A 39 kDa protein mediates adhesion of avian Pasteurella multocida to chicken embryo fibroblast cells

To clarify the role of avian Pasteurella multocida capsule in pathogenesis, adhesion of capsulated strains P-1059, X-73 and Pm-18, and noncapsulated strains P-1059B, Pm-1 and Pm-3 to chicken embryo fibroblast (CEF) cells was compared. Number of adherent organisms of the capsulated strains to CEF cells were approximately three times as much as noncapsulated strains indicating that adhesive properties were enhanced by the presence of bacterial capsule. Pretreatments of the bacterial cells with heat, trypsin, or with antiserum caused a marked decrease in adhesion of capsulated strain P-1059 and its noncapsulated variant P-1059B. However, depolymerization of capsular hyaluronic acid with high dose of hyaluronidase enhanced adhesion of these strains. Combined treatments of the bacterial cells with both hyaluronidase and trypsin significantly (P < 0.05) inhibited the adherence of strain P-1059 as compared to the treatment only with trypsin, but strain P-1059B was not affected. SDS-PAGE profiles of crude capsular extract (CCE) prepared from capsulated strain P-1059 and its noncapsulated variant P-1059B grown on dextrose starch agar (DSA) plates by heating at 56 degrees C in a 2.5% NaCl solution demonstrated eight protein bands of 28, 34, 36, 39, 52, 56, 63 and 93 kDa. The 28, 34 and 36 kDa proteins were commonly major for both strains, and the 39 kDa protein was major only for strain P-1059 but poor in strain P-1059B. Outer membrane protein (OMP) profiles were identical with a major protein at 34 kDa and four minor proteins between the two strains. The adhesion of strain P-1059 and strain P-1059B to CEF cells was inhibited significantly (P < 0.01) by treatment with rabbit antisera against P-1059, P-1059B, CCE or 39 kDa protein of strain P-1059 as compared to the treatment with either PBS or with normal rabbit serum. These results indicated that an antigenic 39 kDa protein in the capsule may be responsible for adhesion of avian P. multocida type A strains to CEF cells as a virulence factor.     

Tumor necrosis factor-alpha (TNF alpha) inhibits progesterone and estradiol-17beta production from cultured granulosa cells: presence of TNFalpha receptors in bovine granulosa and theca cells

The aim of this study was to investigate whether functional tumor necrosis factor-alpha (TNFalpha) receptors are present in the granulosa cells and the cells of theca interna (theca cells), obtained from bovine follicles classified into one of three groups. Each group was defined as either small vesicular ovarian follicles (small follicles; 3-5 mm in diameter), preovulatory mature ovarian follicles (preovulatory follicles) or atretic follicles (12-18 mm) according to gross examination of the corpus luteum in the epsilateral or contralateral ovary and the uterus (size, color, consistency and mucus), and the ratio of progesterone (P(4)) and estradiol-17beta (E(2)) concentrations in follicular fluid. A Scatchard analysis showed the presence of a high-affinity binding site on both granulosa and theca cells from all follicles examined (dissociation constant: 4.7 +/- 0.15 to 6.9 +/- 1.40 nM). Moreover, TNFalpha receptor concentrations in granulosa and theca cells obtained from atretic follicles were significantly higher than those in the cells from preovulatory follicles (P<0.05). Exposure of cultured granulosa cells from small antral follicles to recombinant human TNFalpha (rhTNFalpha; 0.06-6 nM) inhibited E(2) secretion in a dose-dependent fashion (P<0.01), but did not affect P(4) secretion. In addition, rhTNFalpha inhibited follicle stimulating hormone-, forskolin- or dibutylyl cyclic AMP-induced P(4) and E(2) secretion by the cells (P<0.01). These results indicate the presence of functional TNFalpha receptors in bovine granulosa and theca cells in small, preovulatory and atretic follicles, and suggest that TNFalpha plays a role in regulating their secretory function.     

A passage and storage system for isolated bovine endometrial epithelial and stromal cells

To establish a storage system for isolated endometrial cells, we investigated the basal, oxytocin (OT)- and tumor necrosis factor (TNF) alpha-stimulated production of prostaglandin (PG) F(2alpha) in bovine-passaged and frozen-thawed endometrial cells. Stromal and epithelial cells obtained from cows in the early stage of the estrous cycle (Days 2-5) were frozen at -80 C or further cultured and/or passaged until passage 4 in DMEM/Ham's F-12 supplemented with 10% calf serum. A fresh-unfrozen primary culture and one-time passaged fresh-unfrozen cells were used as the control. When both unfrozen and frozen cells reached confluence, the culture medium was replaced with fresh medium with 0.1% BSA and the cells were stimulated with OT (100 ng/ml) or TNFalpha (1 ng/ml) for 4 h. The passage and freezing of the endometrial cells did not affect their morphology. In primary culture of frozen and unfrozen endometrial cells, OT strongly stimulated PGF(2alpha) production in epithelial cells, and TNFalpha strongly stimulated PGF(2alpha) production in stromal cells (P<0.05). The basal output of PGF(2alpha) in frozen stromal cells was similar to that in unfrozen stromal cells. However, the basal output of PGF(2alpha) in frozen epithelial cells was significantly lower than that unfrozen cells (P<0.05). On the other hand, in passaged cells, the basal level of PGF(2alpha) production was retained until passage 1 in epithelial cells, whereas it was retained until passage 4 in stromal cells. Although epithelial cells responded to OT in PGF(2alpha) production until passage 2 (P<0.05), the stromal cells showed a significant response to TNFalpha until passage 4 (P<0.05). These results suggest that stored cells could be used for studying the physiology of bovine endometrium in vitro until passage 1 in endometrial epithelial cells, and until passage 4 in stromal cells.     

Pfaffia paniculata-induced changes in plasma estradiol-17beta, progesterone and testosterone levels in mice

The present study undertook chemical analysis of components of Pfaffia paniculata roots. In addition, an animal experiment was conducted in which mice had ad libitum access to water enriched with powdered P. paniculata root for 30 days. Changes in plasma concentrations of estradiol-17beta and progesterone in female mice and of testosterone in male mice were ascertained. The results revealed that P. paniculata roots contain two types of phytosteroids, beta-sitosterol and stigmasterol, in addition to other compounds such as pfaffic acid, allantoin, saponins, beta-sitosteryl-beta-D-glucoside, and stigmasteryl-beta-D-glucoside. Regarding changes in plasma concentrations of hormones, levels of the sex hormones estradiol-17beta, progesterone and testosterone were clearly higher for mice that drank P. paniculata root-enriched water than for mice that drank plain water. Powdered P. paniculata root is easily dissolved in feed or water, and as no adverse reactions were seen in mice within 30 days of oral intake, consumption of P. paniculata for long periods of time appears safe.     

Properties of the tumor suppressor gene brca2 in the cat

Mammary tumors are common in cats. As mutations in human Brca2 confer an increased risk of breast cancer, the full-length cDNA of the feline homologue of Brca2 was sequenced to obtain a basis for studying the relationship between its function and susceptibility to mammary tumors. The feline Brca2 cDNA is 10 kb long, and encodes 3,371 amino acids. The amino acid sequence of feline Brca2 shares low homology with the Brca2 of other mammals, e.g., 53% homology with the murine protein. Analysis of the expression pattern of the feline Brca2 gene revealed that, as previously reported for other mammals, it is transcribed in various tissues, including the mammary gland.     

Comparison of the cardiovascular effects of intracellular cyclic adenosine 3',5'-monophosphate (cAMP)-modulating agents in isoflurane-anesthetized cats

The inotropic, chronotropic, and vasodilatory effects of five commonly used cyclic adenosine 3',5'-monophosphate (cAMP)-modulating agents were evaluated. Hemodynamic functions were measured continuously in isoflurane-anesthetized cats during infusion of the following: dobutamine (DOB; 2.5, 5 and 10 microg/kg/min; n=8), dopamine (DOP; 1.25, 2.5, 5 and 10 microg/kg/min; n=5), milrinone (MIL; 2.5, 5 and 10 microg/kg/min; n=8), 6-(3-dimethyl-aminopropionyl) forskolin hydrochloride (COL; 0.2, 0.4, 0.8, and 1.6 microg/kg/min; n=7), and bucladesine sodium (BUC; 10, 20, and 40 microg/kg/min; n=9). At the highest infusion rate, DOB and DOP produced the greatest positive inotropic (increase in left ventricular (LV) dP/dt = 89 +/- 4% and 75 +/- 6%, respectively) and chronotropic (increase in heart rate (HR) = 42 +/- 4% and 22 +/- 6%, respectively) effects. MIL and COL produced similar albeit less pronounced positive inotropic (increase in LV dP/dt = 18 +/- 3% and 22 +/- 6%, respectively) and chronotropic (increase in HR = 13 +/- 4% and 21 +/- 4%, respectively) effects. Both also had significant vasodilatory effects (decrease in peripheral resistance (PR) = -30 +/- 2% and -35 +/- 7%, respectively). In contrast, BUC produced only vasodilatation (decrease in PR = -33 +/- 6%). Hence, MIL, COL, and BUC had significant vasodilatory effects and less-pronounced inotropic effects than the catecholamines DOB and DOP. The vasodilatory effects of non-catecholamine drugs for treatment of congestive heart failure should translate into beneficial decreases in both pre-load and after-load. In contrast, the strong inotropic effects of DOB and DOP should be beneficial in the treatment of acute heart failure and anesthetic crisis.     

Dose-dependent inhibition of angiotensin converting enzyme by enalapril in cats

Although heart failure in cats is treated with angiotensin converting enzyme (ACE) inhibitors, data on the effects of different doses of enalapril on hemodynamics and the inhibition of ACE activity have not been published. To evaluate the effect of enalapril, 0.25, 0.5, or 1.0 mg/kg was given once (s.i.d., p.o.) or twice (b.i.d., p.o.) a day, and plasma ACE activity, indirect blood pressure, and heart rate were measured. Plasma ACE activity and blood pressure fell dose-dependently. There was a biphasic effect on blood pressure with twice daily administration. Enalapril 0.25 mg/kg b.i.d. inhibited plasma ACE activity by 40% after 24 hr, which was almost the same as the effect of 0.5 and 1.0 mg/kg s.i.d., and 0.5 and 1.0 mg/kg b.i.d., while 0.25 mg/kg s.i.d. inhibited it by 23%. Thus, enalapril with a daily dose exceeding 0.5 mg/kg may provide similar efficacy of ACE inhibition in cats.     

Bacterial lipopolysaccharide induces mRNA expression of an IkappaB MAIL through toll-like receptor 4

Molecule possessing ankyrin-repeats induced by lipopolysaccharide (MAIL) is a nuclear IkappaB protein recently identified as a molecule appearing in immunocompetent organs after administration of bacterial lipopolysaccharide (LPS). Participation of Toll-like receptor (TLR) 4, which is a major form of LPS receptors, in the LPS-induced MAIL expression was investigated. When a human myelomonocytic cell line U937 was treated with phorbol 12-myristate 13-acetate for 3 days, the LPS-induced MAIL expression was much potentiated in parallel with an increase in TLR4 expression. The MAIL induction was attenuated when the cells were treated with a neutralizing antibody against TLR4. The in vivo induction of MAIL in the spleen was smaller in mice having a missense mutation of the Tlr4 gene than in normal control mice. These results collectively indicate that TLR4 contributes, at least in part, MAIL induction after LPS stimulation.     

The identification and properties of chitin-binding protein b01 (CBPb01)

Bovine serum contains N-acetyl-D-glucosamine (GlcNAc)-sensitive opsonin inhibitory factors. In the present study, a major component of chitin-binding protein (chitin-binding protein b01, CBPb01) was purified from bovine serum, and identified CBPb01 as bovine IgM by its subunit structure, antigenic properties, and partial sequences. The results of a lectin-binding assay showed that the heavy chain of CBPb01 had a GlcNAc structure, but the commercial IgM did not. It is possible that CBPb01 interconnects through its GlcNAc structure, subsequently forming complexes. We also demonstrated that CBPb01 had opsonin-inhibitory activity, and that this activity was dependent on the binding of CBPb01 to GlcNAc on the zymosan surface. These findings indicate the presence of a kind of IgM that recognizes GlcNAc structure in the regulation of opsonization.     

Similarity between Copper Resistance Genes from Pseudomonas syringae pv. actinidiae and P. syringae pv. tomato

Twenty-eight strains of Pseudomonas syringae pv. actinidiae isolated in 1984, 1987 and 1988 from kiwifruit orchards in Japan were tested for their resistance to copper sulfate. All strains isolated in 1984 were copper sensitive with a minimum inhibitory concentration (MIC) of cupric sulfate of 0.75 mM. However, some strains isolated in 1987 and 1988 were resistant, with the MIC ranging from 2.25 to 3.0 mM. All copper-resistant strains contained at least one of two plasmids, pPaCul (about 70.5 kb) or pPaCu2 (about 280 kb), or both. In a copper-resistant strain Pa429, the location of the copper-resistance gene(s) was examined by insertional inactivation with Tn5. The MIC of copper sulfate in the copper-sensitive mutant obtained by Tn5 tagging decreased from 2.75 to 0.75 mM. The 14.5 kb BamHI fragment, designated pPaCuB14, containing the same locus mutagenized with Tn5 was cloned from pPaCu1. However, pPaCuB14 did not confer copper resistance in the transformant of copper-sensitive strain Pa21R, suggesting that this clone did not contain a full set of copper-resistance gene(s). Then a cosmid library of pPaCu1 was constructed and six cosmid clones hybridized with pPaCuB14 were selected. One of the six cosmids, designated pPaCuC1, conferred a near wild-type level of copper resistance in the transformant of the copper-sensitive strain. pPaCuC1 had a homologous region that hybridized with all of the PCR-amplifled fragments of copA, copB, copR, and copS genes of P. syringae pv. tomato. DNA sequence analysis of the homologous region revealed the existence of four open reading frames (ORF A, B, R and S) oriented in the same direction. The predicted amino acid sequences of ORF A, B, R and S had 80, 70, 97 and 95% identity with CopA, B, R and S of P. syringae pv. tomato, respectively. Received 5 July 2001/ Accepted in revised form 27 September 2001