Effect of active immunization with recombinant-derived goose INH-α, AMH, and PRL fusion protein on broodiness onset and egg production in geese (Anser cygnoides).

This study was conducted to investigate the potential effects of active immunization against recombinant-derived goose inhibin-α (INH-α), anti-Müllerian hormone (AMH), and prolactin (PRL) fusion protein on broodiness onset and egg production in geese. The purified fusion proteins (INH-α, AMH, and PRL) were prepared using a prokaryotic expression system. Female Zhedong geese (10 mo old) were randomly assigned to one of 4 treatments and raised in separate pens. The geese were actively immunized with the recombinant goose INH-α, AMH, or PRL, respectively, and phosphate-buffered saline as control. The results showed the corresponding antibodies were produced when the geese were immune INH-α, AMH-, and PRL-recombinant proteins. The significantly higher luteinizing hormone contents were observed in the INH-α, AMH, and PRL recombinant protein-immunized geese, while the lower AMH hormone content only in PRL-immunized birds. AMH recombinant protein immunized geese had more large yellow follicles of ovary, while the INHα-treated birds with more other follicles compared with control geese. In addition, the geese receiving INH-α recombinant protein, the broodiness onset was about 6 d, which significantly shorter than did PBS immunization (16 d). The INHα- and PRL-immunization also resulted in 12.5 and 8.5 d shorter broody duration intervals compared to the control birds. Moreover, the lower new broodiness rate was observed in three recombinant proteins treated birds. Finally, the PRL recombinant protein-immunization resulted in an average increase of 1.34 eggs during a 40-d observation. Collectively, the data demonstrated that active immunization against recombinant proteins INH-α or AMH could promote LH hormone secretion, regulate follicle development and decrease the broodiness rate. Also, active immunization with a recombinant-derived goose PRL protein might improve egg laying performance.

[Effect of silencing FABP3 gene on LPS-induced apoptosis and endoplasmic reticulum stress in alveolar epithelial cells].

Objective: To investigate the effect of silencing fatty acid binding protein 3 (FABP3) gene on lipopolysaccharide (LPS)-induced apoptosis and endoplasmic reticulum stress in alveolar epithelial cells A549. Methods: According to the processing method, A549 cells were divided into control group(A549 cells cultured for 24 h), LPS group (10 mg/L LPS treated A549 cells for 24 h), LPS+si-con group (10 mg/L LPS was used to treat A549 cells transfected with si-con for 24 h) and LPS+si-FABP3 group (10 mg/L LPS was used to treat A549 cells transfected with si-FABP3 for 24 h). Then quantitative real-time PCR was used to detect the level of FABP3, methylthiazoletrazolium was used to detect the cell proliferation, flow cytometry was used to detect the apoptosis, and Western Blot was used to detect the levels of FABP3, CyclinD1, cleaved-caspase-3, GRP78, ATF4, CHOP, cleaved-caspase-12 and p-Akt and PI3Kp110α protein expression. Enzyme-linked immunosorbent assay was used to detect the levels of IL-6, IL-8 and TNF-αlevels. Results: In the LPS group, FABP3 protein level (1.00±0.09) and mRNA (2.15±0.22), apoptosis rate [(26.1±2.6)%], inflammatory factor IL-6 [(554.4±55.4) ng/L], IL-8 [(389.3±38.5) ng/L] and TNF-α [(601.3±60.0) ng/L], cleaved-caspase-3 (1.00±0.11), GRP78 (1.05±0.11), ATF4 (1.20±0.12)), CHOP (1.05±0.10), cleaved-caspase-12 (1.10±0.11), p-Akt (0.88±0.08) and PI3Kp110α (0.75±0.08) protein levels were significantly higher than the control group [(0.53±0.05), (1.00±0.10), (4.5±0.5)%, (75.4±7.5) ng/L, (25.2±2.5) ng/L, (66.5±6.7) ng/L, (0.34±0.05), (0.35±0.05), (0.43±0.05), (0.37±0.04), (0.45±0.05), (0.16±0.04), (0.35±0.05)] (all P<0.05). Cell viability [(50.1±5.4)%] and CyclinD1 protein level (0.40±0.05) in LPS group were significantly lower than those in the control group [(100.1±12.4)%, (1.25±0.12)] (both P<0.05). Cell viability [(89.1±8.5)%] and CyclinD1 protein level (1.15±0.11) in LPS+si-FABP3 group were significantly higher than those in LPS+si-con group [(53.1±5.4)%, (0.42±0.05)] (both P<0.05). Apoptosis rate [(10.5±1.1)%], IL-6[(301.3±30.0) ng/L], IL-8[(189.4±19.0) ng/L], TNF-α [(400.1±40.1) ng/L], cleaved-caspase-3 (0.45±0.05), GRP78 (0.48±0.05), ATF4 (0.60±0.06), CHOP (0.55±0.05), cleaved-caspase-12 (0.60±0.06), p-Akt (0.50±0.05) and PI3Kp110α(0.45±0.05) in LPS+si-FABP3 group were significantly lower than those in LPS+si-con group [(28.1±2.8)%, (536.3±53.6) ng/L, (400.2±40.2) ng/L, (623.1±62.3) ng/L, (0.96±0.10), (1.02±0.10), (1.15±0.12), (1.10±0.11), (1.15±0.12), (0.90±0.09), (0.72±0.07)] (all P<0.05). Conclusion: Silencing FABP3 gene can inhibit LPS-induced alveolar epithelial cell apoptosis and endoplasmic reticulum stress, which may act by inhibiting the PI3K/Akt signaling pathway.

Comparison of the broody behavior characteristics of different breeds of geese.

A low laying performance in goose is one of the key factors preventing the industrial development, and the laying performance is related to broody behavior. However, the characteristics of broody behavior in geese remain unclear. In this study, the total 144 geese (300 day old), including Zhedong geese (Anser cygnoides), Sichuan geese (Anser cygnoides), and Carlos geese (Anser anser) were selected and assigned to 1 of 3 groups/breed (including 4♂+12♀). Laying and broody behaviors were recorded using the infrared video cameras from 2016 November 11 to 2017 June 15. The broody behavior was detected in 19.4% of Carlos geese, 33.3% of Sichuan geese, and 100% of Zhedong geese. Different goose breeds showed similar behavior characteristics. The low frequency of feeding, drinking, and low body weight were observed in the middle of broodiness. As the brooding progressed, the body temperature showed a downward trend and then recovered, whereas no difference was observed in Carlos goose. In addition, the plasma hormone concentration from different breeds and stages of broodiness were compared. The contents of FSH (follicle-stimulating hormone) and LH (luteinizing hormone) in geese were greater in the laying stage than that in the broody stage. Fewer FSH and LH were detected in Zhedong geese and Carlos geese, more in Sichuan geese. In broody goose, the PRL (prolactin) concentrations of the 3 goose breeds peaked in the middle of broodiness, and greater PRL was detected in Sichuan geese than those in Carlos geese and Zhedong geese. Finally, we compared egg production between the broody and non-broody geese in the observation period. The egg production of broody Carlos geese was 27, which was significantly higher than non-broody geese (14 eggs), while in Sichuan geese there was no significant difference between broody (24 eggs) and non-broody geese (26 eggs). Finally, the higher egg production was found with the more broody times in Zhedong geese. Taken together, although the different goose breeds showed similar broody behavior characteristics, the broody rate and hormone secretion were dissimilar, and the Zhedong geese exhibited strong broody feature.

Histological characteristics of follicles and reproductive hormone secretion during ovarian follicle development in laying geese.

Egg production in different goose breeds vary significantly, which is related with the physiology of reproduction. However, the knowledge of physiology of goose reproduction is not well documented. In the present study, the 3 breeds with significantly different egg production were selected to investigate the histological characteristics of follicles and reproductive hormone secretion during follicle development, which included Carlos geese (Anser anser), Zhejiang geese (Anser cygnoides), and Yangzhou geese (Anser cygnoides). The results indicated that there were significant differences in the morphology of ovary and follicles among different goose breeds. The mode of hierarchical follicles in Yangzhou geese was 5, and those were 3 and 4 in Zhejiang and Carlos geese, respectively. The numbers of prehierarchical follicles were 61 to 70, 69 to 75, and 28 to 39 in Yangzhou geese, Zhejiang geese, and Carlos geese, respectively. The thickness of granulosa layer of follicles was higher in the large yellow follicle than those in the other prehierarchical and hierarchical follicles, and Yangzhou geese were the highest among the 3 breeds. The concentration of follicle stimulating hormone (FSH) ranged from 12.17 to 28.06 U/L, and 17ß-Estradiol ranged from 27.01 to 49.39 pmol/L by the enzyme-linked immuno sorbent assay. The level of FSH of Yangzhou geese reached to the highest in the hierarchical follicle (F1), while the other 2 geese did not show the similar feature. In addition, the level of luteinizing hormone (LH) and progesterone (PROG) in the prehierarchical follicles of Yangzhou geese was higher than those in Carlos and Zhejiang geese. In summary, the difference of histological characteristics of follicles and reproductive hormone in different goose breeds was not only reflected in the number of follicles and the thickness of the granulose cell layer, but also embodied the secretion LH and PROG. The more thickness of the granulose cell layer and high secretions of LH and PROG contributed to the development of prehierarchical follicles to hierarchical follicles, which may be due to the fact that Yangzhou geese (Anser cygnoides) has more egg production.

Comparisons in geese of the courtship, mating behaviors and fertility of the Carlos and Sichuan breeds and the breed crosses.

The Chinese goose originated from the swan goose (Anser cygnoides) and the European goose originated from the greylag goose (Anser anser). The Chinese and European geese have the potential to crossbreed. Whether interspecific differences in mating behaviors affect successful hybridization is unknown. In this study, 10-month-old Carlos geese (n = 120; Anser anser) and Sichuan geese (Anser cygnoides) were selected, and 12 multi-male parent families (3♂+12♀) were established. The courtship and mating behaviors of pure and cross-bred combinations of the Carlos and Sichuan geese were recorded using video cameras. Initiative courtship by males was the main type of courtship. Fixed mating, mating interference, and uncooperative mating were common in the flocks. The frequencies of some courtship and mating behaviors were less in the cross-bred groups (Carlos ganders × Sichuan geese, Sichuan ganders × Carlos geese) compared with the Sichuan pure-bred groups (P < 0.05). The Carlos male geese had some unique mating behaviors (i.e., one-to-one mating, formation of distinct hierarchies, and competition interference). The fertility rate had a significant correlation with the frequency of successful mating (rp = 0.992, P < 0.05), rather than with the courtship behavior. These results indicate there were lesser frequencies of courtship and successful matings in the cross-breeding than purebreeding groups. Furthermore, the fertility rate depended largely on the successful mating behavior and was independent of the courtship behavior.

Effect of two conserved amino acid residues on DREB1A function.

Transcription factors of the DREBP subgroup and the EREBP subgroup contain conserved DNA-binding domains called AP2/EREBP domains, which specifically bind to DRE cis-element and GCC-box, respectively. The 14th and 19th amino acid residues of AP2/EREBP domains are absolutely conserved in the transcription factors of the DREBP subgroup as well as in the EREBP subgroup. However, these two residues of transcription factors of the DREBP subgroup are different from those of the EREBP subgroup. To assess the functional significance of these two residues in binding to the target sequence, the Val (14th residue) and Glu (19th residue) of the AP2/EREBP domain of DREB1A (a transcription factor of the DREBP subgroup) were mutated individually or doubly to Ala and Asp, respectively. This made the 14th and 19th amino acid residues of mutant DREB1A identical to the corresponding residues of transcription factors of the EREBP subgroup. Yeast in vivo analysis showed that: 1) on a selective medium plate of SD/His- Ura- Trp- + 30 mM approximately 60 mM 3-AT, the growth of yeast cells containing HIS and lacZ double reporter genes was normal in the transformation of the 19th singly mutated DREB1A, obviously inhibited in the transformation of the 14th singly mutated DREB1A, and seriously inhibited in the transformation of the 14th/19th doubly mutated DREB1A; 2) quantitative assay of beta-galactosidase activity showed that the intensities of lacZ expression decreased in the transformations of the 14th singly mutated and 14th/19th doubly mutated types. The experimental results revealed that the 19th site mutation did not affect the binding of the DREB1A transcription factor to the DRE cis-element; the 14th site mutation obviously inhibited their binding; and the double mutation of the 14th/19th sites seriously inhibited their binding. This suggests that the conserved Val (14th) and Glu (19th) residues are crucial in the regulation of the binding activity of DREB1A to the DRE cis-element.

Reactivation and refolding of rabbit muscle creatine kinase denatured in 2,2,2-trifluoroethanol solutions.

The unfolding and refolding of creatine kinase (ATP:creatine N-phosphotransferase (CK), EC 2.7.3.2) during denaturation and reactivation by trifluoroethanol (TFE) have been studied. Significant aggregation was observed when CK was denatured at TFE concentrations between 10% and 40% (v/v). 50% TFE (v/v) was used to study the denaturation and unfolding of CK. The activity loss of CK was a very quick process, as was the marked conformational changes during denaturation followed by fluorescence emission spectra and far-ultraviolet CD spectra. DTNB modification and size exclusion chromatography were used to find that CK dissociated and was in its monomer state after denaturation with 50% TFE. Reactivation and refolding were observed after 80-fold dilution of the denatured CK into 0.05 M Tris-HCl buffer, pH 8.0. The denatured CK recovered about 38% activity following a two phase course (k(1)=4.82+/-0.41x10(-3) s(-1), k(2)=0.60+/-0.01x10(-3) s(-1)). Intrinsic fluorescence maximum intensity changes showed that the refolding process also followed biphasic kinetics (k(1)=4.34+/-0.27x10(-3) s(-1), k(2)=0.76+/-0.02x10(-3) s(-1)) after dilution into the proper solutions. The far-ultraviolet CD spectra ellipticity changes at 222 nm during the refolding process also showed a two phase course (k(1)=4.50+/-0.07x10(-3) s(-1), k(2)=1.13+/-0.05x10(-3) s(-1)). Our results suggest that TFE can be used as a reversible denaturant like urea and GuHCl. The 50% TFE induced CK denaturation state, which was referred to as the 'TFE state', and the partially refolded CK are compared with the molten globule state. The aggregation caused by TFE during denaturation is also discussed in this paper.

High affinity binding of receptor-associated protein to heparin and low density lipoprotein receptor-related protein requires similar basic amino acid sequence motifs.

The 39-kDa receptor-associated protein (RAP) is a specialized chaperone for members of the low density lipoprotein receptor gene family, which also binds heparin. Previous studies have identified a triplicate repeat sequence within RAP that appears to exhibit differential functions. Here we generated a series of truncated and site-directed RAP mutants in order to define the sites within RAP that are important for interacting with heparin and low density lipoprotein receptor-related protein (LRP). We found that high affinity binding of RAP to heparin is mediated by the carboxyl-terminal repeat of RAP, whereas both the carboxyl-terminal repeat and a combination of amino and central repeats exhibit high affinity binding to LRP. Several motifs were found to mediate the binding of RAP to heparin, and each contained a cluster of basic amino acids; among them, an intact R(282)VSR(285)SR(287)EK(289) motif is required for high affinity binding of RAP to heparin, whereas two other motifs, R(203)LR(205)R(206) and R(314)ISR(317)AR(319), also contribute to this interaction. We also found that intact motifs of both R(203)LR(205)R(206) and R(282)VSR(285)SR(287)EK(289) are required for high affinity binding of RAP to LRP, with the third motif, R(314)ISR(317)AR(319), contributing little to RAP-LRP interaction. We conclude that electrostatic interactions likely contribute significantly in the binding of RAP to both heparin and LRP and that high affinity interaction with both heparin and LRP appears to require mostly overlapping sequence motifs within RAP.

Reactivation kinetics of guanidine hydrochloride-denatured creatine kinase measured using the substrate reaction.

Guanidine hydrochloride-denatured creatine kinase (CK) can very quickly form a dimer with reactivity when the denaturant is diluted into the reaction system in the presence of DTT or EDTA. Tsou's method and its applied equation [Tsou (1988), Adv. Enzymol. Rel. Areas Mol. Biol. 61, 381-436; Yang and Zhou (1998), Biochim. Biophys. Acta 1388, 190-198] were used to measure the kinetic reactivation rate constants and the reactivation degree for reassociated CK dimers. Partial reactivation (about 50% at best) occurred following a monophasic course during the substrate reaction when compared with previous time interval measurements. The reactivation degree increased with increasing DTT (0.1-5 mM) and EDTA (0.1-1 mM) concentrations. The apparent forward rate constants do not change with concentration, showing that the reactivation is a reversible first-order reaction, but not of complex formation type. However, the apparent forward rate constants do change with EDTA concentration, showing that the reactivation with EDTA is a reversible first-order reaction as well as of complex formation type. Excess DTT concentrations have an inhibitory effect, indicating that the excessive EDTA acts as a metal chealate not only for free Mg2+, but also for MgATP during the enzyme catalysis. This study shows that additional information about the reactivation of CK can be obtained from examining the substrate reaction. The possible refolding pathway of CK is discussed.

Comparison of inactivation and unfolding of methanol dehydrogenase during denaturation in guanidine hydrochloride and urea.

The activity and the conformational changes of methanol dehydrogenase (MDH), a quinoprotein containing pyrrolo-quinoline quinone as its prosthetic group, have been studied during denaturation in guanidine hydrochloride (GdnHCl) and urea. The unfolding of MDH was followed using the steady-state and time resolved fluorescence methods. Increasing the denaturant concentration in the denatured system significantly enhanced the inactivation and unfolding of MDH. The enzyme was completely inactivated at 1 M GdnHCl or 6 M urea. The fluorescence emission maximum of the native enzyme was at 332 nm. With increasing denaturant concentrations, the fluorescence emission maximum red-shifted in magnitude to a maximum value (355 nm) at 5 M GdnHCl or 8 M urea. Comparison of inactivation and conformational changes during denaturation showed that in general accord with the suggestion made previously by Tsou, the active sites of MDH are situated in a region more flexible than the molecule as a whole.

Effect of Mg2+ on the thermal inactivation and unfolding of creatine kinase.

The effect of Mg2+ on the thermal inactivation and unfolding of rabbit muscle creatine kinase has been studied for various temperatures and Mg2+ concentrations. Increasing the Mg2+ concentration in the denatured system significantly enhanced the inactivation and unfolding of creatine kinase during thermal denaturation. The analysis of the kinetic course of substrate reaction during thermal inactivation showed that at 47 degrees C the increased free Mg2+ concentration caused the creatine kinase inactivation rate to increase. Increasing the temperature strengthened the effect of Mg2+ on the thermal inactivation. Control experiments showed that treating native creatine kinase with different concentrations of Mg2+ did not change the enzymatic activity. The fluorescence emission spectra showed that the emission maximum for creatine kinase red-shifted from 335 to 337 nm during thermal denaturation at 47 degrees C for 10 min, while the presence of 3 mM Mg2+ caused the enzyme emission maximum to red-shift from 335 to 342.5 nm for the same thermal denaturation conditions. In addition, Mg2+ also enhanced the unfolding of the equilibrium state and decreased the time required to reach the equilibrium state of creatine kinase at 47 degrees C. The potential biological significance of these results are discussed.

[Heat shock response induces resistance to hydrogen peroxide and increases synthesis of interleukin-6 in rat astrocyte in vitro].

Cell viability and cell membrane disruption of rat astrocyte after heat shock response (HSR) were assessed by the analysis of MTT reduction and lactate dehydrogenase (LDH) release. We studied whether HSR would modulate the susceptibility of astrocyte to H2O2-induced oxidative stress. Cell viability was assessed by reduction of MTT. HSR in 43 degrees C water bath for 30 min decreased H2O2 toxicity (P < 0.01) to astrocyte. HSR induced decrease in H2O2 (50 mumol/L) toxicity was also shown by the reduction in the release of LDH, which was a marker of cell membrane disruption. The result also showed that prior to the incubation in 43 degrees C water bath for 30 min strongly increased IL-6 release 6 h (P < 0.05) after HSR. The above data suggest that the enhanced release of IL-6 from astrocyte may be one of the mechanisms underlying the cell protective effect induced by HSR.

Scavenging effect of tetrandrine of active oxygen radicals.

The scavenging effects of tetrandrine (TD) on active oxygen radicals were investigated through chemiluminescence experiments and oxidation of ferrocytochrome c. The superoxide anions (O2-.) were generated either in hypoxanthine/xanthine oxidase (HX-XOD) system or by means of auto-oxidation of erythrocytes. It was found that TD (7.5 microM) could remarkably scavenge O2-. in the HX-XOD system and that the effect was concentration-dependent. The scavenging effect of TD on O2-. generated from auto-oxidation in erythrocytes was not as strong as in the HX-XOD system. The hydroxyl radicals (.OH) were generated in aqueous solution of H2O2 irradiated by UV, it was also found that TD (15 microM) effectively scavenged .OH in that system. This study led to the conclusion that the antioxidant property of TD may account for some of its pharmacological effects.

[Scavenging effects of ginger on superoxide anion and hydroxyl radical].

Ginger can significantly scavenge O2-. in hypoxanthinexanthine oxidase system and .OH in ultraviolet exposure of H2O2 system. The scavenging effects of ginger on O2-. and .OH may contribute to explaining some of the pharmacological mechanisms of this drug.