Large-scale vibrating coil magnetometer for the magnetic characterization of bulk superconductors.

This work presents the design and validation of a vibrating coil magnetometer for the characterization of the field dependence of the critical current density of centimeter-sized bulk superconductors as an alternative to the destructive methods typically used. The magnetometer is also shown to be capable of measuring the magnetic moment in an applied field of up to 5 T for diverse magnetic materials, such as soft and hard ferromagnets and high-temperature superconducting pellets. The vibrating coil magnetometer was first optimized using finite element simulations and calibrated using a commercial vibrating sample magnetometer. The vibrating coil magnetometer was benchmarked with hysteresis measurements of a Nd2Fe14B disk made with a commercial hysteresisgraph, showing good agreement between the different setups. The magnetic hysteresis of a YBa2Cu3O7-x superconducting pellet was measured at 77 K, showing a penetration field of 1 T and an irreversibility field of 4 T. The field dependent critical current density of the superconductor was then inferred from the magnetic hysteresis measurements and extrapolated at low fields. Finally, the resulting critical current density was used to successfully reproduce the measured magnetization curve of the pellet at 2 T with finite element simulations.

Effects of source of supplementary trace minerals in pre- and postpartum diets on reproductive biology and performance in dairy cows.

Our objectives were to evaluate the effects of complete replacement of inorganic salts of trace minerals (STM) with organic trace minerals (OTM) in both pre- and postpartum diets on ovarian dynamics, estrous behavior measured by sensors, preimplantation conceptus development, and reproductive performance in dairy cows. Pregnant cows and heifers (n = 273) were blocked by parity and body condition score and randomly assigned to either STM or OTM diets at 45 ± 3 d before their expected calving. Pre- and postpartum diets were formulated to meet 100% of recommended levels of each trace mineral in both treatments, taking into consideration both basal and supplemental levels. The final target concentrations of Co, Cu, Mn, Se, and Zn were, respectively, 0.25, 13.7, 40.0, 0.3, and 40.0 mg/kg in the prepartum diet, and 0.25, 15.7, 40.0, 0.3, and 63.0 mg/kg in the postpartum diet. The STM group was supplemented with Co, Cu, Mn, and Zn sulfates and sodium selenite, while the OTM group was supplemented with Co, Cu, Mn, and Zn proteinates and selenized yeast. Treatments continued until 156 d in milk (DIM) and were assigned to individual cows using automatic feeding gates. Starting at 21 DIM, ultrasonography examinations of the ovaries were performed weekly to determine the presence of a corpus luteum and postpartum resumption of ovarian cyclicity. Cows were presynchronized with 2 injections of PGF2α at 42 and 56 DIM. Estrous behavior was monitored using electronic activity tags that indirectly measured walking activity. Cows detected in estrus after the second PGF2α were inseminated, and those not detected in estrus by 67 DIM were enrolled in a synchronization program. Cows that returned to estrus after artificial insemination (AI) were reinseminated. Pregnancy diagnosis was performed 33 d after AI, and nonpregnant cows were resynchronized. Transcript expression of interferon-stimulated genes in peripheral blood leukocytes was performed in a subgroup of cows (STM, n = 67; OTM, n = 73) on d 19 after AI. A different subgroup of cows (28 STM, 29 OTM) received uterine flushing 15 d after AI for recovery of conceptuses and uterine fluid for analyses of transcriptomics and metabolomics, respectively. In addition, dominant follicle diameter, luteal size and blood flow, and concentration of progesterone in plasma were measured on d 0, 7, and 15 relative to AI. After flushing, PGF2α was given and the dominant follicle was aspirated 2 d later to measure the concentration of trace minerals by mass spectrometry. Estrous behavior, size of the dominant follicle and corpus luteum, concentration of progesterone, time to pregnancy, and proportion of cows pregnant by 100 d of the breeding period did not differ between treatments. A greater proportion of cows supplemented with OTM had a corpus luteum detected before presynchronization (64.3 vs. 75.2%), and primiparous cows supplemented with OTM tended to resume cyclicity earlier than their STM counterparts. Cows supplemented with OTM had a greater concentration of Cu in follicular fluid than cows supplemented with STM (0.89 vs. 0.77 µg/mL, respectively). In pregnant multiparous cows, expression of receptor transporter protein 4 in peripheral blood leukocytes was 42% greater in the OTM group. Conceptuses of the 2 treatments had 589 differentially expressed transcripts, with many indicating advanced conceptus elongation and greater transcript expression of selenoproteins in the OTM group. In pregnant cows, 24 metabolites were more abundant in the uterine fluid of OTM, including spermidine, sucrose, and cholesterol. In conclusion, replacing STM with OTM caused modest improvements to resumption of ovarian cyclicity and important changes in preimplantation conceptus development, but it did not alter conception risk and pregnancy rate.

PIWIs, piRNAs and Retrotransposons: Complex battles during reprogramming in gametes and early embryos.

Gamete and embryo development are indispensable processes for successful reproduction. Cells involved in these processes acquire pluripotency, the ability to differentiate into multiple different cell types, through a series of events known as reprogramming that lead to profound changes in histone and DNA methylation. While essential for pluripotency, this epigenetic remodelling removes constraints that normally limit the expression of genomic sequences known as transposable elements (TEs). Unconstrained TE expression can lead to many deleterious consequences including infertility, so organisms have evolved complex and potent mechanistic arsenals to target and suppress TE expression during reprogramming. This review will focus on the control of transposable elements in gametes and embryos, and one important TE suppressing system known as the PIWI pathway. This broadly conserved, small RNA-targeted silencing mechanism appears critical for fertility in many species and may participate in multiple aspects of gene regulation in reproduction and other contexts.

Expression of microRNAs miR-21 and miR-181c in cerebrospinal fluid and serum in canine meningoencephalomyelitis of unknown origin.

The potential of microRNAs (miRNAs) as biomarkers for canine meningoencephalomyelitis of unknown origin (MUO) was investigated by using quantitative real-time (qRT)-PCR to determine the expression of microRNA-21 (miR-21) and microRNA-181c (miR-181c) in the cerebrospinal fluid (CSF) of dogs. Dogs with MUO (n = 10) had higher levels of expression of miR-21 and miR-181c in the CSF than dogs with non-inflammatory neurological diseases (n = 8). There was a positive correlation between CSF cellularity and expression of miRNAs in the CSF, particularly for miR-21 in the MUO group.

Telomere length and telomerase activity in bovine pre-implantation embryos in vitro.

Telomeres are specialized structures that cap the ends of chromosomes and help to maintain genomic integrity and stability. Telomeres undergo dynamic changes during embryo development, which also represents an important stage for telomere elongation through telomerase enzyme activity. The objectives of this study were to examine changes in telomere length and telomerase activity from the early oocyte, through to the blastocysts stage of development, and the expression of factors with the potential to directly regulate telomeres. In vitro-produced bovine embryos were lysed and analysed for either relative telomere length, or telomerase activity using quantitative real-time PCR protocols. Our results reveal that relative telomere length is the shortest in the presumptive zygote stage of development and gradually increases to the blastocyst stage. We also demonstrate that differences between the mean telomere lengths throughout these stages are statistically significant (p < 0.05). Telomerase activity in the stages examined appears relatively constant until the blastocyst, where the highest level of activity is detected, leading to a significant difference in telomerase activity across embryonic stages (p < 0.005). Bovine telomerase RNA component (bTERC) expression levels were highest in the blastocyst, TERF1 transcripts showed little change in expression, and TERF2 expression decreased in the blastocysts (p < 0.05). Our results suggest that a complex integration of telomere-related RNA and proteins influences the regulatory mechanisms involved in 'reprogramming' of telomeres during early embryonic stages.

Characterization of a rat subcutaneous malignant fibrous histiocytoma and its tumor lines, with reference to histiocytic features.

Malignant fibrous histiocytoma (MFH) is regarded as soft tissue-derived undifferentiated pleomorphic sarcoma, of which the histogenesis remains to be proven. To investigate the cellular characteristics, a homotransplantable tumor line (KJ) was established from a spontaneous MFH that developed in the subcutis of an aged F344 rat. KJ tumors have been produced in syngeneic rats by serial subcutaneous implantation of tissue fragments. The original and KJ tumors consisted of oval and fusiform cells arranged in interlacing bundles with fibrous stroma. Occasional giant cells with bizarre nuclei were observed. Enzyme/immunohistochemically, neoplastic cells reacted to ED1 and ED2 (antibodies specific for rat histiocytes/macrophages), and showed a positive reaction to vimentin and lysosomal enzyme markers such as acid phosphatase (ACP) and nonspecific esterase (Non-SE). Electron microscopically, neoplastic cells possessed lysosomal granules in cytoplasm. A cloned cell line (KJ-A) was isolated from a KJ tumor. KJ-A cells showed positive reactions to ED1, ED2, ACP, and Non-SE, and had cytoplasmic lysosomal granules. Tumors induced by KJ-A cells exhibited histologic and enzyme/immunohistochemical findings similar to those of KJ tumors. Lipopolysaccharide (LPS) treatment increased the number of ED1-positive cells and the expression of tumor necrosis factor-alpha mRNA by reverse transcription-polymerase chain reaction. Collectively, it is likely that rat MFH cells originally possess histiocyte/macrophage-like features that may be enhanced by LPS. Because tumor lines are useful for in vivo and in vitro studies concerning different characteristics of the original neoplasms. KJ and KJ-A should prove useful for studies concerning the morphogenesis of MFH.

The annual effective dose from natural sources of ionising radiation in Canada.

A review and analysis of published information combined with the results of recent gamma ray surveys were used to determine the annual effective dose to Canadians from natural sources of radiation. The dose due to external radiation was determined from ground gamma ray surveys carried out in the cities of Toronto, Ottawa, Montreal and Winnipeg and was calculated to be 219 microSv. A compilation of airborne gamma ray data from Canada and the United States shows that there are large variations in external radiation with the highest annual outdoor level of 1424 microSv being found in northern Canada. The annual effective inhalation dose of 926 microSv from 222Rn and 220Rn was calculated from approximately 14,000 measurements across Canada. This value includes a contribution of 128 microSv from 222Rn in the outdoor air together with 6 microSv from long-lived uranium and thorium series radionuclides in dust particles. Based on published information, the annual effective dose due to internal radioactivity is 306 microSv. A program developed by the Federal Aviation Administration was used to calculate a population-weighted annual effective dose from cosmic radiation of 318 microSv. The total population-weighted average annual effective dose to Canadians from all sources of natural background radiation was calculated to be 1769 microSv but varies significantly from city to city, largely due to differences in the inhalation dose from 222Rn.

Characterization of newly established tumor lines from a spontaneous malignant schwannoma in F344 rats: nerve growth factor production, growth inhibition by transforming growth factor-beta1, and macrophage-like phenotype expression.

Transplantable tumor (KE) and clone cell (KE-F11) lines were established from a spontaneous malignant schwannoma found in an aged F344 rat. The primary tumor and KE tumors consisted of oval or spindle cells arranged in ill-defined bundles. Cultured KE-F11 cells exhibited polygonal or spindle configurations. Immunohistochemically, neoplastic cells in KE and KE-F11 reacted to vimentin, S-100 protein, neuron-specific enolase, myelin basic protein, and glial fibrillary acidic protein in varying degrees, indicating neurogenic features; occasional cells reacted to alpha-smooth muscle actin. Cells positive for lysosomal enzymes (acid phosphatase and non-specific esterase), and ED1 (rat macrophage specific) were observed in KE-F11, and electron microscopically, cells with many lysosomes were frequently present, indicating expression of macrophage-like phenotypes. Bioassay analysis revealed that KE-F11 cells produced high levels of nerve growth factor. DNA synthesis was inhibited by addition of transforming growth factor-beta1 (TGF-beta1), and Northern blot analysis revealed that expression of c-myc, a cell cycle-related immediate early gene, was depressed by TGF-beta1. Likely, TGF-beta1 is a factor capable of inhibiting cellular growth of Schwann cells. mRNA expression of the low-density lipoprotein receptor-related protein (LRP) was seen in KE-F11 cells by Northern blot analysis, and the level was decreased by lipopolysaccharide (LPS) treatment. LRP may be attributable to regulation of Schwann cell functions. KE-F11 cells seeded on laminin-coated dishes exhibited more extended cytoplasmic projections than on collagen type I-coated dishes. The present study provides evidence that biological properties of malignant schwannoma-derived cells might be affected by exogenous factors such as TGF-beta1, LPS and laminin. These tumor lines may be useful for studies on pathobiological characteristics of Schwann cells.

A fence line noble gas monitoring system for nuclear power plants.

A noble gas monitoring system has been installed at Ontario Power Generation's Pickering Nuclear Generating Station (PNGS) near Toronto, Canada. This monitoring system allows a direct measure of air kerma from external radiation instead of calculating this based on plant emission data and meteorological models. This has resulted in a reduction in the reported effective dose from external radiation by a factor of at least ten. The system consists of nine self-contained units, each with a 7.6 cm x 7.6 cm (3 inch x 3 inch) NaI(TI) detector that is calibrated for air kerma. The 512-channel gamma ray spectral information is downloaded daily from each unit to a central computer where the data are stored and processed. A spectral stripping procedure is used to remove natural background variations from the spectral windows used to monitor xenon-133 (133Xe), xenon-135 (135Xe), argon-41 (41Ar), and skyshine radiation from the use of radiography sources. Typical monthly minimum detection limits in air kerma are 0.3 nGy for 133Xe, 0.7 nGy for 35Xe, 3 nGy for 41Ar and 2 nGy for skyshine radiation. Based on 9 months of continuous operation, the annualised air kerma due to 133Xe, 135Xe and 41Ar and skyshine radiation were 7 nGy, 8 nGy, 26 nGy and 107 nGy respectively.

Identification of a 43-kDa protein in human liver cytosol that binds to the 3'-untranslated region of CYP2A6 mRNA.

Hepatic expression of cytochrome P450 2A6 (CYP2A6) varies widely in humans and is induced during hepatitis; however, the mechanism regulating CYP2A6 has not been established. The murine orthologue Cyp2a5 is regulated post-transcriptionally by mRNA stabilization. A 43-kDa protein that binds to the 3'-untranslated region (3'-UTR) of Cyp2a5 mRNA has been identified, but its role in mRNA stabilization is unclear. We hypothesized that similar interactions occur between cytosolic proteins in human liver and CYP2A6 3'-UTR mRNA. We identified, by RNA electrophoretic mobility shift assay, an hepatic cytosolic protein that binds specifically to sequences in the 3'-UTR of CYP2A6. Complexes did not form with denatured proteins and were eliminated with proteinase K digestion. Complex formation was inhibited with a molar excess of unlabeled CYP2A6 RNA but not by non-specific competitor RNA. Protein-mRNA interactions were not affected by probe denaturation, suggesting that RNA secondary structure is not essential for binding. UV cross-linking of complexes revealed RNA-binding proteins in both human and mouse liver cytosols with molecular masses of approximately 43 kDa. Using truncated RNA probes corresponding to various lengths of CYP2A6 mRNA, the protein-binding site was localized to a 50-nucleotide region between bases 1478 and 1527 of the 3'-UTR. Complex formation with hepatic cytosolic protein from four human subjects correlated with levels of hepatic CYP2A6 microsomal protein, suggesting a possible regulatory role. Further characterization of the RNA-binding protein, the primary binding site, and the influence of this interaction on CYP2A6 mRNA stability will help to elucidate the relevance of these findings to the post-transcriptional control of CYP2A6.

Differential effects of transforming growth factor-beta1, a fibrogenic factor, on macrophage-like cells (HS-P) and myofibroblastic cells (MT-9) in vitro.

Transforming growth factor-beta1 (TGF-beta1) produced by infiltrating macrophages plays a role in fibrotic disorders through the induction of myofibroblasts. To explore possible mechanisms by which TGF-beta1 may act in this context, we investigated effects of TGF-beta1 on macrophage-like (HS-P) and myofibroblastic (MT-9) cells, two novel cell lines developed by us. Immunocytochemically, the addition of TGF-beta1 (0, 0.1, 0.5, and 1.0 ng/ml) dose-dependently suppressed the expressions of antigens recognized by macrophage/histiocyte-specific antibodies (ED1 and ED2) in HS-P cells, whereas the addition concomitantly increased the number of anti-alpha-smooth muscle actin antibody-positive myofibroblastic cells, suggesting a possible phenotypical modulation of macrophages into myofibroblasts in the fibrotic lesions. By contrast, MT-9 cells did not show such immunophenotypical changes following TGF-beta1 addition. DNA synthesis, measured by tritiated thymidine-incorporation, was inhibited in a dose-dependent manner in MT-9 cells by TGF-beta1 addition (0, 0.1, 0.2, 0.5, 1.0, 5, and 10 ng/ml), but that in HS-P cells was unchanged. Northern blot analysis revealed that expressions of cell cycle-related early genes, c-jun and c-myc, were increased in HS-P cells after TGF-beta1 (1 ng/ml) addition, with c-jun showing peak expression prior to c-myc. By contrast, the peak expressions of c-jun and c-myc were delayed in TGF-beta1 (1 ng/ml)-added MT-9 cells, and their levels were less in MT-9 cells than in HS-P cells. Furthermore, TGF-beta1 (1 and 10 ng/ml) induced DNA laddering in MT-9 cells, but did not in HS-P cells. Based on these findings, it was speculated that TGF-beta1 could have induced G1 arrest in cell cycle and apoptosis in MT-9 cells. The present study showed that there were significant differences in the effects of TGF-beta1 between macrophage-like HS-P cells and myofibroblastic MT-9 cells, presumably depending on divergent susceptibilities to TGF-beta1 between both cell types. Because such cell types are key cells in the fibrogenesis, HS-P and MT-9 might be useful models for investigating the pathogenesis of fibrosis in vitro.

Calibration of a 7.6 cm x 7.6 cm (3 inch x 3 inch) sodium iodide gamma ray spectrometer for air kerma rate.

An experimental procedure is described for converting a gamma ray spectral measurement from a 7.6 cm x 7.6 cm (3 inch x 3 inch) sodium iodide (NaI) detector to air kerma rate. The calibration procedure involves measuring the energy deposited in the detector using 10 radioactive sources of known activity covering an energy range from 60 keV to 1,836 keV. For each of the 10 sources, gamma ray spectra were measured with the source at different angles to the detector axis. The total energy deposited in the detector for the ten sources was confirmed by Monte Carlo calculations. The spectra measured at different angles were combined to produce a spectrum that would represent a homogeneous semi-infinite source of radiation. The resultant spectrum was then subdivided into 10 energy regions. Based on the known air kerma rates due to the sources, a calibration coefficient was calculated for each of the 10 energy regions. These calibration coefficients could then be used to convert the energy deposited in the 10 regions of an unknown spectrum to air kerma rate. The calibration procedure was confirmed by comparing the results from the detector with those from calibrated collimated beams of 137Cs and 60Co. A comparison of measurements using a calibrated pressurised ionisation chamber with those from a similar Nal spectrometer in Finland provided additional confirmation of the calibration procedure.

Effects of lipopolysaccharide on a macrophage-like cell line (HS-P) from a rat histiocytic sarcoma.

Lipopolysaccharide (LPS) is a major modulator of macrophage functions. To characterize a newly established rat histiocytic sarcoma-derived cell line (HS-P), immunophenotypic changes and cellular growth responses of HS-P cells exposed to LPS were investigated and compared with those of MT-9 cells isolated from a rat malignant fibrous histiocytoma. MT-9 cells have somewhat histiocytic features, because occasional cells react to rat macrophage-specific antibodies. Addition of LPS to cultured HS-P cells increased the numbers of cells immunopositive to ED1 (rat macrophage-specific antibody) and ED2 (rat histiocyte-specific antibody) and stimulated the phagocytosis of latex beads, whereas LPS-treated MT-9 cells did not show such immunophenotypic changes. LPS-treated HS-P cells showed enhanced immunolabelling of alpha-smooth muscle actin, suggesting a possible modulation of macrophages towards myofibroblastic cells. To evaluate cellular growth after the addition of LPS or fetal bovine serum, DNA synthesis was examined by measuring tritiated thymidine incorporation, and the mRNA expression of c- jun and c- myc (immediate early genes in the cell cycle) was examined by Northern blot analysis. In HS-P cells, the addition of serum greatly increased DNA synthesis and induced high expression of c- jun and c- myc; in contrast, LPS markedly depressed DNA synthesis and reduced the expression of c- jun and c- myc. HS-P cells were more sensitive than MT-9 cells to the growth-promoting effect of serum and the growth-inhibiting effect of LPS. The study demonstrated that HS-P cells are highly LPS-responsive, indicating that they would be useful for studies of macrophage functions.

Divergent Pseudomonas exotoxin A sensitivity in normal and transformed liver cells is correlated with low-density lipoprotein receptor-related protein expression.

Pseudomonas exotoxin A (PEA) is an extracellular virulence factor produced by the opportunistic human pathogen Pseudomonas aerguinosa. PEA intoxification begins when PEA binds to the low-density lipoprotein receptor-related protein (LRP). The liver is the primary target of systemic PEA, due largely to the high levels of functional LRP expressed by liver cells. Using a 3H-leucine incorporation assay to measure inhibition of protein synthesis we have demonstrated that normal (BNL CL.2) and transformed (BNL 1ME A7R.1) liver cells exhibit divergent PEA sensitivity; with BNL 1ME A7R.1 cells demonstrating greater PEA sensitivity than their non-transformed counterparts. The receptor-associated protein, a LRP antagonist, decreased PEA toxicity in BNL 1ME A7R.1 cells, confirming the importance of the LRP in PEA intoxification in this cell type. Increased PEA sensitivity in BNL 1ME A7R.1 cells was associated with increased functional cell surface LRP expression, as measured by alpha2-macroglobulin binding and internalization studies, and increased LRP mRNA levels, as determined by Northern blot analysis. Interestingly, BNL CL.2 cells were more sensitive than BNL 1ME A7R.1 cells to conjugate and mutant PEA toxins that do not utilize the LRP for cellular entry. These data demonstrate that increased LRP expression is an important mechanism by which PEA sensitivity is increased in BNL 1ME A7R.1 transformed liver cells.

Application of the ethoxyresorufin-O-deethylase (EROD) assay to mixtures of halogenated aromatic compounds.

The ethoxyresorufin-O-deethylase (EROD) assay monitors the induction of the xenobiotic-metabolizing enzyme cytochrome P-450 (CYP) 1A1 and is a widely used biomarker for exposure of wildlife to substances that bind the aryl hydrocarbon (Ah) receptor. In this work the induction of EROD activity by single compounds and binary mixtures in primary rat hepatocytes was compared with the predictions of a kinetic model involving mixtures of inducers. The inducing agents were also examined for their ability to activate the Ah receptor to its DNA-binding form and for their ability to act as competitive inhibitors for CYP 1A1. Xenobiotics that failed to activate the Ah receptor did not induce EROD activity. Competitive inhibition for CYP 1A1 between the xenobiotic and 7-ethoxyresorufin caused EROD activity to fall at high xenobiotic concentrations. Competition for a limited number of Ah receptor sites depressed the EROD activity of a strong inducer such as 2,3,7,8-tetrachlorodibenzo-p-dioxin at high concentrations of a weak inducer. Application of the kinetic model to the example of a mixture of low concentrations of dibenzo-p-dioxins and much higher concentrations of polychlorinated biphenyls indicated that EROD assays often seriously underestimate the true potency of an environmental sample. Hence the EROD assay cannot be used for determining dioxin equivalent concentrations using the toxic equivalence factor approach.

Activating transcription factor 3 induces DNA synthesis and expression of cyclin D1 in hepatocytes.

Activating transcription factor 3 (ATF3) is an early response gene that is induced rapidly during in vivo situations of cellular growth such as liver regeneration. However, neither the physiological function nor the potential target genes of this transcription factor related to cellular proliferation have been identified in the liver or other tissues. We demonstrate here that endogenous ATF3 mRNA expression is rapidly induced up to 4-fold upon mitogenic stimulation of quiescent Hepa 1-6 mouse hepatoma cells. Overexpression of exogenous ATF3 results in a significant, dose-dependent increase in DNA synthesis of up to 140% over control cells. ATF3-transfected cells also display significantly higher rates of [(3)H]thymidine incorporation in comparison with nontransfected controls in the presence of serum. Northern blot analysis and co-transfection experiments demonstrate that overexpression of ATF3 enhances cyclin D1 mRNA expression and activates the cyclin D1 promoter 2.5-fold when activating protein-1 (AP-1) and cyclic AMP response element (CRE) sites within the promoter are intact. ATF3-mediated promoter activation is reduced to 1.3-fold and 1.6-fold respectively when the AP-1 or CRE sites are mutated, and mutation of both sites simultaneously leads to the complete abrogation of promoter activation. Furthermore, DNA-binding studies demonstrate that ATF3 binds directly to the AP-1 site within the cyclin D1 promoter. These results indicate that ATF3 expression stimulates hepatocellular proliferation, suggesting that this effect is mediated, at least in part, by the ATF3-dependent activation of cyclin D1 transcription.

Macrophage-like cell line (HS-P) from a rat histiocytic sarcoma.

With future exploration of macrophage properties in mind, we established a novel cell line (HS-P) from a transplantable histiocytic sarcoma, derived originally from a tumour in an aged F344 rat. HS-P was subjected to 70 serial passages, in which the mean doubling time was 15.7 h. The cells, which were round, oval or polygonal in shape, were arranged in a compact sheet. They reacted to varying degrees for lysosomal enzymes (acid phosphatase and non-specific esterase) and with the following antibodies: ED1/ED2 (rat macrophage/histiocyte-specific), OX6 (rat MHC class II-specific), lysozyme antibody and alpha1-antichymotrypsin antibody. Electron microscopically, HS-P cells showed lysosomes and prominent cell projections. These findings indicated that the cultured cells were macrophage-like. Syngeneic rats inoculated subcutaneously or intraperitoneally with HS-P cells invariably developed sarcomatous tumours consisting of monomorphic mononuclear cells, which exhibited cytochemical properties similar to those of cultured HS-P cells. Bioassay and reverse transcription-polymerase chain reaction methods revealed that tumour necrosis factor-alpha increased on addition of lipopolysaccharide (LPS), indicating that HS-P cells remained LPS-responsive. HS-P cells may prove to be a useful tool for in-vitro studies of macrophage function.

Cyclooxygenase expression in canine platelets and Madin-Darby canine kidney cells.

OBJECTIVE: To examine cyclooxygenase (COX) expression in canine platelets and Madin-Darby canine kidney (MDCK) cells in culture. SAMPLE POPULATION: Canine platelets and MDCK cells. PROCEDURE: Total RNA was recovered from isolated canine platelets and MDCK cells. Northern blot analysis and reverse transcription-polymerase chain reaction (RT-PCR), using complementary DNA probes and primers designed from the human COX sequences, were used to determine COX-1 and -2 (cyclooxygenase isoforms 1 and 2) messenger RNA (mRNA) expression. RESULTS: Following northern blot analysis, canine platelets were found to express only the 2.8-kb COX-1 transcript; COX-2 was not detected. Canine MDCK cells expressed the 4.5-kb COX-2 transcript, in addition to the 2.8-kb COX-1 transcript. A single DNA band of 270 base pairs was identified following gel electrophoresis of the product obtained from RT-PCR of mRNA from canine platelets. Sequencing revealed that this PCR product was 90% homologous to a portion of the human COX-1 gene (Genbank M59979). CONCLUSIONS AND CLINICAL RELEVANCE: Detection of COX-1 by RT-PCR of RNA obtained from canine platelets is a novel finding. The 90% homology of the PCR product with the human sequence suggests strong conservation between the canine and human COX-1 gene. Cloning and sequencing of the canine gene will be required to fully characterize homologous regions. Because of the importance of COX in the inflammatory process and as a potential target of currently available nonsteroidal anti-inflammatory drugs (NSAID), a better understanding of canine COX may improve our ability to use NSAID appropriately, achieve efficacy, and avoid potential adverse drug effects in dogs.

Amounts of selected coagulation factors in pre- and post-mortem follicular fluid are similar and do not correlate with molecular mass.

This study was designed to evaluate the amounts of coagulation factors and to determine whether the protein profile in pre-ovulatory ovarian follicular fluid aspirated from ovaries collected from mares at slaughter are representative of that in follicular fluid collected from live animals. The proteins evaluated included, (i) albumin, ceruloplasmin and fibronectin, (ii) the procoagulant plasma proteins, Factor V (FV), Factor VII (FVII), Factor X (FX) and prothrombin, and (iii) the anticoagulant plasma proteins, antithrombin and alpha2-macroglobulin. The amounts of the individual proteins were similar in both types of follicular fluid. There was no correlation between the activity of FV, FVII, FX or prothrombin in follicular fluid and their molecular size although a correlation was found for the other proteins. These results suggest that the procoagulant proteins in follicular fluid are not likely derived from plasma. The total protein content of follicular fluid samples collected from both sources was similar and the results determined with the Biuret, Lowry and Biorad methods were also not significantly different (P>0.05).

In vitro effects of nonsteroidal anti-inflammatory drugs on cyclooxygenase activity in dogs.

OBJECTIVE: To establish an in vitro assay and determine the differential suppressive activity of non steroidal anti-inflammatory drugs (NSAID) on cyclooxygenase (COX)-1 and COX-2 isoenzymes in dogs. PROCEDURE: COX activity was evaluated in the presence and absence of 4 NSAID (meloxicam, tolfenamic acid, carprofen, and ketoprofen), using a canine monocyte/macrophage cell line that constitutively expresses COX-1, but can be induced to express COX-2 when incubated with lipopolysaccharide. Inhibition of prostaglandin E2 TPGE2) synthesis by each NSAID was measured by enzyme immunoassay and attributed to specific COX-1 or COX-2 activity through assessment of COX messenger RNA expression by use of northern blot analysis and reverse transcription-polymerase chain reaction (RT-PCR). The COX selectivity of each drug was evaluated from dose-response curves by calculating a ratio (COX-1:COX-2) of inhibitory concentration values on the basis of concentrations that reduced PGE2 by 50% in each COX model. RESULTS: Meloxicam and tolfenamic acid preferentially inhibited COX-2, with meloxicam inhibiting COX-2 activity 12 times more effectively than COX-1 activity. Carprofen was only 1.75 times more selective for COX-2 than for COX-1, and ketoprofen was slightly more selective for COX-1. CONCLUSIONS: COX-1 and COX-2 were differentially sensitive to inhibition in vitro by NSAID. Meloxicam and tolfenamic acid were selective for COX-2. Effects of carprofen and ketoprofen approached equipotency against both isoenzymes. Selective COX-2 inhibitors are a new class of drugs with anti-inflammatory effects similar to conventional NSAID but with fewer adverse effects. Development of these agents for veterinary use would be facilitated by the convenience of using a canine cell line as a model system to screen COX-1 and COX-2 inhibitor activities in vitro.