6-Formylindolo[3,2-b]carbazole reduces apoptosis induced by benzo[a]pyrene in a mitochondrial-dependent manner.

Benzo[a]pyrene (B[a]P), a potent carcinogen, has been proved that it can induce apoptosis via activation of the aryl hydrocarbon receptor (AhR) pathway. The metabolite of tryptophan 6-formylindolo[3,2-b]carbazole (FICZ), an endogenous activator of AhR, plays bifunctional roles in cell growth and apoptosis. However, whether and how FICZ can reduce the toxicity of B[a]P and the mechanism underlying this remain unclear. In this study, FICZ interfered with the toxicity of B[a]P in mouse hepatocarcinoma cell line Hepa1-6. The results of the MTT assay indicated that FICZ and B[a]P made opposite effects on cell proliferation. The scratch-wound healing assay showed that B[a]P (1 µM for 24 hr) exposure triggered cell migration and that was inhibited by FICZ (10 nM). In addition, FICZ ameliorated B[a]P-induced apoptosis by inhibiting reactive oxygen species generation and caspase-3 activation, as well as increasing reduced glutathione level in mitochondria. Furthermore, gene expression analyses indicated that FICZ competed with B[a]P, which reduced the transcriptional activation of the cyp1a1 and cyp1b1 genes, as well as Bcl2 and P53. Accordingly, the interaction between FICZ and B[a]P in the AhR pathway inhibited apoptosis in a mitochondrial-dependent manner, suggesting that endogenous compound may reduce the toxicity of exogenous pollutant in vivo and providing an available way to improve health condition related to the hepatic metabolic disorder.

Effects of spongioplasty on neourethral function following hypospadias repair: an experimental study in rabbits.

PURPOSE: Spongioplasty (mobilization and midline approximation of the two branches of the bifid dysplastic distal corpus spongiosum) can form a covering layer for the neourethra to prevent urethrocutaneous fistula in hypospadias repair surgery. However, it remains unclear whether spongioplasty affects neourethral function. The objective of this study was to compare neourethral function after hypospadias repair with and without spongioplasty. MATERIALS AND METHODS: Fourteen congenital hypospadiac New Zealand male rabbits were randomly allocated into two groups, seven animals underwent Duplay hypospadias repair and spongioplasty (experimental group), while seven underwent Duplay surgery alone (control group). Functional differences between groups were assessed by comparing neourethral compliance and flow rate. Two months after surgery, in vivo neourethral compliance was assessed by measuring intraluminal pressure with a digital pressure meter of an isolated neourethral segment, following progressive distension with 1, 2, and 3mL of air. Penises were harvested for uroflowmetry test using a simple device. RESULTS: Postoperatively, fistula developed in one and zero rabbits in the control and experimental groups, respectively. Mean pressures tended to be higher in the experimental group than in the control group (82.14 vs. 69.57, 188.43 vs. 143.26, and 244.71 vs. 186.29mmHg for 1, 2, and 3mL of air, respectively), but the difference was not statistically significant. Mean flow rates also did not significantly differ between the experimental and control groups (2.93mL/s vs. 3.31mL/s). CONCLUSION: In this congenital rabbit model, no obvious functional differences were found between reconstructed urethras after hypospadias repair with and without spongioplasty.

A method for efficient expression of Pseudomonas aeruginosa alginate lyase in Pichia pastoris.

As an eco-friendly biocatalyst for alginate hydrolysis, bacteria-derived alginate lyase (AlgL) has been widely used in research and industries to produce oligosaccharides. However, the cost of AlgL enzyme production remains high due to the low expression and difficulty in purification from bacterial cells. In this study we report an effective method to overexpress the Pseudomonas aeruginosa AlgL (paAlgL) enzyme in Pichia pastoris. Fused with a secretory peptide, the recombinant paAlgL was expressed extracellularly and purified from the culture supernatant through a simple process. The purified recombinant enzyme is highly specific for alginate sodium with a maximal activity of 2,440 U/mg. The enzymatic activity remained stable below 45°C and at pH between 4 and 10. The recombinant paAlgL was inhibited by Zn(2+), Cu(2+), and Fe(2+) and promoted by Co(2+) and Ca(2+). Interestingly, we also found that the recombinant paAlgL significantly enhanced the antimicrobial activity of antibiotics ampicillin and kanamycin against Pseudomonas aeruginosa. Our results introduce a method for efficient AlgL production, the characterization, and a new feature of the recombinant paAlgL as an enhancer of antibiotics against Pseudomonas aeruginosa.

Effects of Dietary Supplementation with Glycerol Monolaurate (GML) or the Combination of GML and Tributyrin on Growth Performance and Rumen Microbiome of Weaned Lambs.

Our objective was to evaluate the effects of dietary supplementation with glycerol monolaurate (GML) or the combination (Solider, SOL) of GML and tributyrin (TB) on the growth performance and rumen microbiome of weaned lambs. Thirty-six male Hu lambs (11.46 ± 0.88 kg BW and 40 ± 5 days of age) were divided into three treatment groups: (1) CON: basal diet, (2) GML: basal diet supplemented with GML at 1.84 g/kg DM, and (3) SOL: basal diet supplemented with SOL at 3 g/kg DM. GML increased the final BW (p = 0.04) and ADG (p = 0.02) compared with CON. There were no significant differences in the DMI (p > 0.10) among the three treatment groups. GML and SOL tended to decrease the dry matter intake/average daily gain (p = 0.07) compared with CON. GML tended to increase the apparent digestibility of CP (p = 0.08) compared with CON. SOL increased the apparent digestibility of NDF (p = 0.04) compared with CON. The Chao1 and Shannon indexes of SOL were both significantly higher than those of the other groups (p = 0.01). LefSE analysis showed that Bifidobacteriaceae of the Bifidobacteriales was enriched in the GML group. In addition, compared with GML, SOL reduced the relative abundance of Actinobacteria (p < 0.01) and increased the relative abundance of Verrucomicrobia (p = 0.05), and GML reduced the relative abundance of Ruminococcus (p = 0.03). Our results indicated that dietary supplementation with GML or SOL improved growth performance and feed conversion, and changed the rumen microbiome of weaned lambs.

Effects of daidzein on antioxidant capacity in weaned pigs and IPEC-J2 cells.

Our previous study found that soybean isoflavones in soybean meal play an important role in improving growth performance and antioxidant capacity in pigs. However, it is still unknown whether long-term supplementation with daidzein, an active molecule deglycosylated from daidzin, in a corn-soybean meal diet can enhance growth performance in pigs. Thus, in the present study, an animal trial was carried out to investigate the effects of dietary supplementation with daidzein on the growth performance and antioxidant capacity of pigs. A total of 80 weaned piglets (40 barrows and 40 females) were assigned to 4 treatments with 5 pens per treatment and 4 piglets per pen and fed a diet supplemented with 0, 25, 50 and 100 mg/kg daidzein for a 72-day trial. In addition, porcine intestinal epithelial cells (IPEC-J2) were used as an in vitro model to explore the underlying antioxidant mechanisms of daidzein. IPEC-J2 cells were treated with 0.6 mM hydrogen peroxide (H2O2) in the presence or absence of 40 µM daidzein. The results showed that adding 50 mg/kg of daidzein to the diet significantly improved body weight on day 72, average daily gain (ADG) during days 0 to 72 and plasma superoxide dismutase (SOD) activity on day 42 (P < 0.05). Treatment with 0.6 mM H2O2 for 1 h significantly decreased cell viability and catalase (CAT) activity and increased intracellular reactive oxygen species (ROS) levels and malondialdehyde (MDA) content (P < 0.05), while pretreatment with 40 µM daidzein prevented the decrease in cell viability and CAT activity and the increase in intracellular ROS levels and MDA content caused by H2O2 (P < 0.05). In addition, H2O2 stimulation significantly suppressed the expression of nuclear factor erythroid-2-related factor 2 (Nrf2), CAT, occludin and zonula occludens-1 (ZO-1), while pretreatment with daidzein preserved the expression of Nrf2, CAT and occludin in H2O2-stimulated IPEC-J2 cells (P < 0.05). In conclusion, our results suggested that long-term dietary supplementation with 50 mg/kg daidzein improved growth performance in pigs and was beneficial to the antioxidant capacity of pigs. Daidzein exerted protective effects against H2O2-induced oxidative stress in IPEC-J2 cells and the underlying mechanism may be related to the activation of the Nrf2 signaling pathway.

Effect of Age and Weaning on Growth Performance, Rumen Fermentation, and Serum Parameters in Lambs Fed Starter with Limited Ewe-Lamb Interaction.

Sixty neonatal Hu lambs were weaned at either 21 (n = 30) (early weaning, EW) or 49 days (n = 30) of age (control, CON). The starter intake and body weight (BW) of lambs was recorded weekly from birth to 63 days of age. Diarrhea rate of lambs was measured from birth to 35 days. Six randomly selected lambs from each treatment were slaughtered at 26, 35, and 63 days of age, respectively. Ruminal pH, NH3-N, and volatile fatty acid (VFA) concentration, as well as serum parameters including immunity, antioxidant status, and inflammatory parameters from randomly selected lambs from each treatment were measured. There was no difference in BW at birth and day 21 between the two groups of lambs (p > 0.05). However, BW of the lambs in the EW group was significantly lower than those in the CON group (p < 0.01) from 28 to 49 days of age. Average daily gain (ADG) of the lambs in the EW group was significantly lower than those in the CON group (p < 0.01) at three weeks after early weaning. Starter intake of the lambs in the EW group was obviously higher than that in the CON group (p < 0.01) from day 28 to 49. In addition, the diarrhea rate was significantly higher than that in the CON group from day 5 to 14 after weaning (p < 0.01). The EW group had heavier carcasses (p < 0.01) and rumen relative to whole stomach weights (p < 0.01). Rumen pH was increased by age (p < 0.01) and was not affected by early weaning (p > 0.05). Early weaning decreased abomasum relative to whole stomach weight (p < 0.01) and increased total VFA concentrations (p < 0.01) at day 26. There was no difference in lambs' immunity and stress indicators (p > 0.05). The results indicated that lambs weaned at 21 days of age had decreased ADG and higher diarrhea rate, although the overall immunity was not compromised. Long-term study is needed to further validate the feasibility of early weaning strategy in lambs.

Effects of spongioplasty on neourethral function following hypospadias repair: an experimental study in rabbits

ABSTRACT Purpose: Spongioplasty (mobilization and midline approximation of the two branches of the bifid dysplastic distal corpus spongiosum) can form a covering layer for the neourethra to prevent urethrocutaneous fistula in hypospadias repair surgery. However, it remains unclear whether spongioplasty affects neourethral function. The objective of this study was to compare neourethral function after hypospadias repair with and without spongioplasty. Materials and Methods: Fourteen congenital hypospadiac New Zealand male rabbits were randomly allocated into two groups, seven animals underwent Duplay hypospadias repair and spongioplasty (experimental group), while seven underwent Duplay surgery alone (control group). Functional differences between groups were assessed by comparing neourethral compliance and flow rate. Two months after surgery, in vivo neourethral compliance was assessed by measuring intraluminal pressure with a digital pressure meter of an isolated neourethral segment, following progressive distension with 1, 2, and 3mL of air. Penises were harvested for uroflowmetry test using a simple device. Results: Postoperatively, fistula developed in one and zero rabbits in the control and experimental groups, respectively. Mean pressures tended to be higher in the experimental group than in the control group (82.14 vs. 69.57, 188.43 vs. 143.26, and 244.71 vs. 186.29mmHg for 1, 2, and 3mL of air, respectively), but the difference was not statistically significant. Mean flow rates also did not significantly differ between the experimental and control groups (2.93mL/s vs. 3.31mL/s). Conclusion: In this congenital rabbit model, no obvious functional differences were found between reconstructed urethras after hypospadias repair with and without spongioplasty.

Cloning, expression and characterization of a trehalose synthase gene from Rhodococcus opacus.

Trehalose is a unique disaccharide capable of protecting proteins against environmental stress. A novel trehalose synthase (TreS) gene from Rhodococcus opacus was cloned and expressed in Escherichia coli Top10 and BL21 (DE3) pLysS, respectively. The recombinant TreS showed a molecular mass of 79 kDa. Thin layer chromatography (TLC) result suggested that this enzyme had the ability to catalyze the mutual conversion of maltose and trehalose. Moreover, high-performance liquid chromatography (HPLC) result suggested that glucose appeared as a byproduct with a conversion rate of 12 %. The purified recombinant enzyme had an optimum temperature of 25 °C and pH optimum around 7.0. Kinetic analysis revealed that the K m for trehalose was around 98 mM, which was a little higher than that of maltose. The preferred substrate of TreS was maltose according to the analysis of k cat/K m. Both 1 and 10 mM of Hg(2+), Cu(2+) and Al(3+) could inhibit the TreS activity, while only 1 mM of Ca(2+) and Mn(2+) could increase its activity. Five amino acid residues, Asp(244), Glu(286), Asp(354), His(147) and His(353), were shown to be conserved in R. opacus TreS, which were also important for α-amylase family enzyme catalysis.

Molecular cloning, expression, purification, and characterization of soluble full-length, human interleukin-3 with a baculovirus-insect cell expression system.

We report gene cloning, plasmid construction, baculovirus expression, purification, and biological activity testing of the human hematopoietic cytokine interleukin-3. cDNA was constructed from extracted total RNA of Jurkat cells. Both signal and structural fragment of interleukin-3 were cloned from this cDNA library, modified by adding a hexahistidine-tag at the C-terminus, and introduced into the pBacPAK9 transfer vector to generate recombinant baculoviruses. For protein expression High Five cells were infected either in spinner flasks or 2.5-L bioreactors in batch culture yielding levels of 1.5-3 mg L(-1) interleukin-3 in the cell culture supernatant. Interleukin-3 was purified by a single step chromatography using cobalt metal affinity resins, which yielded a highly stable and soluble protein. N-terminal amino acid sequencing of the purified interleukin-3 showed correct cleavage of the signal peptide during protein processing. The two N-glycosylation sites were found to be occupied by 100 and 35%, respectively, with an N-glycan pattern of paucimannosidic structures, which are typical for recombinant glycoproteins produced by High Five lepidopteran cells. The specific biological activity of purified interleukin-3 was several times higher when compared with different lots of commercially available material from Escherichia coli. The results indicate that the strategy we used in this experiment is a straightforward and convenient way for recombinant protein preparation and can be adapted to produce other recombinant cytokines.