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1.
Theriogenology ; 209: 170-177, 2023 Oct 01.
Article in English | MEDLINE | ID: mdl-37393747

ABSTRACT

As a functional fatty acid, α-linolenic acid (ALA) is essential in promoting animal testosterone biosynthesis. This study investigated the effects of ALA on testosterone biosynthesis and the possible mechanism underlying the signaling pathway in primary Leydig cells of the rooster. METHODS: Primary rooster Leydig cells were treated with ALA (0, 20, 40, or 80 µmol/L) or pretreated with a p38 inhibitor (50 µmol/L), a c-Jun NH2-terminal kinase (JNK) inhibitor (20 µmol/L), or an extracellular signal-regulated kinase (ERK) inhibitor (20 µmol/L) before ALA treatment. Testosterone content in the conditioned culture medium was detected using an enzyme-linked immunosorbent assay (ELISA). The expression of steroidogenic enzymes and JNK-SF-1 signaling pathway factors was detected using real-time fluorescence quantitative PCR (qRT-PCR). RESULTS: Supplementation with ALA significantly increased testosterone secretion within culture media (P < 0.05), and the optimized dose was 40 µmol/L. Compared with the control group, steroidogenic acute regulatory protein (StAR), cholesterol side-chain cleavage enzyme (P450scc), and 3ß-hydroxysteroid dehydrogenase (3ß-HSD) mRNA expression significantly increased (P < 0.05) in the 40 µmol/L ALA group; 17-hydroxylase/c17-20 lyase (P450c17) and p38 mRNA expressions were not significantly different in the 40 µmol/L ALA group; ERK and JNK mRNA expressions were significantly upregulated (P < 0.05) in 40 µmol/L ALA group. In the inhibitor group, testosterone levels were significantly downregulated (P < 0.05). Compared with the 40 µmol/L ALA group, StAR, P450scc, and P450c17 mRNA expressions were significantly decreased (P < 0.05), and 3ß-HSD mRNA expression in the p38 inhibitor group did not change; StAR, P450scc, and 3ß-HSD mRNA expressions were significantly decreased (P < 0.05), and P450c17 mRNA expression in ERK inhibitor group did not change; StAR, P450scc, 3ß-HSD, and P450c17 mRNA expressions were significantly decreased (P < 0.05) in JNK inhibitor group. Additionally, the increased steroidogenic factor 1 (SF-1) gene expression levels induced by ALA were reversed when the cells were pre-incubated with JNK and ERK inhibitors. The levels in the JNK inhibitor group were significantly lower than those in the control group (P < 0.05). CONCLUSION: ALA may promote testosterone biosynthesis by activating the JNK-SF-1 signaling pathway to upregulate StAR, P450scc, 3ß-HSD, and P450c17 expression in primary rooster Leydig cells.


Subject(s)
Leydig Cells , alpha-Linolenic Acid , Male , Animals , Leydig Cells/metabolism , Steroidogenic Factor 1/metabolism , Steroidogenic Factor 1/pharmacology , alpha-Linolenic Acid/pharmacology , Chickens/genetics , 3-Hydroxysteroid Dehydrogenases/metabolism , RNA, Messenger/metabolism , Testosterone/metabolism , Signal Transduction , Cholesterol Side-Chain Cleavage Enzyme/genetics , Cholesterol Side-Chain Cleavage Enzyme/metabolism
2.
Appl Microbiol Biotechnol ; 103(11): 4575-4584, 2019 Jun.
Article in English | MEDLINE | ID: mdl-31001745

ABSTRACT

The identification of animal species in feed and feedstuffs is important for detecting contamination and fraudulent replacement of animal components that might cause health and economic problems. A novel multiplex assay, based on xMAP technology and the generic detection of closely related species, was developed for the simultaneous differential detection of avian, fish, and ruminant DNA in products. Universal primers and probes specific to avian, fish, or ruminant species were designed to target a conserved mitochondrial DNA sequence in the 12S ribosomal RNA gene (rRNA). The assay specificity was validated using samples of 27 target and 10 nontarget animal species. The limits of detection of the purified DNA were determined to be 0.2 pg/µL-0.1 ng/µL by testing the meat samples of six species and four feedstuffs. The detection sensitivity of the experimental mixtures was demonstrated to be 0.01% (weight percentage). The assay's suitability for practical application was evaluated by testing feed samples; unlabeled animal ingredients were detected in 32% of the 56 samples. The assay differentially detected the three targeted categories of animal species in less than 2 h, reflecting improvements in speed and efficiency. Based on these results, this novel multiplex xMAP assay provides a reliable and highly efficient technology for the routine detection of animal species in feed and other products for which this information is needed.


Subject(s)
Animal Feed/analysis , DNA/isolation & purification , Food Contamination , Nucleic Acid Hybridization/methods , Polymerase Chain Reaction/methods , Animals , Birds , DNA/genetics , DNA Primers/genetics , Fishes , Oligonucleotide Probes/genetics , RNA, Ribosomal/genetics , Ruminants , Sensitivity and Specificity
3.
J Cell Mol Med ; 23(4): 2731-2743, 2019 04.
Article in English | MEDLINE | ID: mdl-30770623

ABSTRACT

Lipid metabolism disorders lead to vascular endothelial injury. Matrine is an alkaloid that has been used to improve obesity and diabetes and for the treatment of hepatitis B. However, its effect on lipid metabolism disorders and vascular injury is unclear. Here, we investigated the effect of matrine on high-fat diet fed mice and oxidized low-density lipoprotein (ox-LDL)-induced human umbilical vein endothelial cells (HUVECs). Computational virtual docking analyses, phosphoinositide 3-kinase (PI3K) and protein kinase C-α (PKCα) inhibitors were used to localize matrine in vascular injuries. The results showed that matrine-treated mice were more resistant to abnormal lipid metabolism and inflammation than vehicle-treated mice and exhibited significantly alleviated ox-LDL-stimulated dysfunction of HUVECs, restored diminished nitric oxide release, decreased reactive oxygen species generation and increased expression phosphorylation of AKT-Ser473 and endothelial nitric oxide synthase (eNOS)-Ser1177. Matrine not only up-regulates eNOS-Ser1177 but also down-regulates eNOS-Thr495, a PKCα-controlled negative regulator of eNOS. Using computational virtual docking analyses and biochemical assays, matrine was also shown to influence eNOS/NO via PKCα inhibition. Moreover, the protective effects of matrine were significantly abolished by the simultaneous application of PKCα and the PI3K inhibitor. Matrine may thus be potentially employed as a novel therapeutic strategy against high-fat diet-induced vascular injury.


Subject(s)
Alkaloids/pharmacology , Diet, High-Fat/adverse effects , Endothelium, Vascular/drug effects , Gene Expression Regulation/drug effects , Lipoproteins, LDL/toxicity , Quinolizines/pharmacology , Vascular System Injuries/prevention & control , Animals , Endothelium, Vascular/metabolism , Endothelium, Vascular/pathology , Human Umbilical Vein Endothelial Cells , Humans , In Vitro Techniques , Male , Mice , Mice, Inbred C57BL , Nitric Oxide/metabolism , Nitric Oxide Synthase Type III/genetics , Nitric Oxide Synthase Type III/metabolism , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/metabolism , Protein Kinase C-alpha/genetics , Protein Kinase C-alpha/metabolism , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction , Vascular System Injuries/etiology , Vascular System Injuries/metabolism , Vascular System Injuries/pathology , Matrines
4.
J Virol Methods ; 228: 60-6, 2016 Feb.
Article in English | MEDLINE | ID: mdl-26615807

ABSTRACT

A novel multiplex PCR assay using non-homologous oligonucleotides with locked nucleic acid (LNA) modifications as universal primers was developed and validated for the simultaneous detection of five swine viruses. The assay utilizes five virus-specific primer pairs modified at the 5' end through the addition of the universal primer sequence. In the reaction, small amounts of target templates with the 5' tail were generated and subsequently amplified through the extension of a LNA universal primer set. To validate the specificity of this assay, 27 viral target strains and 12 non-target pathogens were tested. The lower limit of detection of viral nucleic acids was 1.1-1.9 pg per reaction or 11-32 pg in a five-plex viral nucleic acid mixture. The LNA mPCR assay displayed higher analytical sensitivity and efficiency for the detection of plasmid standards compared with the conventional assay, which uses standard primers without the 5' tail. A total of 207 field samples were tested using both assays. The LNA mPCR assay provided numerically higher detection rates for all pathogens in independent samples. Moreover, the LNA mPCR assay had significantly higher detection rates in independent samples compared with the conventional assay.


Subject(s)
Multiplex Polymerase Chain Reaction/methods , Oligonucleotides , Swine Diseases/diagnosis , Virus Diseases/veterinary , Viruses/genetics , Viruses/isolation & purification , Animals , Circovirus/genetics , Circovirus/isolation & purification , Classical Swine Fever Virus/genetics , Classical Swine Fever Virus/isolation & purification , DNA Primers , Herpesvirus 1, Suid/genetics , Herpesvirus 1, Suid/isolation & purification , Limit of Detection , Porcine respiratory and reproductive syndrome virus/genetics , Porcine respiratory and reproductive syndrome virus/isolation & purification , Sensitivity and Specificity , Swine , Swine Diseases/virology
5.
Sci Rep ; 5: 16093, 2015 Nov 05.
Article in English | MEDLINE | ID: mdl-26537450

ABSTRACT

As a nutritional factor, folic acid can prevent cardiac and neural defects during embryo development. Our previous study showed that arsenic impairs embryo development by down-regulating Dvr1/GDF1 expression in zebrafish. Here, we investigated whether folic acid could protect against arsenic-mediated embryo toxicity. We found that folic acid supplementation increases hatching and survival rates, decreases malformation rate and ameliorates abnormal cardiac and neural development of zebrafish embryos exposed to arsenite. Both real-time PCR analysis and whole in-mount hybridization showed that folic acid significantly rescued the decrease in Dvr1 expression caused by arsenite. Subsequently, our data demonstrated that arsenite significantly decreased cell viability and GDF1 mRNA and protein levels in HEK293ET cells, while folic acid reversed these effects. Folic acid attenuated the increase in subcellular reactive oxygen species (ROS) levels and oxidative adaptor p66Shc protein expression in parallel with the changes in GDF1 expression and cell viability. P66Shc knockdown significantly inhibited the production of ROS and the down-regulation of GDF1 induced by arsenite. Our data demonstrated that folic acid supplementation protected against arsenic-mediated embryo toxicity by up-regulating the expression of Dvr1/GDF1, and folic acid enhanced the expression of GDF1 by decreasing p66Shc expression and subcellular ROS levels.


Subject(s)
Arsenic/adverse effects , Arsenites/adverse effects , Embryonic Development/drug effects , Folic Acid/pharmacology , Transforming Growth Factor beta/metabolism , Zebrafish Proteins/metabolism , Zebrafish/metabolism , Animals , Cell Survival/drug effects , Down-Regulation/drug effects , Oxidative Stress/drug effects , Reactive Oxygen Species/metabolism , Up-Regulation/drug effects
6.
Appl Microbiol Biotechnol ; 99(2): 919-28, 2015 Jan.
Article in English | MEDLINE | ID: mdl-25557628

ABSTRACT

A novel multiplex detection array based on Luminex xMAP technology was developed and validated for simultaneous detection of five major viruses causing swine reproductive diseases. By combining one-step asymmetric multiplex reverse transcription polymerase chain reaction (RT-PCR) with xMAP bead-based hybridization and flow cytometry analysis, the resulting multiplex assay was capable of detecting single and mixed infections of PRRSV, PCV-2, PRV, CSFV, and PPV in a single reaction. The assay accurately detected and differentiated 23 viral strains used in this study. The low detection limit was determined as 2.2-22 copies/µL (corresponding to 0.5-6.8 fg/µL DNA template) on plasmid constructs containing viral fragments. The intra-assay and inter-assay variances (CV%) were low that ranged from 2.5 to 5.4 % and 4.1 to 7.6 %, respectively. The assay was applied to test field samples and detected single and mixed viral infections. The detection rate was higher than that of uniplex conventional PCR and RT-PCR methods. The detection of PRRSV by the bead-based multiplex assay was comparable with a commercially available real time RT-PCR kit. The test procedure on purified DNA or RNA samples could be completed within 2 h. In conclusion, the bead-based suspension array presented here proved to be a high-throughput practical tool that provided highly specific and sensitive identification of single and multiple infections of five major viruses in pigs and boar semen.


Subject(s)
Circovirus/isolation & purification , Classical Swine Fever Virus/isolation & purification , DNA, Viral/isolation & purification , Parvovirus, Porcine/isolation & purification , Porcine respiratory and reproductive syndrome virus/isolation & purification , RNA, Viral/isolation & purification , Animals , Circovirus/classification , Classical Swine Fever/diagnosis , Classical Swine Fever/virology , DNA Primers , DNA, Viral/genetics , Multiplex Polymerase Chain Reaction , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction , Reproducibility of Results , Sensitivity and Specificity , Specimen Handling , Swine/virology , Swine Diseases/diagnosis , Swine Diseases/virology
7.
Diagn Microbiol Infect Dis ; 77(1): 53-7, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23867326

ABSTRACT

A new assay with the combination of multiplex polymerase chain reaction and denaturing high-performance liquid chromatography analysis was developed for simultaneous detection of Mycobacterium genus and identification of the Mycobacterium tuberculosis complex (MTC). Targeting at genus-specific 16S rRNA sequence of Mycobacterium and specific insertion elements IS6110 and IS1081 of MTC, the assay was validated with 84 strains covering 23 mycobacteria species and 30 strains of non-mycobacteria species. No cross reactivity was observed. Clinical application was carried out on 198 specimens (155 human sputum and 43 bovine tissue samples) and compared with culture. The multiplex assay detected all culture-positive (36 in number) and 35.2% (57/162) culture-negative specimens. The molecular assay was fast that could be completed within 1 h on purified DNA, with the limit of detection as 0.8-1.6 pg per reaction on DNA template. This work provided a useful laboratory tool for rapid identification of Mycobacterium and differentiation of MTC and nontuberculous mycobacteria.


Subject(s)
Bacteriological Techniques/methods , Chromatography, High Pressure Liquid/methods , Molecular Diagnostic Techniques/methods , Multiplex Polymerase Chain Reaction/methods , Mycobacterium tuberculosis/isolation & purification , Tuberculosis/diagnosis , Tuberculosis/microbiology , DNA Transposable Elements , Humans , Mycobacterium tuberculosis/classification , Mycobacterium tuberculosis/genetics , RNA, Ribosomal, 16S/genetics , Sensitivity and Specificity , Time Factors
8.
Shanghai Kou Qiang Yi Xue ; 22(2): 233-6, 2013 Apr.
Article in Chinese | MEDLINE | ID: mdl-23708044

ABSTRACT

PURPOSE: To investigate the therapeutic effects of pingyangmycin injection as a primary therapy of macrocystic lymphatic malformations in cervical region. METHODS: Thirty-six patients with macrocystic lymphatic malformations in cervical region underwent the therapy of pingyangmycin injection between 2009 and 2012 at School of Stomatology, China Medical University. Among them, 16 patients had unilateral submandibular lesions, 20 patients had lesions in anterior cervical regions. The age of patients was from 6 months to 25 years old. The concentration of the drug was 1.6 mg /mL with an addition of lidocaine. The dose and cycle of treatment were dependent on the lesions' size and patients' age. The follow-up period was 12 months to 2 years after the last treatment. RESULTS: The total effective rate was 100%, and the curative rate was 94.4%. No serious complications were encountered. CONCLUSIONS: Intralesional Injection of pingyangmycin provides a safe and effective treatment for macrocystic lymphatic malformations in cervical region as a primary treatment.


Subject(s)
Bleomycin/analogs & derivatives , Lymphatic Abnormalities , Adolescent , Adult , Child , Child, Preschool , Humans , Infant , Injections, Intralesional , Neck , Oral Medicine , Treatment Outcome , Young Adult
9.
Br J Oral Maxillofac Surg ; 51(3): e29-33, 2013 Apr.
Article in English | MEDLINE | ID: mdl-22475367

ABSTRACT

Reduction of intracapsular condylar fractures is difficult, so we have based our technique on preoperative simulation using computer-aided design (CAD), which has proved useful in other surgical specialties. We have treated 11 patients with intracapsular condylar fractures. Before the operation the procedure was shown on the computer using a three-dimensional simulation system. The relation between the stump and the fragment of the condyle, and assessment of the position and the size of the screw, were made preoperatively to obtain a perfect fit. The displaced fragment was reduced by elevators, and fixed with a bicortical screw through a minimised preauricular incision under general anaesthesia. The fragments and the location of the screws were similar on the preoperative simulation and on the postoperative computed tomographic (CT) scan. The reduction and fixation of the fracture showed a perfect fit on the same view in the preoperative CAD simulation in the Mimics 10.01 software and postoperatively. Postoperative clinical examinations showed good occlusion and satisfactory mouth opening. Two patients had temporary paralysis of the occipitofrontalis muscle that recovered within 3 months. All patients regained normal mandibular movements and had short and invisible scars at 6 months' follow up. The technique of CAD simulation could help to improve the accuracy during open treatment for intracapsular condylar fractures.


Subject(s)
Computer Simulation , Computer-Aided Design , Fracture Fixation, Internal/methods , Joint Capsule/injuries , Mandibular Condyle/injuries , Mandibular Fractures/surgery , Adult , Bone Screws , Cicatrix/classification , Dental Occlusion , Facial Muscles/physiopathology , Facial Paralysis/etiology , Female , Follow-Up Studies , Humans , Image Processing, Computer-Assisted/methods , Imaging, Three-Dimensional/methods , Joint Capsule/surgery , Joint Dislocations/surgery , Male , Mandibular Condyle/surgery , Middle Aged , Minimally Invasive Surgical Procedures , Models, Anatomic , Postoperative Complications , Range of Motion, Articular/physiology , Temporomandibular Joint/injuries , Temporomandibular Joint/surgery , Tomography, Spiral Computed/methods , Treatment Outcome
10.
Food Chem Toxicol ; 50(8): 2854-63, 2012 Aug.
Article in English | MEDLINE | ID: mdl-22583652

ABSTRACT

Selenium (Se) is an essential micronutrient, but also a potential toxin, which may be absorbed in excess. Relatively little is known about selenium embryotoxicity in zebrafish. In this study, we evaluated the effect of selenite exposure in zebrafish embryos. Selenite treatment decreased survival and resulted in abnormal development in a dose- and time-dependent manner. We observed irregular growth of neurons in selenite treated embryos, characterized by the absence of neurons in the brain, trunk and tail. Selenite exposure also induced defects in heart function, such as bradycardia and cardiac dysplasia with irregular and smaller chamber shape. In addition, selenite exposure caused ectopic cell proliferation, apoptosis, and a change in the pattern of DNA methylation. Our results suggested that supplementation with folic acid (FA) ameliorated the cardiac and neural defects in selenite-treated embryos. In conclusion, we demonstrated that selenite exposure caused cardiac and neural defects in zebrafish embryos and that folic acid protected against this embryotoxicity. It will give insight into the risk assessment and prevention of Se-mediated embryotoxicity.


Subject(s)
Embryo, Nonmammalian/drug effects , Folic Acid/pharmacology , Sodium Selenite/toxicity , Teratogens/toxicity , Zebrafish/embryology , Animals , DNA Methylation , Heart/drug effects , Heart/embryology
12.
Zhongguo Yi Xue Ke Xue Yuan Xue Bao ; 33(6): 615-9, 2011 Dec.
Article in Chinese | MEDLINE | ID: mdl-22509542

ABSTRACT

OBJECTIVE: To study the role of extracellular signal-regulated protein kinase 5 (ERK5) during the biosynthesis of follicle-stimulating hormone (FSH)-mediated progesterone in primary granulosa cells. METHODS: The expressions of phosphorylated and general forms of ERKS in primary granulosa cells after the treatment of FSH were detected by Western blot analysis. The subcellular localization of ERK5 was observed by confocal microscopy. The effect of ERK5 on FSH-mediated progesterone biosynthesis in primary granulosa cells was analyzed using recombinant adenovirus vectors. RESULTS: ERK5 activation was induced by FSH in a time-dependent manner in primary cultured granulosa cells, although the general ERK5 protein level decreased also in a time-dependent manner. The treatment of FSH showed no remarkable effect on the subcellular distribution of endogenous ERK5, which was mainly in the cytoplasm of granulosa cells. The co-infection of Ad-caMEK5 and Ad-wtERK5 increased the progesterone production and StAR expression in primary cultured granulosa cells, whereas inhibition of ERK5 activation suppressed the FSH-stimulated progesterone production. CONCLUSION: ERK5 may stimulate FSH-mediated progesterone production in primary cultured granulosa cells.


Subject(s)
Granulosa Cells/metabolism , Mitogen-Activated Protein Kinase 7/physiology , Progesterone/biosynthesis , Animals , Cells, Cultured , Female , Follicle Stimulating Hormone/pharmacology , Granulosa Cells/drug effects , Mitogen-Activated Protein Kinase 7/metabolism , Rats , Rats, Sprague-Dawley
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