Search

1. Over the past decade, rapid advancement in isolation methods for identifying markers of the once elusive intestinal stem cell (ISC) populations has laid the foundation for unravelling their complex interrelationships during homeostasis. Study on ISC in avian intestinal tissue might play a pivotal foundation for further studies on the epithelial-to-mesenchymal transition (EMT) in gastrointestinal disease and cell-based therapy as well as intestinal tissue engineering. 2. The following experiment isolated a population of fibroblast-like, plastic adhering cells derived from chick embryo intestine, showing a strong self-renewing and proliferative ability, which was maintained in vitro up to passage 25. The findings included growth characteristics, detected expression of cell surface markers and characterised the capability of these cells to differentiate towards the osteogenic, adipogenic, and chondrogenic cell lineages. 3. RT-PCR analysis showed that these cells from chick embryos expressed mesenchymal stromal cell markers CD44, CD90 and VIMENTIN as well as ISC-specific genes LGR5, MI1, SMOC2, BMI1, and HOPX. Immunofluorescence and flow cytometry confirmed this biology characterisation further. 4. In conclusion, cells were isolated from the intestine of 18-day-old chicken embryos that exhibited the biological characteristics of mesenchymal stromal cells as well as markers of intestinal stem cells. Our findings may provide a novel insight for in vitro cell culture and characteristics of ISCs in avian species, which may also indicate a benefit for obtaining cell source for intestinal tissue engineering as well as cell-based investigation for gastrointestinal disease and treatment.

Low temperature has become a major abiotic stress factor that can reduce maize yield and cause a number of economic loss. This study was designed to identify key genes and pathways associated with coldresistance of maize. The gene expression profile GSE46704, including 4 control temperature treated plants and 4 low temperature treated plants, was downloaded from the Gene Expression Omnibus database. Differentially-expressed genes (DEGs) were identified by limma package. Then, protein-protein interaction (PPI) network and module selection were constructed using Cytoscape. Moreover, the DEGs were re-matched based on the Zea mays L. gene ID and symbol data from PlantRegMap. Finally, the re-matched DEGs were performed functional and pathway enrichment analyses by the DAVID online tool. A total of 750 DEGs were screened (including 387 up-regulated and 363 down-regulated genes) In the PPI network, GRMZM2G070837_P01 and GRMZM2G114578_P01 had higher degrees. Besides, carbohydrate metabolic process, starch and sucrose metabolism and biosynthesis of secondary metabolites were significantly enriched in functional and pathway enrichment analysis. GRMZM2G070837_P01 and GRMZM2G114578_P01 might play a critical role in cold-resistance of maize. Meanwhile, carbohydrate metabolic process, starch and sucrose metabolism and biosynthesis of secondary metabolites might function in cold-resistance of maize.

Seedlings of the hypoxia-sensitive cucumber cultivar were hydroponically grown under hypoxia for 7 d in the presence or absence of 24-epibrassinolide (EBR, 2.1 nM). Hypoxia significantly inhibited growth, while EBR partially counteracted this inhibition. Leaf net photosynthetic rate (PN), stomatal conductance, transpiration rate, and water-use efficiency declined greatly, while the stomatal limitation value increased significantly. The maximum net photosynthetic rate was strongly reduced by hypoxia, indicating that stomatal limitation was not the only cause of the PN decrease. EBR markedly diminished the harmful effects of hypoxia on PN as well as on stomata openness. It also greatly stimulated CO2 fixation by the way of increasing the carboxylation capacity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), ribulose-1,5-bisphosphate regeneration, Rubisco activity, and the protection of Rubisco large subunit from degradation. Our data indicated that photosystem (PS) II was damaged by hypoxia, while EBR had the protective effect. EBR further increased nonphotochemical quenching that could reduce photodamage of the PSII reaction center. The proportion of absorbed light energy allocated for photochemical reaction (P) was reduced, while both nonphotochemical reaction dissipation of light energy and imbalanced partitioning of excitation energy between PSI and PSII increased. EBR increased P and alleviated this imbalance. The results suggest that both stomatal and nonstomatal factors limited the photosynthesis of cucumber seedlings under hypoxia. EBR alleviated the growth inhibition by improving CO2 asimilation and protecting leaves against PSII damage.

Hu sheep is one of the most important species in China; it is also listed as one of the 78 nationally protected domestic animals by the Chinese government in 2000. The construction of cDNA expression library of Hu sheep is of great significance for protecting individual genomes, generating transgenic sheep, and conducting clinical research using cDNA from Hu sheep. In this study, the total RNA from the ear tissue of Hu sheep was extracted, and a cDNA expression library was constructed using the SMART(TM) technique. The titer of amplified cDNA library was 1.09 x 10(10) PFU/mL, the rate of recombination was above 91.6%, and the average size of fragments was 1.1 kb. This study has an important significance for the preservation of Hu sheep resources at the genome level.

To investigate the genetic diversity of seven Chinese indigenous meat goat breeds (Tibet goat, Guizhou white goat, Shannan white goat, Yichang white goat, Matou goat, Changjiangsanjiaozhou white goat and Anhui white goat), explain their genetic relationship and assess their integrity and degree of admixture, 302 individuals from these breeds and 42 Boer goats introduced from Africa as reference samples were genotyped for 11 microsatellite markers. Results indicated that the genetic diversity of Chinese indigenous meat goats was rich. The mean heterozygosity and the mean allelic richness (AR) for the 8 goat breeds varied from 0.697 to 0.738 and 6.21 to 7.35, respectively. Structure analysis showed that Tibet goat breed was genetically distinct and was the first to separate and the other Chinese goats were then divided into two sub-clusters: Shannan white goat and Yichang white goat in one cluster; and Guizhou white goat, Matou goat, Changjiangsanjiaozhou white goat and Anhui white goat in the other cluster. This grouping pattern was further supported by clustering analysis and Principal component analysis. These results may provide a scientific basis for the characteristization, conservation and utilization of Chinese meat goats.

A Silkie Bantam embryo fibroblast line (named SBF59 line) was successfully established by using direct explant culture and cryopreservation techniques. Cell morphology, viability, dynamic growth and contamination were tested and the Karyotype and levels of isoenzymes of lactic dehydrogenase and malic dehydrogenase were analyzed. Four kinds of fluorescent protein extro-genes, including pEGFP-N 3 , pECFP-N 1 , pEYFP-N 1 and pDsRedl-Ni were transfected into the cells. The results showed that the cells were healthy and possessed a fibrous structure without a change in morphology. The average viability of the cells was 96% before freezing and 90.5% after thawing. The growth curve appeared as typical "S" shape and the cell growth passed through a detention phase, a logarithmic phase and a platform phase; the estimated population doubling time (PDT) was 38.5 h; assays for the presence of bacteria, fungi, viruses and mycoplasmas were negative; the cell line showed no cross contamination when assessed by isoenzyme analysis; the chromosome number was 2n = 78 on more than 88% of occasions; four kinds of fluorescent protein extro-genes appeared to be expressed effectively with a high transfection efficiency between 18.3% and 42.3%. The cell line met the required quality control standard. It not only preserves the genetic resources of the important Silkie Bantam at the cellular level but also provides valuable materials for genomic, post-genomic, somatic cell cloning research and other applications.

Lipopolysaccharide binding protein (LBP) and CD14 protein play important roles in the defense against infection of Gram-negative bacteria. In the present study, tissue distribution and polymorphism of porcine LBP and CD14 genes were analyzed. Real-time PCR results showed that the porcine LBP gene was especially highly expressed in liver, while CD14 gene was highly expressed in liver and spleen tissues. A 1,732 bp cDNA fragment of porcine LBP gene and a 1,682 bp genomic DNA fragment of CD14 gene were isolated. Polymorphisms were identified in these two fragments and showed that there were 14 potential SNPs in the porcine LBP gene and 3 potential SNPs in the porcine CD14 gene. Three SNPs, 292G/A (Gly/Ser), 1168G/A (Ala/Thr) of the LBP gene and -61G/A of the CD14 gene, were genotyped using restriction fragment length polymorphism (RFLP) method. Association analyses indicated that polymorphism of the 292G/A locus was significantly associated with porcine immune traits hematocrit (HCT), IgG and delayed-type hypersensitivity (DTH) (p

Egg production traits are economically important both for egg-laying and broiler lines of chicken. In sheep, the Q249R mutation in BMPR-IB is associated with ovulation rate. The present study cloned a partial chicken BMPR-IB fragment which contained the corresponding ovine Q249R mutation, including partial exon 6 and exon 7 and full-length intron 6. Five nucleotide changes were identified by alignment of the fragment amplified from Jining Bairi and Zang chickens. Among these nucleotide substitutions, the C/T transition at the base position of 35 and the A/G transition at the base position of 287 were found to be highly polymorphic, and named as SNPs C35T and A287G, respectively. For the SNP C35T, 331 hens of a synthetic broiler line were genotyped by a PCR-SSCP approach and allele C was found to be dominant. For the SNP A287G, 604 birds from the synthetic broiler line, a commercial egg-laying line, as well as three Chinese indigenous chicken breeds were genotyped by a PCR-RFLP technique. The associations of these two SNPs with egg production traits in the broiler line were analyzed. The results indicated that both the C35T and the A287G SNPs were not associated with egg production at 33wks and from 33wks to 42 wks (p>0.1), whereas the SNP A287G was associated with egg production from 47 to 56 wks (p

The Debao pony ear marginal tissue fibroblast cell line (NDPEM 2/2) was successfully established using either primary explant technique or collagenase technique. The characterizations of the cell line were identified as following: the cells were adherent and of density limitation; population doubling time (PDT) of cells made with the two techniques were 35.9 h and 48 h, respectively; chromosome analysis showed that the frequency of cell chromosome number to be 2n=64 was 91.3%-92.8%. Confirmed by isoenzyme analysis, this cell line had no cross- contamination. Tests for microbial contamination from bacteria, fungi, virus or mycoplasma were negative. This newly established cell line meets all the standard quality controls of ATCC. It will provide a precious genetic resource for the conservation of the Debao pony breed, as well as effective experimental material for genetic studies on Debao ponies. (Asian-Aust. J. Anim. Sci. 2004. Vol 17, No. 10 : 1338-1343)

In the present study, real time-polymerase chain reaction was applied to analyze the expression of IGF-I and MyoG genes in Hu sheep longissimus dorsi at different growth stages and their association with meat traits. Expression of the IGF-I gene in Hu sheep differed significantly between males and females at the two day-old (0.01P0.05), one-month old (0.01P0.05), and three month-old (P0.01) stages. IGF-I gene expression in male longissimus muscles was higher than that of females at all growth stages, except for the three month-old stage. There was no significant difference (P0.05) between males and females at any growth stage in expression of the MyoG gene. MyoG gene expression in male longissimus muscles tended to be higher than that of females at all growth stages, except for the six month-old stage. IGF-I gene expression was significantly and positively correlated with live weight (P0.01) and carcass weight (0.01P0.05), and was non-significantly positively correlated with net meat weight (P0.05). In contrast, MyoG gene expression was non-significantly and positively correlated with live weight, carcass, and net meat weight (P0.05). Carcass traits showed highly significant positive correlations (P0.01). Furthermore, expressions of IGF-I and MyoG genes showed highly significant positive correlations (P0.01). We conclude that the expressions of IGF-I and MyoG genes are significantly and positively correlated with early muscle traits of Hu sheep.

Besides stimulating GH release, some GH secretagogues also release ACTH and adrenal steroids. Several novel classes of potent GH secretagogues have recently been described, and we have now tested their ability to release corticosterone in conscious normal rats. All analogs that released GH also stimulated corticosterone release to some degree, though the relative effects on GH and corticosterone varied somewhat. The corticosterone responses for some analogs were in the range of those obtained with CRF (2 μg, iv), whereas closely related analogs inactive for GH release failed to release corticosterone. Activation of the hypothalamic-pituitary-adrenal axis with GH release by GHRPs could be a highly diabetogenic combination in susceptible individuals. Therefore, a potent GHRP pentapeptide analog (G7039, 100μ g/day, sc, bid) was given to young obese male Zucker diabetic fatty rats (ZDF, n = 8/group) for 24 days. Other groups received hGH (500 μg/day, sc, bid), recombinant human insulin-like growth factor (rhI...

5 pages; International audience; This article documents the addition of 411 microsatellite marker loci and 15 pairs of Single Nucleotide Polymorphism (SNP) sequencing primers to the Molecular Ecology Resources Database. Loci were developed for the following species: Acanthopagrus schlegeli, Anopheles lesteri, Aspergillus clavatus, Aspergillus flavus, Aspergillus fumigatus, Aspergillus oryzae, Aspergillus terreus, Branchiostoma japonicum, Branchiostoma belcheri, Colias behrii, Coryphopterus personatus, Cynogolssus semilaevis, Cynoglossus semilaevis, Dendrobium officinale, Dendrobium officinale, Dysoxylum malabaricum, Metrioptera roeselii, Myrmeciza exsul, Ochotona thibetana, Neosartorya fischeri, Nothofagus pumilio, Onychodactylus fischeri, Phoenicopterus roseus, Salvia officinalis L., Scylla paramamosain, Silene latifo, Sula sula, and Vulpes vulpes. These loci were cross-tested on the following species: Aspergillus giganteus, Colias pelidne, Colias interior, Colias meadii, Colias eurytheme, Coryphopterus lipernes, Coryphopterus glaucofrenum, Coryphopterus eidolon, Gnatholepis thompsoni, Elacatinus evelynae, Dendrobium loddigesii Dendrobium devonianum, Dysoxylum binectariferum, Nothofagus antarctica, Nothofagus dombeyii, Nothofagus nervosa, Nothofagus obliqua, Sula nebouxii, and Sula variegata. This article also documents the addition of 39 sequencing primer pairs and 15 allele specific primers or probes for Paralithodes camtschaticus.

Objective: We needed to establish an embryonic fibroblast cell line from the Langshan chicken (LSCEF61) to preserve their important genetic resources at the cellular level. Material and methods: The cell line was established from 9‐day‐old embryos by direct explant culture and cryopreservation techniques. Cell morphology, dynamic proliferation and any contamination present were tested, and the karyotype and levels of isoenzymes of lactic dehydrogenase and malic dehydrogenase were analysed. Four types of fluorescent protein exogenous genes for pEGFP‐C(1), pEGFP‐N(3), pEYFP‐N(1) and pDsRed1‐N(1) were transfected into the cells. Results: Showed that the cells were healthy and were of spindle shaped structure, without change in morphology. Cell growth curves were of typical S‐shape. Assays for microbial contamination were negative. The LSCEF61 line showed no cross‐contamination when assessed by isoenzyme analysis. Chromosome number (2n) = 78 on more than 90% of occasions. The four types of fluorescent protein extro‐genes appeared to be expressed effectively with high transfection efficiency between 15.6% and 38.6%. Conclusion: The cell line met each of the quality control standards required for the American Type Culture Collection. It had not only preserved the genetic resources of the important Langshan chicken at the cellular level, but also provided valuable material for genomic, post‐genomic and somatic cell cloning research and other applications.

The product of the OTUB1 gene is a member of the OTU superfamily of predicted cysteine proteases and inhibits cytokine gene transcription via its interaction with a ubiquitin protease and E3 ubiquitin ligase. To further understand the functions of the porcine OTUB1 gene, the subcellular localization of porcine OTUB1 protein was analyzed. We first cloned a partial DNA sequence of porcine OTUB1 which contained an 816 bp ORF encoding 271 amino acids. The deduced protein product was found to contain an OTU domain. The corresponding porcine OTUB1 protein was subsequently demonstrated to localize predominantly in the nucleus by confocal fluorescence microscopy. By spatial expression analysis, we further found that OTUB1 is highly expressed in the brain, liver, spleen, lung, kidney, large intestine, small intestine, stomach, ovary, uterus and thymus. In contrast, only low levels of this gene were evident in the heart, dorsal muscles and leg muscle of the pig. This is the first report to show the subcellular localization of porcine OTUB1, and our current data provides us with an important basis for conducting further studies on the functions and regulatory mechanisms underlying the role of OTUB1 gene in the immune system.

Nepal borders India in the south and China in the north. Four distinct indigenous domestic sheep breeds, i.e. Bhyanglung in the alpine region, Baruwal in the high hills, Kage in the mid hills and Lampuchhre in the low lands are distributed in the country. In this study, the mitochondrial DNA control region of 111 sheep from these four breeds was directly sequenced to determine their genetic variations and phylogenetic relationships. High mitochondrial DNA diversity among these breeds was observed and all haplotypes were classified into three haplogroups (A to C). Among the four breeds, three residing in middle to high hills had all three haplogroups while Lampuchhre sheep in low land only carried haplogroups A and B. This study revealed that a south-western route of gene flow in sheep might have come from China to India via Nepal. It can be concluded that these indigenous sheep breeds have isolated breeding paths. Lack of crossbreeding among Nepalese sheep breeds is unique, and it is important for the decision making on utilization and conservation of Nepalese sheep genetic resources. Tropical Agricultural Research Vol. 26 (4): 632 – 641 (2015)

Several high frequency restriction fragment length polymorphisms (RFLPs) associated with the human gene for apolipoprotein B have been previously reported by Priestly et al. The EcoRI RFLP here was shown to be very strongly associated with the Ag(t/z) immunochemical polymorphism of human low density lipoproteins, allowing correct Ag(t/z) phenotyping of 17 (out of 17 tested) unrelated individuals. The Xbal RFLP was associated with the Ag(g/c) immunochemical polymorphism, permitting correct phenotyping of 14 (out of 17 tested) unrelated individuals. Its close association with an RFLP permitted localization of the Ag(t/z) polymorphism to the C-terminal end of the apolipoprotein B peptide, and allowed detailed discussion of its probable molecular basis.

Apolipoprotein B (apoB) is a major protein component of low density and very low density lipoproteins. Because of its large size and heterogeneity, molecular studies of apoB have been difficult, and its structure and regulation remain poorly understood. We now report the identification of human apoB cDNA clones by antibody screening of hepatoma libraries in the expression vector lambda gt11. Both oligo(dT) primed and random primed libraries were constructed and screened with polyclonal antibodies to intact apoB, as well as with antibodies raised against a synthetic peptide based on the limited amino acid sequence available for apoB. The identity of the clones was unambiguously established by comparisons of the cloned cDNA sequences with apoB amino acid sequences. The clones hybridize to an exceptionally large 20 kb mRNA that is present in liver and intestine but not other tissues examined, consistent with the distribution expected from protein biosynthetic studies. The properties of the mRNA have implications for the biogenesis of the multiple apoB molecular weight forms secreted by liver and intestine.

Apolipoprotein B (apoB) is the major protein component of low-density and very-low-density lipoproteins. We have recently isolated nonoverlapping cDNA clones for apoB and confirmed their identity by sequence comparisons. We now report the mapping of the human apoB gene (APOB) to the p23-p24 region of chromosome 2 by examination of human-mouse somatic cell hybrids and by in situ hybridization to human chromosomes. Thus, APOB is unlinked to members of the dispersed gene family encoding other apolipoprotein species or to the gene encoding the low-density lipoprotein receptor. Hybridization analysis with genomic DNA and liver and intestinal mRNA suggests that APOB encodes both the high-molecular-weight form of apoB (apoB100) incorporated into very-low-density lipoproteins in liver and the lower-molecular-weight form (apoB48) incorporated into chylomicrons in intestine. Restriction fragment length polymorphisms of APOB have been identified and should prove useful in examining the possibility that genetic variations of APOB are involved in dyslipoproteinemias and atherosclerosis.

Berg et al. (Clin Genet 1986;30:515-520) have reported that an Xba I DNA polymorphic site in exon 26 of the apolipoprotein (apo) B gene is associated both with the Ag(x/y) immunochemical polymorphism and with elevated serum lipoprotein levels. Ma et al. (Arteriosclerosis 1987;7:301-305) have reported that the same Xba I polymorphism is associated with a different immunochemical polymorphism, Ag(c/g). To extend and clarify these observations, we have determined the Ag and Xba I polymorphism for 106 individuals. We find that the Xba I restriction fragment length polymorphism is in linkage disequilibrium with both Ag(x/y) and Ag(c/g) loci; thus, all 31 Xba I(X1/X1) genotypes observed in this study are also Ag(y/y). All but one of 22 Xba I(X2/X2) genotypes are also Ag(g/g). For individuals homozygous at either two or three of these loci, it was possible to determine the haplotypes for 128 apo B alleles. Of the eight possible apo B haplotypes, only four were represented in this unambiguous subpopulation, although other minor haplotypes were present in the total population from which it was derived. The identification of major apo B haplotypes in human populations may simplify the search for significant correlations between certain apo B alleles and lipid levels and atherosclerosis.

Southern blot analysis of the genomic DNA from 17 individuals showed perfect correspondence between a Bsp 12861 restriction fragment length polymorphism (RFLP) and the Ag(c/g) locus on human apolipoprotein (apo) B. The RFLP polymorphism is caused by a C--------T transition at nucleotide 421 on the cDNA, resulting in a threonine-to-isoleucine conversion. Thus, three of the five Ag sites have now been tentatively located on the 4536 residue apo B peptide at amino acyl residues 71: Ag(c/g), 591 (Ag(a1/d), and 4154 Ag(t/z).

Short cDNA fragments covering the entire coding region for apolipoprotein (apo) B have been cloned in the pING expression vector. A monoclonal antibody specific for the Ag(d) epitope on apo B has been used, together with the expressed apo B peptides, to locate this epitope to a stretch of 26 amino acids. Sequencing of this region from several genomic DNAs of known Ag(a1/d) genotype showed a single T-to-C substitution at nucleotide 1981, creating an Alu I restriction site and resulting in a val to ala residue change in the corresponding peptide (at position 591 in the mature protein). Southern blots, using the Alu I restriction endonuclease and a short probe for this region of the cDNA, showed perfect correspondence between the restriction fragment length polymorphism and the Ag(a1/d) immunochemical polymorphism in all 17 persons examined.