Transcriptional regulation of the MAIL gene in LPS-stimulated RAW264 mouse macrophages.

IkappaB inhibits nuclear factor kappa B (NF-kappaB), which is known to regulate the expression of various genes, including genes involved in inflammation. Recently, a novel IkappaB family protein, 'molecule possessing ankyrin repeats induced by lipopolysaccharide' (MAIL), was identified. MAIL is a nuclear-acting, inducible protein, unlike typical IkappaB proteins. However, the mechanism of its induction by lipopolysaccharide (LPS) is unclear. Using the LPS-reactive region located upstream from the MAIL gene, we investigated the mechanism of MAIL induction. MAIL expression was strongly regulated by NF-kappaB and partly regulated by CREB. Furthermore, deletion, point mutation and binding analyses revealed that the NF-kappaB binding site located at -229 to -220 bp is an essential target of MAIL expression. Overexpression of MAIL protein suppressed the LPS-induced promoter activity of the MAIL gene. These data indicate that MAIL expression is strongly upregulated by NF-kappaB, and it is controlled, at least in part, by an autoregulation mechanism.

Quantitative changes in serum concentration of bovine gut chitinase in Theileria infection.

Bovine gut chitinase is exclusively produced in the liver and secreted into the blood. In the present study, we established a semi-quantitative method by Western blot analysis for measurement of the chitinase content in blood and examined its alteration during postnatal development and experimental infection with hemoprotozoan parasite in cattle. Its serum levels from 1 week to 1 year of age showed a significant increase only in 3-4-month-old group. The plasma concentration of the gut chitinase was not changed during acute inflammation caused by lipopolysaccharide but increased gradually after a Theileria injection and peaked at 52 days post-infection. It appears that the increase in the blood chitinase levels might be a defensive response in cattle against protozoan infection.

Role of CXCR4 and SDF-1 in mammary tumor metastasis in the cat.

It has recently been suggested that the chemokine receptor CXCR4 and its ligand SDF-1 (CXCL12) promote metastasis of various cancers in humans. Since feline mammary tumors also metastasize to distant organs frequently, we used real-time quantitative PCR to examine the expression of feline CXCR4 (fCXCR4) in ten feline mammary tumor cell lines and seven feline mammary tumor tissues, and also the expression of feline SDF-1 (fSDF-1) in various organs. Cell lines derived from metastatic regions expressed more fCXCR4 than those derived from primary tumors. Mammary tumor tissues overexpressed more fCXCR4 than normal mammary tissues. Organs with high levels of fSDF-1 expression represent common sites of metastasis. Migration assays using the feline mammary tumor cell line NAC were also performed to test the activity of TN14003 and TC14012, antagonists of human CXCR4, to antagonize fCXCR4 expressed on NAC cells. TN14003 and TC14012 inhibited migration of NAC cells. We conclude that fCXCR4 may be a therapeutic target for feline mammary tumors.

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.

The plasma superoxide scavenging activity in canine cancer and hepatic disease.

To clarify the relationship between plasma antioxidant activity and diseases in dogs, plasma samples were collected from 6 healthy dogs and 16 diseased dogs (6 dogs with cancer, 5 dogs with hepatic disease, and 5 dogs with inflammation ), and measured superoxide anion scavenging activities. Antioxidant activities of canine plasma were evaluated by measuring their superoxide anion (O(2)(-.)) scavenging activities with electron spin response spectroscopy combined with spin trapping reagent, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). Total O(2)(-.) scavenging activities in the presence of plasma of diseased dogs tended to be higher than those in healthy controls, especially significant higher activities in the presence of canine plasma of hepatic disease and inflammation were observed. In diseased dogs, KCN-insensitive activities, suggesting the activity of manganese-containing superoxide dismutase (Mn-SOD), were significantly higher than those in healthy controls. Therefore, it seems that there is a possibility of utilizing of plasma O(2)(-.) scavenging activity as one of clinical indicators for oxidative-related diseases such as cancer, hepatic disease and inflammation in dogs.

Serological differentiation of Brucella-vaccinated and -infected domesticated animals by the agar gel immunodiffusion test using Brucella polysaccharide in mongolia.

To investigate Brucella infection in cattle, sheep, goat, reindeer and yak in Mongolia, serological reactions of Brucella-infected and -vaccinated domestic animals were compared by the agar gel immunodiffusion (AGID) test with a polysaccharide (poly-B) of the B. Abortus strain S-19. The sensitivity and specificity were compared with conventional serological tests that are commonly used in Mongolia, such as the rose Bengal test, the tube agglutination test and the compliment fixation test. A total of 73.3, 100, 100, 95.8 and 61.9% of the sera from suspected cattle, yak, goat, sheep and reindeer, respectively, that were positive in the compliment fixation test, were also positive in the AGID test. Sera from vaccinated cattle, sheep and goat were positive over 90% by conventional tests 3 months after vaccination, but were negative by the AGID. These results suggest that the AGID test may be useful to differentiate infected and vaccinated animals in the field.

Cellular expression of gut chitinase mRNA in the gastrointestinal tract of mice and chickens.

Recently, the second mammalian chitinase, designated acidic mammalian chitinase (AMCase), has been identified in human, mouse, and cow. In contrast to the earlier identified macrophage-derived chitinase (chitotriosidase), this chitinase is richly expressed in the gastrointestinal (GI) tract, suggesting its role in digestion of chitin-containing foods as well as defense against chitin-coated microorganisms and parasites. This in situ hybridization study first revealed cellular localization of the gut-type chitinase in the mouse and chicken. In adult mice, the parotid gland, von Ebner's gland, and gastric chief cells, all of which are exocrine cells of the serous type, expressed the gut chitinase mRNA. In the chicken, oxyntico-peptic cells in glandular stomach (proventriculus) and hepatocytes expressed the chitinase mRNA. Because cattle produce the gut chitinase (chitin-binding protein b04) only in the liver, the gut chitinases in mammals and birds have three major sources of production, i.e., the salivary gland, stomach, and liver. During ontogenetic development, the expression level in the parotid gland and stomach of mice increased to the adult level before weaning, whereas in the stomach of chickens intense signals were detectable in embryos from incubation day 7.

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.

Clinical availability of urinary N-acetyl-beta-D-glucosaminidase index in dogs with urinary diseases.

Urinary excretion of N-acetyl-beta-D-glucosaminidase (NAG) was examined in healthy dogs and dogs with urinary diseases, and its clinical usefulness as an indicator of urinary diseases was discussed. Twenty-eight healthy dogs and 20 dogs with urinary diseases were used. Urinary NAG activity was measured using p-nitrophenyl N-acetyl-beta-D-glucosaminide as substrate, and expressed as units per gram of urinary creatinine (NAG index). Urinary NAG index in urine of healthy dogs was 3.2+/-2.4 U/g, and NAG index in the dogs with chronic renal failure or lower urinary tract infection accompanied by pyelonephritis was higher than that in healthy dogs. However, the dogs with lower urinary tract infection without pyelonephritis showed normal values of NAG index. Some dogs with diabetic mellitus showed elevated values of NAG index when control of blood sugar was not successful. Increase of NAG index was observed in some dogs with pyometra before increases of BUN and serum creatinine concentration. Therefore, NAG index in urine seems to be a good indicator for urinary diseases in dogs.

Urinary excretion of N-acetyl-beta-D-glucosaminidase and its isoenzymes in cats with urinary disease.

The present study was designed to assess the clinical usefulness of measurement of urinary N-acetyl-beta-D-glucosaminidase (NAG) activity and its isoenzymes in cats with urinary disease. Thirty-five healthy cats and 9 cats with renal disease were used. Furthermore, a 5-year-old female cat was administered a large amount of sulfonamide in order to induce acute renal failure, and urine samples were collected for the assay of NAG activity and its isoenzymes. Urinary NAG activity was measured using p-nitrophenyl N-acetyl-beta-D-glucosaminide, and expressed as units per gram of urinary creatinine (NAG index). Urinary NAG isoenzymes were assayed by use of the mini-column method and electrophoresis. The overall mean value of urinary NAG index in healthy cats was 1.6+/-1.5 U/g. Urinary NAG index varied from 6.2 to 35.5 U/g in cats with chronic renal failure. There was no significant correlation between BUN, serum creatinine concentration and urinary NAG index. In cats with feline lower urinary tract disease, normal values of urinary NAG index were observed. In the urine samples of healthy cats, the proportions of NAG isoenzyme A (NAG-A) and isoenzyme B (NAG-B) were 79.1+/-4.4% and 21.0+/-4.4%, respectively, as assayed by the mini-column method. In the assay of NAG isoenzymes by electrophoresis, the proportions of NAG-A and NAG-B in healthy cats were 66.6+/-5.8% and 33.4+/-5.8%, respectively. The proportion of NAG-B as measured by electrophoresis was significantly larger (p<0.05) than that obtained with the mini column method. A feline case of acute renal failure experimentally-induced by sulfonamide showed elevation of urinary NAG index, NAG-A and NAG-B after injection of sulfonamide. The increase in NAG-B was larger than that of NAG-A. From the results reported here, measurement of urinary NAG and its isoenzymes seems to yield information about tubular damage at an early stage in cats with urinary disease.