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Cyadox is a good antimicrobial growth-promoter of quinoxalines. However, the molecular mechanism of action remains unclear. A growth performance study and mRNA differential display reverse transcription polymerase chain reaction (DDRT-PCR) in combination with Northern dot-blot and reverse Northern dot-blot analysis were conducted to determine the differentially expressed genes in liver tissues of piglets after treatment with cyadox. Transcription levels of the differentially expressed genes were quantificated by realtime RT-PCR in porcine primary hepotocytes. Cyadox could significantly promote body weight of piglets via feed with average daily gain (ADG) improved by 24.7% and 64.8% in 100 and 500mg/kg group, compared with control. A total of eight differentially expressed genes were found, of which the expression levels of five genes had positive correlation with cyadox dose. One gene expression had a negative correlation with cyadox dose and it was a new gene. The other two genes were up-regulated by cyadox, but the expression quantity was invariably when the cyadox doses were increased. Among the up-regulated genes, one was transcriptional regulating factor, two were growth-related factors, one was involved in immune defense and immune-regulation and three might be involved in the maintenance of normal development. In primary cultured pig hepatocytes, cyadox treatment evoked a significant time-dependent effect of eight genes expression. The results suggest, at the transcriptional level in vitro and in vivo, that growth factor and metabolism may be associated with cyadox growth-promoting activity, whereas immune defense and immune-regulation could play major roles in prophylaxis of cyadox in piglets., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: Recently identified porcine circovirus-like virus P1 has the smallest DNA viral genome. In this study, we identified the viral genes and their corresponding mRNA transcripts., Results: The RNAs of P1, synthesized in porcine kidney cells, were examined with northern blotting and PCR analyses. Eight virus-specific RNAs were detected. Four mRNAs (open reading frames (ORFs) 1, 2, 4, and 5) are encoded by the viral (-) strand and four (ORFs 3, 6, 7, and 8) are encoded by the viral (+) strand. All proteins encoded by the ORFs of the P1 virus are less than 50 amino acids in length, except that encoded by ORF1 (113 amino acids)., Conclusions: We show a very complex viral transcription pattern in P1-infected cells.

Salicornia brachiata Roxb., an extreme halophyte, is a naturally adapted higher plant model for additional gene resources to engineer salt tolerance in plants. Ascorbate peroxidase (APX) plays a key role in protecting plants against oxidative stress and thus confers abiotic stress tolerance. A full-length SbpAPX cDNA, encoding peroxisomal ascorbate peroxidase, was cloned from S. brachiata. The open reading frame encodes for a polypeptide of 287 amino acid residues (31.3-kDa protein). The deduced amino acid sequence of the SbpAPX gene showed characteristic peroxisomal targeting sequences (RKRAI) and a C-terminal hydrophobic region of 39 amino acid residues containing a transmembrane domain (TMD) of 23 amino acid residues. Northern blot analysis showed elevated SbpAPX transcript in response to salt, cold, abscisic acid and salicylic acid stress treatments. The SbpAPX gene was transformed to tobacco for their functional validation under stresses. Transgenic plants over-expressing SbpAPX gene showed enhanced salt and drought stress tolerance compared to wild-type plants. Transgenic plants showed enhanced vegetative growth and germination rate both under normal and stressed conditions. Present study revealed that the SbpAPX gene is a potential candidate, which not only confers abiotic stress tolerance to plants but also seems to be involved in plant growth.

The characterization and quantitative analyses of the key transcription factors for spermiogenesis are necessary in the identification of causal factors for the production of the seemingly normal sperm with dysfunctions in Japanese Black bulls and further elucidation of whole aspect of molecular mechanisms for spermiogenesis in livestock. The objective of this study was to obtain the information regarding the characterization and individual changes of an activator cAMP-responsive element modulator (CREM), which is necessary to the normal progress of spermiogenesis and is required for the transcriptional activity of genes coding essential factors for the sperm fertilization ability in rodents, using testes from 21 Japanese Black bulls with the ability to produce sperm indicating the normal motility and morphology. The bull CREM ταγ (one of activator variants) was detected in testes more strongly than livers by reverse transcription-polymerase chain reaction and Northern blotting. This variant was localized in the nuclei of spermatids as shown by indirect immunofluorescence with the homemade mouse antiserum. The motility and morphology of the cauda epididymal sperm from 16 Japanese Black bulls were examined before the quantitative analyses of testicular activator CREM to confirm the ability to produce sperm with normal motility and morphology in these males. The percentages of the motile sperm, those of the sperm with the normal acrosomes, and those of morphologically normal sperm were 60.0% to 90.0%, 88.0% to 100%, and 83.0% to 97.9%, respectively. The quantitative analyses with real-time polymerase chain reaction using the testicular RNA from the same bulls revealed that the relative expression levels of activator CREM variants in testes varied significantly among these bulls in the range from 0.56 to 1.64 (P < 0.05). These results are consistent with the suggestions that CREM ταγ are involved in the spermiogenesis in the testes of Japanese Black bulls and that the expression levels of the activator CREM variant mRNAs in the testes are varied significantly among individual bulls that have the ability to produce sperm with the normal motility and morphology., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Marek's disease virus (MDV) encodes a ribonucleotide reductase (RR), a key regulatory enzyme in the DNA synthesis pathway. The gene coding for the RR of MDV is located in the unique long (UL) region of the genome. The large subunit is encoded by UL39 (RR1) and is predicted to comprise 860 amino acids whereas the small subunit encoded by UL40 (RR2) is predicted to be 343 amino acids long. Immunoprecipitation analysis of MDV-1 (GA strain)-infected cells with T81, a monoclonal antibody specific for RR of MDV, identified two major proteins of 90,000 and 40,000 daltons, corresponding to RR1 and RR2, respectively. In addition, RR was abundantly expressed in the cytoplasm of cells infected with 51 strains of MDV belonging to MDV serotypes 1, 2, and 3 as demonstrated by immunofluorescence staining. Northern blot analysis of RNA extracted from MDV-infected cells showed a major band of around 4.4 kb in size corresponding to the RR1 and RR2 transcripts. In vivo, RR was abundantly expressed in lymphoid organs and feather follicle epithelium of MDV-infected chickens during early cytolytic infection, as determined by immunohistochemistry. There was, however, no expression of RR in MDV-induced tumors in lymphoid organs. The abundant expression of RR in MDV-infected chicken may suggest an important role of RR in the conversion of ribonucleotides to deoxyribonucleotides for MDV DNA synthesis.

The characterization of the immune response of chickens to Salmonella infection is usually limited to the quantification of expression of genes coding for cytokines, chemokines or antimicrobial peptides. However, processes occurring in the cecum of infected chickens are likely to be much more diverse. In this study we have therefore characterized the transcriptome and proteome in the chicken cecum after infection with Salmonella Enteritidis. Using a combination of 454 pyrosequencing, protein mass spectrometry and quantitative real-time PCR, we identified 48 down- and 56 up-regulated chicken genes after Salmonella Enteritidis infection. The most inducible gene was that coding for MMP7, exhibiting a 5952 fold induction 9 days post-infection. An induction of greater than 100 fold was observed for IgG, IRG1, SAA, ExFABP, IL-22, TRAP6, MRP126, IFNγ, iNOS, ES1, IL-1β, LYG2, IFIT5, IL-17, AVD, AH221 and SERPIN B. Since prostaglandin D2 synthase was upregulated and degrading hydroxyprostaglandin dehydrogenase was downregulated after the infection, prostaglandin must accumulate in the cecum of chickens infected with Salmonella Enteritidis. Finally, above mentioned signaling was dependent on the presence of a SPI1-encoded type III secretion system in Salmonella Enteritidis. The inflammation lasted for 2 weeks after which time the expression of the "inflammatory" genes returned back to basal levels and, instead, the expression of IgA and IgG increased. This points to an important role for immunoglobulins in the restoration of homeostasis in the cecum after infection.

ERp57 is a member of a protein disulfide isomerase family and is a chaperone responsible for the correct folding of newly synthesized glycoproteins in the endoplasmic reticulum and in the assembly of the major histocompatibility complex class I in the endogenous pathway of antigen presentation. This study reports the identification of a full length ERp57 cDNA in rainbow trout that encodes a putative 477aa mature protein with an additional signal sequence of 16aa. The trout protein shared 75% identity with the human homolog, but interestingly did not include either a C terminal endoplasmic reticulum retention signal, Q/KEDL in humans, or a nuclear localization signal which is highly conserved in mammals. Amino acid sequence alignment revealed conservation of four classical domains in trout ERp57 and two conserved active CXXC redox motifs. Trout ERp57 protein was identified as a single band around 57 kDa. Southern blotting analysis revealed that there two copies of the ERp57 gene in the trout genome and northern blotting showed a wide tissue distribution of gene expression in various tissues with the highest expression in liver and egg. This study showed for the first time in teleost that ERp57 transcript is upregulated in response to immune stimuli such as double stranded RNA or phytohemagglutinin. Furthermore, upon treatment with ER stress inducer A23187, trout ERp57 protein expression levels were increased both in peripheral blood leukocytes and the RTS11 macrophage like cell line after 6 and 8 h respectively. These findings suggest a possible conserved function for trout ERp57 in the ER and during the activation of the immune response., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

A full-length cDNA sequence of canine L-type amino acid transporter 1 (Lat1) was determined from a canine brain. The sequence was 1828 bp long and was predicted to encode 485 amino acid polypeptides. The deduced amino acid sequence of canine Lat1 showed 93.2% and 91.1% similarities to those of humans and rats, respectively. Northern blot analysis detected Lat1 expression in the cerebellum at 4 kb, and Western blot analysis showed a single band at 40 kDa. RT-PCR analysis revealed a distinct expression of Lat1 in the pancreas and testis in addition to the cerebrum and cerebellum. Notably, Lat1 expression was observed in the tissues of thyroid cancer, melanoma and hemangiopericytoma. Although the cancer samples examined were not enough, Lat1 may serve as a useful biomarker of cancer cells in veterinary clinic.

Objective: To compare in vitro expansion, explant colonization, and matrix synthesis of equine tendon- and bone marrow-derived cells in response to insulin-like growth factor-I (IGF-I) supplementation., Sample: Cells isolated from 7 young adult horses., Procedures: Tendon- and bone marrow-derived progenitor cells were isolated, evaluated for yield, and cultured on autogenous cell-free tendon matrix for 7 days. Samples were analyzed for cell viability and expression of collagen type I, collagen type III, and cartilage oligomeric matrix protein mRNAs. Collagen and glycosaminoglycan syntheses were quantified over a 24-hour period., Results: Tendon- and bone marrow-derived cells required 17 to 19 days of monolayer culture to reach 2 passages. Mean ± SE number of monolayer cells isolated was higher for tendon-derived cells (7.9 ± 0.9 × 10(6)) than for bone marrow-derived cells (1.2 ± 0.1 × 10(6)). Cell numbers after culture for 7 days on acellular tendon matrix were 1.6- to 2.8-fold higher for tendon-derived cells than for bone marrow-derived cells and 0.8- to 1.7-fold higher for IGF-I supplementation than for untreated cells. New collagen and glycosaminoglycan syntheses were significantly greater in tendon-derived cell groups and in IGF-I-supplemented groups. The mRNA concentrations of collagen type I, collagen type III, and cartilage oligomeric matrix protein were not significantly different between tendon- and bone marrow-derived groups., Conclusions and Clinical Relevance: In vitro results of this study suggested that tendon-derived cells supplemented with IGF-I may offer a useful resource for cell-based strategies in tendon healing.

Objective: To compare in vitro expansion of equine tendon- and bone marrow-derived cells with fibroblast growth factor-2 (FGF-2) supplementation and sequential matrix synthesis with pulverized tendon and insulin-like growth factor-I (IGF-I)., Sample: Cells from 6 young adult horses., Procedures: Progenitor cells were expanded in monolayers with FGF-2, followed by culture with autogenous acellular pulverized tendon and IGF-I for 7 days. Initial cell isolation and subsequent monolayer proliferation were assessed. In pulverized tendon cultures, cell viability and expression of collagen types I and III and cartilage oligomeric matrix protein (COMP) mRNAs were assessed. Collagen and glycosaminoglycan syntheses were quantified over a 24-hour period., Results: Monolayer expansion with FGF-2 significantly increased the mean ± SE number of tendon-derived cells (15.3 ± 2.6 × 10(6)), compared with bone marrow-derived cells (5.8 ± 1.8 × 10(6)). Overall, increases in collagen type III and COMP mRNAs were seen in tendon-derived cells, compared with results for bone marrow-derived cells. After IGF-I supplementation, increases in collagen type I and type III mRNA expression were seen in bone marrow-derived cells, compared with results for unsupplemented control cells. Insulin-like growth factor-I significantly increased collagen synthesis of bone marrow-derived cells. Monolayer expansion with FGF-2 followed by IGF-I supplementation significantly increased glycosaminoglycan synthesis in tendon-derived cells., Conclusions and Clinical Relevance: Tendon-derived cells had increased cell numbers and matrix synthesis after monolayer expansion with FGF-2, compared with results for bone marrow-derived cells. In vivo experiments with FGF-2-expanded tendon-derived cells are warranted to evaluate effects on tendon healing.

In mammals, several cell surface molecular markers have been characterized in order to identify the mitotic germ cells. However, little is known in fish about their cell surface antigen. In this study, we identified lymphocyte antigen 75 (Ly75/CD205) as a germ cell-specific cell surface marker by combination expressed sequence tag analysis of purified type A spermatogonia (A-SG) from immature testis, in silico prediction of membrane proteins, and expression studies. The ly75 transcripts were abundant in the testis and gills, and weak signals were detected in the head kidney and brain. In addition, ly75 mRNA was predominantly localized in the primordial germ cells of newly hatched embryos, A-SG in testis, oogonia, and chromatin nucleolus-stage oocytes in the ovary. In contrast, ly75 mRNA was not detected in spermatocytes, spermatids, spermatozoa, vitellogenic oocytes, or gonadal somatic cells from either males or females. The expression profile of Ly75 protein was similar to that of the mRNA. Furthermore, identification of various fish homologs of ly75 confirmed that their amino acid sequences are well conserved. Therefore, Ly75 may be appropriate for use as a versatile cell surface marker for mitotic germ cells in fish.

Estradiol increases basal growth hormone (GH) concentrations in sheep and cattle. This study sought to determine the effects of estradiol on GH-releasing hormone (GRH)-stimulated GH release in sheep. Growth hormone secretory characteristics, the GH response to GRH, and steady-state GH mRNA concentrations were determined in castrated male lambs treated with 2 different doses of estradiol 17-beta for a 28-d experimental period. Although no differences between treatments in mean GH, basal GH, or GH pulse number were observed after 28 d of estradiol treatment, GH pulse amplitude was greater (P < 0.05) in the 2.00-cm implant-treated animals than in the control and 0.75-cm implant group. The effect of estradiol treatment on GRH-stimulated GH release revealed differences between the control and estradiol-treated animals (P < 0.05). The 15-min GH responses to 0.075 microg/kg hGRH in the control, 0.75-cm, and 2.00-cm implant groups, respectively, were 76 +/- 10, 22.6 +/- 2.1, and 43.6 +/- 15.0 ng/mL. Growth hormone mRNA content was determined for pituitary glands from the different treatment groups, and no differences in steady-state GH mRNA levels were observed. There were no differences in the mean plasma concentrations of IGF-I, cortisol, T(3), or T(4) from weekly samples. Growth hormone release from cultured ovine pituitary cells from control sheep was not affected by estradiol after 72 h or in a subsequent 3-h incubation with estradiol combined with GRH. These data suggest that estradiol has differing actions on basal and GRH-stimulated GH concentrations in plasma, but the increase in pulse amplitude does not represent an increased pituitary sensitivity to GRH.

The avian inner perivitelline layer (IPVL) contains zona pellucida protein-B1 (ZPB1), zona pellucida protein-C (ZPC) and zona pellucida protein-D (ZPD). These three proteins may be involved in sperm binding to the IPVL. ZPB1 is produced by the liver and transported to the developing preovulatory follicle, while ZPC and ZPD are synthesized and secreted by the granulosa cells of the preovulatory follicle. The mRNA of ZPB1, ZPC, and ZPD was investigated in two lines of turkey hens selected for over 40 generations for either increased egg production (E line) or increased body weight (F line). Total RNA was extracted from the liver and from 1cm(2) sections of the granulosa layer around the germinal disc and a nongerminal disc area of the F(1) and F(2) follicles of hens from each genetic line. Northern analysis was performed using chicken cDNA probes for all three ZP proteins. Hepatic mRNA for ZPB1 was greater (P<0.05) in turkey hens from the E line than the F line. Although, there was no difference in ZPC mRNA between the germinal disc and nongerminal disc region of the two largest follicles in E line hens, ZPC mRNA was greater in the nongerminal disc region compared to the germinal disc region in the two largest follicles obtained from the F line hens. There were no differences in ZPD mRNA between the germinal disc and nongerminal disc regions of the F(1) and F(2) follicles for either genetic line. The results suggest that the greater rates of fertility previously observed in eggs from the E line hens compared with the F line of hens may be related to differential amounts of the potential sperm binding proteins ZPB1 and ZPC.

Expressed sequence tag (EST) analysis is an efficient tool for gene discovery and profiling gene expression. Aeromonas hydrophila, a ubiquitous waterborne bacterium, is one of the most frequent pathogens isolated from diseased aquatic organisms. In order to understand the molecular mechanism of anti-bacteria immune response in reptile, we have investigated the differentially expressed genes in Chinese soft-shelled turtle (Trionyx sinensis) experimentally infected with A. hydrophila by suppression subtractive hybridization (SSH). Forty-two genes were identified from more than 200 clones, of which 25 genes are found for the first time in reptiles, and classified into 6 categories: 18 in defense/immunity, 4 in catalysis, 2 in retrotransposon; 2 in cell signal transduction, 5 in cell metabolism, 10 in protein expression, and 1 in cell structure. Of the 42 differentially expressed genes, 6 genes, IL-8, serum amyloid A (SAA), CD9, CD59, activating transcription factor 4 (ATF4) and cathepsin L genes, were further observed to be up-regulated in the infected turtles by virtual Northern hybridization and RT-PCR assays.

C1q, a subunit of the C1 complex, plays a key role in the recognition of immune complexes to initiate the classical complement pathway. In this study, we reported two C1q-like cDNAs from mandarin fish (Siniperca chuatsi), mC1q-like-1 (mC1qL1) and mC1q-like-2 (mC1qL2). The full-length cDNA of mC1qL1was 990bp, containing a 71bp 5'-untranslated region (UTR), an open reading frame (ORF) of 723bp, and a 196bp long 3'-UTR. mC1qL2 cDNA was 1193bp, containing a 100bp 5'-UTR, followed by an ORF of 756bp and a 3'-UTR of 337bp. mC1qL1 and mC1qL2 share 29% identity in amino acid sequence. Both mC1qL1 and mC1qL2 contained three parts: a short amino-terminal region, a collagen-like region and a carboxyl-terminal globular C1q domain. The phylogenetic analysis showed that mC1qL1 clustered with two Danio rerio hypothetical proteins and further grouped with C1q proteins, while mC1qL2 clustered with C1qA proteins from other species. In healthy mandarin fish, mC1qL1 and mC1qL2 were expressed in all tissues tested, including liver, spleen, head kidney, caudal kidney, intestine and gill. mC1qL1 was highly expressed in head kidney, while mC1qL2 was mainly expressed in spleen. The expression level of mC1qL1 and mC1qL2 in liver were not changed obviously and mC1qL2 was significantly changed (p<0.05) in spleen after infectious spleen and kidney necrosis virus (ISKNV) infection. Mandarin fish C1q may play a role in response to ISKNV infection.

The meiotic maturation of oocyte and spermatocyte in animals is controlled by the maturation promotion factor (MPF), a complex of Cdc2 and cyclin B proteins. To better understand the mechanism of oocyte and spermatocyte maturation in fish, the expression of cyclin B1 (CB1), B2 (CB2) and Cdc2 kinase during oogenesis and spermatogenesis in rainbow trout were examined at both the mRNA and protein levels. Quantitative real-time PCR analysis showed that the amount of CB1 and CB2 mRNA was greater at previtellogenesis and late vitellogenesis stages, but less at early vitellogenesis stage and during early embryogenesis. Cdc2 mRNA was continuously present throughout the processes of oogenesis and early embryogenesis except for a decline at early vitellogenesis. In situ hybridization analysis indicated that CB1, CB2 and Cdc2 transcripts were present in oocytes of different developmental stages as well as in all spermatogenic cells except for spermatogonia. Immunohistochemical analysis revealed that CB1 protein was absent in vitellogenic oocytes, but present in young previtellogenic and mature oocytes. In contrast, CB2 and Cdc2 proteins were present at all stages oocyte development. Similarly, CB2 and Cdc2 proteins were present throughout spermatogenesis, whereas CB1 protein was only detected in spermatogonia and spermatocytes, but not in spermatids. Thus, it appears that CB1, CB2 and Cdc2 transcripts have similar expression patterns during oogenesis and spermatogenesis, but CB1 protein varies in amount during these processes. These data suggest that CB1 may have a leading role in the regulation of meiotic maturation of oocytes and spermotocytes.

Atrazine is an extensively used triazine herbicide in agricultural and residential areas and has been routinely detected in many surface and ground waters. This study reveals various up- and down-regulated genes associated with hypoxic stress in atrazine-treated fourth-instar Chironomus tentans larvae (midges) by using restriction fragment differential display-PCR. Two down-regulated hemoglobin cDNAs were isolated from the midges. Northern blot analysis indicated CteHb-IIbeta and CteHb-III mRNA expressions decreased by 36 and 21%, respectively, in midges exposed to atrazine at 1 microg/L for 96h. Decreased hemoglobin gene expression was associated with elevated oxygen consumption in atrazine-treated midges. Midges exposed to atrazine at 1 microg/L increased their oxygen consumption by 47%, whereas midges exposed to atrazine at 1000 microg/L for 48h increased their oxygen consumption by 66%. Our study demonstrates for the first time that atrazine, at environmentally relevant concentrations, can elevate respiration, possibly eliciting counteractive measures at the transcriptional level to adapt to oxygen deficiency in an ecologically important aquatic insect. Our results further suggest that the ability to modulate both the quantity and quality of Hb serves as an adaptive response to counteract the initial onset of oxygen deficiency induced by atrazine in midges.

beta-Catenin signaling has been reported to initiate feather bud development. In the present study, beta-catenin gene was isolated and identified from a cDNA library constructed using embryonic goose skin. Expression patterns of beta-catenin gene in the dorsal skin of goose embryos were investigated using the methods of semi-quantitative reverse transcription PCR, Northern blot analysis, and in situ hybridization. The sequence of beta-catenin was found highly conserved at the amino acid level, sharing 100, 99, and 99% identity with chicken, Chinese soft-shell turtle, and human sequences, respectively. Relatively high levels (62.51 +/- 7.11% to 101.74 +/- 7.29%) of beta-catenin mRNA were detected in the dorsal skin samples. The levels of beta-catenin expression were most prominent at the early stage from embryo day (E)10 to E20 and then significantly declined with the embryonic development. In situ hybridization demonstrated that at E10, beta-catenin expression was mainly observed at the surface periderm cells and the localized region of the epidermal layer. Because feather bud forms with an anterior-posterior orientation, strong staining was observed in the periderm layer and in the ectoderm and epidermis with a diffuse distribution within the internal area of the buds. The stronger staining was seen in the barb ridges than in the center pulp of the feather follicles at E18 and E20. In this study, expression of Shh as a marker gene for the bud development was examined paralleling with expression patterns of beta-catenin. It was found that the expression pattern of beta-catenin was almost similar spatially and temporally to that of Shh mRNA at the later stages of bud development. The differential beta-catenin mRNA expression in the goose dorsal skin may be essential for promoting the normal development of embryonic feather bud.

The ferric uptake regulator (Fur) is an iron-dependent transcriptional regulator that regulates genes related to iron acquisition, oxidative stress response, and various other functions. Transcription of fur is typically self-regulating and sensitive to iron and oxidative stress. Following the identification of a fur gene in the genome of the bovine pathogen Mannheimia haemolytica, an attempt was made to characterize the transcriptional control of M. haemolytica fur. Northern blotting, RT-PCR, and primer extension were done to determine that M. haemolytica fur is transcribed using three distinct promoters, two of which are located within the upstream fldA gene. The third promoter is located upstream of a conserved hypothetical protein and drives transcription of a tricistronic message. Quantitative real time PCR experiments indicated that unlike current models of Fur regulation, M. haemolytica fur transcription is unchanged by iron depletion at logarithmic phase and repressed by iron depletion at stationary phase.

Prolactin (PRL) regulates various functions in pigs including reproduction, mammary development and lactation. We used 5'-rapid amplification of cDNA ends (5'-RACE) to clone three full-length alleles of the porcine PRL receptor (pPRLR) from Landrace (alleles LR2 and LR4) and Yucatan miniature (MP) pigs, corresponding to the A and B alleles previously reported to be associated with reproductive traits. When expressed in Chinese hamster ovary (CHO-K1) cells, all three pPRLRs transduced differentiation signals to a beta-casein promoter with the same effectiveness, where human growth hormone (hGH) and porcine PRL (pPRL) were more effective ligands than ovine PRL (oPRL). The pPRLR had a lower binding affinity for oPRL than pPRL while binding affinity for hGH was not different between the three pPRLR variants. The pPRLRs primarily localized to the cytoplasm with perinuclear concentration. In conclusion, we have cloned three allelic variants of the pPRLR and have functionally characterized these as different from the hPRLR. However, our data do not support the proposal that allelic variation of the pPRLR confers functional differences in vivo.

Pleurochrysis haptonemofera is a unicellular marine coccolithophorid that has calcified scales, coccoliths, on the cell surface. Some coccolithophorids including P. haptonemofera have a coccolith-bearing stage and a naked stage in their life cycles. To characterize genes involved in the coccolithogenesis, we generated a total of 9550 expressed sequence tags (EST) from a normalized cDNA library that was prepared using both coccolith-bearing cells (C-cells) and naked cells (N-cells), constructed a cDNA macroarray using the EST clones, and then analyzed the gene expression specificity in C-cells and N-cells. When cDNA clones whose expression ratio exceeded 3-fold were selected, as many as 180 clones were identified as C-cell-specific ones, while only 12 were found to be N-cell-specific ones. These clones were sequenced, assembled, and homology-searched against a public nonredundant protein database. As a result, they were grouped into 54 C-cell-specific and 6 N-cell-specific genes, and 59% and 50% of these genes exhibited significant similarity to those of other known proteins, respectively. To assess mRNA expression further, Northern hybridization was performed for 12 of the C-cell-specific genes and one of the N-cell-specific ones. These clones, together with the new cDNA macroarray, will provide a powerful tool for the future genome-wide functional analysis of uncharacterized genes related to the regulation of the calcification and life cycle of coccolithophorids.

We previously demonstrated that six genes involved in ecdysteroid signaling are expressed preferentially in Kenyon-cell subtypes in the mushroom bodies of the honeybee (Apis mellifera L.). To further examine the possible involvement of ecdysteroid signaling in honeybee brain function, we isolated a cDNA for the A isoform of the ecdysone receptor gene homolog AmEcR-A and analyzed its expression in the brain. In situ hybridization revealed that AmEcR-A is expressed selectively in the small-type Kenyon cells of the mushroom bodies in the worker and queen brain, like AmE74 and AmHR38, suggesting a possible association of these gene products. Analysis of AmEcR-A expression in queen and worker abdomens demonstrated that AmEcR-A is strongly expressed in nurse cells of the queen ovary, suggesting that ecdysteroid and ecdysteroid signaling have roles in oogenesis. Our present results further support the possible involvement of ecdysteroid signaling in brain function, as well as in regulating queen reproductive physiology in the adult honeybee.

Purpose: Canine multifocal retinopathy (cmr) is an autosomal recessive disorder of multiple dog breeds. The disease shares a number of clinical and pathologic similarities with Best macular dystrophy (BMD), and cmr is proposed as a new large animal model for Best disease., Methods: cmr was characterized by ophthalmoscopy and histopathology and compared with BMD-affected patients. BEST1 (alias VMD2), the bestrophin gene causally associated with BMD, was evaluated in the dog. Canine ortholog cDNA sequence was cloned and verified using RPE/choroid 5'- and 3'-RACE. Expression of the canine gene transcripts and protein was analyzed by Northern and Western blotting and immunocytochemistry. All exons and the flanking splice junctions were screened by direct sequencing., Results: The clinical phenotype and pathology of cmr closely resemble lesions of BMD. Canine VMD2 spans 13.7 kb of genomic DNA on CFA18 and shows a high level of conservation among eukaryotes. The transcript is predominantly expressed in RPE/choroid and encodes bestrophin, a 580-amino acid protein of 66 kDa. Immunocytochemistry of normal canine retina demonstrated specific localization of protein to the RPE basolateral plasma membranes. Two disease-specific sequence alterations were identified in the canine VMD2 gene: a C(73)T stop mutation in cmr1 and a G(482)A missense mutation in cmr2., Conclusions: The authors propose these two spontaneous mutations in the canine VMD2 gene, which cause cmr, as the first naturally occurring animal model of BMD. Further development of the cmr models will permit elucidation of the complex molecular mechanism of these retinopathies and the development of potential therapies.

Mannheimia haemolytica is the major causative agent of shipping fever, a severe pneumonia in cattle causing high morbidity and mortality. A prerequisite of successful lung colonization by M. haemolytica is the necessity to adapt to the paucity of iron. The lack of genome information has precluded an assessment of the genetic repertoire available to M. haemolytica to adapt to low iron environments. To close this knowledge-gap, we have determined 90% of a virulent M. haemolytica serotype A1 genome sequence and produced a microarray in order to study gene expression under iron-limiting growth for 15, 30 and 60 min. M. haemolytica responded to iron limitation by the up-regulation of transcripts coding for receptors and ABC-type transporters of transferrin, haemoglobin, haem and siderophores. Real time PCR analysis of lung tissue from Mannheimia-infected calves demonstrated the in vivo transcription of two potential haemoglobin receptors, hmbR1 and hmbR2. The relative hmbR1 and hmbR2 transcript levels in the infected lung tissue were comparable to the induced levels observed under iron-limiting growth, demonstrating in vivo induction of receptor transcription in the context of an infection. When the iron response of M. haemolytica was compared to the iron response of Pasteurella multocida, another pathogen colonizing the bovine lung, only few homologous genes were induced in both organisms. These included the haemoglobin receptor hmbR2 and the periplasmic transport systems yfeABCD and fbpABC. The comparative analysis suggests that the two pathogens use different strategies to adapt to the iron-limiting environment in the bovine host.

Vascular endothelial growth factor (VEGF) is a major regulator of angiogenesis and a potential autocrine growth factor for neoplastic cells in various malignancies. In the present study, we have investigated expression of VEGF and VEGF receptors in canine mastocytomas and the canine mastocytoma cell line C2. As assessed by immunostaining of tissue sections and cytospin slides, primary neoplastic mast cells (MC) and C2 cells were found to express the VEGF protein. In Northern blot and RT-PCR experiments, C2 cells expressed VEGF mRNA in a constitutive manner. VEGF mRNA expression in C2 cells was counteracted by LY294002 and rapamycin, suggesting involvement of the PI3-kinase/mTOR pathway. Moreover, C2 cells were found to express VEGF receptor-1 (Flt-1) and VEGF receptor-2 (KDR). However, recombinant VEGF failed to promote (3)H-thymidine uptake in C2 cells, and a neutralizing anti-VEGF antibody (bevacizumab) failed to downregulate spontaneous proliferation in these cells. In addition, rapamycin decreased the expression of VEGF in C2 cells at the mRNA and protein level without suppressing their proliferation. Together, canine mastocytoma cells express VEGF as well as VEGF receptors. However, despite co-expression of VEGF and its receptors, VEGF is not utilized as an autocrine growth regulator by canine mastocytoma cells.

Like IGF-I, progranulin (pgrn) is a growth factor involved in tumorigenesis and wound healing. We report here the identification and characterization of pgrn cDNA in tilapia and the regulation of its expression by growth hormone (GH). The tilapia pgrn cDNA was cloned by RT-PCR amplification, using gene specific oligonucleotides as amplification primers. The cDNA contains an open reading frame encoding a peptide of 206 amino acid residues (aa) that contains a presumptive signal peptide (23 aa) and two repeat units of granulin (grn, 51 and 52 aa, respectively) franked by a GAP of 49 aa and the carboxyl terminus with 31 aa. The two predicted grn peptides are arranged in tandem repeats interrupted by a GAP peptide. RT-PCR analysis revealed that high levels of prgn mRNA were present in several tissues such as spleen, gastric cecum, intestine, fat tissue, gill, kidney, eye and pancreas, and lower levels in liver, muscle, heart, brain, skin and stomach. Administration of a single dose (500 ng/g body weight) of recombinant seabream growth hormone (rbGH) by intraperitoneal (ip) injection into one-month-old tilapia resulted in an obvious increase of IGF-I and pgrn mRNA (2.7-fold and 2.5-fold, respectively) in the liver at three hours post-GH treatment. The peptide levels of pgrn in the liver of GH-treated fish also were substantially induced over controls at 12h post-GH treatment as detected by western immuno-blot analysis. The co-induction of IGF-I and pgrn following GH treatment may suggest the involvement of pgrn in GH regulated growth in tilapia.

Based on the known partial cDNA sequence of dragline silk protein an artificial gene monomer, a 360 bp sequence, was designed and polymerized to encode an analog of dragline silk protein. Six tandem copies of monomer were cloned into pBC1 vector and microinjected into the pronuclei of fertilized Kunming White eggs. Transgenic mice were screened by Polymerase Chain Reaction (PCR) and Southern blot which revealed that 10 mice (5 male, 5 female) among 58 mice were transgenic positive. Milk of five F0 mice and eight F1 mice was analyzed by Western blot, and two F0 mice and seven F1 mice expressed recombinant dragline silk protein. In transgenic mice milk a maximum of concentration of recombinant dragline silk protein was 11.7 mg/L by radioimmunoassay.

Requiring only simple heating devices, isothermal nucleic acid-based amplification (NASBA) is a potential detection platform to be developed for on-site diagnosis of aquaculture pathogens. In this report, an NASBA assay has been developed for the Taura syndrome virus (TSV), one of the most devastating RNA virus pathogens for several penaeid shrimp species. The NASBA amplicons were detected by agarose gel electrophoresis and confirmed by Northern-blotting and dot-blotting analysis, using a biotinylated TSV-specific primer. The sensitivity of the TSV NASBA coupled with dot-blotting detection was approximately 5-fold less sensitive than that of the commercially available RT-nested, PCR-based IQ2000 TSV Detection and Prevention System that was also confirmed to be more sensitive than the RT-PCR-based TSV detection protocol recommended by the OIE (Office International des Epizooties). The specificity of the TSV NASBA reaction was substantiated by the results that RNA of non-target viruses did not generate any signals. Furthermore, a simple colorimetric microtiter plate assay employing TSV-specific capture and detection primers was developed as a simple alternative approach for the detection of NASBA amplicons. Taken together, the combination of the isothermal NASBA and colorimetric solid phase-based assays should allow sensitive, straightforward, and speedy on-site detection of TSV.

Respiration rate, time to pupation and the expression patterns of selected genes were examined during the diapause to post-diapause transition in the alfalfa leafcutting bee, Megachile rotundata held at constant 4 degrees C in winter storage. Respiration quotients were at or below 0.7 from December to May and then increased to over 0.8 in June and July. The time required for prepupae to reach the pupal stage following transfer to 29 degrees C decreased from 23 days in December to 10 days in July. HSP70 was expressed at a consistently high level in all the diapausing prepupae stored at 4 degrees C. In contrast, we demonstrated previously that HSP70 expression in diapausing prepupae maintained under field conditions began decreasing after December and was expressed at trace levels in the June samples. Transferring prepupae stored at 4 to 25 degrees C at monthly intervals from December to July induced a significant decrease in HSP70. Levels of HSC70 showed no changes during the transition to post-diapause development in prepupae maintained at 4 degrees C. Transferring the prepupae to 25 degrees C during the April-June time interval elicited an increase in HSC70 expression. HSP90 was expressed at a consistent level in prepupae stored at 4 degrees C but decreased to very low levels after being transferred to 25 degrees C in December-February prepupae: no decrease was noted in the April-July prepupae. Actin was expressed at trace levels in the diapausing prepupae maintained at 4 degrees C and increased slightly in the post-diapausing pupae. Transferring prepupae stored at 4 to 25 degrees C at monthly intervals from December to July induced an increase in actin expression. These results indicate that the level of gene expression for selected genes in diapausing and post-diapause bees is highly influenced by their thermal history.

Two actin genes cloned from Culex pipiens L. are upregulated during adult diapause. Though actins 1 and 2 were expressed throughout diapause, both genes were most highly expressed early in diapause. These changes in gene expression were accompanied by a conspicuous redistribution of polymerized actin that was most pronounced in the midguts of diapausing mosquitoes that were exposed to low temperature. In nondiapausing mosquitoes reared at 25 degrees C and in diapausing mosquitoes reared at 18 degrees C, polymerized actin was clustered at high concentrations at the intersections of the muscle fibers that form the midgut musculature. When adults 7-10 days post-eclosion were exposed to low temperature (-5 degrees C for 12 h), the polymerized actin was evenly distributed along the muscle fibers in both nondiapausing and diapausing mosquitoes. Exposure of older adults (1 month post-eclosion) to low temperature (-5 degrees C for 12 h) elicited an even greater distribution of polymerized actin, an effect that was especially pronounced in diapausing mosquitoes. These changes in gene expression and actin distribution suggest a role for actins in enhancing survival of diapausing adults during the low temperatures of winter by fortification of the cytoskeleton.

Fatty acid-binding proteins belong to a superfamily of lipid-binding proteins that exhibit a high affinity for long-chain fatty acids and appear to function in metabolism and intracellular transportation of lipids. The current study was designed to investigate expression characterization and association with growth and composition traits of the liver fatty acid-binding protein (L-FABP) gene in the chicken. Northern blot analysis indicated that the gene, similar to the mammal L-FABP gene, was expressed only in liver and intestinal tissues. The mRNA levels of the chicken L-FABP gene in liver and intestine had significant differences between the broilers and Baier layers. The China Agricultural University F(2) population was used in the present study. Body weight and body composition traits were measured in the populations. Primers for the coding region and 5' upstream region of the L-FABP gene were designed according to chicken genomic and cDNA sequence. Polymorphisms were detected by DNA sequencing, and the PCR single-strand conformation polymorphisms method was developed to genotype the F(2) population. The results indicated that the L-FABP gene polymorphisms were associated with abdominal fat weight and percentage of abdominal fat, and the L-FABP gene could be a candidate locus or linked to a major gene(s) that affects fatness traits in the chicken. The results of the current study provided basic molecular information for studying the role of the L-FABP gene in the regulation of lipid metabolism in avian species.

Small proline-rich proteins (SPRR) are known to construct the cornified cell envelope (CE) in the stratified squamous epithelial cell. Their functions in the simple epithelium such as the uterine epithelium are not clear hitherto. In the present study, the mRNA expression patterns of sprr2 family members in the mouse uterus and vagina during the estrous cycle and pregnancy as well as their regulation by steroids were investigated. Using semi-quantitative RT-PCR, it was revealed that the transcripts of sprr2b, 2e and 2g genes were up-regulated in the proestrous and estrous uteri, and sprr2d was up-regulated only in the estrous uterus. In the vagina, transcription of sprr2a, 2b, 2d, 2e and 2k genes were up-regulated at the metestrous stage. Northern blot analysis demonstrated that the overall expression of sprr2 was highly up-regulated in the estrous uterus and the metestrous vagina. During pregnancy, the sprr2 mRNA in the uterus was sharply repressed from day 3 postcoitus on, and began to be induced around labor time. In situ hybridization showed that the sprr2 transcripts were localized in uterine luminal and glandular epithelial cells as well as vaginal stratified epithelial cells. In ovariectomized mice, the expression of sprr2a, 2d, 2e and 2f genes in the uterus were induced by estrogen, and the effect of estrogen on sprr2d and 2e expression could be partly abolished by progesterone. The data indicate that the sprr2 genes have unique regulation patterns in different reproductive tissues under different physiological conditions, and the encoded proteins might play diverse functions in the female reproductive system.

The phosphorylated polypeptide (pp)38 of oncogenic Marek's disease (MD) herpesvirus (MDV) is expressed during lytic infections in vivo and in vitro, but its functions have not been fully elucidated. The quail cell line QT-35, latently infected with MDV, was used to generate QTP32 in which pp38 is expressed under control of a tetracycline controlled promoter to examine possible functions of pp38. Induction of pp38 did not influence late MDV genes expression, but it enhanced mitochondrial dehydrogenase activity significantly. Two new pp38 splice variants were found in induced QTP32 cells, in additional in vitro systems and MDV-infected chickens. Differential expression of full-length pp38 and splice variants suggests that the splice variants are important during latency and perhaps transformation. Polypeptides of 40 and 20kDa were detected by Western blot using monoclonal antibody H19. These polypeptides were also produced in DF-1 cells transfected with a pp38 construct in which the splice acceptor sites had been mutated. Our results add important new information to the role of pp38 in the pathogenesis of MD. The data suggest that pp38 and the two newly described splice variants may influence metabolic activity, which may have important consequences for the understanding of latency and tumor development.

Adipose tissue plays a critical role in metabolism, storage, and release of fatty acids in mammals. Construction of a full-length cDNA library is an effective way to understand the functional expression of genes in adipose tissue, and in addition, novel genes for further research can be found in the library. In this study, adipose tissue RNA was extracted from three 18-mo-old Lee-Sung pigs. The mRNA was isolated, reverse transcribed, and used to construct a cDNA library. After transformation, 2,880 clones were selected and sequenced. Cluster analysis was performed, and the assembled contig of each cluster was subjected to search against DNA sequences in the nucleotide databases (NCBINR/TIGRGI). These sequences were clustered into 1,527 unique sequences; 80% of the sequences were categorized as known genes, and 20% of the sequences were categorized as unknown genes. In this adipose tissue cDNA library, approximately 16% of the genes contained full-length sequences with start and stop codons. Gene ontology analysis was performed to indicate the possible functions of these genes. Genes associated with mitochondrial function were abundant and represented 10% of the total. Several fatty acid transport genes and stearoyl coenzyme A desaturase were among the most abundant genes expressed. Tissue distribution of several abundant genes was analyzed by northern analysis, and many of these genes were transcribed in porcine adipose tissue in high copy number. Our full-length sequence data and tissue distribution data can be used to decipher the functional roles exhibited by the adipocyte under various perturbations via endocrine, environmental, genetic, nutritional, pharmacological, or physiological manipulations.

The phosphatase and TENsin homolog deleted on chromosome 10 (PTEN) is a lipid and protein phosphatase able to inhibit significant actors of cell signaling (i.e. phosphatidylinositol-3'kinase and mitogen-activated protein kinase pathways). The aim of this study was to characterize PTEN and to investigate its regulation during ontogenesis in chicken muscle. Pectoralis major muscle was sampled on day 18 of the embryonic period (E18), at hatching (d0) and in fed chickens at 2, 7 and 43 days after hatching (d2, d7 and d43). We first cloned the totality of chicken PTEN cDNA; its translation into a putative protein showed more than 95% sequence identity with that characterized in mammals (humans, mice). PTEN was expressed under two major transcripts in the majority of tissues, including muscles where the expression of PTEN mRNA increased with age (P < 0.05). Surprisingly, the protein levels of PTEN (protein characterized with an apparent molecular weight of 55kDa) and its activity were considerably decreased between the E18 and d43 stages (approximately 8-10-fold reduction, P < 0.001). An association between these decreases and higher phosphorylation levels of two potential indirect downstream targets of phosphatase (i.e. AKT and ERK) was observed only in the early growth phases. It was concluded that phosphatase PTEN was expressed in chicken muscle and that its expression was regulated during ontogenesis.

Finfish in the wild are regularly subjected to low temperatures, which have been shown to cause a loss of Major Histocompatibility receptor expression in common carp kept at 6 degrees C. This is similar to what was seen in a mammalian cell line cultured at 26 degrees C. Loss of expression of this critical viral recognition protein may provide one mechanism for the increased frequency of fish diseases at low temperatures. This report demonstrates that unlike carp and mammals, beta(2)m transcript levels in both rainbow trout and Atlantic salmon do not decrease after 10 days at temperatures as low as 2 degrees C. Reverse transcriptase (RT)-PCR indicated that transcript steady-state levels of trout beta(2)m were maintained in both tissues and peripheral blood leucocytes, whether freshly isolated or in primary culture. Polyclonal antibodies raised against a recombinant form of trout beta(2)m, demonstrated cross-reactivity to both rainbow trout and Atlantic salmon protein lysates. Use of these antibodies in western blot analyses indicated that cellular protein levels are also maintained at low temperatures in both species while qualitative epifluorescence analysis of freshly isolated peripheral blood leucocytes indicated persistent cell surface expression of trout beta(2)m even after 10 days at 2 degrees C. Rainbow trout and Atlantic salmon may therefore utilise an alternative mode of immune gene regulation than the common carp and mammals allowing them to maintain viral recognition machinery at low temperatures, possibly due to selection for survival in cold climates.

In the present study, we constructed gender-enriched cDNA libraries for the adult stage of the parasitic nematode Oesophagostomum dentatum (order Strongylida) using suppressive-subtractive hybridization (SSH), sequenced clones from the female-library and male-library (480 from each) and conducted bioinformatic and microarray analyses of the expressed sequence tags (ESTs). In total, 873 ESTs (440 male and 433 female) were obtained, achieving a sequencing success of 91%The nucleotide sequences reported in this article (Tables 1-5) have been deposited in the EMBL, GenBank and DDJB databases under the Accession nos. AM157797-AM158083. Microarray analyses of 516 unique ESTs representing both gender-enriched libraries revealed differential hybridization for 391 of them (75.8%). Of these, 220 (56.3%) had significantly greater signal intensities in the female than in the male, and 154 (70%) of these were predicted to have homologues in C. elegans. These homologues were predicted to be involved in key biological processes, including embryonic nutrition, gametogenesis, molecular binding/transport or metabolism, nucleic acid synthesis and function, and signal transduction. Of the 171 ESTs with statistically higher signal intensities in male O. dentatum, 43.8% had homologues in C. elegans. These homologues included major sperm proteins (MSPs) or MSP-like molecules, keratin-like molecules, molecules involved in metabolism, PDZ domain-containing proteins, sugar binding proteins, protein kinases, serine proteases or protease inhibitors, molecules involved in proteolysis and other proteins, such as enzymes and various putative proteins. Of the 287 ESTs (from both gender-enriched cDNA libraries) with no known homologues in C. elegans, 50 (17.4%) had homologues in other nematodes, 8 had homologues in various other organisms and 104 (36.2%) had no homology to any sequence in current gene databases. The present study lays a foundation for the isolation and molecular, biochemical and functional characterization of selected genes from the gender-enriched cDNA archives established for O. dentatum.

Background: Hypoxia-inducible factors (HIFs) are involved in adaptive and survival responses to hypoxic stress in mammals. In fish, very little is known about the functions of HIFs., Results: We have cloned and characterized two distinct HIF-alpha cDNAs--gcHIF-1alpha and gcHIF-4alpha--from the hypoxia-tolerant grass carp. The deduced gcHIF-1alpha protein is highly similar to the HIF-1alphas (57-68%) from various vertebrate species, while gcHIF-4alpha is a novel isoform, and shows an equivalent degree of amino acid identity (41-47%) to the HIF-1alpha, HIF-2alpha and HIF-3alpha proteins so far described. Parsimony analysis indicated that gcHIF-4alpha is most closely related to the HIF-3alpha proteins. Northern blot analysis showed that mRNA levels of gcHIF-1alpha and gcHIF-4alpha differ substantially under normoxic and hypoxic conditions, while Western blot studies demonstrated that the endogenous protein levels for both gcHIF-1alpha and gcHIF-4alpha are similarly responsive to hypoxia. Our findings suggest that both gcHIF-1alpha and gcHIF-4alpha are differentially regulated at the transcriptional and translational levels. HRE-luciferase reporter assays show that both proteins function as transcription activators and play distinct roles in modulating the hypoxic response in grass carp., Conclusion: There are at least two distinct HIF-alpha isoforms--gcHIF-1alpha and gcHIF-4alpha--in the hypoxia-tolerant grass carp, which are differentially expressed and regulated in different fish organs in response to hypoxic stress. Overall, the results suggest that unique molecular mechanisms operate through these two HIF-alpha isoforms, which underpin the hypoxic response in the hypoxia-tolerant grass carp.

Leptin is reported to have direct effects on lipogenesis in peripheral tissues that are independent of its central effects on food intake and body weight. These experiments have been mainly carried out in rodents (different models of obesity) in which lipogenesis occurs in both adipose tissue and liver. Such effects are unknown in birds in which lipogenesis occurs essentially in the liver. In this study, leptin gene expression and circulating leptin levels were examined in two chicken lines, selected for high (FL) or low (LL) abdominal fat pad size, at different nutritional states (fasted and fed state). In addition, effects of recombinant chicken leptin on liver metabolism were investigated. Hepatic leptin and fatty acid synthase (FAS) gene expression and plasma leptin levels were significantly higher in FL than in LL chickens (P < 0.05). In both lines, fasting significantly reduced hepatic leptin and FAS mRNA levels (P < 0.05). Continuous administration of recombinant chicken leptin (8 microg/kg/h) during 6 h significantly inhibited food intake (51%) and increased leptinemia (23-fold) compared to untreated group. Despite the decrease of food intake, leptin significantly induced the expression of FAS in chicken liver. These changes were accompanied by a significant down-regulation of leptin receptor gene expression, however SREBP-1, the main transcription factor of lipogenic genes, remained unchanged. This result suggests a local potential role of leptin in the regulation of avian hepatic lipogenesis, and explain, at least partly, the metabolic changes evolved during the divergent selection of FL and LL chickens.

We report the nucleotide and deduced amino acid sequences of the pituitary glycoprotein hormone common alpha-subunit, and the FSH and LH beta-subunits of the elk (Cervus elaphus). These sequences were obtained by RT-PCR of pituitary gland polyadenylated RNA. A partial genomic fragment of the elk follicle stimulating hormone beta-subunit was also amplified from genomic DNA and sequenced. These sequences show high similarity to other related domestic species, including sika deer, ovine, bovine, porcine and equine. The presently reported cDNAs were used as probes for Northern analysis of pituitary gland and muscle samples from elk and sheep. Mature mRNA transcripts of approximately 700, 1700 and 550 bases were expressed in the pituitary gland but not in muscle tissue, for the glycoprotein hormone common alpha-subunit and FSH and LH beta-subunits, respectively. These observations are consistent with reports for other related species. The genomic fragment of elk FSH beta-subunit revealed an intronic microsatellite of 6 CT repeats, which differs from the 19 CT repeats documented for the sheep genomic sequence. We propose that differences in the CT repeats of the elk FSH beta-subunit gene may be used as a marker for elk parentage testing. The elk DNA sequences presently reported represent a unique addition to the growing collection of the pituitary hormone genes for various vertebrates, which will facilitate evolutionary and phylogenetic studies of the gonadotropin genes. In addition, this information and the cloned cDNAs will be useful for studying seasonal expression of the elk pituitary glycoprotein genes.

Effects of beta-glucan administration on survival and immune modulations were studied in Cyprinus carpio against the bacterial pathogen, Aeromonas hydrophila. Beta-glucan was extracted from Saccharomyces cervisiae and purified. A virulent strain of the pathogen A. hydrophila was collected from infected fish. Different concentrations of beta-glucan were administered to test animals on day 1, 3 and 5 through different routes (intraperitoneal injection (ip), bathing and oral administration). Control and test animals were challenged by ip injection of LD50 concentration of A. hydrophila on day 7 and mortality was observed and Relative Percent Survival (RPS) was calculated. Intraperitoneal injection of 500 microg of glucan significantly enhanced the RPS; bathing and oral administration of glucan did not influence the RPS. On day 7, test animals injected with 100, 500 and 1000 microg of glucan had a significant increase in total blood leucocyte counts and an increase in the proportion of neutrophils and monocytes. Superoxide anion production by kidney macrophages was also elevated. RT-PCR and northern blot analysis of interleukin-1 mRNA showed elevated expression in kidney on day 7 in fish injected with glucan. Glucan had an adjuvant effect on antibody production as pretreatment by injection of 100-1000 microg glucan/fish resulted in the highest antibody titer against A. hydrophila following vaccination. Classical and alternative complement pathways were not affected by glucan administration by any of the three routes.

The ovine genome contains approximately 20 copies of endogenous betaretroviruses (enJSRVs) that are highly related to two exogenous oncogenic viruses, Jaagsiekte sheep retrovirus (JSRV) and Enzootic nasal tumor virus. The cellular receptor for both JSRV and the enJSRVs is hyaluronidase 2 (HYAL2). In this study, we assessed expression of enJSRVs envelope (env) and HYAL2 mRNAs in the ovine uterus and conceptus (embryo/fetus and extraembryonic membranes) throughout gestation. By reverse transcription-polymerase chain reaction analyses, enJSRVs env were found to be expressed beginning in the Day 12 conceptus, whereas HYAL2 was expressed from Day 16. HYAL2 mRNA was detected throughout gestation in the placentome but not in the endometrium, whereas enJSRVs env expression was detected throughout gestation in endometrium and placentomes. The enJSRVs env mRNA was specifically expressed in the endometrial lumenal epithelium (LE) and glandular epithelium (GE) as well as the trophoblast giant binucleate cells (BNC) and multinucleated syncytia of the placenta. HYAL2 mRNA was only detected in the BNC and multinucleated syncytial plaques of the placentome. Partial sequencing of the transcriptionally active enJSRVs from sheep endometrium, placentomes, and placenta revealed expression of many enJSRV loci. Cloning of the expressed enJSRVs env mRNA from ovine uteroplacental tissues found sequences similar to the previously identified enJS5F16 and enJS56A1 gene with an intact open reading frame, although the polypeptides they encode were not studied. Collectively, results provide further support for our hypothesis that the enJSRVs Env have been beneficial to the host and are involved in protection of the uterus from viral infection and regulators of placental morphogenesis and function.

Uteroplacental prostaglandins (PGs) play pivotal roles in the maintenance and termination of pregnancy in mammals. In the present study, we have characterized the expression of prostaglandin transporter (PGT) in placentome caruncles, intercaruncular tissues, fetal membranes, and utero-ovarian plexus during pregnancy in cattle. Pregnant bovine uteri were collected and classified into six groups covering the entire gestational length. In caruncles and intercaruncular tissues, PGT mRNA (also known as SLC02A1) and PGT protein were highly expressed at the late stage of pregnancy compared to the early and mid stages, whereas the level of expression is constant and low in fetal membranes throughout pregnancy. PGT mRNA and PGT protein were expressed at a constant level in the utero-ovarian plexus both ipsilateral and contralateral to corpus luteum throughout the course of pregnancy. Overall, the relative expression of PGT mRNA and PGT protein were higher in caruncles than in intercaruncular tissue and fetal membranes, whereas no differences were detected between intercaruncular tissues and fetal membranes at any stage of gestation. Immunohistochemistry indicated that PGT was preferentially expressed in caruncular epithelial cells of placentomes and endometrial luminal epithelial and myometrial smooth muscle cells of the intercaruncular regions. The level of PGT expression was comparatively higher in maternal components than in fetal components. In conclusion, differential spatiotemporal tissue-specific expression of PGT in uterine and intrauterine tissues suggests a role for this transporter in the exchange of PGs between the maternal and the fetal compartments, as well as for intrauterine metabolism of PGs during pregnancy.

Molecular determinants and mechanisms involved in ovarian follicular growth, ovulation, and luteinization are not well understood. The objective of this study was to identify genes expressed in bovine granulosa cells (GC) of dominant follicles (DF) and downregulated after hCG-induced ovulation, using the suppression subtractive hybridization (SSH). GC were collected from DF at Day 5 of the estrous cycle and from ovulatory follicles (OF) obtained 23 h following injection of hCG. A subtracted cDNA library (DF-OF) was generated and screened using unsubtracted (DF, OF) and subtracted (DF-OF, OF-DF) cDNAs as complex (32)P-probes. A total of 32 nonredundant cDNAs were identified: 23 cDNAs matched with sequences of known biological function and 9 cDNAs with complete or partial sequences of undefined biological function. Detection of genes known to be downregulated during the periovulatory period in the bovine species, such as CPD, CYP11A1, CYP19A1, FSHR, LRP8/ ApoER2, and SERPINE2, validated the physiological model and analytical techniques used. For a subset of genes, such as ARFGAP3, CYP11A1, CYP19A1, FSHR, FST, GJA1, IDH3, INHBA, LHCGR, LHCGR lacking exon 10, PRC1, PRG1, RPA2, SCD, and TRIB2, gene expression profiles were compared by virtual Northern blot or reverse transcriptase-polymerase chain reaction from follicles obtained at different developmental stages. Results confirmed a downregulation of the respective mRNAs in GC of OF compared with that of DF. We conclude that we have identified novel genes that are downregulated by hCG in bovine GC of DF during the periovulatory period, which may contribute to follicular growth, ovulation, and/or luteinization.

To investigate the photoperiodic entrainment of peripheral rhythms in ruminants, we studied the expression of clock genes in the liver in the highly seasonal Soay sheep. Animals were kept under long (LD 16:8) or short photoperiod (LD 8:16). Daily rhythms in locomotor activity were recorded, and blood concentrations of melatonin and cortisol were measured by RIA. Per2, Bmal1, and Cry1 gene expression was determined by Northern blot analyses using ovine RNA probes in liver collected every 4h for 24h. Liver Per2 and Bmal1, but not Cry1, expression was rhythmic in all treatments. Under long days, peak Per2 expression occurred at end of the night with a similar timing to Bmal1, whereas, under short days the Per2 maximum was in the early night with an inverse pattern to Bmal1. There was a photoperiodxtime interaction for only Per2 (P < 0.001). The 24-h pattern in plasma cortisol matched the observed phasing of Per2 expression, suggesting that it may act as an endocrine entraining factor. The clock gene rhythms in the peripheral tissues were different in timing compared with the ovine suprachiasmatic nucleus (SCN, central pacemaker) and pars tuberalis (melatonin target tissue), and the hepatic rhythms were of lower amplitude compared with photoperiodic rodents. Thus, there are likely to be important species differences in the way the central and peripheral clockwork encodes external photoperiod.

The expression of the growth hormone receptor (GHR) gene was investigated in semitendinosus muscle during bovine foetal development in both normal and double-muscled Charolais foetuses which differ with respect to muscle development. Northern-blot analysis of foetal muscle RNA preparations with a GHR cDNA probe identified the 4.5 kb GHR mRNA as early as 130 days post-conception. In double-muscled animals, the expression of GHR mRNA increased from 130 to 210 days of gestation while it stayed stable in normal ones. It was significantly higher (P < 0.05) in double-muscled foetuses compared to normal ones from the second third of gestation. Northern-blot analysis of foetal muscle RNA preparations from both genotypes with a beta-actin cDNA probe, revealed lower beta-actin gene expression in double-muscled foetuses than in normal ones, suggesting a delay in the differentiation of muscle cells. In situ hybridisation revealed the localisation of specific GHR mRNA in muscle cells at all gestation stages analysed (130, 170, 210 days post-conception) but not in connective tissue surrounding the muscle cells. At the adult stage, the hybridisation signal was also very high and observed in muscle cells only. These results show the ontogeny of GHR mRNA in bovine muscle and demonstrate a difference between normal and double-muscled animals.

Angiogenesis is an essential process during follicular development and corpora lutea (CL) formation. Recent studies have shown that vascular endothelial growth factor (VEGF) is an essential regulator of ovarian angiogenesis. Several lines of evidence have indicated that the production of VEGF is regulated by hypoxia inducible factor-1alpha (HIF-1alpha), especially under hypoxic conditions, but the expression of HIF-1alpha has not been well characterized in the porcine ovary. The present study examined the expression of HIF-1alpha mRNA and its localization in porcine ovaries at different stages of the estrous cycle. Northern blot analyses of total CL RNA indicated hybridization of the porcine HIF-1alpha probe to transcripts of approximately 3.8 kb. The mRNA expression of HIF-1alpha was highest in CL during the early luteal phase, followed by a decrease during the mid- and late-luteal phases. Using in situ hybridization, abundant HIF-1alpha mRNA was evident in follicles and CL. Within non-atretic follicles, HIF-1alpha mRNA was highly expressed in the granulosa cell layer, while weaker labeling was evident in the theca interna. These results suggest that HIF-1alpha may play a role in the regulation of cellular metabolism and blood supply during follicular growth and CL formation.

Adipocyte determination and differentiation-dependent factor 1 (ADD1) drives the expression of several lipogenic genes in mammals. Polyunsaturated fatty acids decrease ADD1 mRNA abundance in differentiating porcine adipocytes. The current study was designed to explore the mechanisms by which PUFA inhibit the expression of ADD1 in porcine adipocytes. Porcine preadipocytes were differentiated for 24 h with 0 or 100 microM of docosahexaenoic acid (DHA) and mixtures of different concentrations of antioxidants to investigate the effect of DHA and antioxidants on the ADD1 mRNA abundance. We found the relative mRNA abundance was decreased by the addition of 100 microM DHA to the medium for porcine differentiating adipocytes, and adding an antioxidant mixture to the medium prevented part of the decrease in ADD1 mRNA abundance. These data suggest that DHA decreased the steady-state transcription factor ADD1 mRNA through a mechanism related to fatty acid peroxidation. Indeed, adding 7.5 microM vitamin E (a natural antioxidant) also restored the concentrations of ADD1 and fatty acid synthase mRNA, which were decreased by DHA treatment; however, the DHA or the antioxidant treatment did not change the expression of antioxidation genes (superoxide dismutase 1 and glutathione peroxidase 1) in porcine stromal vascular cells. When supplemented with the eicosanoid synthesis pathway inhibitors, the inhibition of the expression of ADD1 by arachidonic acid was partially recovered. These results suggest that the mechanism by which PUFA decrease ADD1 mRNA is due to the metabolic product of eicosanoids and peroxidation of these PUFA.

Reasons for Performing Study: Colic is a serious disease syndrome in horses. Much of the mortality is associated with ischaemic-injured intestine during strangulating obstruction, yet there is limited understanding of the associated molecular events. Identification of differentially expressed genes during ischaemic injury should expand our understanding of colic and may lead to novel targeted therapeutic approaches in the future., Objective: To isolate and identify differentially expressed genes in equine jejunum following a 2 h ischaemic event compared to normally perfused jejunum., Methods: Suppressive subtractive hybridisation was used to clone genes that are differentially expressed in equine jejunum injured by 2 h of complete ischaemia as compared to time-matched control jejunal tissues. Expression of selected clones was further evaluated by northern blot analysis., Results: Of the 384 clones selected, 157 were confirmed to possess cDNAs corresponding differentially expressed genes by dot blot analysis. Two genes, fatty acid binding protein 2 and calcium-activated chloride channel 4 were further confirmed to be differentially expressed by northern blot analysis., Conclusions: Suppressive subtractive hybridisation can be used to detect changes in expression of a broad array of genes, as confirmed by northern blot analysis of selected genes., Potential Relevance: These initial results have identified a pool of equine intestinal epithelial genes that are differentially expressed following a 2 h ischaemic event. In particular, genes indicative of deranged metabolic activity and those potentially involved in early repair events were identified and may ultimately provide clues as to the nature of epithelial ischaemic injury in horses.

In sheep, fetal cortisol stimulates the conversion of progesterone to estradiol in late gestation initiating labor. It is unclear whether an intact fetal hypothalamic-pituitary-adrenal (HPA) axis is required to induce the estradiol-triggered subsequent endocrine changes including enhanced intrauterine prostaglandin (PG) synthesis associated with the onset of labor. We have shown that maternal estradiol administration stimulates PG H synthase (PGHS)-2 expressions in pregnant ovine intra-uterine tissues. The current study was undertaken to determine whether the fetal adrenal mediates estradiol's stimulation of the intrauterine PGHS-2 in pregnant sheep. Placenta, myometrium, and endometrium were collected from two groups of ewes at 123-127 d of gestational age (dGA) after fetal adrenalectomy and vehicle treatment (ADX; n = 5); or fetal ADX and maternal estradiol administration (5 mg twice a day for 2 d, ADX+E2, n = 5). PGHS-2 mRNA and protein were analyzed by Northern and Western Blot analyses in both groups and presented as the ratios to beta actin mRNA for Northern and G protein beta subunit for Western blot analysis. Fetal plasma cortisol was measured by radioimmunoassay. Data were analyzed by Student's t test. Fetal plasma cortisol levels were low in ADX and ADX+ E2 groups (<6 ng/mL). The cervix of all ADX+E2 treated ewes was dilated at necropsy. Three out of five ADX+ E2-treated ewes delivered within 48 h. The cervix was closed in all fetal ADX ewes at necropsy. PGHS-2 mRNA and protein increased (p < 0.05) in myometrium and endometrium, but not placenta in ADX+E2-treated ewes compared with ADX group. These data provide the first in vivo evidence for estradiol upregulation of intrauterine PGHS-2 in late gestation in the absence of an intact fetal HPA axis. Thus, the fetal adrenal is not required to mediate estradiol's stimulation of uterine PGHS-2 expression associated with the onset of labor.