Mu-Chiou Huang

Caveolin-1 sensitizes rat pituitary adenoma GH3 cells to bromocriptine induced apoptosis

BackgroundProlactinoma is the most frequent pituitary tumor in humans. The dopamine D2 receptor agonist bromocriptine has been widely used clinically to treat human breast tumor and prolactinoma through inhibition of hyperprolactinemia and induction of tumor cell apoptosis, respectively, but the molecular mechanism of bromocriptine induction of pituitary tumor apoptosis remains unclear. Caveolin-1 is a membrane-anchored protein enriched on caveolae, inverted flask-shaped invaginations on plasma membranes where signal transduction molecules are concentrated. Currently, caveolin-1 is thought to be a negative regulator of cellular proliferation and an enhancer of apoptosis by blocking signal transduction between cell surface membrane receptors and intracellular signaling protein cascades. Rat pituitary adenoma GH3 cells, which express endogenous caveolin-1, exhibit increased apoptosis and shrinkage after exposure to bromocriptine. Hence, the GH3 cell line is an ideal model for studying the molecular action of bromocriptine on prolactinoma.ResultsThe expression of endogenous caveolin-1 in GH3 cells was elevated after bromocriptine treatment. Transiently expressed mouse recombinant caveolin-1 induced apoptosis in GH3 cells by enhancing the activity of caspase 8. Significantly, caveolin-1 induction of GH3 cell apoptosis was sensitized by the administration of bromocriptine. Phosphorylation of caveolin-1 at tyrosine 14 was enhanced after bromocriptine treatment, suggesting that bromocriptine-induced phosphorylation of caveolin-1 may contribute to sensitization of apoptosis in GH3 cells exposed to bromocriptine.ConclusionOur results reveal that caveolin-1 increases sensitivity for apoptosis induction in pituitary adenoma GH3 cells and may contribute to tumor shrinkage after clinical bromocriptine treatment.

Female-specific DNA sequences in geese

1. The OPAE random primers (Operon Technologies, Inc., CA) were used for random amplified polymorphic DNA (RAPD) fingerprinting in Chinese, White Roman and Landaise geese. One of these primers, OPAE-06, produced a 938-bp sex-specific fragment in all females and in no males of Chinese geese only. 2. A novel female-specific DNA sequence in Chinese goose was cloned and sequenced. Two primers, CGSex-F and CGSex-R, were designed in order to amplify a 912-bp sex-specific polymerase chain reaction (PCR) fragment on genomic DNA from female geese. 3. It was shown that a simple and effective PCR-based sexing technique could be used in the three goose breeds studied. 4. Nucleotide sequencing of the sex-specific fragments in White Roman and Landaise geese was performed and sequence differences were observed among these three breeds.

Application of the SNaPshot minisequencing assay to species identification in the Lactobacillus casei group.

This study used group-specific PCR combined with SNaPshot minisequencing for species identification within the Lactobacillus casei group. The L. casei group-specific PCR primer pair was designed using the rpoA gene sequence. A SNaPshot minisequencing assay using dnaK as a target gene was developed, and five SNP primers were designed by analysing the conserved regions of the dnaK sequences. The specificity of the minisequencing assay was evaluated using 63 strains of L. casei group species. The results showed that the group-specific PCR could assign Lactobacillus strains into the L. casei group, and the SNaPshot minisequencing assay was able to unambiguously and simultaneously discriminate strains belonging to the species L. casei, Lactobacillus paracasei, and Lactobacillus rhamnosus. In conclusion, we have successfully developed a rapid, accurate and cost-effective assay for species identification of members of the L. casei group.

AFLP fingerprinting for paternity testing in ducks

1. The accuracy and reproducibility of AFLP fingerprinting was investigated in the duck (Anas Platyrhynchos), using a multicolour fluorescent labeling technique. The fluorescent labelling fragments were separated on a capillary electrophoresis-base ABI PRISM 3100 Genetic Analyzer. 2. A total of 337 AFLP peaks with 103 of them being polymorphic markers were generated by 16 sets consisting of EcoRI/TaqI primer pair combinations. The number and size range of AFLP polymorphisms detected per primer pair varied from 3 to 11 and 58 to 290 bp, respectively. About 30·6% (103/337) of AFLP peaks were detected polymorphisms, with an average of 6·4 polymorphic markers per primer pair. 3. The clear polymorphic peaks were amplified with EcoR+AC/Taq+AC primer combinations. The AFLP peaks showed high reproducibility. From the family testing, we found that the fingerprints of all the offspring were derived from one or other parent. Therefore, we conclude that AFLP fingerprinting might be a suitable method for duck paternity testing.

A novel genetic marker of the ovomucoid gene associated with hatchability in Tsaiya ducks (Anas platyrhynchos)

Transcriptome analysis using a cDNA microarray was performed to identify differentially expressed genes that are correlated with hatchability, and a new PCR-RFLP marker of high hatchability among the identified genes was observed. We used the cDNA microarray technique for gene expression profiling of the magnum epithelium of laying Tsaiya ducks, and several regulated genes associated with hatchability were found. The results of real-time PCR and Western blotting analysis confirmed that the mRNA and protein levels of ovomucoid in the magnum epithelium of animals in the low-hatchability group were significantly higher than the levels in the high-hatchability group (P < 0.05). Primers TovF1 and TovR1, designed according to the ovomucoid EST sequence, were used to amplify genomic DNA samples of different individual Tsaiya ducks, and sequence analysis of the amplified DNA products showed deletion among the ducks from the low-hatchability group. Primers TovF2 and TovR2 were used to perform PCR-RFLP analysis on the amplified DNA products to classify the ducks into +/+, +/- and -/- genotypes. The animals of +/+ and +/- genotypes were identified as having significantly higher hatchability than those of the -/- genotype (P < 0.05). In contrast, no differences were observed between genotypes in terms of fertility, duration of fertility, egg weight or total number of eggs. Our results indicated that a novel PCR-RFLP marker of high hatchability, an ovomucoid gene polymorphism, can be used as a genetic marker for marker-assisted selection to improve hatchability in Tsaiya ducks.

Duck (Anas platyrhynchos) linkage mapping by AFLP fingerprinting

Amplified fragment length polymorphism (AFLP) with multicolored fluorescent molecular markers was used to analyze duck (Anas platyrhynchos) genomic DNA and to construct the first AFLP genetic linkage map. These markers were developed and genotyped in 766 F2 individuals from six families from a cross between two different selected duck lines, brown Tsaiya and Pekin. Two hundred and ninety-six polymorphic bands (64% of all bands) were detected using 18 pairs of fluorescent Taq I/Eco RI primer combinations. Each primer set produced a range of 7 to 29 fragments in the reactions, and generated on average 16.4 polymorphic bands. The AFLP linkage map included 260 co-dominant markers distributed in 32 linkage groups. Twenty-one co-dominant markers were not linked with any other marker. Each linkage group contained three to 63 molecular markers and their size ranged between 19.0 cM and 171.9 cM. This AFLP linkage map provides important information for establishing a duck chromosome map, for mapping quantitative trait loci (QTL mapping) and for breeding applications.

Alteration of endogenous antioxidant enzymes in naturally occurring hypertrophic cardiomyopathy

We have recently developed a porcine model with naturally occurring hypertrophic cardiomyopathy (HCM). Similar to humans, occluded intramural coronary artery and damaged mitochondria are frequently observed in these animals in which the disease is thought to be associated with the local ischemia of myocardium. In view of antioxidant functions involved in the ischemic injury, we measured the expression of endogenous antioxidant enzymes in the tissues with and without HCM. The results showed a significant increase of Cu,Zn‐superoxide dismutase (SOD), but not Mn‐SOD, and decrease of catalase (CAT) activities in the various areas of HCM hearts. It was demonstrated that SOD/CAT ratios in the HCM hearts were significantly higher than those in normals and were found to be dramatically correlated with the severity of cardiac hypertrophy. The altered SOD/CAT ratio was also consistent with increase in lipid damage. We hypothesize that the elevated SOD combined with an inadequate amount of H2O2 scavenging enzyme may lead HCM heart at oxidative stress risk. However, the pathogenic role of imbalanced antioxidant enzyme needs to be further explored.

Discrimination of the Lactobacillus acidophilus group using sequencing, species‐specific PCR and SNaPshot mini‐sequencing technology based on the recA gene

BACKGROUND To clearly identify specific species and subspecies of the Lactobacillus acidophilus group using phenotypic and genotypic (16S rDNA sequence analysis) techniques alone is difficult. The aim of this study was to use the recA gene for species discrimination in the L. acidophilus group, as well as to develop a species-specific primer and single nucleotide polymorphism primer based on the recA gene sequence for species and subspecies identification. RESULTS The average sequence similarity for the recA gene among type strains was 80.0%, and most members of the L. acidophilus group could be clearly distinguished. The species-specific primer was designed according to the recA gene sequencing, which was employed for polymerase chain reaction with the template DNA of Lactobacillus strains. A single 231-bp species-specific band was found only in L. delbrueckii. A SNaPshot mini-sequencing assay using recA as a target gene was also developed. The specificity of the mini-sequencing assay was evaluated using 31 strains of L. delbrueckii species and was able to unambiguously discriminate strains belonging to the subspecies L. delbrueckii subsp. bulgaricus. CONCLUSION The phylogenetic relationships of most strains in the L. acidophilus group can be resolved using recA gene sequencing, and a novel method to identify the species and subspecies of the L. delbrueckii and L. delbrueckii subsp. bulgaricus was developed by species-specific polymerase chain reaction combined with SNaPshot mini-sequencing.

The SNP Genotypes of Growth Hormone Gene Associated with Reproductive Traits in Tsaiya Ducks

Our previous cDNA microarray study showed that the growth hormone (GH) gene may involve in the duck egg formation process. The purpose of this study was to investigate the relationship between GH genotypes of single nucleotide polymorphisms (SNPs) and reproductive traits of Tsaiya ducks. Primer pairs for the coding region in the GH were designed based on the duck genomic sequence. Polymorphisms were detected by polymerase chain reaction (PCR)-single strand polymorphism (SSCP) and were verified by DNA sequencing. Nineteen SNPs were identified in the duck GH gene, of which three coding SNPs (C3169T, C3700T and C5058G) were genotyped to investigate the associations with reproductive traits. The results showed that each SNP was associated with at least one duck fertility-related trait (p < 0.05). Haplotypes constructed on these three SNPs were associated with fertility rate (FR) and maximum duration of fertility (MDF) (p < 0.05). In particular, diplotype H1H1 was dominant for FR and MDF. This suggested that GH gene polymorphisms are associated with duck fertility-related traits. The SNPs in this gene may be used as potential markers for marker-assisted selection.

Association of prolactin haplotypes with reproductive traits in Tsaiya ducks

A previous cDNA microarray study showed that the prolactin (PRL) gene may be involved in the duck ovarian follicle development and egg formation process. The purpose of this study was to investigate the relationship between PRL genotypes of single nucleotide polymorphism (SNP) and reproductive traits of Tsaiya ducks. Primer pairs for the coding regions in the PRL were designed based on the duck genomic sequence database. Polymorphisms were detected by polymerase chain reaction (PCR)-single strand polymorphism (SSCP) and were verified by DNA sequencing. Six novel SNPs (T233C, T295C, G309T, C381A, G3941T and A3975C) were identified in the 1972 bp region of duck PRL gene, and all of them were located in non-coding regions. Single SNP-trait association analysis showed that each SNP was associated with at least one duck reproductive trait (P<0.05). Haplotype combinations constructed on these SNPs were associated with egg weight at 40 weeks of age (EW40), fertility rate (FR) and maximum duration of fertility (MDF) (P<0.0001). In particular, diplotype H1H2 had positive effect on EW40, whereas it had negative effect on FR and MDF (P<0.05). Positive effects of the diplotype H1H5 were observed for FR and MDF, but a negative effect was observed for EW40 (P<0.05). This suggested that the PRL gene plays an important role in the regulation of egg weight and fertility-related traits and could be a potential marker in a marker assisted selection program during duck balancing selection. Further investigations on more duck populations with large sample sizes are needed to confirm this finding.