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Journal Article 2010; 31(1): 67-72 PubMed PMID: 20150874 Citation  Keywords:  Animals, Animals, Genetically Modified, Cell Proliferation:drug effects, Chick Embryo:metabolism, Down-Regulation:drug effects, Embryonic Development:drug effects, Ganglia, Spinal:drug effects, Gene Expression Regulation, Developmental:drug effects, Gene .   

OBJECTIVE: Protein Isl-1 RNA interference and over expression in early chicken embryo dorsal root ganglia (DRG) were used to investigate the function of Isl-1 in DRG cell proliferation.

METHODS: Isl-1 targeted shRNA expression vector and Isl-1 over-expression vector were transfected into chicken embryo DRG by in ovo electroporation. Then, the DRG proliferation rate was detected by BrdU immunohistochemistry.

RESULTS: The rate of DRG cell proliferation increased after Isl-1 knock-down and decreased after Isl-1 over-expression.

CONCLUSIONS: In this study, we found that Isl-1 negatively modulates DRG cell proliferation....

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Journal Article 2009; 30(1): 56-60 PubMed PMID: 19300392 Citation  Keywords:  Adult, Body Mass Index, Case-Control Studies, China, Cholesterol Side-Chain Cleavage Enzyme:genetics, Female, Gene Frequency, Genetic Linkage, Genetic Predisposition to Disease, Genotype, Humans, Microsatellite Repeats:genetics, Polycystic Ovary Syndrome:.   

: Evidence indicates that CYP11alpha is a positional and functional candidate for genetic study in polycystic ovarian syndrome (PCOS). In the present study, we have evaluated the association between the CYP11alpha promoter pentannucleotide (tttta)n polymorphism and PCOS among Han Chinese women. Subjects include 125 patients with PCOS and 121 healthy controls, and all were Han Chinese women. Clinical characteristics of patients with PCOS and control subjects were examined according to the Rotterdam consensus criteria. The CYP11alpha promoter pentannucleotide (tttta)n polymorphism was genotyped with PCR and fluorescent capillary electrophoresis protocol. Results indicate that common alleles of the CYP11alpha promoter pentannucleotide (tttta)n polymorphism in this population of Han Chinese women were P4, P6 and P8, and allele P6 was the most common one. Frequencies of those three common alleles between PCOS cases and controls (24.8, 65.6, 6.8% and 23.6, 65.7, 9.9%, respectively) were similar. No significant allelic association of this polymorphism with PCOS was found. However, the carriers with allele P6 among patients with PCOS had increased WHR (0.85+/-0.05 vs 0.82+/-0.08, P=0.039) and decreased AUCG (9.5+/-2.1 vs 11.4+/-2.8, P=0.021) compared to the patients carrying other alleles. Therefore, the most common allele of the CYP11alpha promoter pentannucleotide (tttta)n polymorphism in the population of Han Chinese women is P6, while the most common allele in European Caucasians, as previously reported, is P4. This polymorphism is an ethnic and racial variant, and may have the risk susceptibility in abnormal metabolism of patients with PCOS in Han Chinese women....

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Journal Article 2009; 30(3): 357-362 PubMed PMID: 19855359 Citation  Keywords:  Adult, Alleles, Catalase:genetics, Chi-Square Distribution, Depressive Disorder:genetics, European Continental Ancestry Group:genetics, Female, Gene Frequency, Genetic Predisposition to Disease, Genotype, Humans, Male, Oxidative Stress:genetics, Patient S.   

: Numerous studies have provided information indicating the involvement of oxidative stress in the pathophysiology of depressive disorder (DD). The antioxidative system protects against the effects caused by reactive oxygen species (ROS). Catalase (CAT) is one of antioxidative enzymes observed to change their levels in the course of depression. The enzyme decomposes hydrogen peroxide (H(2)O(2)), whose overproduction is a result of many processes taking place in depression. Therefore, functional polymorphism of the CAT gene can be a candidate marker of the risk of depression. The presented study assessed the correlation between -262C>T polymorphism of the CAT gene, which influences the increase of CAT expression and activity, and the risk of depression development. The study, carried out on a homogeneous group recruited from the Polish population, enrolled 149 healthy subjects and 149 depressive patients. The groups were age-matched. The obtained results indicate no correlation between -262C>T polymorphism of the CAT gene (both with respect to genotype distribution and allele frequency) and the risk of depression. Nevertheless, further studies assessing the correlations between depression and polymorphism of the genes encoding antioxidative enzymes on larger groups of subjects should be undertaken....

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Journal Article 2009; 30(2): 221-226 PubMed PMID: 19675522 Citation  Keywords:  5' Untranslated Regions, Animals, Female, Gene Expression, Genotype, Mutation, Polymerase Chain Reaction, Polymorphism, Single Nucleotide, Prolactin:blood, Reproduction:genetics, Sequence Analysis, DNA, Swine:genetics, Thyrotropin-Releasing Hormone:metabo.   

OBJECTIVES: The recent genetics and molecular biology progress seems to be a fascinating challenge for the interdisciplinary studies on the effects of genetic changes in gene structure that causes the modification of physiological functions of many important proteins including hormones. Pig prolactin is one of the interesting hormones for this study.

AIM OF THE STUDY: The aim of the study was to analyze the mutation in 5'UTR region of the pig prolactin (PRL) gene and to evaluate the effect of this polymorphism on changes in plasma prolactin concentration.

RESULTS: It was found that only two individual groups of animals differed by the genotype in examined PRL gene locus - homozygote C/C and heterozygote C/T. PRL plasma concentration was 38.4 ng/ml (for C/T animals) or 42.7 ng/ml (for C/C animals). Animals with C/C genotyped exhibited a tendency to elevate PRL concentration as compared to the C/T group (p< 0.07).

CONCLUSIONS: This research combines the genetic, molecular and, in vivo, physiological study which allows focus on the possible relationship between the gene polymorphism and physiological status of animal....

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Journal Article 2008; 29(6): 1015-120 PubMed PMID: 19112418 Citation  Keywords:  Adaptation, Physiological, Adrenal Medulla:enzymology, Analysis of Variance, Animals, Catecholamines:biosynthesis, Dopamine beta-Hydroxylase:genetics, Gene Expression Regulation:physiology, Immobilization, Male, Phenylethanolamine N-Methyltransferase:gene.   

OBJECTIVE: Isolation of adult animals represents a form of psychsocial stress that produces sympatho-adrenomedullar activation. The aim of this work was to investigate the changes in gene expression and protein levels of catecholamine biosynthetic enzymes: tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT) in the adrenal medulla of naive control and chronically (12 weeks) socially isolated adult Wistar rat males and the response of these animals to additional immobilization stress (2 h).

METHODS: TH, DBH and PNMT mRNA levels were quantified by quantitative real-time RT-PCR (qRT-PCR). TH, DBH and PNMT immunoproteins were assayed by Western Blot.

RESULTS: In chronically isolated rats, gene expression levels of catecholamine biosynthetic enzymes in the adrenal medulla were decreased, but only TH mRNA was significantly decreased. However, protein levels of TH, DBH and PNMT of these animals were elevated by 55%, 20% and 18%, respectively, in relation to the corresponding control. Naive control and chronically socially isolated rats exposed to additional 2-h-immobilization showed increased gene expression of the examined enzymes, the increase being greater in socially isolated rats as compared to the controls. Additional immobilization of naive controls did not affect TH, DBH and PNMT protein levels. In contrast, this stress produced increased TH, DBH and PNMT protein levels in long-term socially isolated rats.

CONCLUSION: We can conclude that psychosocial stress expressed a differential influence on gene expression and protein levels of catecholamine biosynthetic enzymes in the adrenal medulla of adult rats. The results indicate a possible adaptation of catecholamine-synthesizing system at the level of TH gene expression in adrenal medulla of chronically isolated animals....

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Journal Article 2008; 29(6): 958-964 PubMed PMID: 19112413 Citation  Keywords:  Analysis of Variance, Animals, Estrous Cycle:metabolism, Female, Follicle Stimulating Hormone, beta Subunit:analysis, Follistatin:analysis, Gene Expression Regulation:physiology, Gonadotropin-Releasing Hormone:antagonists & inhibitors, Hormone Antagonists.   

OBJECTIVES: Neural control of the anterior pituitary function consists of the interplay of neuropeptides action, gonadal steroid hormones and many other factors. The physiological effect of this regulatory action is the release and synthesis of protein hormones in the precise time and quantity. The main factor responsible for the gonadotropins release and synthesis is the gonadotropin-releasing hormone (GnRH). We must still study the modulation of the synthesis of the gonadotropins subunits - LHbeta, FSHbeta and alpha subunit by different forms of GnRH and by its analogs, in order to better understand the regulation of gonadotropin release and synthesis. THE AIM of this study was to develop real-time PCR assays of five candidate reference genes for normalization purposes in order to quantify target transcripts in anterior pituitary cells during the preovulatory period. Moreover, we focused on the influence of GnRH receptor antagonist (antide) treatment on mRNA expression levels of GPalpha, LHbeta, FSHbeta, FST(follistatin) and PRL(prolactin) genes in these cells.

MATERIAL AND METHODS: Anterior pituitary cells were obtained from pituitary glands of four mature pigs at the preovulatory phase. Cells were incubated with or without antide and relative mRNA level of target genes was measured using the Applied Biosystems 7500 Real Time System. For an exact comparison of mRNA quantity, the stability of five reference genes, ACTB, B2M, GAPDH, RPL1, and TOP2B was evaluated to choose the most appropriate reference gene for qRT-PCR normalization in the pituitary cells. Expression stability of reference genes was calculated using the geNorm application. The developed method of PCR assay was applied to study gene expression in pig pituitary cells in short culture.

RESULTS: The most stably expressed genes in the pituitary cells were GAPDH and TOP2B. The expression of ACTB, B2M and RPL1 appeared to be highly unstable. After normalization to the GAPDH/TOP2B, results showed that the mRNA expression of the FSHbeta gene was highest in comparison with LHbeta, GPalpha, FST and PRL genes (p<0.005). Pre-treatment of cells by the antide resulted in lower mRNA expression of these genes, while FSHbeta mRNA had a significantly lower expression (p<0.05) in comparison with control.

CONCLUSIONS: Real-time PCR analysis of the expression of LHbeta, FSHbeta, alpha subunit, follistatin and prolactin genes in porcine anterior pituitary cells during the preovulatory period is suitable for the study of modulatory action of metal complexes with GnRH on the expression of these genes....

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Journal Article 2008; 29(6): 884-888 PubMed PMID: 19112410 Citation  Keywords:  Analysis of Variance, Animals, Dopamine:genetics, Gene Knockdown Techniques, Inverted Repeat Sequences:genetics, Myelin Proteins:metabolism, Nogo Proteins, PC12 Cells, RNA Interference:physiology, RNA, Messenger:analysis, Rats,.   

OBJECTIVES: Reticulon proteins, which are localized in the endoplasmic reticulum, have recently been shown to be involved in hormone secretion, in particular RTN1 and RTN3. The aim of the present study was to examine the effects of Nogo-A gene expression knockdown by RNA interference (RNAi) on dopamine release in PC12 cells.

METHODS: A small hairpin RNA (shRNA) eukaryotic expression vector, targeting the Nogo-A gene, was constructed and transfected into cultured PC12 cells by lipofecamine2000. Inhibition of Nogo-A gene expression was detected by semi-quantitative reverse transcription PCR and Western blot analysis. Following transfection, dopamine release was detected by high performance liquid chromatography.

RESULTS: The pGenesil-1-Nogo-A-2 plasmid was identified by gene sequencing. After transfection of the recombinant vector in PC12 cells, Nogo-A gene expression was significantly inhibited (p<0.01). Compared with the empty vector control group, dopamine release significantly decreased within 48 hours after transfection.

CONCLUSION: Results from this study suggest that Nogo-A might be involved in the mechanism of DA release in PC12 cells....

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Journal Article 2008; 29(6): 981-989 PubMed PMID: 19112400 Citation  Keywords:  Amino Acid Substitution:genetics, Animals, Cattle:genetics, Female, Gene Expression Regulation:genetics, Haplotypes, Insulin-Like Growth Factor I:genetics, Liver:metabolism, Male, Polymorphism, Single Nucleotide:genetics, Receptors, Somatotropin:genetics,.   

BACKGROUND: Polymorphisms in the bovine ghr and igf1 genes. Ghr and igf1 genes have been associated with milk and meat production of cattle. However, the molecular and physiological mechanisms underlying such associations are unknown. The objective of this study was to examine the effects of polymorphisms in 5'-regions of the bovine ghr and igf1 genes on the igf1 gene expression in the liver and on the level of IGF1 in blood of Polish Holstein-Friesian cattle.

METHODS: Individual and combined effects of single nucleotide polymorphisms (SNPs) in the 5'noncoding regions of the bovine igf1 and ghr genes on the IGF1 level in blood and igf1 gene expression in liver were examined. One SNP in the igf1 gene and four SNPs in the ghr gene were analyzed. IGF1 level in blood was measured by radioimmunoassay (RIA) in 211 heifers and bulls of Polish Holstein-Friesian cattle (of Black-and-White type). The igf1 gene expression was measured in livers of bulls carrying different igf1 and ghr genotypes (from three to nine animals per genotype) using real-time reverse transcription-PCR methods with the gapdh gene as a reference.

RESULTS: We showed that C/T transition in the promoter region of the igf1 gene influences the gene expression; relative igf1 expression was higher for animals with the CC genotype than for those with the TT and CT genotypes. TESS analysis showed that C/T transition in the igf1 gene co-localizes with the NF1 transcription factor binding site. Also, the ghr genotype appeared to significantly influence the igf1 gene expression in the liver, and we found the highest expression for the genotypes: RFLP-AluI (AT), RFLP-Fnu4HI(CC), and RFLP-NsiI(GA), and for the combined ghr genotype: AluI(AT)/ Fnu4HI(CC)/NsiI(GA). We discovered a significant association between the igf1 genotype and the IGF1 blood level. The highest IGF1 content in blood serum was found in CC genotype animals (1024 ng/ml) vs 698 ng/m and 859 ng/min in the TT and CT igf1 genotypes, respectively. Moreover, we noticed significant differences between ghr genotypes. The highest blood levels of IGF1 were for the animals carrying the ghr genotypes: RFLP-AluI(AA), RFLP-Fnu4HI(CC), and RFLP-NsiI(AG). Ghr haplotypes also significantly affected the IGF1 blood level. Animals of the combined ghr genotypes AluI(AA)/AccI(CC)/Fnu4HI(CC)/NsiI(AG) and AluI(AA)/AccI(CT)/Fnu4HI (CC)/ NsiI(AG) had a higher IGF1 concentration in blood than other genotype carriers.

CONCLUSIONS: The present results indicate that the effects of polymorphism in the igf1 and ghr genes on cattle milk or meat production traits could be at least partially mediated through their effects on the igf1 gene expression in the liver and the IGF1 level in blood....

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