Effect of Gallus TGFβ1 on the Proliferation,Apoptosis, Migration and Invasion of MDCC-MSB1 Cells

This study aimed to detect the effect of Gallus TGFβ1 on the biological behavior of MDCC-MSB1 cells. MDCC-MSB1 cells were transiently transfected with Gallus TGFβ1 overexpression vector, interference expression vector, and the corresponding negative control. Then, the expression of Gallus TGFβ1, the cell proliferation, the cell cycle and apoptosis, the migration and invasion of each transfection groups were examined. Results showed that compared with the corresponding control, the MDCC-MSB1 cells transfected with overexpression vector of Gallus TGFβ1 could up-regulate the expression level of TGFβ1, the proliferation of MDCC-MSB1 cells was significantly inhibited, G1 phase cells were increased, S and G2 cells were decreased, the apoptosis rate of the cells was increased, the migration and invasion ability were decreased.However,the MDCC-MSB1 cells transfected with the interference expression vector of TGFβ1 significantly down-regulated the expression level of TGFβ1, cell proliferation was improved,G1 phase cells were decreased, S and G2 cells were increased, the cell apoptosis was decreased, the migration and invasion ability was increased. The results showed that Gallus TGFβ1 could inhibit the proliferation, migration and invasion of MDCC-MSB1 cells, and promote their apoptosis.     

Investigation of serum concentrations and immunohistochemical localization of α1-acid glycoprotein in tumor dogs

The purpose of this study was to measure the dynamics of serum α1-acid glycoprotein (AGP) concentration in dogs with various tumors, and to investigate the localization of AGP in some tissues using immunohistochemical staining. Sera were obtained from 171 dogs bearing tumors of various types. Serum AGP concentration was measured by single radial immunodiffusion. Tumors occurring in the liver and spleen were also investigated immunohistochemically using anti-canine AGP antibody. Mean serum AGP levels were 749 ± 602 mg/L in dogs with carcinoma (n = 39), 1,014 ± 971 mg/L with sarcoma (n = 18), and 887 ± 935 mg/L with round cell tumors (n = 46), all significantly higher than serum AGP level in healthy dogs (n = 137, 364 ± 106 mg/L). Mean serum AGP levels were significantly higher than in healthy dogs in complex mammary gland carcinoma (n = 5, 876 ± 721 mg/L), malignant melanoma (n = 7, 1,010 ± 821 mg/L), and hepatocellular carcinoma (n = 5, 936 ± 741 mg/L) among carcinomas, hemangiosarcoma (n = 5, 1,740 ± 1,323 mg/L) among sarcomas, and lymphoma (n = 19, 1,072 ± 965 mg/L) and histiocytic tumor (n = 6, 1,800 ± 1,387 mg/L) among round cell tumors. In an immunohistochemical investigation of AGP localization, both weak and strong staining for anti-AGP antibody were seen in hepatic tissue in dogs with primary non-tumorous lesions originating in the spleen (hematoma) and elevated serum AGP, but all tumor tissue in the spleen was negative. Among dogs with primary tumor lesions of the spleen (hemangiosarcoma) and elevated serum AGP levels, both weak, moderate and strong staining for anti-AGP antibody were seen in hepatic tissue, while strong positive staining was apparent in all tumorous tissue from the spleen. In primary tumor lesions in the liver (hepatocellular carcinoma), both moderate and strong staining for anti-AGP antibody were seen in normal hepatic tissue, and both weak, moderate and strong staining were seen in tumor tissues of the liver. AGP levels thus appear to be elevated in dogs with carcinomas, sarcomas, and round cell tumors. With some of these malignant tumors, localization of AGP in tumor tissue was seen.     

Characterization of toll-like receptors 1–10 in spotted hyenas

Previous research has shown that spotted hyenas (Crocuta crocuta) regularly survive exposure to deadly pathogens such as rabies, canine distemper virus, and anthrax, suggesting that they have robust immune defenses. Toll-like receptors (TLRs) recognize conserved molecular patterns and initiate a wide range of innate and adaptive immune responses. TLR genes are evolutionarily conserved, and assessing TLR expression in various tissues can provide insight into overall immunological organization and function. Studies of the hyena immune system have been minimal thus far due to the logistical and ethical challenges of sampling and preserving the immunological tissues of this and other long-lived, wild species. Tissue samples were opportunistically collected from captive hyenas humanely euthanized for a separate study. We developed primers to amplify partial sequences for TLRs 1–10, sequenced the amplicons, compared sequence identity to those in other mammals, and quantified TLR expression in lymph nodes, spleens, lungs, and pancreases. Results show that hyena TLR DNA and protein sequences are similar to TLRs in other mammals, and that TLRs 1–10 were expressed in all tissues tested. This information will be useful in the development of new assays to understand the interactions among the hyena immune system, pathogens, and the microbial communities that inhabit hyenas.     

Influence of protein concentration on the sulphur‐containing amino acid requirement of broiler chickens 1

1. Two experiments were conducted with male broiler chickens from 3 to 6 weeks of age to determine the effect of dietary protein content on the requirement for sulphur amino acids (SAA). In experiment 1, 0, 0.5, 1.0 or 1.5 g DL‐methionine/kg were added to diets calculated to contain 200, 240 or 280 g protein/kg. In experiment 2, 0, 0.6, 1.2 or 1.8 g DL‐methionine/kg were added to diets calculated to contain 160, 180 or 200 g protein/kg.

2. In experiment 1, the SAA requirement for body weight gain increased as dietary protein content increased. Regression analysis indicated a requirement of 38 g SAA/kg protein.

3. In experiment 2 in which lysine supplementation provided a minimum of 10 g/kg, the requirement for SAA per unit of diet increased only slightly as protein concentration increased indicating that below 200 g protein/kg of diet, the SAA requirement increases per unit of protein with supplementation of the second‐limiting amino acid.

4. Abdominal fat percentage declined in a linear manner with each increment of SAA added to diets containing 160 to 200 g protein/kg. Adding methionine to diets containing 240 or 280 g protein/kg did not affect abdominal fat content. A lower limit of abdominal fat was achieved with a protein concentration of 240 g/kg.

5. It is concluded that the requirement for SAA of finishing broiler chickens is directly related to protein content at concentrations of 200 or more g protein/kg but increases per unit of protein at lower protein concentrations when a minimum lysine concentration is specified. Abdominal fat content reaches a minimum between 200 and 240 g protein/kg of a maize‐soyabean meal diet regardless of SAA content.     

Role of estrogen receptor-α (ESR1) and the type 1 insulin-like growth factor receptor (IGFR1) in estradiol-stimulated proliferation of cultured bovine satellite cells

Although the exact mechanism(s) by which estradiol (E₂) enhances muscle growth in a number of species, including humans and cattle, is not known, E₂ treatment has been shown to stimulate proliferation of cultured bovine satellite cells (BSCs). This is particularly significant because satellite cells are the source of nuclei needed to support postnatal muscle fiber hypertrophy and are thus crucial in determining the rate and extent of muscle growth. The objective of this study was to assess the role of estrogen receptor-α (ESR1) and the type 1 insulin-like growth factor receptor (IGFR1) in E₂-stimulated proliferation of cultured BSCs. To accomplish this, we have used small interfering RNA (siRNA) to silence expression of ESR1 or IGFR1 and assessed the effects on E₂-stimulated proliferation in BSC cultures. In BSCs treated with nonspecific siRNA, E₂ significantly (P < 0.05) stimulates proliferation under conditions in which neither IGF-1 nor IGF-2 expression is increased; however, treatment of ESR1- or IGFR1-silenced cells with E₂ does not significantly stimulate proliferation. These results indicate that both ESR1 and IGFR1 are required for E₂ to stimulate proliferation in BSC cultures. The fact that this occurs under culture conditions in which neither IGF-1 nor IGF-2 mRNA expression is increased strongly suggests that E₂ activates IGFR1 via a mechanism that does not involve increased IGF-1 or IGF-2 binding to the receptor.     

Unique structure of the M loop region of β1-tubulin may contribute to size variability of platelets in the family Felidae

Background: Platelet size is relatively uniform in mammals except for domestic cats. Uniform platelet production by megakaryocytes can be disrupted if microtubule assembly or dynamics is impaired. Mutations in the gene encoding β1‐tubulin have been documented in dogs and people, and the resulting microtubule effects have been associated with production of large platelets. Objectives: The objectives of this study were to evaluate morphology of platelets on feline blood smears, determine the gene sequences encoding β1‐tubulin in members of the family Felidae, and compare the findings with those in other mammalian species to determine whether predicted structural differences in β1‐tubulin that might affect microtubule stability or assembly were present. Methods: At least 100 platelets/smear on blood smears from 15 domestic cats and 88 big cats were evaluated to assess platelet size variability. Platelet‐derived cDNA obtained from a domestic cat and genomic DNA isolated from blood samples of domestic cats and other members of the family Felidae were analyzed by PCR using primers specific for β1‐tubulin. Gene sequences obtained were compared with those of other common mammals. Results: Two differences in gene sequence were found in a highly conserved region encoding the M loop of β1‐tubulin in members of the family Felidae compared with sequences from other species. Platelet size variation was present in big cats and domestic cats. In addition, a rare amino acid change was documented in the C‐terminal region encoding the H11 helix in domestic cats. Conclusion: Members of the family Felidae have an altered M loop region in β1‐tubulin compared with other mammals. This variation may contribute to the observed platelet size variability.     

Cloning and Sequence Analysis of Major Histocompatibility Complex Ⅰ Gene from Goose F1 of Fast-growth Lines

According to the multiple alignments identified major histocompatibility complex Ⅰ (MHC Ⅰ) gene conserved sequence registered in GenBank from the family ducks (Anatidae) anser waterfowl, a pairs of specific primers for the fragments of MHCⅠgene of goose F1 from fast-growth lines were designed and synthesized by Primer Premier 5.0. Using the genome DNA of goose F1 from fast-growth lines, the target gene fragment was obtained by PCR. To conduct sequencing of the fragments of MHCⅠgene of goose F1 from fast-growth lines and make sequence alignment and analysis of protein structure and function by bioinformatics, and research the characteristics of MHCⅠgene of goose F1 and the physicochemical properties of the protein. Bioinformatics was analyzed the nucleic acid data, deduced amino acid sequence and phylogenetic trees. The result of sequence analysis showed that the fragments of MHCⅠgene of goose F1 from fast-growth lines was 1036 bp in length, which coded 96 amino acids polyprotein. The homology were 93% and 83% with MHC Ⅰ gene and coding sequence of Wulong goose in NCBI respectively. There were 72 different bases sequence and 16 amino acids change. There also was higher homology with other poultry, and existed genetic relationship of Siji goose > chickens > ducks.The homology segment sequences corresponding to the fragments of MHCⅠ gene of goose F1 coded 96 amino acids protein, which molecular weight, PI, positively or negatively charged amino acid, estimated half-life, instability index, aliphatic index and average hydrophobicity were 11.342 ku, 5.32, 14, 17, 2.8 h, 34.92, 42.81, -1.066, respectively, and appeared 9 B cell epitopes, but contained no signal peptide. These results indicated that the protein for hydrophilic non-secreted proteins, had the high immunogenicity. In addition, The protein structure study indicated that alpha-helix, beta-sheet, beta-turn and random coil were 31.25%,16.67%, 14.58% and 37.50%, respectively. There existed amino terminal domain and carboxyl terminal domain in the tertiary structure. Therefore, MHC gene had significant difference between species and populations of individuals by the pathogen pressure in environment, and there were the interaction between polymorphism of MHC molecules and the diversity of antigenic peptide. MHC determined the differences of individual susceptibility to disease, and could be treated as a candidate gene for disease resistance.     

Central injection of exogenous IL-1β in the control activities of hypothalamic-pituitary-gonadal axis in anestrous ewes

This study was performed to determine the effect of intracerebroventricular (icv) injection of interleukin (IL)-1β on the gene expression, translation and release of gonadotropin-releasing hormone (GnRH) and the GnRH receptor (GnRHR) gene expression in the hypothalamus of anestrous ewes. In the anterior pituitary gland (AP), the expression of genes encoding: GnRHR, β subunits of luteinizing hormone (LH) and folliculotropic hormone (FSH) was determined as well as the effect of IL-1β on pituitary gonadotropins release. The relative mRNA level was determined by real-time PCR, GnRH concentration in the cerebrospinal fluid (CSF) was assayed by ELISA and the plasma concentration of LH and FSH were determined by radioimmunoassay. Our results showed that icv injection of IL-1β (10 or 50 μg/animal) decreased the GnRH mRNA level in the pre-optic area (POA) (35% and 40% respectively; p ≤ 0.01) and median eminence (ME) (75% and 70% respectively; p ≤ 0.01) and GnRHR gene expression in ME (55% and 50% respectively; p ≤ 0.01). A significant decrease in GnRHR mRNA level in the AP in the group treated with the 50 μg (60%; p ≤ 0.01) but not with the 10 μg dose was observed. The centrally administrated IL-1β lowered also GnRH concentration in the CSF (60%; p ≤ 0.01) and reduced the intensity of GnRH translation in the POA (p ≤ 0.01). It was not found any effect of icv IL-1β injection upon the release of LH and FSH. However, the central injection of IL-1β strongly decreased the LHβ mRNA level (41% and 50%; p ≤ 0.01; respectively) and FSHβ mRNA in the case of the 50 μg dose (49%; p ≤ 0.01) in the pituitary of anestrous ewes. These results demonstrate that the central IL-1β is an important modulator of the GnRH biosynthesis and release during immune/inflammatory challenge.     

Role of interleukin-1β in the regulation of porcine corpora lutea during the late luteal phase of the cycle and during pregnancy

Interleukin-1β (IL-1β) may regulate ovarian physiology. In this study, the influence of IL-1β on secretory activity within the corpora lutea (CL) of cyclic and gravid pigs was determined in vitro during different stages of the CL lifespan, e.g. on Days 10-11, 12-13 and 15-16 of the oestrous cycle and pregnancy. IL-1β (10 ng/ml) increased prostaglandin E2 (PGE2) secretion from CL of the cyclic and gravid pigs during studied days of the oestrous cycle and pregnancy. Increase (P < 0.05) of prostaglandin F2α (PGF2α) in IL-1β-treated CL was demonstrated only on Days 10-11 of the oestrous cycle. More potent stimulatory effect of IL-1β on PGE2 than PGF2α secretion resulted in the enhancement of the PGE2:PGF2α ratio in cyclic and early pregnant CL. IL-1β increased (P < 0.05) progesterone (P4) secretion only in gravid CL and had no effect on oestradiol-17β (E2) release. Expression of cyclooxygenase-2 (COX-2) mRNA was stimulated (P < 0.05) in IL-1β-treated cyclic and gravid CL. Expression of prostaglandin synthase mRNAs in response to IL-1β did not increase. In conclusion, IL-1β modulates PGE2, PGF2α and P4 secretion from porcine CL, depending on luteal stage and the surrounding hormonal milieu. The cytokine may act locally in porcine CL for luteotrophic support throughout the PGE2-mediated synthesis and secretion.     

Effect of dietary supplementation of probiotic on the performance of F1 crossbred (Rhode Island red male × Fayoumi female) cockerels

A study was conducted to evaluate four different probiotic levels, using diets supplemented with 0 (control), 0.1%, 0.2%, 0.3% or 0.5% of organic-green culture-zs (probiotic). The cockerels of 6-weeks old were randomly divided into 15 separate floor pens each comprising 25 birds and three pens (replicates) per treatment group following completely randomized design. At 12 weeks of age BW and feed to gain ratio (FCR) were determined. At the end of 42 days of experiment, nine birds per treatment were sacrificed to evaluate carcass characteristics, abdominal fat contents and the internal organs. Blood haemato-biochemical parameters were also determined. Haemagglutination inhibition antibody titres against Newcastle disease virus and lymphoid organs weight/body weight ratio were also determined. The BW of birds fed 0.2-0.5% of probiotic was significantly greater than birds fed without probiotic diet. Similarly, better FCR was observed in birds those fed diets of high level of probiotic. There was no mortality recorded at higher levels of probiotics. Differences in carcass characteristics, organs weight, meat composition, haematological values and HDL and LDL concentrations among all the diets were non-significant. However, abdominal fat contents reduced significantly in supplemented groups in relation to control and cholesterol contents were reduced significantly in 0.3% or 0.5% supplemented groups in relation to control. Feeding levels of 0.2-0.5% of probiotic, did positively affect the immune system within the parameters measured. It may be concluded that performance, blood chemistry and immunity against disease in cockerels could be maintained when supplementing 0.3% or 0.5% level of probiotic incorporated in crossbred cockerel's diets.     

The effect of tonicity of storage media for fowl semen on the occurrence of neck‐bending spermatozoa,fertility and hatchability 1

1. Semen of White Leghorn and Rhode Island Red males was stored for 24 h in diluents hypertonic (460 mOsm/kg H₂O) and isotonic (340 mOsm/kg H₂O) to cock seminal plasma.

2. Compared with the fertility results with semen that had been stored in the hypertonic diluent or was fresh, the fertility of the White Leghorns was not affected after storage in the isotonic diluent; a decrease (P < 0.lb05) was observed, however, using Rhode Island Red semen and isotonic diluent.

3. Fresh RIR semen contained 2.lb31% “neck‐bent spermatozoa” (NBS) which was increased to 4.lb23% and 5.lb76% after dilution in hypertonic and isotonic diluents respectively and stored for 24 h. It is doubtful whether this increase (P < 0.lb05) is the sole reason for the lowered fertility obtained with this breed after storage in the isotonic diluent.     

IFN-γ renders human intestinal epithelial cells responsive to lipopolysaccharide of Vibrio cholerae by down-regulation of DMBT1

Although intestinal epithelial cells (IECs) are continuously exposed to high densities of enteric bacteria, they are not highly responsive to microbe-associated molecular patterns (MAMPs). However, inflammatory cytokines such as interferon-γ (IFN-γ) are potentially capable of priming IECs to enhance responsiveness to MAMPs. In this study, we observed that heat-killed Vibrio cholerae (HKVC) and its lipopolysaccharide (LPS) poorly induced IL-8 production in a human IEC line, HT-29. However, both HKVC and the LPS showed a substantial induction of IL-8 production in IFN-γ-primed HT-29 cells. LPS-induced IL-8 production was proportional to the IFN-γ-priming period and LPS could not induce IL-8 production in the presence of polymyxin B. Moreover, LPS-induced IL-8 production in the IFN-γ-primed HT-29 cells was mediated through signaling pathways requiring p38 kinase and ERK, but not the JNK/SAPK pathway. Since deleted in malignant brain tumor 1 (DMBT1) is known to interact with and antagonize the action of LPS, we hypothesized that IFN-γ enhanced the responsiveness to LPS in HT-29 through down-regulation of DMBT1. We found that IFN-γ indeed attenuated DMBT1 expression at both the mRNA and protein levels in HT-29 cells. Conversely, when the cells were transfected with small interfering RNA to specifically silence DMBT1, IL-8 expression was augmented even in the absence of IFN-γ and the augmentation was further enhanced by treatment with V. cholerae LPS. Since IFN-γ is known to increase IFN-β expression in the IECs, we examined if IFN-β functioned similar to IFN-γ. Although IFN-β alone was able to induce IL-8 expression, it failed to render HT-29 cells responsive to V. cholerae LPS. In conclusion, our study suggests that IFN-γ primes IECs to become responsive to V. cholerae and its LPS by suppressing the expression of DMBT1.     

A Comparison of the Concentrations of C-reactive Protein and α1-Acid Glycoprotein in the Serum of Young and Adult Dogs with Acute Inflammation

The concentrations of C-reactive protein (CRP) and ₁-acid glycoprotein (AAG) were evaluated in 1-, 3- and 18-month-old dogs (four of each age) that had been inoculated with turpentine oil. The CRP and AAG in 3-month-old and younger dogs subjected to surgery or inoculated with either Staphylococcus aureus or a viral vaccine were also evaluated. The average CRP concentration in the sera peaked 2 days after inoculation of turpentine oil. The peak CRP concentrations in 3- and 18-month-old dogs were significantly (p<0.05) greater than those in 1-month-old dogs. The average AAG concentration in the sera peaked 4 days after inoculation of turpentine oil. No significant difference was found in AAG concentrations between any of the age groups. When experimentally inoculated with S. aureus or subjected to oophorohysterectomy, the CRP and AAG concentrations increased in 3-month-old dogs, but they increased little in 1-month-old dogs. The CRP and AAG in dogs inoculated with the viral vaccine did not increase. In dogs with fractures or subjected to percutaneous gastrostomy, the CRP and AAG concentrations correlated with the condition of dogs.