Promoter identification and analysis of key glycosphingolipid biosynthesis-globo series pathway genes in piglets.

Glycosphingolipid biosynthesis-globo series pathway genes (FUT1, FUT2, ST3GAL1, HEXA, HEXB, B3GALNT1, and NAGA) play an important regulatory role in the defense against Escherichia coli F18 in piglets. In this study, we identified the transcription initiation site and promoter of this gene cluster by mined previous RNA-seq results using bioinformatics tools. The FUT1 transcription initiation region included five alternative splicing sites and two promoter regions, whereas each of the six other genes had one promoter. Dual luciferase reporter results revealed significantly higher transcriptional activity by FUT1 promoter 2, indicating that it played a more important role in transcription. The promoters of glycosphingolipid biosynthesis genes identified contained a CpG island within the first 500 bp, except for the B3GALNT1 promoter which included fewer CpG sites. These results provide a deeper insight into methylation and the regulatory mechanisms of glycosphingolipid biosynthesis-globo series pathway genes in piglets.

Nramp1 gene expression in different tissues of Meishan piglets from newborn to weaning.

Natural resistance-associated macrophage protein gene 1 (Nramp1) plays an important role in the innate immune response of swine, and is believed to influence disease resistance. In this study, a real-time quantitative polymerase chain reaction technique was used to investigate Nramp1 expression in 12 different tissues in newborn and 7-, 14-, 21-, 28-, and 35-day-old Meishan piglets. Results indicated that Nramp1 was expressed to varying degrees in all sample tissues, although expression differed among growth stages. For example, Nramp1 was highly expressed in the spleen, but minimally expressed in heart, liver, and muscle tissues among the various piglet age classes. Overall, Nramp1 expression increased with age, reaching significant levels in 21- and 28-day-old animals. Nramp1 was expressed in all 12 tissues tested; however, expression in spleen, lung, kidney, and thymus tissues was highest among newborns, which is consistent with this gene's role in innate immunity improvement. Before and after weaning, Nramp1 was highly expressed in digestive (stomach) and intestinal (duodenum, jejunum, and ileum) tissues, further indicating a genetic role in both immune regulation to compensate for weaning stress and enhanced development of intestinal immunity.

Developmental expression of LTßR and differential expression in Escherichia coli F18 resistant/sensitive piglets.

We analyzed LTßR mRNA expression in piglets from birth to weaning and compared the differential expression between Escherichia coli F18-resistant and sensitive populations to determine whether this gene could be used as a genetic marker for E. coli F18 resistance. Sutai piglets of different age groups (8, 18, 30, and 35 days; N = 4 each) and piglets demonstrating resistance/sensitivity to E. coli F18 were used. LTßR expression levels were determined by real-time PCR. The LTßR expression levels in the lymph node, duodenum, and jejunum were significantly higher in 8-day-old piglets than in the other age groups (P < 0.01), and the expression levels were significantly higher in the lungs of 8-day-old piglets than in 35-day-old piglets (P < 0.01) and 30 day-old piglets (P < 0.05). In liver tissue, the expression level was significantly higher in the 35-day-old piglets than in other age groups (P < 0.01). In the stomach tissue, the expression level was significantly higher in 35-day-old piglets than in 18-day-old piglets (P < 0.05). LTßR expression in the lymph nodes was significantly higher in the resistant group than in the sensitive group (P < 0.01), but there was no significant difference in the other tissues (P > 0.05). These results indicate that 8 days after birth is a crucial stage in the formation of mesentery lymph nodes and immune barriers in pigs, and increased expression of LTßR may be beneficial for developing resistance to E. coli F18.

Isolation and characterization of microsatellite loci in Quercus fabri (Fagaceae).

Quercus fabri is a pioneer species of secondary succession in evergreen broadleaved forests in China. In this study, we isolated and developed 12 polymorphic and 2 monomorphic microsatellite loci for Q. fabri using the biotin-streptavidin capture method. We characterized 12 polymorphic loci in 52 individuals from two populations. The number of alleles per locus ranged from 3 to 23. The observed and expected heterozygosities per locus were 0.033-0.773 and 0.138-0.924, respectively. These microsatellite loci will facilitate the studies on genetic variation, mating system, and gene flow of Q. fabri.

Differential expression of FUT1 and FUT2 in Large White, Meishan, and Sutai porcine breeds.

To assess the relationship between the expression of a(1,2)-fucosyltransferase (FUT1 and FUT2) genes and resistance to Escherichia coli F18 in weaned pigs, FUT1 and FUT2 expression levels in Large White, Meishan, and Sutai pigs (with resistance to E. coli F18) were determined using real-time PCR. The results revealed that FUT1 and FUT2 expression levels were higher in the liver, lungs, kidneys, stomach, duodenum, and jejunum than in the muscle and heart. Medium FUT2 expression levels were detected in the spleen, thymus, and lymph nodes. Intestinal FUT1 expression levels were higher in Sutai pigs than in Large White and Meishan pigs (P < 0.05). However, intestinal FUT2 expression levels were lower in Sutai pigs than in Large White and Meishan pigs (P < 0.05). FUT1 and FUT2 expression levels did not differ between Large White and Meishan pigs (P > 0.05). The results revealed that high FUT1 expression levels and low FUT2 expression levels in the intestines of Sutai pigs affected FUT1 and FUT2 enzymes, the synthesis of type 2 H and type 1 H antigens, and E. coli F18 adhesion. Moreover, low FUT2 expression levels conferred resistance to E. coli F18.

Molecular cloning and expression analysis of MAT1 gene in black tiger shrimp (Penaeus monodon).

MAT1 (ménage à trois 1), an assembly factor and targeting subunit of the CDK-dependent kinase (CAK), can regulate the cell cycle, transcription, and DNA repair. This study was intended to investigate the role of MAT1 in the reproductive maturation of black tiger shrimp (Penaeus monodon). In this study, the P. monodon MAT1 (PmMAT1) gene was identified and characterized. The full-length cDNA of PmMAT1 was 1490 bp in length with an open-reading frame of 993 bp corresponding to 330 amino acids. The temporal expression of PmMAT1 in various tissues was measured by quantitative real-time PCR with the highest expression observed in ovaries. In the ovaries, the PmMAT1 gene was continuously but differentially expressed during the maturation stages. Comparative analyses of MAT1, CDK7, and cyclin H in the CAK complex of P. monodon indicated that the expression of CDK7 and cyclin H coincided with that of MAT1 during the ovary maturation stages. Serotonin (5-HT) injection promoted the expression level of PmMAT1 in the ovaries of shrimp at 6-48 h post-injection. These results indicate that PmMat1 plays a prominent role in the process of ovarian maturation.

Cloning, molecular characterization, and expression pattern of FGF5 in Cashmere goat (Capra hircus).

Fibroblast growth factor 5 (FGF5) is a secreted signaling protein that belongs to the FGF family, and was found to be associated with hair growth in humans and other animals. The Inner Mongolia Cashmere goat (Capra hircus) is a goat breed that provides superior cashmere; this breed was formed by spontaneous mutation in China. Here, we report the cloning, molecular characterization, and expression pattern of the Cashmere goat FGF5. The cloned FGF5 cDNA was 813 base pairs (KM596772), including an open reading frame encoding a 270-amino-acid polypeptide. The nucleotide sequence shared 99% homology with Ovis aries FGF5 (NM_001246263.1). Bioinformatic analysis revealed that FGF5 contained a signal peptide, an FGF domain, and a heparin-binding growth factor/FGF family signature. There was 1 cAMP- and cGMP-dependent protein kinase phosphorylation site, 11 protein kinase C phosphorylation sites, 4 casein kinase II phosphorylation sites, 1 amidation site, 1 N-glycosylation site, and 1 tyrosine kinase phosphorylation site in FGF5. Real-time polymerase chain reaction showed that FGF5 mRNA levels were higher in testis than in the pancreas and liver. These data suggest that FGF5 may play a crucial role in Cashmere goat hair growth.

Association between polymorphisms in exons 4 and 10 of the BPI gene and immune indices in Sutai pigs.

The bactericidal/permeability-increasing protein (BPI) gene has been identified as a candidate gene for disease-resistance breeding. We evaluated whether polymorphisms in exons 4 and 10 of the BPI gene are associated with immune indices [interleukin-2 (IL-2), IL-4, IL-6, interferon-b (IFN-b), IL-10, and IL-12]. In this study, we identified one mutation (C522T) in the BPI exon 4 site and two mutations (A1060G and T1151G) in the BPI exon 10 site. Correlation analysis revealed that in the Sutai pig population, the effect of genotypes at the BPI exon 4 site on the level of IL-6 was significant (P < 0.05), with an effective genotype of CD; moreover, the effect of genotypes at the BPI exon 10 site on the level of IL-12 was significant (P < 0.05), and the effective genotype was AB. The optimal combined genotype was CD-AB, which was more effective regarding the IL-6 and IL-12 levels compared to the other combined genotypes (P < 0.05). These results indicate that single nucleotide polymorphisms and the combined genotypes of BPI exons 4 and 10 affect immune indices in Sutai pigs. Therefore, these genotypes should be further examined as effective markers for disease-resistant breeding of pigs.

Identification of BPI protein produced in different expression system and its association with Escherichia coli F18 susceptibility.

The super antibiotic bactericidal/permeability-increasing (BPI) protein is a member of a new generation of proteins that have been implicated as endotoxin-neutralizing agents. In this study, recombinant porcine BPI protein was obtained by generating porcine BPI encoding prokaryotic, eukaryotic, and yeast expression vectors. Recombinant protein expression was detected in yeast GS115, Escherichia coli, and 293-6E cells by gel electrophoresis and Western blotting. Escherichia coli F18 is the primary Gram-negative bacteria in the gut and the main pathogen leading to diarrhea and edema dis-ease in weaning piglets. Therefore, E. coli F18-resistant and -sensitive Sutai piglets were used to test differential expression of BPI protein by Western blotting and to investigate the potential correlation between BPI protein expression and E. coli F18-susceptibility. Recombinant porcine BPI protein expression was not detected in the prokaryotic and yeast expression systems; however, soluble protein was detected in the eukaryotic expression system. These data indicate the strong bacterio-static action of the BPI protein and confirm the feasibility of obtaining large amounts of recombinant porcine BPI recombinant protein using this eukaryotic expression system. In addition, the BPI protein expres-sion levels in the E. coli F18-resistant group were significantly higher than those in the sensitive group, indicating that high BPI protein ex-pression is associated with resistance to E. coli F18. Our findings pro-vide a basis for further investigations into the development of a drug designed to confer resistance to E. coli F18 in weaning piglets.

Overexpression of protein kinase B/AKT induces phosphorylation of p70S6K and 4E-BP1 in goat fetal fibroblasts.

Protein kinases regulate many processes, including cell growth, metabolism, molecular interactions, and cell proliferation. Protein kinase B (PKB)/AKT (v-AKT mouse thymoma viral oncogene homolog) is an upstream component of mammalian target of rapamycin (mTOR) signaling and mediates pathophysiological processes in several signaling pathways. This study aimed to construct and overexpress a eukaryotic goat AKT expression vector in goat fetal fibroblasts and examine the effects of AKT on the phosphorylation of p70S6K and 4E-BP1. AKT was subcloned into the expression vector pIRES2-DsRed2 to generate pIRES2-DsRed2-AKT, which was transfected into goat fetal fibroblasts with LipofectamineTM 2000. AKT was measured by reverse transcription-polymerase chain reaction in the transgenic cells, and the expression of AKT and phosphorylation of p70S6K (Thr389) and 4E-BP1 (Thr37/46) were analyzed by Western blot. Cell clones that stably emitted red fluorescence were obtained after transfection for 48 h, and the exogenous gene was verified. Exogenous AKT was transcribed, and AKT was overexpressed, inducing the phosphorylation of p70S6K (Thr389) and 4E-BP1 (Thr37/46) in goat fetal fibroblasts. Thus, the overexpression of AKT activates mTOR signaling in goat cells.

Dynamic changes in TAP1 expression levels in newborn to weaning piglets, and its association with Escherichia coli F18 resistance.

The transporter associated with antigen processing (TAP) transports peptides from the cytosol into the endoplasmic reticulum for subsequent loading onto the major histocompatibility complex (MHC) class I molecules. This study showed the dynamic changes in the TAP1 expression level in newborn to weaning piglets. Tissue expression profiles revealed that the TAP1 gene was expressed at low levels in all tissues, and the expression levels were relatively higher in the lung, spleen, lymph, and thymus; further, no significant difference was observed in the expression in each tissue among the 3 unweaned stages (8, 18, and 30 days). Nevertheless, the postweaning (35 days) expression levels in tissues, including the spleen, lung, lymph, duodenum, and jejunum were significantly higher than those in the unweaned stages. Furthermore, gene ontology and pathway analysis showed that TAP1 took part in 38 biological functions and 5 pathway processes, including ABC transporters and antigen processing and presentation. These analyses showed that the TAP1 gene, which was related to MHC I immune regulation, had a stable and low expression level in unweaned stages; however, its expression increased in the postweaning stages. The high expression level of TAP1 indicated that the gene might play an important role in Escherichia coli F18 resistance.

TLR4 gene expression in pig populations and its association with resistance to Escherichia coli F18.

TLR4 is the main recognition receptor of bacterial lipopolysaccharides, which play an important role in innate and adaptive immunity. We used real-time PCR to analyze the tissue expression profile and differential expression of TLR4 in 4 pig populations (Escherichia coli F18-resistant Sutai, E. coli F18-sensitive Sutai, Large White, Meishan), in order to determine the role that the TLR4 gene plays in resistance to E. coli F18. We found that TLR4 expressed consistently in the 4 populations, with relatively high levels in immune tissues and the highest level in the lung. Generally, the expression of TLR4 in E. coli F18-sensitive individuals was the highest, followed by that in E. coli F18-resistant, Large White and Meishan. In the spleen, lung, kidney, lymph nodes, and thymus gland, TLR4 expression is significantly higher in the E. coli F18-sensitive than in the other 3 populations; there were no significant differences among E. coli F18-resistant Sutai, Large White, and Meishan. In addition, Gene Ontology and pathway analysis showed that TLR4 takes part in the inflammatory response. We found that porcine TLR4 has consistent tissue specificity in each breed, and downregulation of expression of the TLR4 gene is related to resistance to E. coli F18 in weaning piglets.

Age-dependent expression of the BPI gene in Sutai piglets.

We compared and analyzed the expression of the BPI gene of Sutai piglets ranging from newborn to post-weaning days 8, 18, 30, and 35 by the real-time PCR method, in order to determine if it is involved in protection against disease caused by ETEC F18. There was a significant difference between 18 and 35-day expression in the jejunum. There were also significant differences between 35-day expression and expression at the other development stages in the duodenum. There were no significant differences in expression at 8, 18, and 30 days in the jejunum. We conclude that the porcine BPI gene may be the direct factor that resisted the ETEC F18 in weaning piglets, and that the resistance to ETEC F18 in weaning piglets is related to up-regulation of mRNA expression of BPI gene to a certain extent.

Effects of secular trends in obesity on coronary risk factors in children: the Bogalusa Heart Study.

OBJECTIVE: To determine whether a secular trend toward increased weight gain was present in children examined 11 years apart in the Bogalusa Heart Study and, if present, the association of this trend with the cardiovascular risk status of the children. STUDY DESIGN: Two biracial cohorts (approximately 65% white, 35% black) were identified. One cohort was examined first in 1973 at 7 to 9 years of age and was reexamined in 1981 (n = 417). The second was examined first in 1984 at the same age and reexamined in 1992 (n = 235). Measurements made at each assessment included age, gender, race, height, weight, blood pressure, and lipoproteins. The two cohorts were then compared. RESULTS: The two cohorts were comparable at their first assessment. However, at follow-up 8 years later the more recent cohort was 5 to 7 kg heavier without any difference in linear growth; this increased ponderosity was associated with adverse changes in lipids and lipoproteins: the high-density lipoprotein-cholesterol concentration was 0.15 to 0.35 mmol/L (6 to 13 mg/dl) lower, triglyceride values were 0.09 to 0.40 mmol/L (8 to 36 mg/dl) higher, and there were small increases in total cholesterol and low-density lipoprotein-cholesterol concentrations in white girls. Multivariate analyses showed that in the more recent cohort these changes were related more to a change in ponderosity than to ponderosity. Although blood pressure was generally lower in the recent cohort, increasing ponderosity was associated with higher blood pressure. CONCLUSIONS: There is a secular trend toward increased ponderosity in children examined in the Bogalusa Heart Study. This trend is associated with worsening cardiovascular risk, particularly with regard to lipoproteins.