Distribution patterns of mast cells in the uterus of pregnant Meishan pigs.

The present study was performed to investigate the numerical distribution of mast cells (MCs) in the uteri of pregnant Meishan pigs to explore the functions of MCs in pig pregnancy. The uterine samples from pregnant (on days 15, 26 and 50 of gestation) pigs were obtained respectively and stained with toluidine blue. The results were as follows: MCs were constitutively located in the uterus of the Meishan pig, with the distribution varying with gestational stages. On days 15 and 26 of gestation, MCs were mainly distributed around the blood vessels and uterine glands within the endometrium. On day 50 of gestation, MCs were mostly distributed in the myometrium. These results indicated that uterine MCs possibly have versatile functions in pig pregnancy.

cDNA cloning of ghrelin and ontogeny of ghrelin mRNA expression in the gastrointestinal tract of African ostrich chicks.

Ghrelin is the endogenous ligand for the growth hormone secretagogue receptor (GHS-R). The sequence of ghrelin has been determined in many species ranging from fish to mammals. The ostrich is the largest herbivorous bird in the world. Although the distribution, morphological characteristics, and developmental changes of ghrelin-producing cells in the gastrointestinal tract of African ostrich chicks have recently been determined, the sequence and structure of ghrelin and its expression in the gastrointestinal tract of African ostrich chicks have not been studied. In the present study, the sequence and structure of ghrelin and its expression in the gastrointestinal tract of African ostrich chicks were investigated by reverse-transcriptase polymerase chain reaction (RT-PCR). Results of cDNA cloning revealed that African ostrich ghrelin is composed of 28 amino acid residues and the sequence of the 7 amino acids of the N-terminal region of African ostrich ghrelin was identical with that of other birds. Ninety-day-old female African ostriches were used to investigate the expression of ghrelin in the gastrointestinal tract. The results showed that ghrelin mRNA existed in the proventriculus, gizzard, duodenum, ileum, cecum, and rectum; there was no expression in the jejunum and colon. We observed developmental changes in the ghrelin mRNA expression in the stomach and small intestine of African ostriches. The results of the present study showed that ghrelin mRNA existed on day 1 in the proventriculus, but there was no expression in other tissues. On day 45, ghrelin mRNA existed in the proventriculus, gizzard, and ileum; however, there was no expression in the duodenum and jejunum. On day 90 and 334, we detected ghrelin mRNA in the proventriculus, gizzard, duodenum, and ileum, but there was no expression in the jejunum. The results of the present study clearly demonstrate that ghrelin mRNA exists and the distribution of ghrelin mRNA in the gastrointestinal tract of African ostriches changes with age (from postnatal day 1 to day 334).

Sequence characterization and promoter identification of porcine APC10 gene.

APC10 protein, a subunit of the anaphase-promoting complex (APC), plays an essential role in the progression of cells from mitosis to G1. In this study, we cloned and sequenced partial cDNA, intron 1 and 5'-flanking sequences of porcine APC10. The partial cDNA is 595 bp long and has an open reading frame of 558 bp which encodes 185 putative amino acids. Real-time PCR analysis revealed that the porcine APC10 mRNA expression shows a wide distribution and expression levels varies within a small different range in detected tissues. The deduced protein has a high identity with other eight species and comprises a conserved DOC domain. The phylogenetic tree indicated that porcine APC10 has the closest genetic relationship with human, monkey and dog. Promoter activity was demonstrated by transient transfection of 5'-deletion promoter/luciferase constructs into PK15 cells, and indicated that the proximal region from -2,052 to -1,764 is necessary for basal promoter activity. Positive cis-regulatory elements are present from -2,544 to -2,052 and from -3,114 to -2,774, while negative cis-regulatory elements may be present from -2,774 to -2,544. Yeast one-hybrid assay revealed Sp1 can interact with proximal promoter region of porcine APC10.

Expression and SNP association analysis of porcine FBXL4 gene.

As a kind of E3 ligase, the product of FBXL4 gene belongs to a member of FBLs which is the biggest eukaryotic subfamily of F-BOX proteins, it can recognize some substrate through particular protein-protein interaction domains. To investigate its functions, the polymorphism and association analysis was analyzed. The partial cDNA of porcine FBXL4 with 2384 bp long was first cloned; the deduced protein comprises a conserved F-BOX domain at position from the 277th to 332nd amino acid. The phylogenetic tree indicated porcine FBXL4 has the closest genetic relationship with bovine FBXL4 than other selected animal species. Ten tissue expression level of porcine FBXL4 mRNA fluctuated remarkably in a large range by quantitative RT-PCR analysis. For two identified SNPs, the genotyping analysis of Tail showed TT genotype owned dominance in introduced Landrace pig and miniature Guizhou and Wuzhishan breeds, but CC genotype was more than two other genotypes in miniature Laiwu breed. While in another genotyping analysis of BsaJI, CC genotype was obviously more than other genotypes in two kinds of Chinese miniature pig breeds and introduced Landrace pig breeds. Furthermore, the association analysis with immune traits and blood parameters revealed that SNP Tail was significantly associated with the lymphocyte percentage (P = 0.0166) and the antibody levels for pseudorabies virus vaccination (P = 0.0001) of neonate piglets at 0 day. Meanwhile, SNP BsaJI was significantly associated with lymphocyte percentage of individuals at 32 days (P = 0.0351), neutrophil percentage (P = 0.0005), the absolute lymphocyte count (P = 0.0458), and the mixed cells (P = 0.0010) of neonate piglets at 0 day.

Molecular characterization and expression analysis of nuclear oligomerization domain proteins NOD1 and NOD2 in grass carp Ctenopharyngodon idella.

Nuclear oligomerization domains (NODs) are cytosolic pattern recognition receptors (PRRs) to detect bacterial component. In this study, the molecular cloning and genomic characterization of grass carp NOD1 (gcNOD1) and grass carp NOD2 (gcNOD2) were reported. The complete open reading frame of gcNOD1 contains 2814 bp, encoding a 937-amino acid polypeptide. The gcNOD2 cDNA sequence encodes 982-amino acid polypeptide. Both gcNOD1 and gcNOD2 possess three conserved domains: carboxy terminal leucine rich repeat (LRR) domains, a central NOD, NBS or NACHT domain, and an amino terminal CARD domain (two in the case of NOD2). At the genomic level, gcNOD1 consists of 11 exons, with 10 intervening introns, spanning approximately 9 kb of genomic sequence. Whereas gcNOD2 has a length of approximately 5 kb with 9 intervening introns. Real time PCR analysis showed gcNOD1 and gcNOD2 were ubiquitously expressed in adult tissues. The highest transcript level of gcNOD1 was detected in brain, but in head kidney for gcNOD2. Grass carp reovirus significantly induced the expression of gcNOD1 and gcNOD2 in spleen (from days 1 to 6). However, expression profiles differed in time course response. Induction experiments with lipopolysaccharide (LPS), peptidoglycan (PGN) and polyI:C revealed the differential expression and regulation of gcNOD1 and gcNOD2 in blood, head kidney, trunk kidney and spleen. All these data suggest a potential role of NOD1 and NOD2 in fish innate immune protection to bacterial and virus infections.

Molecular characterization and expression analysis of the IFN-gamma related gene (IFN-gammarel) in grass carp Ctenopharyngodon idella.

Interferon gamma (IFN-gamma), the only member of the type II class of interferons, has been identified in teleost fish. In addition to the IFN-gamma gene, fish possess an IFN-gamma related gene (IFN-gammarel) neighbouring the authentic IFN-gamma gene in the genome. In the present study, the cDNA sequence encoding 167 amino acids of IFN-gammarel and its genomic organization were identified in grass carp Ctenopharyngodon idella. The predicted protein sequence of grass carp IFN-gammarel (gcIFN-gammarel) showed 63% and 50% identities to zebrafish and common carp IFN-gammarel (previously termed as IFN-gamma1), respectively. The IFN-gammarel gene consists of 4 exons, with 3 intervening introns, spanning approximately 2kb of genomic sequence. The gcIFN-gammarel gene did not contain any polymorphic DNA repeats in the introns. Realtime PCR analysis showed that grass carp reovirus induced a high and long lasting (from days 1 to 7) expression of gcIFN-gammarel in spleen. The expression of gcIFN-gammarel in blood, head kidney, trunk kidney and spleen was also increased by bacterial peptidoglycan (PGN), lipopolysaccharide (LPS) and the interferon inducer polyI:C. The highest induction of gcIFN-gammarel expression by PGN was observed in spleen, then in blood and head kidney. Further analysis of the expression patterns of gcIFN-gammarel and PGN receptors, nucleotide oligomerization domains (NOD) 1 and 2, may suggest that IFN-gammarel was possibly activated in a NOD2-dependent mechanism.

Partial molecular characterization, expression pattern, polymorphism and association analysis of porcine SKP2 gene.

As a component of E3 ubiquitin protein ligases called SCFs, SKP2 protein belongs to a member of FBLs protein which is the biggest eukaryotic subfamily of F-BOX proteins with 12 members. In this study, we cloned and sequenced partial cDNA, intron 1 and intron 6 of porcine SKP2 gene. The partial cDNA is 1,402 bp long and has an open reading frame of 1,272 bp which encodes 424 putative amino acids. The deduced protein comprises a conserved F-BOX domain at position from the 90th to 140th amino acid. The phylogenetic tree indicated that porcine SKP2 has the closest genetic relationship with bovine SKP2 than other selected animal species. Quantitative RT-PCR analysis displayed that the tissue expression level of porcine SKP2 fluctuated remarkably in a large range, and it expressed in thymus with the highest level and in longissimus dorsi muscle with the lowest level. Two SNPs were identified, meanwhile, further polymorphism analysis with Cfr42I showed that AA genotype was in dominance absolutely among four kinds of unrelated Chinese indigenous miniature and one introduced Landrace pig breeds. In addition, association analysis with immune traits and blood parameters revealed that the SNP Cfr42I in intron 1 was significantly associated with red cell distribution width of neonate piglets at 0 day (P = 0.027).

Strategic localization of toll-like receptor 4 in the digestive tract of blunt snout bream (Megalobrama amblycephala).

This study was performed to determine the localization strategies of Toll-like Receptor 4 (TLR4) in digestive tract (oesophagus, bulbodium, foregut, midgut and hindgut) of Blunt snout bream (Megalobrama amblycephala) using immunohistochemical staining method. TLR4 positive cells were observed throughout the digestive tract. In the oesophagus, some positive reactions in lamina propria were found around small blood vessels and there were also some positive cells within the stratified squamous epithelium. Lots of positive cells were observed in the muscular layer of the oesophagus. In bulbodium, foregut and hindgut, the expression of TLR4 was mainly restricted to the apical surface of epithelial cells located at the bottom of the mucosal folds and the mesenchymal cells in lamina propria. It was very interesting that epithelial cells in the midgut, but none in other parts, had many TLR4 positive cytoplasmic granular structures which were also periodic acid Schiff positive. These findings suggested that TLR4 was expressed in a compartmentalized manner in the Blunt snout bream (M. amblycephala) digestive tract and provided novel information about the in vivo localization of pattern recognition receptors.

Distribution and developmental changes in ghrelin-immunopositive cells in the gastrointestinal tract of African ostrich chicks.

Ghrelin, the endogenous ligand for the growth hormone secretagogue receptor (GHS-R), has been found in the gastrointestinal tract of many vertebrates, but little is known about its distribution in the gastrointestinal tract of African ostrich chicks. In the present study, the distribution, morphological characteristics, and developmental changes of ghrelin-producing cells in the gastrointestinal tract of African ostrich chicks were investigated using immunohistochemistry. Ghrelin-immunopositive (ghrelin-ip) cells were found to be localized in the mucous membrane of the entire gastrointestinal tract, but not in the myenteric plexus. The greatest number of ghrelin-ip cells was found in the proventriculus, and the ghrelin-ip cell density gradually decreased from the proventriculus to the rectum. Interestingly, from postnatal day 1 to day 45 in the proventriculus, and from postnatal day 1 to day 90 in the gizzard and small intestine, there was a steady increase in the number of ghrelin-ip cells. By day 45 in the proventriculus and day 90 in the gizzard and small intestine, the number of cells reached a plateau and remained steady. These results clearly demonstrate that ghrelin-ip cells exist and the number of ghrelin-ip cells increases with age in the African ostrich gastrointestinal tract from postnatal day 1 to day 90; ghrelin may be involved in gastrointestinal tract development.

Developmental morphology of the small intestine of African ostrich chicks.

The objective of this study was to investigate the morphological development of the small intestine of African ostrich chicks and to examine the changes in the number of goblet cells therein by observing the gross anatomy and performing histochemistry and morphometry. The BW; length, height, and width of the villi; muscle thickness; depth of the crypts; and number of goblet cells in the intestinal villi and crypts were measured on neonatal d 1, 45, 90, and 334. Our results revealed that the weights of the duodenum, jejunum, and ileum (relative to the BW) peaked on d 90, 45, and 45, respectively, and tended to decline thereafter. The villus height and width and muscle thickness in the small intestine were positively correlated with the age of the birds. The ratio of the villus height to the crypt depth differed among the segments of the small intestine and at the different time points. The number of goblet cells in the intestinal villi and crypts increased rapidly up to postnatal d 45 and then decreased rapidly between d 45 and 90. The number of goblet cells in the villi was greatest in the jejunum on d 1 and in the ileum on d 45, whereas that in the crypt was greatest in the ileum on d 1 and 90 and in the duodenum on d 45. These results suggest that the small intestine develops gradually from postnatal d 1 to 90 and that the period up to postnatal d 45 is marked by significant developmental changes in the parameters reflective of the digestive capacity, such as the weight, length, and surface area of the intestine and the number of goblet cells. Therefore, in reared African ostrich chicks, feed management should be enhanced between postnatal d 1 and 45.

Study of the morphology of the olfactory organ of African ostrich chick.

The anatomy and histology of the olfactory organ of African ostrich chick were carefully observed by gross anatomy observation, paraffin sectioning and haematoxylin-eosin (HE) staining. The results showed that there were no keratotic nose lids at the entrance of the external naris, and that the nasal cavity was separated into two imperforate compartments by the nasal septum. The posterior conchae were connected with the middle conchae without cohering to nasal walls, and appeared to be part of a palinal elongation of the middle conchae. Olfactory cells were distributed in the mucosal epithelium of middle and posterior conchae. Nasal glands were shaped like irregular rectangles, and their connective tissue extended to the parenchyma, which was divided into many glandular lobules. The layers of the olfactory bulb were indistinct, the globular structure was inconspicuous and the granular cells were scattered relatively in the lamina granularis externa.

Identification of an 85-kb DNA fragment containing pms1, a locus for photoperiod-sensitive genic male sterility in rice.

Photoperiod-sensitive genic male-sterile rice has a number of desirable characteristics for hybrid rice production. Previous studies identified pms1, located on chromosome 7, as a major locus for photoperiod-sensitive genic male sterility. The objective of this study was to localize the pms1 locus to a specific DNA fragment by genetic and physical mapping. Using 240 highly sterile individuals and a random sample of 599 individuals from an F2 population of over 5000 individuals from a cross between Minghui 63 and 32001S, we localized the pms1 locus by molecular marker analysis to a genetic interval of about 4 cM, 0.25 cM from RG477 on one side and 3.8 cM from R1807 on the other side. A contig map composed of seven BAC clones spanning approximate 500 kb in length was constructed for the pms1 region by screening a BAC library of Minghui 63 DNA using RFLP markers and chromosomal walking. Analysis of recombination events in the pms1 region among the highly sterile individuals reduced the length of the contig map to three BAC clones. Sequencing of one BAC clone, 2109, identified two SSR markers located 85 kb apart in the clone that flanked the pms1 locus on both sides, as indicated by the distribution of recombination events. We thus concluded that the pms1 locus was located on the fragment bounded by the two SSR markers.