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     

Chronological analysis of canine parvovirus type 2 isolates in Japan

Fifty-five canine parvovirus type 2 (CPV) samples, 12 fecal specimens and 43 cell culture isolates, were examined for their genetic characteristics of VP2 gene. They were collected from the diseased dogs at various districts of Japan during 27 years from 1980 to 2006. A fragment of VP2 gene was analyzed by restriction fragment length polymorphism assay and DNA sequencing. The original antigenic type 2 of CPV (CPV-2) was no longer found in the samples since 1984, and two antigenic variants CPV-2a and CPV-2b replaced CPV-2 as predominant types for about 5 years from 1982. A new genetic variant of prototype CPV-2a with non-synonymous substitution at the VP2 amino acid residue 297 from Ser to Ala was first detected in 1987. New CPV-2b with the same amino acid substitution at position 297 as new CPV-2a was also detected from the samples collected in 1997. Since then new CPV-2b has been the predominant CPV over the field of Japan. Several additional amino acid substitutions were detected in the VP2 gene of some recent CPV strains. Neither CPV-2c(a), CPV-2c(b), nor "Glu-426" of the antigenic variants previously found outside the country was detected in any samples tested. Reactivity of new CPV-2a and 2b variants against antibodies produced by the current vaccine products was determined by a cross hemagglutination-inhibition test. The recent field CPV isolates reacted more efficiently to the antibodies produced in dogs vaccinated with the new CPV-2b vaccine strain than the conventional CPV-2 vaccine strain.     

Novel variations and loss of heterozygosity of BRCA2 identified in a dog with mammary tumors

OBJECTIVE: To establish novel polymorphic markers for analysis of loss of heterozygosity (LOH), so as to study the possible involvement of BRCA2 in mammary tumors obtained from dogs. SAMPLE POPULATION: Blood samples, mammary gland specimens, or mammary tumors from 3 tumor-bearing dogs and 10 tumor-free dogs. PROCEDURES: Nucleotide sequence analysis was performed with a DNA autosequencer. Loss of heterozygosity analysis was performed for markers established in the present study. The expression level of canine BRCA2 was quantified by real-time PCR analysis. RESULTS: 3 novel microsatellite markers with high heterozygosity rates (> 50%) were established, and the previously reported marker for canine BRCA2 gene locus was improved. These markers were used for the analysis of DNA from formalin-fixed and paraffin-embedded samples. By use of these markers, LOH in canine BRCA2 was identified as a result of recombination. In mammary tumor DNA that corresponded to the LOH-positive dog, the level of canine BRCA2 expression was decreased compared with that of nonneoplastic mammary gland tissue; the open reading frame contained 4 missense variations, 1 insertion variation, and 1 silent variation, some of which were localized to functional domains. CONCLUSIONS AND CLINICAL RELEVANCE: 3 novel polymorphic markers were developed for LOH analysis of canine BRCA2 and identified a dog with LOH with some variations in the functional domains. These markers could be useful for assessing the relevance of BRCA2 variation in mammary tumors of dogs.