NF-κB was a positive regulator for the CBR1 gene promoter, but was not necessary for the basic expression.

Profiling the genome-wide DNA methylation pattern of porcine ovaries using reduced representation bisulfite sequencing.

Substantial evidence has shown that DNA methylation regulates the initiation of ovarian and sexual maturation. Here, we investigated the genome-wide profile of DNA methylation in porcine ovaries at single-base resolution using reduced representation bisulfite sequencing. The biological variation was minimal among the three ovarian replicates. We found hypermethylation frequently occurred in regions with low gene abundance, while hypomethylation in regions with high gene abundance. The DNA methylation around transcriptional start sites was negatively correlated with their own CpG content. Additionally, the methylation level in the bodies of genes was higher than that in their 5' and 3' flanking regions. The DNA methylation pattern of the low CpG content promoter genes differed obviously from that of the high CpG content promoter genes. The DNA methylation level of the porcine ovary was higher than that of the porcine intestine. Analyses of the genome-wide DNA methylation in porcine ovaries would advance the knowledge and understanding of the porcine ovarian methylome.

Complete mitochondrial genome of the Algerian honeybee, Apis mellifera intermissa (Hymenoptera: Apidae).

In this study, the complete mitochondrial genome sequence of Algerian honeybee, Apis mellifera intermissa, is analyzed for the first time. The results show that this genome is 16,336 bp in length, and contains 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes, and 1 control region (D-loop). The overall base composition is A (43.2%), C (9.8%), G (5.6%), and T (41.4%), so the percentage of A and T (84.6%) is considerably higher than that of G and C. All the genes are encoded on H-strand, except for four subunit genes (ND1, ND4, ND4L, and ND5), two rRNA genes (12S and 16S rRNA), and eight tRNA genes. The complete mitochondrial genome sequence reported here would be useful for further phylogenetic analysis and conservation genetic studies in A. m. intermissa.

[Effects of synchronous treatment of bilateral scalp acupuncture and rehabilitation training on activities of daily life in patients with cerebral infarction at acute phase].

OBJECTIVE: To observe the improvements of synchronous treatment of bilateral scalp acupuncture and rehabilitation training on activities of daily life in patients with cerebral infarction at acute phase, so as to compare the efficacy differences between scalp acupuncture at bilateral and affected side as well as differences between synchronous and non-synchronous treatment. METHODS: Ninety patients of acute-phase cerebral infarction with motor dysfunction were randomly divided into three groups. The observation group was treated with synchronous treatment of scalp acupuncture at the Dingzhongxian (middle line of vertex), bilateral Dingnieqianxiexian (anterior oblique line of vertex-temporal) and bilateral Dingniehouxiexian (posterior oblique line of vertex-temporal) and rehabilitation training; the control group A was treated with synchronous treatment of affected scalp acupuncture at the Dingzhongxian, affected Dingnieqianxiexian and affected Dingniehouxiexian and rehabilitation training; the control group B was treated with bilateral scalp acupuncture for 4 h, followed by rehabilitation training. All the patients took the treatment once a day, and 6 days for a course of treatment for total of 4 courses. The modified Barthel index (MBI), activities of daily living (ADL) and Fugl-Meyer motor assessment (FMA) were used to perform efficacy assessment before treatment, in the 14th days of treatment and in the 28th days of treatment in three groups. RESULTS: After treatment, three indices at each time point were superior to those before the treatment in three groups (all P<0.01) ; the improvements of ADL and FMA in the observation group after 28 days of treatment were superior to those in the control group A and control group B (all P<0.05), and the improvement of MBI was superior to that in the control group B (P<0.05). CONCLUSION: The synchronous treatment of bilateral scalp acupuncture and rehabilitation training could significantly improve the activities of daily life and motor function in patients with cerebral infarction at acute phase, which is superior to scalp acupuncture at affected side and non-synchronous treatment.

[Monitoring the thermal plume from coastal nuclear power plant using satellite remote sensing data: modeling, and validation].

Thermal plume from coastal nuclear power plant is a small-scale human activity, mornitoring of which requires high-frequency and high-spatial remote sensing data. The infrared scanner (IRS), on board of HJ-1B, has an infrared channel IRS4 with 300 m and 4-days as its spatial and temporal resolution. Remote sensing data aquired using IRS4 is an available source for mornitoring thermal plume. Retrieval pattern for coastal sea surface temperature (SST) was built to monitor the thermal plume from nuclear power plant. The research area is located near Guangdong Daya Bay Nuclear Power Station (GNPS), where synchronized validations were also implemented. The National Centers for Environmental Prediction (NCEP) data was interpolated spatially and temporally. The interpolated data as well as surface weather conditions were subsequently employed into radiative transfer model for the atmospheric correction of IRS4 thermal image. A look-up-table (LUT) was built for the inversion between IRS4 channel radiance and radiometric temperature, and a fitted function was also built from the LUT data for the same purpose. The SST was finally retrieved based on those preprocessing procedures mentioned above. The bulk temperature (BT) of 84 samples distributed near GNPS was shipboard collected synchronically using salinity-temperature-deepness (CTD) instruments. The discrete sample data was surface interpolated and compared with the satellite retrieved SST. Results show that the average BT over the study area is 0.47 degrees C higher than the retrieved skin temperature (ST). For areas far away from outfall, the ST is higher than BT, with differences less than 1.0 degrees C. The main driving force for temperature variations in these regions is solar radiation. For areas near outfall, on the contrary, the retrieved ST is lower than BT, and greater differences between the two (meaning > 1.0 degrees C) happen when it gets closer to the outfall. Unlike the former case, the convective heat transfer resulting from the thermal plume is the primary reason leading to the temperature variations. Temperature rising (TR) distributions obtained from remote sensing data and in-situ measurements are consistent, except that the interpolated BT shows more level details (> 5 levels) than that of the ST (up to 4 levels). The areas with higher TR levels (> 2) are larger on BT maps, while for lower TR levels (≤ 2), the two methods perform with no obvious differences. Minimal errors for satellite-derived SST occur regularly around local time 10 a. m. This makes the remote sensing results to be substitutes for in-situ measurements. Therefore, for operational applications of HJ-1B IRS4, remote sensing technique can be a practical approach to monitoring the nuclear plant thermal pollution around this time period.

Molecular characterization, tissue expression and nucleotide variation of the porcine AZ1 gene.

Antizyme 1 (AZ1) is a member of the antizyme family that is involved in many biological processes. As a natural inhibitor, AZ1 controls the normal level of polyamines, which is indispensable to cellular function. Our prior research showed that the expression of the AZ1 gene in Longissimus doris of Landrace pigs was higher (P<0.05) than in Lantang pigs. The AZ1 gene might be involved in the development of muscle and potentially serves as an important target for muscle improvement in pigs. In this study, the molecular characterization, tissue expression, and sequence variation of porcine AZ1 gene were analyzed. A 4082 bp sequence including the 5'-flanking region and gene sequence was obtained through RACE and sequencing. The genomic sequence of AZ1 gene consists of six exons and five introns. The mRNA of AZ1 gene contains three elements: 5'-untranslated regions (UTR, 79 bp), CDS (684 or 207 bp), and 3'-UTR (161 or 423 bp for the two transcripts). Three termination signals of AATAAA were found in the longer 3'-UTR. The mRNA sequence of the AZ1 gene contained two ORFs with a frameshifting site at the 69th codon. The amino acid sequence from the porcine AZ1 gene was similar to other vertebrates and exhibited the highest similarity to cattle. The partial 5'-flanking region was 852 bp with typical cis-regulatory elements such as TATA boxes and CAAT boxes. By DNA pooling and sequencing, nine and 12 single nucleotide polymorphisms (SNPs) were detected in the 5'-flanking region and introns, respectively. Except the SNP at -713, the other eight SNPs were found in putative cis-acting elements and might alter the binding of trans-acting factors. Expression patterns showed that in Lantang pig, the AZ1 gene is differentially expressed in various tissues and displayed higher expression in lung and skeletal muscle.

Effects of ghrelin gene genotypes on the growth traits in Chinese cattle.

Ghrelin is an important peptide that stimulates food intake and regulates energy balance of animals. Single nucleotide polymorphisms of ghrelin gene in three Chinese cattle populations were investigated through PCR-SSCP and DNA sequencing. Five over-lapped DNA fragments were analyzed and a total of three ones exhibited different genotypes. Three genotypes and four SNPs (-415 A > G, -414 T > C, -321 C > A, and -172 A > G) were found on the -544 to +35 bp region (G-1) of ghrelin gene. On the locus of -1037 to -509 bp (G-2), two genotypes and one SNP (-726 A > T) were discovered. And in the exon1, exon2, and intron1 (G-4 locus, (+4 to +427)), two genotypes and one SNP were detected (+205 C > T, located in intron1). Positions of the five SNPs in the 5' regulatory region might be the transcription factor binding sites. The SNPs at -415 and -414 in the core binding sequence were found to cause the change of the site. Though the SNP at -172 did not change the binding site, it generated one new site at the same time. The frequencies of the genotypes varied differently in the three breeds. Results of ANOVA showed that G-1 was correlative to the ischium width (IW) of Nanyang cattle aged 18 months (p = 0.043). The least square analysis between genotypes at G-1 locus and growth traits in Nanyang cattle showed that the individuals (aged 18 months) with C genotype had greater IW than that of the other two genotypes. The C genotype might serve as one potential candidate genetic marker for cattle growth and development.

Association analysis between variants in bovine NPY gene and growth traits in Nanyang cattle (Bos tarus).

Neuropeptide Y (NPY) is one of the most potent orexigenic factors that is implicated in the control of feeding behavior and energy homeostasis. Four Chinese indigenous cattle breeds (n=338) were used to detect single nucleotide polymorphisms (SNPs) in the coding regions with their intron-exon boundaries and the proximal flanking regions of NPY gene by PCR-SSCP and DNA sequencing. Five novel SNPs (38017 C>G, 34240 C>A, 34168 G>A, 32463 A>C and 32302 C>G) were identified. Polymorphisms of P3 lous in NPY gene, containing SNP4 and SNP5, were associated with body length and chest girth in Nanyang cattle aged 6, 12 and 18 months (P<0.05), but they had no significant effect on the two growth traits in Nanyang cattle aged 24 months (P>0.05). The results open a new field of study and extend the spectrum of genetic variation of the bovine NPY gene, which might contribute to cattle genetic resources and breeding.

An efficient and rapid method for gene cloning from eukaryotic genomic DNA using overlap-PCR: With an example of cattle Ghrelin gene.

In this study, overlap-PCR, an efficient and rapid method, was used to clone cattle Ghrelin gene CDS (coding sequence) from genomic DNA. The procedure included seven primers and three-step PCRs. Cattle Ghrelin gene consists of four exons and the CDS contains 351 bps. In the first step three PCRs were performed to generate extended exon1, exon2, and exon3 that contained overlapped nucleotides and were used as the templates for second ligation PCR. Secondly, exon1 and exon2 were spliced together. And it was same with exon3 and exon4. Lastly, the four exons were linked together with outermost primers and the templates from the second step. Comparison analysis on the obtained CDS of Ghrelin gene and cDNA by RT-PCR showed that the two sequences were same. As an efficient and rapid method, overlap-PCR is feasible and acceptable for gene cloning from genomic DNA.

5-(4-Bromo-phen-yl)-1,2,3,4-tetra-hydro-benzo[a]phenanthridine.

The title compound, C(23)H(18)BrN, was synthesized by the reaction of 4-bromo-benzaldehyde, naphthalen-2-amine and cyclo-hexa-none in tetra-hydro-furan, catalyzed by iodine. The saturated six-membered ring adopts a half-chair conformation, and the four vicinal rings form a helical conformation, which results in a significant deviation from planarity for the pyridine ring. In the crystal, a weak C-H⋯π inter-action occurs, leading to inversion dimers.

[Molecular genetic characteristic of bovine HTR1B gene].

The aim of the present study was to identify and characterize polymorphisms within the coding region and the 3' flanking region of the bovine serotonin receptor 1B gene among different cattle breeds. Four DNA fragments were amplified by polymerase chain reaction and then used for polymorphism identification by SSCP. The fragments showing different SSCP patterns were sequenced. And a total of six SNPs (G205T, C507T, C546G, C744T, G816A and G942A) were detected. The SNPs were at Hardy-Weinberg equilibrium except C507T and C546G in all genetic population. The frequencies of allele 205T of Holstein were much higher than that of the other six beef cattle populations. Almost the PIC of all SNPs were not more than 0.10 except that of G205T in Holstein cows, which indicated the bovine HTR1B gene was conserved.

[Effect of fertilization condition on mammalian sex determination].

Selection of sex at or before conception is perhaps the most desired biotechnology of all time because many economic traits are related with sex. Sex selection by manipulating the conditions of sperm-oocytes fertilization would be one of the most practicable and widely used methods. This review summarized the progress in the effects of fertilization conditions on mammalian sex. Four main factors which possibly affected sex were described such as arginine concentration in female animal vagina, glucose in the uterus, maturational status of the oocyte at the time they encounter sperm as well as the hormone levels of both parents at the time of conception.

Assay and characterization of a strong promoter element from B. subtilis.

A new strong promoter fragment isolated from Bacillus subtilis was identified and characterized. Using the heat stable beta-galactosidase as reporter, the promoter fragment exhibited high expression strength both in Escherichia coli and B. subtilis. The typical prokaryotic promoter conservation regions were found in the promoter fragment and the putative promoter was identified as the control element of yxiE gene via sequencing assay and predication of promoter. To further verify and characterize the cloned strong promoter, the putative promoter was sub-cloned and the beta-Gal directed by the promoters was high-level expressed both in E. coli and B. subtilis. By means of the isolated promoter, an efficient expression system was developed in B. subtilis and the benefit and usefulness was demonstrated through expression of three heterologous and homogenous proteins. Thus, we identified a newly strong promoter of B. subtilis and provided a robust expression system for genetic engineering of B. subtilis.

[Analysis on therapeutic effect of acupoint-injection on chronic hepatitis B].

OBJECTIVE: To search for an effective method for treatment of chronic hepatitis B. METHODS: One hundred and twenty-three cases were randomly divided into a treatment group (n = 63) and a control group (n = 60). The treatment group were treated with injection of Huangqi injectio and Danshen Injectio into Ganshu (BL 18) and Zusanli (ST 36), once every other day; and the control group were treated with oral administration of Gankangning tablet and fufang yiganling tablet. The clinical symptoms, hepatic function, hepatitis B e antigen (HBeAg), hepatitis B virus-desoxyribose nucleic acid (HBV-DNA) were compared between the two groups. RESULTS: The total effective rate was 93.7% in the treatment group and 76.7% in the control group with significant difference between the two groups (P < 0.01); and the treatment group in hepatic function and the effects of turning negative for HBeAg and HBV-DNA was better than the control group (P < 0.01). CONCLUSION: Acupoint-injection has a better therapeutic effect on chronic hepatitis B.