Activation of the calcineurin-nuclear factor of activated T-cell signal transduction pathway in atrial fibrillation.

STUDY OBJECTIVES: The calcineurin-nuclear factor of activated T-cell (NFAT) signal transduction pathway regulates the expression of a plethora of genes in the myocardium. Cytosolic calcium overloading occurs in atrial fibrillation (AF), and this fulfills the condition needed for activation of this pathway. We therefore investigated the NFAT pathway in atrial tissue in a porcine model of AF. METHODS AND RESULTS: AF was induced in eight adult pigs by rapid atrial pacing. Investigations on the calcineurin and NFAT pathway were performed on transmural left atrial tissue obtained 6 weeks after implantation of the pacemaker (pacing for 4 weeks, and AF without pacing for 2 weeks). In the AF group, the left atrial dimension increased significantly (26 +/- 4 mm vs 31 +/- 4 mm, respectively, p < 0.05 [mean +/- SD]). Calcineurin enzyme activity increased significantly in pigs with AF (n = 8) when compared to control pigs (n = 6) [0.143 +/- 0.034 vs 0.038 +/- 0.063 mmol PO(4)(-) released, p < 0.01]. We found that both NFAT-c3 and NFAT-c4, the downstream effectors of calcineurin, increased significantly in the nuclei in AF tissue using immunoblotting. Translocation of NFAT-c3 and NFAT-c4 into the nuclei was also demonstrated in AF tissue microsections using immunohistochemistry. The electrophoresis mobility shift assay further demonstrated that nuclear extracts from AF tissue had a significantly larger binding capacity for NFAT-specific oligonucleotide probes. CONCLUSIONS: Our results demonstrate that calcineurin activity was increased in AF with subsequent NFAT-c3 and NFAT-c4 translocation into the nucleus. Activation of this signal transduction pathway may play an important role in the pathogenesis of AF.

Gene transfections with p53 and p21 inhibit cell proliferation, collagen type I, leukemia inhibitory factor, and tumor necrosis factor-alpha expression in leiomyoma cells.

OBJECTIVE: To transfect the p53 and p21 gene into the leiomyoma cells isolated from patients and observe their influence on the cell proliferation, leukemia inhibitory factor production, and gene expression of collagen type I as well as tumor necrosis factor-alpha (TNF-alpha) of cultured cells. DESIGN: Prospective study. SETTING: An assisted reproductive technology (ART) and genetic unit of a medical center. PATIENT(S): Leiomyoma cells isolated from leiomyoma tissue of 12 patients were divided into three groups: [1]. vehicle DNA, [2]. p53 gene, and [3]. p21 gene transfections. INTERVENTION(S): The pcDNA3.1 was used as vector to carry p53 and p21 genes for transfer. After gene transfection, RNAs of the leiomyoma cells were extracted for further analyses of gene expression. MAIN OUTCOME MEASURE(S): Relative cell numbers were determined by 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT) assay. The leukemia inhibitory factor (LIF) concentration was determined with ELISA. Gene expressions of collagen type I and TNF-alpha were detected by reverse transcriptase-polymerase chain reaction (RT-PCR). Gene expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. The cell proliferation, LIF production, as well as gene expressions of collagen type I and TNF-alpha in each group were compared. RESULTS: Relative cell numbers (%)/LIF production (in picograms per milliliter) in each group were: [1]. 100/58, [2]. 71/43, and [3]. 106/65. The ratios of gene expression of collagen type I/TNF-alpha with GAPDH in each group were: [1]. 1.64/0.335, [2]. 1.25/0.434, and [3]. 1.77/0.234. CONCLUSION(S): Transfection with p53 significantly inhibits proliferation of leiomyoma cells and decreases collagen type I gene expression and LIF production. The p21 transfection inhibits TNF-alpha gene expression.

In vitro and ex vivo green fluorescent protein expression in alveolar mammary epithelial cells and mammary glands driven by the distal 5'-regulative sequence and intron 1 of the goat beta-casein gene.

The 5'-regulative sequence and intron 1 of the goat beta-casein gene from -4044 to +2123 bp was cloned and fused with the reporter gene of green fluorescent protein (GFP) to create a plasmid termed pGB562/GFP. To detect GFP expression, pGB562/GFP was transfected in vitro via liposomes into the mammary epithelial cell line NMuMG. Cells could not express GFP unless the transfected NMuMG cells lined up to create functional alveoli. These functional cells were cultured with lactogenic hormones, including insulin, dexamethasone and prolactin, and were grown on a layer of the extracellular matrix Matrigel. Green fluorescent protein expression levels in NMuMG cells were 25-, 55- and 42-fold those in the control group at 24, 48, and 72 h after pGB562/GFP transfection respectively. In addition, pGB562/GFP was transfected ex vivo by electroporation into mammary gland fragments and cells were then cultured in vitro with a supplement of lactogenic hormones. Strong GFP expression localized in fragments of the mammary gland was observed 24 h after gene transfer. The novel strategy of ex vivo gene transfer into mammary tissue using GFP as a reporter gene to detect the function of a tissue-specific promoter is efficient and convenient. The data obtained herein reveal that the 5'-regulative sequence and intron 1 of the 6.2 kb goat beta-casein gene can enhance the efficiency of transgene expression. Thus, the GB562 sequence may act as a good promoter and effectively elevate the production of exogenous protein in mammary glands.

Establishment and application of a fluorescent polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method for identifying porcine, caprine, and bovine meats.

A method of fluorescent Polymerase Chain Reaction-restriction fragment length polymorphism (PCR-RFLP) was applied as an analytical and quantitative tool for meat identification. Following alignments of the nucleotide sequences, an oligonucleotide primer pair was designed to amplify the partial sequences within the 12S ribosomal RNA (12S rRNA) gene of mitochondrial DNA from porcine, caprine, and bovine meats. No fragment can be amplified from dog, cat, fish, duck, goose, turkey, and chicken DNA with the primer pair. Using fluorescence sensor capillary electrophoresis, the species-specific DNA fingerprints of pork, goat, and beef were generated by restriction enzyme digestion following a fluorescence-labeling PCR amplification. Species identification was conducted on the meat mixtures. The reliably semiquantitative levels were below 1% for binary mixtures of pork, goat, and beef. Cooking and autoclaving of meats did not influence the generation of the PCR-RFLP profiles or the analytical accuracy.

Four novel single nucleotide polymorphisms within the promoter region of p53 gene and their associations with uterine leiomyoma.

Dysregulated p53 expression has been implicated as a major contributor to numerous tumorigenesis. Single nucleotide polymorphisms (SNPs) within the functional consequence of the novel p53 promoter region remains obscure. Herein, we aimed to establish the extent of genetic variability within the promoter region of p53 gene as well as their association with leiomyoma susceptibility. Women were divided into two groups, leiomyoma (n = 160) and nonleiomyoma controls (n = 200). Total DNA was isolated from the peripheral blood of subjects. The DNA fragment containing p53 promoter regions (+64 approximately -404 bp) were obtained by amplification of polymerase chain reaction. The variations of DNA fragments were detected by DNA sequencing or restriction fragment length polymorphism (RFLP). Sequence alignment was used to identify sequence variations in p53 promoter regions. Genotypes were analyzed by method of RFLP. Genotypes/allelic frequencies in the leiomyoma and control groups were compared. A total of 15 sequence variations within p53 promoter region were identified, including -408 T/C, -382 A/G, -359 A/G, -325 T/C, -250 A/G, -216 T/C, -205 G/A, -198 G/A, -177 T/C, -103 A/G, -81 G/A, -71 G/A, -51 T/A, -33 A/G, and -17 T/C. Among these variations, four SNPs (-250 A/G, -216 T/C, -103 A/G, and -33 A/G) were established. Allele frequencies of -250*G/-216*C/-103*G/-33*G in the leiomyoma group and control group 6.9/5.0/5.9/3.8% and 3.8/1.8/2.3/4.0%, respectively. Two of them (-216*C and -103*G) are associated with higher leiomyoma susceptibility. We concluded that some sequence variations were observed within the promoter region of p53 gene. The SNPs of -216*C and -103*G among the identical sequence variations are associated with leiomyoma development.

Angiotensin I-converting enzyme insertion-related genotypes and allele are associated with higher susceptibility of endometriosis and leiomyoma.

Endometriosis and leiomyoma display features similar to malignancy, requiring neovascularization to proliferation and growth. Altered vascular-related genes might be related to the development of endometriosis and leiomyoma. Polymorphisms of the angiotensin-converting enzyme (ACE) insertion/deletion (I/D) genes have been linked with some vascular diseases. This study investigates whether ACE I/D gene polymorphisms could be used as markers of susceptibility in endometriosis and leiomyoma. Women were divided into three groups: (1) endometriosis (n = 125); (2) leiomyoma (n = 120); (3) normal controls (n = 128). Genomic DNA was obtained from peripheral leukocyte. ACE I/D gene polymorphisms in intron 16 were amplified by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) Genotypes and allelic frequencies in both groups were compared. We observed the genotype distribution and allele frequency of ACE I/D gene polymorphisms in both groups were significantly different. Proportions of ACE*I homozygote/heterozygote/D homozygote in both groups were: (1) 50.4/24/25.6%; (2) 25/23.33/51.67%; (3) 10.2/29.7/60.1%. Proportions of I/D alleles in each group were: (1) 62.4/37.6%; (2) 36.7/63.3%; (3) 25/75%. We concluded that ACE*I/D gene polymorphisms are associated with endometriosis and leiomyoma susceptibilities. ACE*I-related genotypes and allele are strongly related to the occurrence of endometriosis and moderately related to the occurrence of leiomyoma.

P53 codon 11, 72, and 248 gene polymorphisms in endometriosis.

OBJECTIVE: Mutated p53 gene is related to the instability of cell growth and cell cycle progression. We aimed to evaluate the association between endometriosis and p53 codon 11, 72 and 248 gene polymorphisms. PATIENTS AND METHODS: Women were divided into two groups: (1) moderate/severe endometriosis (n=148), and (2) non-endometriosis groups (n=150). P53 gene polymorphisms include codon11 Glu/Gln or Lys (GAG->CAG or AAG), codon 72 Arg/Pro (CGC->CCC), and codon 248 Arg/Thr (CGG->TCG). These gene polymorphisms were amplified by polymerase chain reaction and detected by electrophoresis after restriction enzyme (Taq I, BstU I, Hap II) digestions. Associations between the endometriosis and p53 polymorphisms were evaluated. RESULTS: The distributions of p53 codon 72 polymorphisms in both groups were significantly different. The proportions of Arg homozygotes/heterozygotes/Pro homozygotes in both groups were 9.5/66.2/24.3% and 30.7/50/19.3%. The proportions of Arg/Pro alleles were 42.6/57.4% and 56/44%. The distributions of p53 codon 11 and 248 polymorphisms in both groups were non-significantly different. All individuals appeared the wild genotypes (Glu11 and Arg248 homozygotes). CONCLUSION: Association between endometriosis and p53 codon 72 polymorphism exists. P53 codon 72*Pro-related genotype and allele are related with higher susceptibility of endometriosis. P53 codon 11 and 248 polymorphisms are not related with endometriosis susceptibility.

Seminal malondialdehyde concentration but not glutathione peroxidase activity is negatively correlated with seminal concentration and motility.

OBJECTIVES: Reactive oxygen species (ROS) induced lipid peroxidation is associated with sperm function. Malondialdehyde (MDA) concentration and glutathione peroxidase (GPx) activity represent the lipid peroxidation and spermicidal antioxidant, respectively. We aimed to evaluate the relationship of MDA and GPx levels with sperm parameters. PATIENTS AND METHODS: Specimens were divided into two groups: group 1. normospermia (n=20); group 2. oligoasthenospermia (n=31). Seminal MDA concentration was measured by thiobarbituric acid reaction method. Seminal GPx activity was measured by oxidation of reduced nicotinamide-adenine dinucleotide. Seminal MDA levels and GPx activities in both groups were compared. RESULTS: MDA concentrations in both groups were significantly different (1.52 +/- 0.75 vs. 2.25 +/- 0.88 nM, p = 0.0021). GPx activities in both groups were non-significantly different (0.48 +/- 0.11 vs. 0.47 +/- 0.12 U/ml). MDA levels were negatively correlated with the sperm motility (MDA = -0.014 x motility + 2.62, p =0.017) and concentration (MDA = -0.0045 x concentration + 2.23, p = 0.0166). GPx activities were positively but non-significantly correlated with the sperm concentration and sperm motility. CONCLUSIONS: Seminal MDA concentrations are negatively correlated with sperm concentration and motility, which might provide a simple and useful tool in predicting sperm parameters. GPx activity is non-significantly correlated with the seminal quality. Roles of seminal MDA upon spermatogenesis merits further surveys.

PROGINS Alu sequence insertion is associated with hyperprolactinaemia but not leiomyoma susceptibility.

OBJECTIVE: Leiomyoma and hyperprolactinaemia are both progesterone-dependent diseases. Hormone-related genes, such as the progesterone receptor (PGR), might be involved in their pathogenesis. DESIGN AND MEASUREMENTS: Subjects were divided into three groups: (i) leiomyoma (n = 120); (ii) hyperprolactinaemia (n = 101); (iii) normal controls (n = 140). We investigated the Alu (306-bp DNA) insertion in intron G of the PGR gene in all individuals. PGR gene polymorphisms [T1 (wild-type); T2 (PROGINS, with Alu insertion)] were determined by PCR and electrophoresis. Genotype and allele frequencies of the PROGINS in each group were detected and compared. RESULTS: We observed no significant difference of the PGR*T1/T2 genotypes and allele frequencies between leiomyoma and other two groups. The proportions of T1 homozygote/heterozygote/T2 homozygote in each group were (i) 90/8.3/1.7%; (ii) 84.2/9.9/5.9%; (iii) 92.9/6.4/0.7%. In contrast, a higher percentage of T2-related genotype and allele were noted in hyperprolactinaemic women compared to other two groups. The proportions of T1/T2 alleles in each group were: (i) 94.2/5.8%; (ii) 89.1/10.9%; (iii) 96.1/3.9%. CONCLUSIONS: The PROGIN*T2-related genotype and allele are related to a higher susceptibility to hyperprolactinaemia. The PROGINS polymorphism is not associated with leiomyoma development.

Superoxide dismutase activities of spermatozoa and seminal plasma are not correlated with male infertility.

Abnormal reactive oxygen species (ROS) production is associated with defective sperm function. Superoxide dismutase (SOD) is related with the scavenging of seminal ROS. We aimed to determine the effect of SOD activities of spermatozoa and seminal plasma on sperm quality. Semen samples from infertile couples who consented to the analyses were divided into two groups: 1) normospermia (n = 20); and 2) oligoasthenozoospermia (n = 31). The SOD activities of the spermatozoa and seminal plasma were measured by determining the inhibition of pyrogallol autoxidation. The SOD activities of spermatozoa and seminal plasma in both groups were compared. The relationships between the SOD activities and the sperm qualities were determined. We noted that SOD activities of sperm/seminal plasma in both groups were nonsignificantly different (group 1 vs. 2 = 0.77 +/- 0.33/0.84 +/- 0.40 U/mg protein for sperm, and 0.66 +/- and 0.36/0.83 +/- 0.47 U/ml for seminal plasma). SOD activities of sperm/seminal plasma were positively but nonsignificantly correlated with the sperm motility (SOD of sperm = 0.0008 x motility + 0.67; SOD of seminal plasma = 0.0006 x motility + 0.81) and concentration (SOD of sperm = 0.0006 x concentration + 0.67; SOD of seminal plasma = 0.0021 x concentration + 0.73). We concluded that SOD activities of sperm and seminal plasma were nonsignificantly correlated with the seminal quality. It appears that the SOD survey is not a useful tool for determining sperm fertilization potential.

In vivo gene transfer of leukemia inhibitory factor (LIF) into mouse endometrium.

PURPOSE: Leukemia inhibitory factor (LIF) is important for embryogenesis and implantation. We aimed to transfect LIF gene into the mouse endometrium. MATERIALS AND METHODS: Expression plasmids carried LIF and luciferase genes for transfer. After superovulation, 100 ICR mice were mated with vasectomized mice. Then LIF-liposome (Group 1) and luciferase-liposome complexes (Group 2) were injected into their uterine lumen (Day 0). Endometrial LIF and luciferase expressions were detected by reverse transcription-polymerase chain reaction on Days 0-4 post gene transfer. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control to normalize the gene transfection. RESULTS: LIF mRNA and luciferase activities reached the peak expression on Day 3. In Group 1, the ratios of LIF/GADPH on Days 1-4 were 0.414, 1.096, 1.162, and 0.782. In Group 2. LIF/GADPH on Days 1-4 were 0.24, 0.22, 0.35, and 0.32. CONCLUSIONS: Mouse endometrium could be effectively transfected with liposome-DNA mixtures. Endometrial LIF transfer via liposome may be effective in human.

Functional genomic study on atrial fibrillation using cDNA microarray and two-dimensional protein electrophoresis techniques and identification of the myosin regulatory light chain isoform reprogramming in atrial fibrillation.

INTRODUCTION: Functional and structural changes of atrial tissue occur during the natural course of atrial fibrillation (AF), and these changes may contribute to further AF. We investigated the changes in AF tissue using cDNA microarray and two-dimensional protein electrophoresis techniques. METHODS AND RESULTS: We established a porcine model of AF by rapid right atrial appendage pacing at a rate of 600/min. Atrial tissue was obtained after rapid atrial depolarization for 6 weeks. Microarrays containing 6,035 cDNA clones were used to evaluate the alterations of mRNA. Two-dimensional protein electrophoresis was performed to compare protein patterns. In cDNA microarray studies, we identified 387 genes with significant change in the left atrium and 81 genes in the right atrium. Among the genes, the ventricular isoform of the myosin regulatory light chain (MLC-2V) showed the greatest fold of change (9.4 and 7.3 in the left and right atrium, respectively). In protein electrophoresis, the expression levels of three protein spots spanning from 18 to 20 kDa in the acidic region (PI 4.5-5.0) were specifically elevated in the AF group. Interestingly, through tandem mass spectrometric analysis, these three spots were identified as MLC-2V. Thus, MLC-2V expression at the mRNA and protein levels corresponded well, and both indicated a significant increase in AF. CONCLUSION: Both cDNA microarray and two-dimensional polyacrylamide protein electrophoresis studies revealed characteristic changes in AF tissue. We demonstrated the reprogramming of myosin regulatory light chain isoform composition, with a significant increase of its ventricular isoform (MLC-2V).