Impact of high dietary zinc on zinc accumulation, enzyme activity and proteomic profiles in the pancreas of piglets.

The exocrine pancreas plays an important role in zinc homeostasis. Feeding very high (2000-3000mgzinc/kg diet) levels of zinc oxide to piglets for short periods is a common practice in the swine industry to improve performance and prevent diseases. The impact on pancreatic function and possible side effects during long-term feeding of high dietary zinc levels are still poorly understood. A total of 54 weaned piglets were either fed with low (57mg/kg, LZn), normal (164mg/kg, NZn) or high (2425mg/kg, HZn) zinc concentration in the diets. After 4 weeks of feeding, ten piglets per treatment were euthanized and pancreas samples were taken. Tissue zinc concentration and metallothionein abundance was greater with HZn compared with NZn and LZn (P<0.05). Similarly, activity of α-amylase, lipase, trypsin and chymotrypsin was higher with HZn as compared with NZn and LZn diets (P<0.05), whereas elastase activity was unchanged. Total trolox equivalent antioxidative capacity of pancreas tissue was higher with HZn diets compared with the other treatments (P<0.05). Pancreatic protein profiles of NZn and HZn fed piglets were obtained by 2D-DIGE technique and revealed 15 differentially expressed proteins out of 2100 detected spots (P<0.05). The differentially expressed proteins aldose reductase, eukaryotic elongation factor II and peroxiredoxin III were confirmed by immunoblotting. Identified proteins include zinc finger-containing transcription factors and proteins mainly associated with oxidative stress response and signal transduction in HZn compared with NZn pigs. Histologic examination however showed no morphologic changes. The results suggest that long-term supply of very high dietary zinc increases zinc and metallothionein concentration, and digestive enzyme activity, but also triggers oxidative stress reactions in the pancreas of young pigs. The data provide new insights into pancreatic function under outbalanced zinc homeostasis.

Characterization of the native C-reactive protein (cCRP) and the corresponding liver mRNA in dogs.

C-reactive protein (CRP) plays an important role in the acute phase reaction in humans and dogs. For the canine CRP (cCRP) only an in silico deduced preliminary transcript and amino acid sequence is available. The objective of this study was to further characterize the native cCRP protein and its corresponding liver mRNA. Furthermore, immunological similarities of serum CRP in related animal species were investigated. Native cCRP protein was isolated from dog-sera by affinity chromatography and further analyzed by immunodetection, protein sequencing (mass spectrometry and N-terminal Edman sequencing), 2D-gel electrophoresis, and glycoprotein analysis. Furthermore, cCRP cDNA sequence was determined from dog liver total RNA by RT-PCR. Gel electrophoresis, immunodetection and glycoprotein detection revealed two cCRP isotypes with different molecular weights (22 and 25kDa) with the upper band being glycosylated. Selective glycoprotein analysis showed sialic acid terminally linked (2-6) to galactose or N-acetylgalactosamine and subsequent PNGase F treatment identified N-terminal linkage. Mass spectrometry confirmed approximately 45% of the cCRP predicted amino acid sequence and N-terminal amino acid sequencing revealed a shorter native cCRP than expected (204 amino acids). The new canine CRP mRNA sequence confirms 100% of the formerly deduced sequence. Immunological homologies to the canine CRP protein were found in selected dog-related species. This study contributes major molecular details to the knowledge about canine CRP. Such structural information may assist in developing new diagnostic tools for inflammatory-based diseases in dogs as well as other dog-related species.

Evaluation of biomarkers for osteoarthritis caused by fragmented medial coronoid process in dogs.

The aim of the present work was to evaluate whether concentrations of the carboxy-terminal cross-linked fragment of type II collagen (CTX-II), the activities of matrix metalloproteinase-2 and -9 (MMP-2/-9) and Myeloperoxidase (MPO) in canine synovial fluids (SF) can reflect structural alterations of articular cartilage in dogs with fragmented medial coronoid process (FMCP). Elbow joints with FMCP underwent radiographic and arthroscopic examination. Commercially available assays were used to analyze SF for CTX-II concentration and MMP-2/-9 activity. MPO activity was measured by o-dianisidine-assay. The MMPs were further evaluated by zymography. CTX-II concentration and MMP-2 activity showed age-dependent trends in controls. Increased enzyme activities of MPO and MMP-2/-9 were found in diseased dogs. MMP-9activity seems suitable to underline the subjective assessment of the degree of cartilage damage. These initial data of the study suggest that MPO and MMP-2/9 may be used as objective biomarkers in the diagnosis of canine osteoarthritis due to FMCP.

Short-term whole body vibration exercise in adult healthy horses.

The purpose of this study was to analyze the acute effect of whole body vibration exercise (WBVE) on clinical parameters and blood values in horses. Seven horses were exposed to a 10 min WBVE at a frequency of 15-21 Hz. Clinical parameters and venous blood samples were taken before and directly after WBVE. Acute short-term WBVE produced a decrease in serum cortisol (p = 0.02) and creatine-kinase (p = 0.02) values. Clinical parameters, hematology, fibrinogen, lactate, IGF-I, GGT, creatinine, myeloperoxidase activity and bone marker values were not significantly changed by WBVE. In adult sound horses WBVE was well tolerated and did not cause any sign of measured discomfort.

Effects of zinc on epithelial barrier properties and viability in a human and a porcine intestinal cell culture model.

Zinc is an essential trace element with a variety of physiological and biochemical functions. Piglets are commonly supplemented, during the weaning period, with doses of zinc above dietary requirements with positive effects on health and performance that might be attributed to anti-secretory and barrier-enhancing effects in the intestine. For a better understanding of these observations increasing zinc sulfate (ZnSO4; 0-200µM) concentrations were used in an in vitro culture model of porcine (IPEC-J2) and human (Caco-2) intestinal epithelial cells and effects on barrier function, viability, and the mRNA expression of one selected heat shock protein (Hsp) were assessed. When treated apically with zinc sulfate, the transepithelial electrical resistance (TEER) did not change significantly. In contrast, cell viability measured by lactate dehydrogenase (LDH) leakage, by ATP and by WST-1 conversion in postconfluent IPEC-J2 monolayers was affected after a 24-h treatment with 200µM ZnSO4. Caco-2 cells were more resistant to Zn. ZnSO4 did not induce any effect on viability, except when it was used at the highest concentration (200µM), and only in preconfluent cells. Furthermore, ZnSO4 induced Hsp70 mRNA expression at 200µM and was more pronounced in preconfluent cells. The observed dose-related effects of zinc are cell-line specific and depended on the differentiation status of the cells. The IPEC-J2 cell line appears to be a suitable in vitro model to characterize specific effects on porcine intestinal cells.

Tandem duplication of KIT exon 11 influences the proteome of canine mast cell tumours.

Mutations with permanent activation of the stem cell factor receptor KIT have been identified as one potential cause for canine cutaneous mast cell tumours (MCTs). The exact changes in global gene expression patterns associated with permanent activation of KIT in these tumours are unknown. The present study compares, by the use of two dimensional difference gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, the proteomes of canine MCTs, with and without KIT exon 11 tandem duplication. Fifteen differentially expressed proteins were identified in mutated MCTs. These are mainly involved in cytoskeleton structure and cell motility (ACTR2, ACTB and CAPPA1), cell signalling (ARHGDIA) and lipid metabolism (ALOX15 and ACSBG4), or are serum proteins. The results therefore support the notion that KIT mutation is associated with changes in the proteome of affected cells with a major effect on the composition of the cytoskeletal proteome and cell motility proteins. No overlaps were identified when the results were compared with a recent study on the proteomic differences between low- and high-grade tumours, suggesting that KIT-mutated tumours may be regarded as a separate entity of high-grade tumours with potential relevance to therapeutic strategies.

Hyaluronic acid concentrations in synovial fluid of dogs with different stages of osteoarthritis.

To compare hyaluronic acid (HA) concentrations measured in synovial fluid (SF) of joints with different stages of canine secondary osteoarthritis (OA), clinical-orthopedic, radiographic, macroscopic intra-operative and SF findings of 49 joints were assessed. The sum of single findings was correlated to HA concentrations measured by a commercially available ELISA. Joints were categorized into three OA-groups: non-osteoarthritic, mildly osteoarthritic, and severely osteoarthritic. A significant negative correlation was found between severity of OA and HA concentrations (r=-0.696; P<0.001). Median values of HA concentrations decreased with increasing severity of the disease. Statistically significant differences in HA concentrations were observed between the OA-groups (P<0.001). Due to overlapping values between groups, it was concluded that synovial HA concentrations may only indicate a trend of osteoarthritic disease activity, but is not suitable for staging the disease.

Differences in the proteome of high-grade versus low-grade canine cutaneous mast cell tumours.

Cutaneous mast cell tumours (MCTs) are the most common skin tumours in dogs. However, the molecular differences between benign tumours with a good prognosis and highly malignant, invasive and metastatic tumours with short survival times are for the most part unclear. In the present study the proteome of low-grade MCTs with a good prognosis was compared with that of poor-prognosis high-grade tumours independent of their mutational status of exon 11 of the KIT gene. Using two-dimensional difference gel electrophoresis and mass spectrometry, 13 proteins with a significant differential expression between the two groups were identified. Four stress response proteins (HSPA9, PDIA3, TCP1A and TCP1E) were significantly up-regulated in high-grade tumours, while proteins mainly associated with cell motility and metastasis had either increased (WDR1, ACTR3, ANXA6) or decreased (ANXA2, ACTB) expression levels. High-grade tumours also had a paradox down-regulation of transferrin, a protein that is usually up-regulated in neoplastic cells. The histologically observable dedifferentiation of high-grade tumours was reflected by decreased tryptase protein expression levels. Results of quantitative real-time RT-PCR analysis indicated that the differences in protein expression levels of most proteins were regulated at the transcript level. Based on these findings, it is hypothesized that high-grade MCT cells have a higher resistance to cellular stress and thus are able to better cope with the adverse environment in highly proliferating tumours independent of increased KIT signalling. It is noteworthy that some of the proteins identified have been proposed as therapeutic targets for human oncology and it will be interesting to evaluate their therapeutic and diagnostic potential for canine MCTs.

Phospholipid compositions of sera and synovial fluids from dog, human and horse: a comparison by 31P-NMR and MALDI-TOF MS.

Alterations of the phospholipid (PL) compositions of body fluids are assumed to be indicative of inflammatory diseases, e.g. rheumatoid arthritis (RA). Recently, we have shown that particularly the phosphatidylcholine/lysophosphatidylcholine (PC/LPC) ratio determined in human synovial fluids (SF) and sera represents a reliable measure of the inflammatory state in RA patients. However, it is not yet clear to what extent the PC/LPC ratio is also affected by nutrition habits. In the present study, the PL and the corresponding acyl chain compositions of human body fluids (SF and serum of RA patients as well as serum from healthy volunteers) are compared with those of two other mammalian species (horses and dogs suffering from degenerative joint diseases as well as healthy controls) by high-resolution 31P-nuclear magnetic resonance (NMR) spectroscopy and matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS). The most important result of this study is that the PL compositions of SF and serum of horse and dog are comparable with those of human body fluids. Compared with humans, however, the horse body fluid contains less PCs with highly unsaturated arachidonoyl residues, while that of dogs possesses the highest content of arachidonoyl-containing PC. These species-related differences stem primarily from different nutrition habits (meat vs. plants).

Establishment and characterization of an adherent pure epithelial cell line derived from the bovine oviduct.

The oviduct in vivo has to perform various tasks: maturation and transport of the gametes, milieu preparation for fertilization and embryonic development, and transport of the embryo. The complex arrangement of endocrine and paracrine signals being exchanged between the early embryo and the inner cell layers of the oviduct is barely understood. Therefore, a reproducible, well-characterized oviduct epithelial cell line as well as an optimized transfection protocol for DNA vectors and siRNA for this cell line has been established. A bovine oviduct primary cell culture system has been optimized using a selection medium permitting the survival of only epithelial cells. From this we established an adherent bovine oviduct pure epithelial cell line (aBOPEC-1). This cell line maintains some important characteristics of the primary cells such as the expression of estrogen receptors and p450 aromatase but it lacks some characteristics due to the selection and dedifferentiation processes (cilia, expression of progesterone receptor and oviduct specific glycoprotein-1). Optimization of the transfection protocols finally revealed a suitable DNA-transfection procedure yielding transfection efficiencies of over 50%. Additionally, siRNA transfection efficiency reached more than 90%. This new cell line builds an essential basis especially for future functional studies in the oviduct epithelium using distinct knock down experiments.

Measurement of equine myeloperoxidase (MPO) activity in synovial fluid by a modified MPO assay and evaluation of joint diseases - an initial case study.

The aim of this study was to develop a specific myeloperoxidase (MPO) activity assay in the synovial fluid of horses and investigate whether MPO activity is increased in different forms of joint diseases. Synovial fluid samples were taken from affected joints from horses with osteoarthritis, chronic non-septic arthritis and septic arthritis, and from healthy control horses. MPO activity was measured using a specific modified o-dianisidine-assay containing 4-aminobenzoic acid hydrazide as a potent and specific inhibitor of the MPO. This assay is characterized by high reproducibility. The results reveal only a slight elevation of MPO activity in the synovial fluid of horses with osteoarthritis and chronic non-septic arthritis. However, in the cases of septic arthritis a significant increase in MPO activity was found when compared to the controls. In conclusion the first field study suggests that synovial fluid MPO may be used as a marker for septic arthritis in horses.

Determination of MMP-2 and -9 activities in synovial fluid of horses with osteoarthritic and arthritic joint diseases using gelatin zymography and immunocapture activity assays.

REASON FOR PERFORMING STUDY: Matrix metalloproteinases (MMPs)-2 and -9 activities have been found elevated in synovial fluid from various joint diseases in man. However, in the horse few data are available. OBJECTIVES: To explore the clinical significance of MMP-2 and -9 activities in synovial fluid of horses with different forms of joint diseases. METHODS: Gelatin zymography and MMP-2 and -9 immunocapture activity assays were applied on synovial fluids from control joints and joints with aseptic joint disease (AJD) and septic arthritis (SA). Additionally, MMP-2 and -9 activities were measured in samples from SA to monitor the disease process. RESULTS: Zymographic analysis revealed that samples from AJD and SA contained significantly increased latent MMP-2 activity compared to controls. Samples from SA showed significantly increased monomeric latent MMP-9 activity compared with all other affected joints and controls. Trace amounts of MMP-9 activity, due to the active and dimer form, were detected in samples from SA; however, these bands were absent in samples from AJD and controls. Using immunocapture activity assays, MMP-2 and -9 activities were found to be significantly elevated in joints from SA compared to controls and AJD samples. MMP-2 activity in samples from AJD was significantly increased compared to controls. Both MMP activities decreased in the joints from SA in the course of successful therapy. CONCLUSIONS: Data from zymographic analysis confirmed that MMP-2 and -9 were elevated in equine joint diseases. Immunocapture activity assays have been shown to be suitable for the quantitative determination of MMP-2 and -9 activities in synovial fluid of horses. Both MMP-2 and -9 activities seem to be useful to indicate SA, and MMP-2 activity might be a suitable marker for AJD. POTENTIAL RELEVANCE: These findings encourage the potential use of MMP-2 and -9 as additional aids to clinical investigation. Further work is required to validate the clinical significance of MMP activities in the progress of different joint diseases in horses.

Exploring cumulus-oocyte-complex-oviductal cell interactions: gene profiling in the bovine oviduct.

Prostaglandin E(2) (PGE(2)) is present in the bovine oviduct and may play an important role in muscle contraction or as survival factor providing the optimal environment for fertilization and the early embryo. The aim of the present study was to investigate the estrous cycle-dependent changes and local distributions of PGE(2) receptors (EP) and members of the trefoil factor (TFF)-family in the bovine oviduct using real-time RT-PCR. A co-cultivation system of cumulus-oocyte-complexes (COC) with primary oviductal cells was screened for the mRNA expression pattern of selected factors. An oviductal primary cell culture was used for investigating effects of estradiol on signal transduction pathways. Quantitative RT-PCR revealed significant higher expression of EP2 and EP4 in the pre-ovulatory phase compared with the luteal phase. TFF3 mRNAwas expressed during the estrous cycle with highest level in the post-ovulatory phase showing higher expression levels in the isthmus compared with the ampulla. A different mRNA expression pattern was observed for factors involved in extracellular matrix formation in co-cultured oviductal cells compared to untreated controls. In vitro, NF-kappaB was found activated after estradiol treatment. These results suggest that a fine-tuned PGE(2) signal transduction pathway may support fertilization, early embryonic development and gamete transport in the bovine oviduct.

Establishment of a new bovine leukosis virus producing cell line.

Due to the prevalence of different bovine leukosis virus (BLV) species in the cattle population in Europe, problems may arise in the serological diagnosis of BLV infections. In addition, earlier investigations demonstrated that contamination of the BLV antigen-producing cell culture systems by bovine viral diarrhea virus (BVDV) may give rise to misinterpretation of serological test results after BVDV vaccination of cattle. By co-cultivation of peripheral leukocytes of a BLV-infected cow with a permanent sheep kidney cell line, a new BLV-producing cell line named PO714 was established. This line carries a BLV provirus of the Belgian species and has been tested to be free of a variety of possibly contaminating viruses and mycoplasms. Investigations of a panel of well-characterised sera by agar gel immunodiffusion (AGID) and capture ELISA (cELISA) tests using antigen prepared from this new cell line in comparison with antigen of the well-known cell line FLK/BLV yielded comparable results. False positive results caused by BVDV cross-reactions could be eliminated when tests were carried out with antigen derived from the new cell line.

Involvement of intracellular Ca2+ in the regulation of bovine leukemia virus expression.

The calcium ionophore A23187, which was used to increase the intracellular calcium concentration ([Ca2+]i), was analyzed for effects on bovine leukemia virus (BLV) expression in two BLV infected cell lines. To clarify the role of intracellular free calcium in this response, [Ca2+]i was measured during ionophore treatment with the fluorescent calcium indicator Fura-2. Elevation of intracellular calcium under these conditions caused an enhancement of BLV gp51 and p24 synthesis as well as an activation of the BLV long terminal repeat (LTR) in a dose-dependent manner. Furthermore, it was observed that elevated levels of intracellular calcium following A23187 stimulation lead to activation of NF-kappaB. Based on inhibitor studies, we hypothesize that the effect of A23187 on BLV expression appears to be mediated by PKC.

A nucleotide deletion causing a translational stop in the protease reading frame of bovine leukaemia virus (BLV) results in modified protein expression and loss of infectivity.

Bovine leukaemia virus (BLV) is an oncogenic retrovirus that causes B-cell lymphocytosis and in the terminal stage of the disease lymphosarcoma. The comparison of the previously published BLV provirus sequence from Belgium, Australia and Japan showed that the protease gene (prt) of the Australian and the Japanese isolate contain a nucleotide deletion when compared to the Belgian isolate. Because all these proviruses were isolated from tumour tissue, the prt gene of functionally active and infectious proviruses from peripheral blood leucocytes (PBLs) of BLV-infected cattle and from BLV-infected fetal lamb kidney cells were sequenced. The only variations between these sequences and the Belgian isolate consist of nucleotide substitutions. The delection of one nucleotide of the prt gene of the Japanese and the Australian BLV tumour isolate caused a changed reading frame and a premature translational stop. It was shown that the Japanese provirus is non-infectious in transfected cell culture and in injected sheep. To analyse the impact of the prt mutation on viral protein expression and infectivity, the prt region of the Japanese provirus was exchanged with the prt region from the Belgian provirus. The resulting pBLVprtbelg was infectious in transfected cells and enabled the expression of gag and gag-precursor proteins. One sheep was injected with this mutated provirus and became positive in BLV-PCR, but no seroconversion was developed. The prt mutation of the Japanese tumour isolates was shown to be responsible for the loss of infectivity and changed viral expression. These results and the occurrence of this mutation in only two isolates from lymphosarcoma indicate a possible relation between the prt mutation and the induction of cell transformation.

Investigations of the Japanese bovine tumour virus (BLV)--its ability to express structural and regulatory BLV proteins.

The mechanism of BLV-induced tumorigenesis has not been clear up to now. Changes of viral protein expression in infected cells may be involved in the molecular events leading to BLV-induced leukaemogenesis. In this study Western blot investigations of cells transfected with plasmid DNA containing the complete Japanese BLV tumour clone provirus demonstrate that this provirus is unable to express gag and env proteins. Following this an attempt was made to express the genes from this provirus in eukaryotic and prokaryotic cells using the phagemid pBK-RSV (Stratagene), but not as fusion proteins. The protein patterns expressed from the 5' and the 3' region of the BLV genome were compared with those of FLK/BLV cells. The results indicate that there is a defect in this provirus located in the genome region between the gag and env gene.

Increase of antigen production in BLV-infected cell lines via additional expression of tax.

The selection of animals infected with the bovine leukaemia virus (BLV) is performed by the immunological detection of antibodies against the virus, commonly using the antigen gp51. Furthermore, research is being carried out to develop protective vaccines against BLV that have gp51 as their main component. Taking both of these factors into account, it is clear that there will be an increasing requirement for the virus antigen gp51 for some time to come. The permanently BLV-infected foetal lamb kidney cell line FLK/BLV (and its sublines) has been proved to be the most useful culture for the mass production of the virus antigen. Stable cell lines producing higher quantities of BLV antigen have not been established, either by subcloning of the FLK/BLV or by infection of other permanent cells with BLV. Here, a report is made on efforts to increase the expression of gp51 in BLV-infected cells via the additional expression of homologous transactivating virus protein tax. Selectable tax expression vectors that integrate into the host cell genome were constructed using BL provirus DNA fragments. Highly productive FLK/BLV cells were transfected with these vectors. Following selection with G 418, gp51-producing cell lines were established and tested for their productivity for several months. Some tax-vector-containing cell lines have produced 1.5-2 times more gp51 than the highly productive parental control cell line FLK/BLV 44-1.

[Different transactivation processes in two bovine leukemia virus producing cell lines]. / Unterschiedliches Transaktivierungsgeschehen in zwei bovines Leukämievirus produzierenden Zellinien.

Comparative studies were conducted through more than six months into quantitative of bovine leukaemia virus (BLV) antigen of FLC/BLV 44 and its FLC/BLV 44-4 subline by means of an enzyme immuno-assay (EIA), using monoclonal antibodies against gp51 and p24. Synthesis of gp51 (factors of two to six) and of p24 (factor of two) by FLC/BLV 44 was clearly higher than that by FLC/BLV 44-4. The transactivation status in either line was determined by transfer of the beta-galactosidase indicator organ under transcription control of BLV-LTR (in pBLV beta Gal plasmid). Transient experiments showed beta-galactosidase activity in the FLC/BLV 44 to be clearly higher than that in subline FLC/BLV 44-4. There is obviously in both cell lines a close correlation between intensity of BLV antigen synthesis and transactivation processes.