Involvement of adhesins (EcpD, FdeC, FimH) expressed in mammary pathogenic Escherichia coli on adhesion to bovine mammary epithelial cells.

Mammary pathogenic Escherichia coli (MPEC) causes mastitis, which results in substantial economic losses to the dairy industry. A high percentage of Escherichia coli isolated from cows with clinical mastitis harbor adhesin genes, such as fimH. However, it is unclear whether these adhesins are important in the adhesion of MPEC to bovine mammary epithelial cells (BMECs). Therefore, we investigated the effect of adhesins (EcpD, FdeC, and FimH) in MPEC on adherence to the bovine mammary epithelium using cultured BMECs. For this purpose, we used wild-type MPEC as well as single- and double-mutants of fimH, ecpD, and fdeC, and performed adhesion assays with BMECs. First, BMECs were cultured in the presence of lactogenic hormones to induce milk component production and tight junction formation. The bacterial count of the wild-type strain that adhered to the BMECs increased in a dose-dependent manner. In deletion mutant strains, the ΔfimH strain showed lower adhesion (P < 0.05), whereas the adhesion ratio of the ΔecpD and ΔfdeC strains was not statistically different compared with that of the wild-type strain (P > 0.05). Additionally, the fimH/fdeC double-deletion mutants showed the lowest adhesion to BMECs. In conclusion, FimH is crucial in the adhesion of MPEC to BMECs. Overall, our work identifies FimH or FimH/FdeC as interesting targets for future drugs or vaccines to improve the treatment, prevention or chronicity of mastitis induced by MPEC.

Ninjurin 1 mediates peripheral nerve regeneration through Schwann cell maturation of NG2-positive cells.

Ninjurin 1 (Ninj1) is identified as a peripheral nerve injury-induced protein. However, the role of Ninj1 in nerve regeneration is unclear. Schwann cells (SCs) and microvasculature are critical for peripheral nerve regeneration. SCs precursors and microvascular pericytes (PCs), which are nerve/glial antigen 2 (NG2)-positive cells are observed in peripheral nervous system. In this study, we investigated the role of Ninj1 in peripheral nerve regeneration using NG2+cell-specific inducible deletion of Ninj1 mouse model. The number of NG2+cells, which were associated with and without microvessels was increased after sciatic nerve crush injury. There was a significant increase in the expression of Ninj1 and EphA7 in the injured nerve tissue. This increase was mostly observed in NG2+cells. Genetic tracing of NG2+cells was performed using tamoxifen (Tam) treatment on NG2CreERT:R26R-tdTomato mice. The sciatic nerve was injured following the Tam-treatment, then tdTomato-expressing SCs were mostly observed in regenerated SCs at 21 days after nerve injury. Ninj1 gene knockout (Ninj1 KO) in NG2+cells was induced using NG2CreERT:Ninj1loxp mice. Tam-treated-NG2CreERT or Tam-nontreated NG2CreERT:Ninj1loxp mice were used as controls. Following Tam-treatment, the sciatic nerve in each group was injured. Ninj1KO significantly attenuated the expression of the myelin binding protein (MBP) as well as the number of myelinated axons. The expression of MBP in cultured SCs was significantly reduced by SiRNA-mediated Ninj1 knockdown (KD). Ninj1KD also attenuated the differentiation of SCs by isolated EphA7+multipotent PCs. The current data indicate that Ninj1 plays a vital role in peripheral nerve regeneration. This is observed particularly in the myelination process of NG2+cells including SCs precursors and multipotent PCs.

Retinoic acid modulates lipid accumulation glucose concentration dependently through inverse regulation of SREBP-1 expression in 3T3L1 adipocytes.

It is well known that retinoic acid (RA) suppresses adipogenesis, although there are some contradicting reports. In this study, we examined the effect of extracellular glucose on RA-induced suppression of adipogenesis in 3T3L1 cell culture. When the cells were cultured in normal glucose medium (NG), the addition of RA suppressed lipid accumulation. However, when cultured in high glucose medium (HG), addition of RA to the cells enhanced lipid accumulation. These changes were accompanied by parallel alterations in fatty acid synthase (FAS) and sterol regulatory element-binding protein (SREBP)-1 gene expression. Transfection of SREBP-1 siRNA suppressed RA-induced enhancement of lipid accumulation and FAS expression in the cells cultured with HG. Transfection of the nuclear form of SREBP-1a cDNA into the cells cultured with NG inhibited RA-induced suppression of lipid accumulation and FAS expression. Moreover, RA- and HG-induced SREBP-1a expression occurred at the early phase of adipogenesis and was dependent on glucocorticoid to induce liver X receptor (LXR) ß, peroxisomal proliferator-activated receptor (PPAR) γ and retinoid X receptor (RXR), the key nuclear factors influencing the SREBP-1a gene expression. These results suggest that RA suppresses and enhances lipid accumulation through extracellular glucose concentration-dependent modulation of SREBP-1 expression.

Relative Contribution of nirK- and nirS- Bacterial Denitrifiers as Well as Fungal Denitrifiers to Nitrous Oxide Production from Dairy Manure Compost.

The relative contribution of fungi, bacteria, and nirS and nirK denirifiers to nitrous oxide (N2O) emission with unknown isotopic signature from dairy manure compost was examined by selective inhibition techniques. Chloramphenicol (CHP), cycloheximide (CYH), and diethyl dithiocarbamate (DDTC) were used to suppress the activity of bacteria, fungi, and nirK-possessing denitrifiers, respectively. Produced N2O were surveyed to isotopocule analysis, and its 15N site preference (SP) and δ18O values were compared. Bacteria, fungi, nirS, and nirK gene abundances were compared by qPCR. The results showed that N2O production was strongly inhibited by CHP addition in surface pile samples (82.2%) as well as in nitrite-amended core samples (98.4%), while CYH addition did not inhibit the N2O production. N2O with unknown isotopic signature (SP = 15.3-16.2‰), accompanied by δ18O (19.0-26.8‰) values which were close to bacterial denitrification, was also suppressed by CHP and DDTC addition (95.3%) indicating that nirK denitrifiers were responsible for this N2O production despite being less abundant than nirS denitrifiers. Altogether, our results suggest that bacteria are important for N2O production with different SP values both from compost surface and pile core. However, further work is required to decipher whether N2O with unknown isotopic signature is mostly due to nirK denitrifiers that are taxonomically different from the SP-characterized strains and therefore have different SP values rather than also being interwoven with the contribution of the NO-detoxifying pathway and/or of co-denitrification.

Inducible deletion of microRNA activity in kidney mesenchymal cells exacerbates renal fibrosis.

MicroRNAs (miRNAs) are sequence-specific inhibitors of post-transcriptional gene expression. However, the physiological functions of these non-coding RNAs in renal interstitial mesenchymal cells remain unclear. To conclusively evaluate the role of miRNAs, we generated conditional knockout (cKO) mice with platelet-derived growth factor receptor-ß (PDGFR-ß)-specific inactivation of the key miRNA pathway gene Dicer. The cKO mice were subjected to unilateral ureteral ligation, and renal interstitial fibrosis was quantitatively evaluated using real-time polymerase chain reaction and immunofluorescence staining. Compared with control mice, cKO mice had exacerbated interstitial fibrosis exhibited by immunofluorescence staining and mRNA expression of PDGFR-ß. A microarray analysis showed decreased expressions of miR-9-5p, miR-344g-3p, and miR-7074-3p in cKO mice compared with those in control mice, suggesting an association with the increased expression of PDGFR-ß. An analysis of the signaling pathways showed that the major transcriptional changes in cKO mice were related to smooth muscle cell differentiation, regulation of DNA metabolic processes and the actin cytoskeleton, positive regulation of fibroblast proliferation and Ras protein signal transduction, and focal adhesion-PI3K/Akt/mTOR signaling pathways. Depletion of Dicer in mesenchymal cells may downregulate the signaling pathway related to miR-9-5p, miR-344g-3p, and miR-7074-3p, which can lead to the progression of chronic kidney disease. These findings highlight the possibility for future diagnostic or therapeutic developments for renal fibrosis using miR-9-5p, miR-344g-3p, and miR-7074-3p.

NG2-positive pericytes regulate homeostatic maintenance of slow-type skeletal muscle with rapid myonuclear turnover.

BACKGROUND: Skeletal muscle comprises almost 40% of the human body and is essential for movement, structural support and metabolic homeostasis. Size of multinuclear skeletal muscle is stably maintained under steady conditions with the sporadic fusion of newly produced myocytes to compensate for the muscular turnover caused by daily wear and tear. It is becoming clear that microvascular pericytes (PCs) exhibit myogenic activity. However, whether PCs act as myogenic stem cells for the homeostatic maintenance of skeletal muscles during adulthood remains uncertain. METHODS: We utilized PC-fused myofibers using PC-specific lineage tracing mouse (NG2-CreERT/Rosa-tdTomato) to observe whether muscle resident PCs have myogenic potential during daily life. Genetic PC deletion mouse model (NG2-CreERT/DTA) was used to test whether PC differentiates to myofibers for maintenance of muscle structure and function under homeostatic condition. RESULTS: Under steady breeding conditions, tdTomato-expressing PCs were infused into myofibers, and subsequently, PC-derived nuclei were incorporated into myofibers. Especially in type-I slow-type myofibers such as the soleus, tdTomato+ myofibers were already observed 3 days after PC labeling; their ratio reached a peak (approximately 80%) within 1 month and was maintained for more than 1 year. Consistently, the NG2+ PC-specific deletion induced muscular atrophy in a slow-type myofiber-specific manner under steady breeding conditions. The number of myonucleus per volume of each myofiber was constant during observation period. CONCLUSIONS: These findings demonstrate that the turnover of myonuclei in slow-type myofibers is relatively fast, with PCs acting as myogenic stem cells-the suppliers of new myonuclei under steady conditions-and play a vital role in the homeostatic maintenance of slow-type muscles.

Plasminogen/plasmin modulates bone metabolism by regulating the osteoblast and osteoclast function.

The contribution of plasminogen (Plg)/plasmin, which have claimed to be the main fibrinolytic regulators in the bone metabolism, remains unclear. This study evaluated how the absence of Plg affects the function of osteoblast (OB) and osteoclast (OC). There was a larger population of pre-OCs in bone marrow-derived cells from the Plg(-/-) mice than the population of that from the WT mice. In addition, the absence of Plg suppressed the expression of osteoprotegerin in OBs. Moreover, an exogenous plasmin clearly induced the osteoprotegerin expression in Plg(-/-) OBs. The osteoclastogenesis of RAW264.7 mouse monocyte/macrophage lineage cells in co-culture with OBs from the Plg(-/-) mice was significantly accelerated in comparison with that in co-culture with OBs from the WT mice. Intriguingly, the accelerated OC differentiation of RAW264.7 cells co-cultured with Plg(-/-) OBs was clearly suppressed by the treatment of an exogenous plasmin. Consequently, Plg(-/-) mice display decreased bone mineral density. These findings could eventually lead to the development of new clinical therapies for bone disease caused by a disorder of the fibrinolytic system.

Alg14 organizes the formation of a multiglycosyltransferase complex involved in initiation of lipid-linked oligosaccharide biosynthesis.

Protein N-glycosylation begins with the assembly of a lipid-linked oligosaccharide (LLO) on the endoplasmic reticulum (ER) membrane. The first two steps of LLO biosynthesis are catalyzed by a functional multienzyme complex comprised of the Alg7 GlcNAc phosphotransferase and the heterodimeric Alg13/Alg14 UDP-GlcNAc transferase on the cytosolic face of the ER. In the Alg13/14 glycosyltransferase, Alg14 recruits cytosolic Alg13 to the ER membrane through interaction between their C-termini. Bioinformatic analysis revealed that eukaryotic Alg14 contains an evolved N-terminal region that is missing in bacterial orthologs. Here, we show that this N-terminal region of Saccharomyces cerevisiae Alg14 localize its green fluorescent protein fusion to the ER membrane. Deletion of this region causes defective growth at 38.5°C that can be partially complemented by overexpression of Alg7. Coimmunoprecipitation demonstrated that the N-terminal region of Alg14 is required for direct interaction with Alg7. Our data also show that Alg14 lacking the N-terminal region remains on the ER membrane through a nonperipheral association, suggesting the existence of another membrane-binding site. Mutational studies guided by the 3D structure of Alg14 identified a conserved α-helix involved in the second membrane association site that contributes to an integral interaction and protein stability. We propose a model in which the N- and C-termini of Alg14 coordinate recruitment of catalytic Alg7 and Alg13 to the ER membrane for initiating LLO biosynthesis.

Bovine milk lactoferrin induces synthesis of the angiogenic factors VEGF and FGF2 in osteoblasts via the p44/p42 MAP kinase pathway.

Lactoferrin (LF) belongs to the transferrin family and is present in several physiological fluids, including milk and colostrum. LF has recently been identified as an anabolic factor for bone. Here we investigated whether bovine LF (bLF) induces synthesis of angiogenic factors by osteoblasts. If so, we examined the underlying mechanism. We found that bLF purified from milk increased the mRNA expression of vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF2) in murine osteoblast-like MC3T3-E1 cells and primary murine osteoblasts in a time- and dose-dependent manner. Furthermore, bLF increased VEGF and FGF2 protein levels in MC3T3-E1 cells. In addition, treatment of MC3T3-E1 cells with bLF rapidly induced phosphorylation of p44/p42 mitogen-activated protein (MAP) kinase. The bLF-mediated increases in VEGF and FGF2 mRNA and protein were inhibited by U0126, a specific inhibitor of the upstream kinase that activates p44/p42 MAP kinase (MEK). Taken together, our results strongly suggest that bLF induces VEGF and FGF2 synthesis in a p44/p42 MAP kinase-dependent manner in MC3T3-E1 cells.

Chemerin Regulates Epithelial Barrier Function of Mammary Glands in Dairy Cows.

Epithelial barrier function in the mammary gland acts as a forefront of the defense mechanism against mastitis, which is widespread and a major disorder in dairy production. Chemerin is a chemoattractant protein with potent antimicrobial ability, but its role in the mammary gland remains unelucidated. The aim of this study was to determine the function of chemerin in mammary epithelial tissue of dairy cows in lactation or dry-off periods. Mammary epithelial cells produced chemerin protein, and secreted chemerin was detected in milk samples. Chemerin treatment promoted the proliferation of cultured bovine mammary epithelial cells and protected the integrity of the epithelial cell layer from hydrogen peroxide (H2O2)-induced damage. Meanwhile, chemerin levels were higher in mammary tissue with mastitis. Tumor necrosis factor alpha (TNF-α) strongly upregulated the expression of the chemerin-coding gene (RARRES2) in mammary epithelial cells. Therefore, chemerin was suggested to support mammary epithelial cell growth and epithelial barrier function and to be regulated by inflammatory stimuli. Our results may indicate chemerin as a novel therapeutic target for diseases in the bovine mammary gland.

Possible involvement of prolactin in the synthesis of lactoferrin in bovine mammary epithelial cells.

Bovine mammary epithelial cells (bMECs) synthesize lactoferrin, which is secreted into milk. Our results suggest that prolactin stimulated secretion of lactoferrin in primary bMECs and their clonal cell line under serum-free conditions. Prolactin also stimulated mRNA expression of lactoferrin in the clonal cell line. This effect was reduced by AG-490, suggesting that the prolactin-stimulated mRNA expression of lactoferrin was mediated by Janus kinase (JAK)2.

Response of the denitrifier community and its relationship with multiple N2O emission peaks after mature compost addition into dairy manure compost with forced aeration.

Animal manure is a source of the greenhouse gas nitrous oxide (N2O), therefore understanding the mechanisms underlying its production is essential for developing mitigating strategies and sustainable livestock production system. In this study, microbial communities potentially involved in multiple emission peaks during initial stage of laboratory-scale dairy manure composting with forced aeration system were investigated. Mature compost was used for the bulking agent. Change of overall bacterial community and nitrification-denitrification gene abundance were monitored by using 16S rRNA gene amoA, nirS, nirK or nosZ genes, respectively. Three N2O emission peaks were observed when the temperature reached at 45, 60 and 72 °C, at the same timing of oxygen consumption peaks. The maximum N2O emission peak was 3.86 mg h-1 kg-1 TS when the temperature reached at 60 °C. The shift of bacterial community among these experimental periods was significant, orders Flavobacteriales, Burkholderiales and Xanthomonadales increased, while orders belong to Bacillales, Lactobacillales, Clostridiales and Bacteroidales decreased. In addition, abundance of two denitrification genes (nirS and nosZ) significantly increased during this period. Clone library analysis of these genes showed that significantly increased sequences belonged to Pseudomonas-like clusters for both genes, indicates that denitrifiers possesses these genes are involved for these N2O emission peaks caused by mature compost addition.

Innate immune response of bovine mammary epithelial cells to Mycoplasma bovis.

Mycoplasma spp. are contagious bacteria, and mycoplasmal mastitis is a serious productivity problem on dairy farms. Bovine mammary epithelial cells (bMECs) have an important role in the elimination of pathogens, but the effect of Mycoplasma bovis on bMECs has not been fully described. To elucidate the immune response against intramammary infection by M. bovis, we undertook microarray analysis to examine and profile mRNA expression in bMECs after stimulation with M. bovis. We also compared the effects of M. bovis, Staphylococcus aureus, and Escherichia coli on immune-related mRNA expression in bMECs. Transcriptome analysis indicated a significant decrease in the level of mRNA-encoding lysine-specific demethylase 4D, suggesting that the immune response is suppressed by a decrease in histone demethylase activity. Interleukin (IL)-1ß, IL-6, tumor necrosis factor alpha, toll-like receptor (TLR) 2, and TLR4 mRNA expression levels were significantly increased in bMECs stimulated with heat-killed M. bovis, but the expression levels were lower than those following stimulation by heat-killed S. aureus or E. coli. Our results suggest that M. bovis weakly affects mRNA expression in bMECs compared to the effects of E. coli or S. aureus. Moreover, live M. bovis may induce suppression of the immune response in bMECs.

Downregulated angiopoietin-like protein 8 production at calving related to changes in lipid metabolism in dairy cows.

Acute physiological adaptation of lipid metabolism during the postpartum transition period of cows facilitates peripheral metabolic regulation. Hepatokines, which are hormones secreted from hepatocytes, are presumed to play a critical role in systemic metabolic regulation. Angiopoietin-like protein 8 (ANGPTL8) has been identified as a novel hepatokine associated with circulating triglyceride concentrations in mice and humans. However, regulation of ANGPTL8 and its physiological effects is still unknown in cattle. The present study aimed to reveal changes in ANGPTL8 expression and secretion during the periparturient period, and to investigate its regulatory effect on adipocytes and mammary epithelial cells. In the peripartum period, liver ANGPTL8 mRNA expression was lesser on the day of parturition and 1 wk postpartum than it was 1 wk before parturition (P < 0.05). Moreover, plasma ANGPTL8 concentrations decreased on the day of parturition as compared with that 1 wk before parturition (P < 0.05). In addition, ANGPTL8 expression in cultured bovine hepatocytes was downregulated after oleate and palmitate treatment but upregulated after insulin treatment (P < 0.05). ANGPTL8 decreased hormone-sensitive lipase (HSL) expression in differentiated adipocytes and cluster of differentiation 36 (CD36), fatty acid synthase (FAS), acetyl-coa carboxylase (ACC), and stearoyl-coa desaturase (SCD) in cultured bovine mammary epithelial cells (P < 0.05). These data suggest that hepatic ANGPTL8 production was downregulated postpartum when the cows experienced a negative energy balance. This downregulation was associated with increased concentrations of NEFA and decreased concentrations of insulin in lactating cows, and it facilitated lipid mobilization from adipose tissue to the mammary glands. We speculate that ANGPTL8 might have beneficial effects in reverting or improving the physiological adaptation and pathological processes of lipid metabolism during the peripartum period.

Differential roles of MAP kinases in atorvastatin-induced VEGF release in cardiac myocytes.

Statins, specific inhibitors of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, are now widely used for treatment of patients with hypercholesterolemia. In addition to the reduction of cholesterol biosynthesis, accumulating evidence indicates that statins have several pleiotropic effects especially on cardiovascular system. However, the exact role of statin in cardiac myocytes remains unclear. In the present study, we investigated whether atorvastatin induces vascular endothelial growth factor (VEGF) release in cardiac myocytes, and the underlying mechanism. We observed that atorvastatin significantly stimulated VEGF release in a dose-dependent manner. It induced the phosphorylation of p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase but not SAPK (stress-activated protein kinase)/JNK (c-Jun N-terminal kinase). The atorvastatin-induced VEGF release was enhanced by PD98059, which is a specific inhibitor of the upstream kinase that activates p44/p42 MAP kinase (MEK). Further, it was significantly reduced by SB203580, a specific inhibitor of p38 MAP kinase. Furthermore, the atorvastatin-induced phosphorylation of p38 MAP kinase was attenuated by SB203580, whereas it was enhanced by PD98059. Taken together, these results suggest that the atorvastatin-induced VEGF release in cardiac myocytes is positively regulated by p38 MAP kinase and negatively regulated byp44/p42 MAP kinase and that the atorvastatin-induced phosphorylation of p38 MAP kinase is regulated by p44/p42 MAP kinase in these cells.

The cell wall component lipoteichoic acid of Staphylococcus aureus induces chemokine gene expression in bovine mammary epithelial cells.

Staphylococcus aureus (SA) is a major cause of bovine mastitis, but its pathogenic mechanism remains poorly understood. To evaluate the role of lipoteichoic acid (LTA) in the immune or inflammatory response of SA mastitis, we investigated the gene expression profile in bovine mammary epithelial cells stimulated with LTA alone or with formalin-killed SA (FKSA) using cap analysis of gene expression. Seven common differentially expressed genes related to immune or inflammatory mediators were up-regulated under both LTA and FKSA stimulations. Three of these genes encode chemokines (IL-8, CXCL6 and CCL2) functioning as chemoattractant molecules for neutrophils and macrophages. These results suggest that the initial inflammatory response of SA infection in mammary gland may be related with LTA induced chemokine genes.

Adenylyl cyclase-cAMP system inhibits thyroid hormone-stimulated osteocalcin synthesis in osteoblasts.

It is generally recognized that thyroid hormone modulates osteoblast cell function. We have previously shown that triiodothyronine (T(3)) activates p38 mitogen-activated protein (MAP) kinase, resulting in the synthesis of osteocalcin in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effect of the adenylyl cyclase-cAMP system on thyroid hormone-stimulated osteocalcin synthesis in these cells. Dibutyryl-cAMP (DBcAMP) reduced the osteocalcin synthesis stimulated by T(3). Forskolin and cholera toxin suppressed the osteocalcin synthesis while dideoxyforskolin, a forskolin derivative that does not activate adenylyl cyclase, had little effect on the synthesis. KT5720, a selective inhibitor of protein kinase A, reversed the inhibitory effect of forskolin or DBcAMP. DBcAMP and forskolin markedly reduced the phosphorylation of p38 MAP stimulated by T(3). Pituitary adenylate cyclase-activating polypeptide (PACAP) significantly inhibited the T(3)-stimulated osteocalcin synthesis. These results strongly suggest that the adenylyl cyclase-cAMP system has an inhibitory role in thyroid hormone-stimulated osteocalcin synthesis via suppression of p38 MAP kinase activation in osteoblasts.

Akt regulates thrombin-induced HSP27 phosphorylation in aortic smooth muscle cells: function at a point downstream from p38 MAP kinase.

We previously reported that p38 MAP kinase takes part in thrombin-induced HSP27 phosphorylation in aortic smooth muscle A10 cells. In the present study, we investigated whether Akt is involved in the phosphorylation of HSP27 and the role of adenylyl cyclase-cAMP system. Thrombin time-dependently induced the phosphorylation of heat shock protein 27 (HSP27) and Akt in aortic smooth muscle A10 cells. SB203580, a p38 MAP kinase inhibitor, significantly suppressed the thrombin-induced phosphorylation of Akt and the Akt inhibitor suppressed the phosphorylation of HSP27. Furthermore, the thrombin-induced phosphorylation of HSP27, p38 MAP kinase and Akt were decreased by dibutyryl-cAMP (DBcAMP). These results strongly suggest that Akt functions the thrombin-induced phosphorylation of HSP27 at a point downstream from p38 MAP kinase in aortic smooth muscle cells and the adenylyl cyclase-cAMP system is upstream regulator of the HSP27 phosphorylation in these cells.

Cytokine mRNA profiling and the proliferative response of bovine peripheral blood mononuclear cells to Mycoplasma bovis.

Mycoplasma bovis is known as a significant pathogen and cause of large economic losses in beef and dairy calves worldwide. Numerous factors appear to play an important role in the development of disease during infection with M. bovis, e.g., inhibition of immune cell proliferation and induction of lymphocyte apoptosis. However, the mechanisms involved in M. bovis infections have not been explored and remain incompletely understood. We investigated the major cytokine mRNA expression in bovine PBMC stimulated with M. bovis, for comparison, Staphylococcus aureus and Escherichia coli, which are the representative mastitis-causing pathogens. Here we demonstrated that live M. bovis significantly induced tumor necrosis factor alpha (TNF-α), interleukin 12p40 (IL-12), and interferon gamma (IFN-γ) mRNA expression in bovine peripheral blood mononuclear cells (PBMC) at a multiplicity of infection (MOI) of 1000 but not at an MOI of 10 and 100. Live M. bovis at MOIs of 1, 10, and 100 induced significant bovine PBMC proliferative responses compared with unstimulated bovine PBMC. Furthermore, we showed that the cultural supernatant of M. bovis induced a significant increase in TNF-α, IL-6, and IL-10 mRNA expression in bovine PBMC. Our results suggest that M. bovis weakly affects the cellular integrity of bovine PBMC and induces clear proliferative responses and associated cytokine production in them. However, large numbers of live M. bovis are required to induce an immune response in bovine PBMC.

Affinity selection of DNA-binding proteins from yeast genomic DNA libraries by improved lambda phage display vector.

Phage display is a useful means of identifying and selecting proteins of interest that bind specific targets. In order to examine the potential of phage display for the genome-wide screening of DNA-binding proteins, we constructed yeast genomic libraries using lambda foo-based vectors devised in this work. After affinity selection using GAL4 UAS(G) as a probe, phages expressing GAL4 were enriched approximately 5 x 10(5)-fold from the library. Approximately 90% of polypeptides encoded in correct translation reading frames by the selected phages were known or putative polynucleotide-binding proteins. This result clearly indicates that the modified lambda phage display vector in combination with our enrichment technique has great potential for the enrichment of DNA-binding proteins in a sequence-specific manner.