Effects of kisspeptin-10 on the reproductive performance of sows in a fixed-time artificial insemination programme.

Kisspeptin (KP) is a major positive regulator of the hypothalamo-pituitary-gonadal axis and affects female reproductive cyclicity in mammals. It offers an attractive alternative strategy to control reproduction in fixed-time artificial insemination (FTAI) protocols. We aimed to evaluate the effects of different doses of kisspeptin-10 (KP-10) on sow reproductive performance in FTAI protocols. One hundred ninety-eight weaned sows were divided into three groups at random. A FTAI-GnRH group of sows (n = 98) received 100 µg (2 mL) gonadotropin-releasing hormone (GnRH; gonadorelin) by intramuscular injection at 96 h after weaning (t = 0 h); FTAI-KPL (KPL: low-dose KP-10, n = 50), and FTAI-KPH groups of sows (KPH: high-dose KP-10, n = 50) received 0.5 or 1 mg KP-10 (2 mL) respectively at 96 h after weaning. Sows were checked twice daily for oestrus. Ultrasonographic evaluations were performed to determine the follicular diameter and time of ovulation; blood samples were collected immediately before injection (t0 = 0 min) and at 15, 30, 45, 60, 75, 90 min, 24 and 48 h postinjection. Sows were inseminated at 112 and 132 h after weaning. The oestrus rates (96 vs 92%; 96 vs 88%) and weaning-to-oestrus intervals (98.9 vs 98.6 h; 98.9 vs 97.1 h) were not affected by treatment, but oestrus in the FTAI-KPL group was significantly longer than in the FTAI-GnRH group (38.7 vs 30.0 h; P < 0.05). The peak LH concentrations were 1.29 times greater than at t0 = 0 in the FTAI-GnRH group, and 1.45 and 1.44 times greater than at t0 = 0 in the FTAI-KPL and FTAI-KPH groups, respectively. Follicular diameters and pregnancy rates (86 vs 88%, 86 vs 80%, respectively) did not differ between the treatments. Moreover, the total numbers of piglets born and those born alive did not differ among the three groups. These findings suggested that 0.5 mg KP-10 given at 96 h after weaning could be used in FTAI programmes to manage batch farrowing in sows.

Menthol in Combination with Iontophoresis Promotes Natamycin Penetration through the Cornea: In Vitro and In Vivo Studies.

We studied whether menthol can promote penetration of natamycin, a representative antifungal macrolide agent, through the cornea. Natamycin penetration was examined using an in vitro iontophoresis system that simulates clinical scenario; menthol (0.1-0.3%, w/v) was added to the donor reservoir of a standard Franz diffusion chambers. In vivo effects of menthol on natamycin penetration were examined in a set of bioassays using rabbits inoculated with Aspergillus fumigatus in the right eye. Potential irritation to the rabbit eye was examined using a standard test. Menthol significantly (p<0.05) potentiated the effects of iontophoresis on natamycin penetration. The optimal combination seemed to be 0.2% menthol in combination with 3 mA/cm2 iontophoresis.

A comparison of the reproductive performance in primiparous sows following two timed artificial insemination protocols.

Timed artificial insemination (TAI) is an efficient reproductive technology in batch farrowing production that aids management in pig farms. However, the effect of TAI on the reproduction performance is still controversial. This study aimed to evaluate the effects of two TAI protocols on the reproductive performance of primiparous sows. A total of 332 weaned sows were randomly allocated into three treatments. Sows assigned to Control (n = 110) were untreated and inseminated on each day in oestrus after weaning. Sows assigned to eG-TAI (n = 112) received equine chorionic gonadotropin (eCG) 24 h after weaning and gonadotropin-releasing hormone (Gonadorelin: GnRH) at oestrus, and were inseminated at 8 and 32 h later if oestrus at 0800, or 16 and 40 h later if oestrus at 1600. Sows assigned to 2e-TAI (n = 110) received eCG and GnRH 24 h and 96 h after weaning, respectively, and were inseminated 16 and 40 h after GnRH administration. Sows showing oestrus at GnRH administration or 64 h after were inseminated immediately, for a total of three inseminations. Ultrasonographic evaluations were performed to determine the follicular diameter and time of ovulation. Most sows in the 2e-TAI and eG-TAI groups ovulated 0-48 h after the GnRH injection. Our results indicated that oestrus rate within seven days after weaning in the experimental groups was higher, and weaning-to-oestrus interval was shorter than in the control group (99.3 h vs 113.5 h, P < 0.05). The breeding and farrowing rates in the experimental groups were significantly higher than in the control group (P < 0.05), while the numbers of total born, live-born and stillborn were not different among the three groups (Control: 12.7, 11.6 and 1.1; 2e-TAI: 12.4, 11.3 and 1.0; eG-TAI: 12.0, 11.4 and 0.4, respectively). These results indicated that TAI could ensure a high farrowing rate in primiparous sows under batch farrowing management.

Increased EWSAT1 expression promotes cell proliferation, invasion and epithelial-mesenchymal transition in colorectal cancer.

OBJECTIVE: Long non-coding RNAs (lncRNAs) have recently been identified as crucial regulators in colorectal cancer (CRC) progression. The aim of the study is to investigate the clinical role and biological effects of long non-coding RNA EWSAT1 in CRC. PATIENTS AND METHODS: The expression of lncRNA EWSAT1 was detected in 106 cases of fresh CRC tissues and matched adjacent normal tissues by qRT-PCR analyses. The Kaplan-Meier analysis and log-rank test were used to assess the association between lncRNA EWSAT1 expression and overall survival (OS) rate of CRC patients. Cell proliferation and invasion capacity were evaluated by CCK8 assay, colony formation, and transwell invasion assays. The protein expression was detected using western blot analysis. RESULTS: LncRNA EWSAT1 expression was abnormally higher in CRC tissues compared to matched adjacent normal tissues. Higher lncRNA EWSAT1 expression significantly associated with depth of invasion, lymph node metastasis, and advanced tumor-node-metastasis (TNM) stage in CRC patients. Patients with higher EWSAT1 expression exhibited shorter OS compared with patients with lower EWSAT1 expression. Furthermore, lncRNA EWSAT1 knockdown significantly suppressed cell proliferation and invasion in CRC. In addition, lncRNA EWSAT1 knockdown suppressed cell epithelial-mesenchymal transition (EMT) through reducing Snail1, Snail2, and N-cadherin expression, but increasing E-cadherin expression in CRC cells. CONCLUSIONS: Downregulation of lncRNA EWSAT1 suppressed cell proliferation and invasion of CRC, which indicated that EWSAT1 may be a potential target of CRC treatment.

N-octanoylated ghrelin peptide inhibits bovine oocyte meiotic resumption.

BACKGROUND: Studies have shown that ghrelin plays an important role in the mammalian reproductive system, including the central, gonadal levels, and also during in vitro maturation of oocytes; however, the functions of ghrelin in bovine oocyte meiosis require further investigation. OBJECTIVE: We aimed to evaluate the effects of an n-octanoylated ghrelin peptide on oocyte meiotic resumption and the developmental competence of mature oocytes in vitro. EXPERIMENTAL: design: The expression of GHRL (encoding ghrelin) mRNA and its receptor (the growth hormone secretagogue receptor, GHSR) in the cumulus-oocyte complex (COCs), denuded oocytes (DOs), and cumulus cells (CCs) was assessed using quantitative real-time reverse transcription PCR (qRT-PCR), and the effects of the n-octanoylated ghrelin peptide on meiotic resumption were studied at four different doses (0, 10, 50, and 100 ng/mL) in a 6 h culture system. RESULTS: qRT-PCR analysis showed that GHRL and GHSR mRNAs were expressed in all tested samples; however, GHRL was predominantly expressed in DOs, and GHSR was predominantly expressed in CCs. Germinal vesicle breakdown was inhibited significantly by 50 ng/mL ghrelin compared with that in the negative control (P < 0.05). Further studies showed that n-octanoylated ghrelin increased the levels of cAMP and cGMP in the CCs and DOs, which inhibited the meiotic resumption of bovine oocytes. And the inhibitory role in the developmental competence of mature oocytes were also included, ghrelin could significantly improve the cleavage rate (P < 0.05) and blastocyst rate (P < 0.05). CONCLUSION: N-octanoylated ghrelin maintained bovine oocytes meiotic arrest and further improved their developmental competence; therefore, n-octanoylated ghrelin could be considered as a potential pharmaceutical inhibitor of meiosis for the in vitro maturation of bovine oocytes.

Effects of N-carbamylglutamate and L-arginine on gonadotrophin-releasing hormone (GnRH) gene expression and secretion in GT1-7 cells.

Recent studies have shown that N-carbamylglutamate (NCG) and arginine (ARG) supplementation improves reproductive performance in livestock. The objectives of the present study were to evaluate the effects of NCG and ARG on GT1-7 cell gonadotrophin-releasing hormone (GnRH) secretion, gene expression and cell proliferation. GT1-7 cells were treated in vitro with different concentrations of NCG (0-1.0mM) or ARG (0-4.0mM) in serum-free medium for 12 or 24h. For GnRH secretion and cell proliferation, GT1-7 cells were more sensitive to NCG than ARG. NCG treatment after 12h increased cell numbers and inhibited GnRH secretion in a dose-dependent manner (P<0.05), although there was no significant effect of NCG on these parameters after 24h culture. ARG treatment decreased GnRH secretion after 24h (P<0.05), whereas it had no effect after 12h. GT1-7 cells express GnRH, Kiss-1 metastasis-suppressor (Kiss1), G-protein coupled receptor 54 (GPR54), neuronal nitric oxide synthase (nNOS) and estrogen receptor α (ERα) genes. High concentrations of NCG (1.0mM) and ARG (4.0mM) inhibited (P<0.05) GnRH and nNOS mRNA abundance in GT1-7 cells. ARG treatment decreased Kiss1 and increased ERα mRNA abundance. Thus, high concentrations of NCG (1.0mM) and ARG (4.0mM) may act both directly and indirectly to regulate GnRH neuron function by downregulating genes related to GnRH synthesis and secretion to slow GnRH production while stimulating GT1-7 cell proliferation.

[Culture medium optimization and primary kinetics analysis for nisin production].

Response surface methodology was used to optimize a medium for nisin production of Lactococcus lactis. In the first optimization step the influence of sucrose, soybean peptone, yeast extract, potassium dihydrogen phosphate, sodium chloride, and magnesium sulfur on nisin production was evaluated using a fractional factorial design. Potassium dihydrogen phosphate influenced nisin production positively while soybean peptone affected nisin production negatively. The other components had no significant effect on nisin production. The path of steepest ascent was used to approach the optimal region of the medium composition. In the third step the optimal concentrations of KH2PO4 and soybean peptone were determined by a central composite design and response surface analysis. The optimized medium allowed nisin production to be increased from 1074 IU/mL to 2150 IU/mL. The kinetic analysis showed that nisin production fashion at optimized and non-optimized media was not changed and maintained partially growth-associated. But the specific growth rates and the specific nisin production rates for the strain at the optimized medium were bigger than the ones at the non-optimized medium after the cells entered the middle of exponential phase.

Aggregation of creatine kinase during refolding and chaperonin-mediated folding of creatine kinase.

The course of refolding and reactivation of urea-denatured creatine kinase (ATP; creatine N-phosphotransferase, EC 2.7.3.2) has been studied in the absence and presence of molecular chaperonin GroEL. The enzyme was denatured in Tris--HCl buffer containing 6 M urea for 1 h. In the refolding studies, the denatured enzyme was diluted 60-fold into the same buffer containing GroEL or not for activity, turbidity, fluorescence measurements and polyacrylamide gel electrophoresis. The results show that the reactivation process is dependent of creatine kinase concentration in the concentration range 2.5--4 microM. The levels of activity recovery decrease with increasing enzyme concentration because of the formation of wrong aggregates. The molecular chaperonin GroEL can bind the refolding intermediate of creatine kinase and thus prevent the formation of wrong aggregates. This intermediate is an inactive dimeric form that is in a conformation resembling the 'molten globule' state.

Preparation and properties of immobilized pig kidney aminoacylase and optical resolution of N-acyl-DL-alanine.

Aminoacylase (EC 3.5.1.14) was immobilized into DEAE-Sephadex A-25 by ion-exchange absorption for optical resolution of N-acyl-DL-alanine. The effects of pH, temperature, and Co2+ concentration on the activity of free and immobilized enzymes were investigated along with the operational and the thermal stability of the immobilized enzyme. The immobilized enzyme retained high catalytic activity. The optimum pH and temperature for the hydrolysis of N-acyl-L-alanine in the DL-isomer mixture were 8.0 and 65 degrees C, respectively. Co2+ was an activator for the immobilized enzyme in a similar role as for the free enzyme. No significant loss of activity was observed for at least 300 h of continuous operation. The yield of L-alanine was about 70% of the theoretical yield. The immobilized aminoacylase column decayed over a very long period of operation, but could be completely reactivated by regeneration.

Evidence for the existence of an unfolding intermediate state for aminoacylase during denaturation in guanidine solutions.

The equilibrium unfolding of pig kidney aminoacylase in guanidinium chloride (GdmCl) solutions was studied by following the fluorescence and circular dichroism (CD). At low concentrations of GdmCl, less than 1.0 M, the fluorescence intensity decreased with a slight red shift of the emission maximum (from 335 to 340 nm). An unfolding intermediate was observed in low concentrations of denaturant (between 1.2 and 1.6 M GdmCl). This intermediate was characterized by a decreased fluorescence emission intensity, a red-shifted emission maximum, and increased binding of the fluorescence probe 1-anilino-8-naphthalenesulfonate. No significant changes of the secondary structure were indicated by CD measurement. This conformation state is similar to a molten globule state which may exist in the pathway of protein folding. Further changes in the fluorescence properties occurred at higher concentrations of GdmCl, more than 1.6 M, with a decrease in emission intensity and a significant red shift of the emission maximum from 340 to 354 nm. In this stage, the secondary structure was completely broken. A study of apo-enzyme (Zn2+-free enzyme) produced similar results. However, comparison of the changes of the fluorescence emission spectra of native (Holo-) enzyme with Zn2+-free (Apo-) enzyme at low GdmCl concentrations showed that the structure of the Holo-enzyme was more stable than that of the Apo-enzyme.

Kinetics of slow reversible inhibition of human muscle creatine kinase by planar anions.

The toxicity of NO3- and NO2- to mammals has been widely publicized. However, the kinetic mechanism of inhibition of human muscle creatine kinase by NO3- and NO2- has not been explored. The kinetic theory of the substrate reaction during the modification of enzyme activity previously described by Tsou (Adv. Enzymol. Related Areas Mol. Biol. 1988, 61, 381-436) has been applied to a study of the kinetics of slow reversible inhibition of human muscle creatine kinase by planar anions (NO3- and NO2-). The kinetic equation of the substrate reaction was derived from theoretical analysis and experimental data, then simplified. The microscopic rate constants for the reaction of the inhibitors with the enzyme were obtained from the simplified equation for the substrate reaction in the presence of the inhibitors. The results show that the apparent forward rate constant A is dependent on ATP concentration, indicating competition between the inhibitor (NO3- or NO2-) and ATP. The results also suggest that binding of creatine-MgADP and the anion with the enzyme is very tight, since their binding constants are much higher than those for normal substrates.

Alkaline-induced unfolding and salt-induced folding of pig heart lactate dehydrogenase under high pH conditions.

The alkaline-induced unfolding and the salt-induced folding of pig heart lactate dehydrogenase under high pH conditions have been followed by fluorescence emission spectra and circular dichroism spectra. The results for alkaline-induced denaturation of lactate dehydrogenase show that at low ionic strength, increasing the pH value increased the extent of unfolding of the enzyme to the maximum ultimate unfolded conformation at about pH 13.0. At pH 12.5, although the enzyme was completely inactivated, most of the ordered structure was retained. Even at pH 13.5, the apparently fully unfolded enzyme still retained some ordered secondary structure. Kinetic analysis showed that at high pH, the inactivation rate constants of the enzyme are an order of magnitude faster than the unfolding rate constants at least. The above results are in accord with the suggestion by Tsou (Trends Biochem Sci 1986;11:427-429 and Science 1993;262:380-381) that the active site is usually more flexible than the enzyme molecule. At pH 13.0, adding salt to the solution caused the relatively unfolded state of the denatured enzyme to change into a compact conformational state by hydrophobic collapsing. The folded state induced by the salt bound ANS strongly, indicating the existence of an increased hydrophobic surface. The above results suggest that the salt-induced folded state at high pH may be the folded intermediate which exists in the general protein folding and that the large residual ordered secondary structure might become folded during the salt-induced folding.

Inactivation of creatine kinase is due to the conformational changes of the active sites during thermal denaturation.

The conformational changes of the active site of creatine kinase (ATP: creatine N-phosphotransferase EC 2.7.3.2.) during thermal denaturation was followed by changes in fluorescence at the active site of the enzyme labeled by o-phthalaldehyde. Conformational changes of the active site occurred at the same time as inactivation of the enzyme. The active site changes occurred before the denaturation of the enzyme molecule as a whole was detected. The above results showed that the thermal inactivation of the creatine kinase was due to the conformational changes of its active sites.

Kinetics of thermal inactivation of lactate dehydrogenase from rabbit muscle.

The kinetics of thermal inactivation of rabbit muscle lactate dehydrogenase at different temperatures has been studied using the kinetic method for the substrate reaction during irreversible inhibition of enzyme activity previously described by Tsou [Adv. Enzymol. Relat. Areas Mol. Biol. (1988), 61, 381-436]. The results show that thermal inactivation of the enzyme is an irreversible reaction. Microscopic rate constants were determined for thermal inactivation of the free enzyme and the enzyme-substrate complex. The inactivation rate constant of the free enzyme is much larger than the rate constant of the enzyme-substrate complex. The results suggest that the presence of the substrate has a certain protective effect against thermal inactivation of the enzyme.

Comparison of inactivation and unfolding of yeast alcohol dehydrogenase during thermal denaturation.

It has been reported that inactivation occurs before noticeable conformational change can be detected during denaturation of creatine kinase (ATP:creatine N-phosphotransferase, EC 2.7.3.2) and other enzymes by guanidinium chloride or urea. It has therefore been suggested that enzyme active sites may display more conformational flexibility than the enzyme molecules as a whole. The present paper compares the inactivation and unfolding of yeast alcohol dehydrogenase during thermal denaturation. Under identical conditions, inactivation takes place before noticeable conformational changes. Kinetics of unfolding can be resolved into two phases. For a given temperature, the fast phase rates are about one order of magnitude slower than the inactivation rates of the free enzyme and approximately the same magnitude as the inactivation rates of enzyme-substrate complexes. This is general accord with the suggestion made previously by Tsou, indicating that the active sites of metal enzymes are situated in a region more flexible than the molecules as a whole.

Ascertaining the number of essential thiol groups for the folding of creatine kinase.

Although the unfolding and refolding of proteins have been extensively studied in the literature, relatively few attempts have been made to see how many residues of the total residues of a certain amino acid in an enzyme can be modified without seriously affecting its folding. Based on a statistical analysis of the quantitative relationship between the extent of modification of protein functional groups and the decrease in their biological activity, a method proposed by Tsou (Sci. Sin. 1962, 11, 1535-1558) is widely used to determine the number of residues essential for the catalytic activity of modified proteins. In the present paper, Tsou's method is applied to determine the number of cystein residues essential for the folding of creatine kinase. The thiol groups of the cysteine residues in fully unfolded creatine kinase were modified by 2-chloromercuri-4-nitrophenol (MNP). The relationship between the number of MNP-groups introduced and the recovery of activity after refolding was determined. Quantitative treatment of the data by Tsou's plot shows that among the cystein residue modified in each subunit of creatine kinase, only three are essential for its folding.