Cloning and sequencing full length of canine Brca2 and Rad51 cDNA.

Mammary tumors are the most common neoplasm in female dogs, Canis canis, and in women. Mutations in human Brca2 confer an increased risk of female breast cancer. Previous studies have shown that the Brca2 tumor suppressor protein interacts with the recombinational repair protein Rad51. We cloned the full-length cDNA of the canine homologues of Brca2 and Rad51 to obtain a basis for studying their relationship with susceptibility to mammary tumors. The canine Brca2 and Rad51 cDNAs are 11 and 1.5 kb long, encoding 3.471 and 339 amino acids, respectively. The amino acid sequence of canine Brca2 showed 68% homology with the human protein, and 58% homology with a murine protein. There were highly conserved regions in the C-terminus of all three proteins, where the Rad51 interacting domain and putative nuclear localization signals are located. Comparing with the partial genomic sequence previously reported, we found possible nuclear polymorphisms in exon 11, some of which result in amino acid substitutions. On the other hand, canine Rad51 protein had extremely high homology (99%) to the human and murine proteins. Expression of both Brca2 and Rad51 was detected in the mammary gland, suggesting that these two genes interact in the canine mammary gland.

A novel serum chitinase that is expressed in bovine liver.

Chitinases are ubiquitous chitin-fragmenting hydrolases. They are synthesized by a vast array of organisms, including those not composed of chitin. Here, we describe a novel serum chitinase (chitin-binding protein b04, CBPb04), which is expressed in bovine liver. Although CBPb04 is secreted as an endocrine chitinase, it shows higher homology with human gastrointestinal tract exocrine chitinase (AMCase) than with macrophage endocrine chitinase (human chitotriosidase). This suggests that cows have a specific defense against chitin-containing microorganisms. CBPb04 mRNA is expressed in hepatocytes. This is the first report of a hepatogenic mammalian chitinase.

Isolation, characterization, and cDNA cloning of chicken turpentine-induced protein, a new member of the scavenger receptor cysteine-rich (SRCR) family of proteins.

Acute-phase serum proteins were induced by administrating a chicken with turpentine oil. One of these proteins was a new protein that appeared in front of albumin in polyacrylamide disc gel electrophoresis using a 4.5-16% gel. To purify this protein, turpentine-administrated chicken serum was fractionated by ammonium sulfate precipitation at 50% saturation, and the supernatant fraction was chromatographed on a DEAE-Toyopearl 650S column. The purified protein is a mannose-glycoprotein, and its N-terminal sequence, determined by the Edoman method, is not homologous from that of other reported acute-phase proteins. An analysis of physiological function with two different test systems, chemiluminescence measurement and electron spin resonance spectroscopy, showed that the purified protein has antioxidant activity and inhibits superoxide (O(2)) mediated by activation of the receptor. In support of these results, the complete amino acid sequence of 18-B is homologous to the scavenger receptor cysteine-rich (SRCR) family of proteins that participate in the regulation of leukocyte function. 18-B is composed of four SRCR domains, which is different from the previously characterized SRCR family of proteins such as Spalpha, CD6, and CD163. These findings indicate that turpentine-induced 18-B, a new member of scavenger receptor cysteine-rich family, may be implicated in regulation of cell function in a manner of inhibition of the overproduction of the reactive oxygen species.

Genomic organization, chromosomal localization, and promoter analysis of the mouse Mail gene.

The Mail (molecule possessing ankyrin repeats induced by lipopolysaccharide) protein is a member of the IkappaB family. It has six ankyrin repeats that are conserved in other IkappaB proteins, such as IkappaB-alpha and Bcl-3. Mail mRNA expression is induced rapidly following lipopolysaccharide (LPS) injection, most notably in the spleen, lung, and lymph nodes of mice, where immune cells, such as lymphocytes and macrophages, are abundant. In this study, we cloned and characterized the Mail gene. The isolated genomic clones span approximately 30 kb and encompass the entire gene. Comparisons with Mail cDNA revealed that the Mail gene consists of 14 exons. Several splice junctions encoding ankyrin repeats are conserved among Mail and other IkappaB family genes. Southern hybridization showed that Mail is a single-copy gene. Using fluorescence in situ hybridization analysis, mouse and rat Mail genes were mapped to Chromosome (Chr) 16C1.2-C1.3 and Chr 11q21.1, respectively. Primer extension determined the transcription start site of Mail. Sequence analysis of the proximal promoter region revealed the presence of a TATA box and putative transcription factor-binding sites, such as those for NF-kappaB and NF-IL6. This region is sufficient to drive high-level reporter gene expression in LPS-stimulated transfected cells.

The evaluation of the potential of botulinum C3 enzyme as an exogenous differentiation inducing factor to neurons.

Botulinum C3 enzyme produced by Clostridium botulinum type C and D strains modifies Rho proteins. In a previous study, we observed that the LDH isozyme pattern of neurons treated with C3 enzyme was different from that induced with endogenous growth factor of neurons such as NGF [21]. This type of change is considered to have an advantage in the medical use of C3 enzyme for neural disorder. To determine the functional similarity of C3-treated neurons to control and NGF-treated neurons, we examined the responses of C3-treated neurons to various drugs, including some neurotransmitters, by measuring the rise of intracellular Ca ions into the neurons. The time course of the rise of intracellular Ca ions induced by high concentration of potassium in the C3-treated neurons was similar to that in the NGF-treated neurons. The C3-treated neurons responded to glutamic acid, aspartic acid, kainic acid, gamma-aminobutylic acid, muscarine and ACh with similar time courses and magnitudes as the control neurons. These results suggest that the C3 enzyme induces the functional differentiation of neurons, and that C3 enzyme has the potential for the medical use as an exogenous differentiation-inducing factor of neurons.

Botulinum C3 enzyme changes the lactate dehydrogenase isozyme pattern of primary culture of neurons.

Changes in the lactate dehydrogenase (LDH) isozyme pattern of primary culture of neurons treated with botulinum C3 enzyme were examined in order to elucidate the functional changes accompanying the morphological change that follows ADP-ribosylation of Rho protein. Primary neurons were prepared from the cerebrum of ICR mouse embryos on day 15. Neurons were cultured in MEM with 10% fetal calf serum at 37 degrees C. In the neurons treated with C3 enzyme, a typical morphological change was observed after 24 hr, and the LDH isozyme pattern was changed after 72 hr. The ratio of H-subunit to M-subunit in LDH was decreased by C3 treatment, suggesting the induction of a state of lower intracellular oxygen consumption in neurons in the primary cultures.

Characterization of olfactory receptor organs in Xenopus laevis Daudin.

Xenopus laevis is highly suitable for studying the mechanisms of olfactory reception for water-soluble odorants and for airborne odorants. However, the functional differences of cells and component protein molecules in the olfactory receptors of Xenopus have remained obscure. In recent studies, the patterns of sugar residues expressed on the cell surface have been utilized to analyze the characteristics of neurons, because the sugar chains in neurons play very important roles in targeting and cell-to-cell communication. In this study, we have determined the distribution of sugar residues and glycoproteins in the olfactory receptor organs of Xenopus using lectins as labeling agents, and characterized the receptors of water-soluble odorants and of airborne odorants. The results of lectin histochemical analysis show distributional differences of GlcNAc, GalNAc and mannose between the middle chamber and the lateral chamber of the main nasal cavity. Furthermore, a 65 kDa glycoprotein containing mannose, GlcNAc and GalNAc was specifically detected in the medial chamber of the main cavity epithelium in receptor organs of airborne odorants by SDS-PAGE and lectin blotting. The characteristics of the epithelia demonstrated in this study should further our understanding of the functional differences between the receptors of water-soluble odorants and of airborne odorants at the molecular level.

Characteristic transfer of colostral components into cerebrospinal fluid via serum in neonatal pigs.

In order to evaluate the possibility of modification of brain function by colostral suckling, the characteristic transfer of colostral components into serum and cerebrospinal fluid (CSF) has been studied by SDS electrophoresis, immunoblot and ELISA methods in nonsuckling pigs. Total protein concentrations in the serum increased immediately after oral administration of bovine colostrum, reaching a peak value (7.0 +/- 0.7 g/dl) at 24 h after administration, corresponding to a 3-fold increase compared to preinfusion levels. IgG and other macromolecular components (MW 19, 000-58,000) were recognized in serum by electrophoretic and ELISA analysis. Total protein concentrations in the CSF collected from the cisterna magna also increased steeply after colostral administration, reaching a maximal value (54.1 +/- 5.0 mg/dl) at 4 h, corresponding to a 4-fold increase compared to preinfusion levels. Two colostral components (MW 19,000 and 31,000) in serum were confirmed to be present in the CSF by electrophoresis. The component of MW 19,000 was identified by immunoblot as beta-lactoglobulin. IgG in serum transferred from colostrum could not be detected in the CSF by ELISA. Lactoferrin administered into the intestine was also detected in the CSF via serum. These results indicate that some components of colostrum can be transported into the CSF via the serum, suggesting the possibility of modification of immature brain functions by colostral suckling in neonatal pigs.

Oral administration of (-)catechin protects against ischemia-reperfusion-induced neuronal death in the gerbil.

The effect of ad libitum oral-administration of (-)catechin solution on ischemia-reperfusion-induced cell death of hippocampal CA1 in the gerbil was histologically examined. When (-)catechin solution instead of drinking water was orally administered ad libitum for 2 weeks, dose-dependent protection against neuronal death following by transient ischemia and reperfusion was observed. To evaluate the involvement of reduction of reactive-oxygen-species (ROIs) by the antioxidant activity of (-)catechin in this protection, the superoxide scavenging activity of the brain in catechin-treated gerbils was measured by ESR and spin-trapping using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). The superoxide scavenging activities of the brains obtained from catechin-treated gerbils were significantly higher than those of catechin-untreated animals. From these results, it was suggested that orally administered (-)catechin was absorbed, passed through the blood-brain barrier and that delayed neuronal death of hippocampal CA1 after ischemia-reperfusion was prevented due to its antioxidant activities.

Polyacrylamide gel electrophoretic serum protein patterns of acute inflammation induced by intramuscular injection of turpentine in young broiler chickens.

Changes in the serum protein patterns accompanying inflammation in young broilers were examined by polyacrylamide gradient gel electrophoresis. Sera were obtained from blood of 4-week-old male broilers after induction of acute inflammation by an injection of 0.5 ml/kg turpentine. Electrophoretic patterns at 48 hr after injection, showed an increase in transferrin (Tf) and a segment containing albumin (Alb) with multiple peaks. The unbound iron binding capacity (UIBC) increased by 10 times, and the serum iron (SI) concentration in acute inflammation was reduced to one-half its initial value. These are considered to be typical changes in serum proteins and iron after acute inflammation.

Molecular cloning of the gene encoding the mosaic neurotoxin, composed of parts of botulinum neurotoxin types C1 and D, and PCR detection of this gene from Clostridium botulinum type C organisms.

The DNA fragment common to the genes encoding botulinum neurotoxin types C1 (BN/C1) and D (BN/D) was amplified by PCR from the culture supernatant of Clostridium botulinum type C strain 6813 (C6813) that was treated with either DNase I or proteinase K but not from the supernatant that was treated with both DNase I and proteinase K, suggesting the neurotoxin gene is located on a certain bacteriophage DNA. Thus, to isolate the neurotoxin gene, we performed PCR with the culture supernatant of C6813 and seven primer pairs designed from the genes encoding BN/C1 and BN/D. The coding region in the connected sequence encodes a neurotoxin composed of 1,280 amino acids with a molecular weight of 147,817. The neurotoxin from C6813 has 95% amino acid identity to BN/C1, except for its C-terminal one-third, which is quite similar to the C-terminal one-third of BN/D (95% identity). When we performed PCRs with four primer pairs designed from the 5'-terminal two-thirds of the BN/C1 gene and two primers from the 3'-terminal one-third of the BN/D gene, DNA fragments of the expected sizes (0.5 to 1.3 kbp) could be amplified from C. botulinum type C strains 6812 and 6814. These results suggest that some strains of C. botulinum type C contain the gene encoding the mosaic neurotoxin composed of parts of BN/C1 and BN/D.

Polydextrose induces precocious cessation of intestinal macromolecular transmission and development of digestive enzymes in the suckling rat.

The effect of repeated oral administration of polydextrose and pectin on intestinal macromolecular transport and digestive enzymes development was examined in the suckling rat. The administration of polydextrose for 7 days resulted in pancreatic hyperplasia, followed by the enhancement of trypsin activity. The length of the small intestine and the wet weight of the cecum were significantly increased by polydextrose treatment. Maltase activity was increased in a dose dependent manner by polydextrose, but not by pectin treatment. Lactase activity was not changed by either treatment. The absorption of bovine IgG was precociously depressed by polydextrose, but not by pectin treatment. These results suggest that oral administration of polydextrose induces precocious maturation of the small intestine and exocrine pancreas in the suckling rat.

Identification of gangliosides as inhibitors of ADP-ribosyltransferases of pertussis toxin and exoenzyme C3 from Clostridium botulinum.

We have previously reported the presence of an endogenous inhibitory activity in bovine brain for the ADP-ribosylation of GTP-binding proteins catalyzed by pertussis toxin (PT) (Hara-Yokoyama, M., and Furuyama, S. (1989) Biochem. Biophys. Res. Commun. 160, 67-71). In the present study, we identified the inhibitor as a ganglioside. The screening of various gangliosides revealed that GQ1b alpha most effectively inhibited the ADP-ribosyltransferase activities of both the holoenzyme and the catalytic subunit of PT. GQ1b alpha is a ganglioside newly identified as one of the antigens recognized by the cholinergic neuron-specific antibody, anti-Chol-1 alpha (Hirabayashi, Y., Nakao, T., Irie, F., Whittaker, V.P., Kon, K., and Ando, S. (1992) J. Biol. Chem. 267, 12973-12978). GQ1b alpha also inhibited the PT-catalyzed NAD+ glycohydrolysis. Unlike PT activity, the ADP-ribosylation and the NAD+ glycohydrolysis catalyzed by the C3 exoenzyme from Clostridium botulinum type C were inhibited by GT1b and GQ1b. The ADP-ribosylation catalyzed by either PT or the C3 exoenzyme was not inhibited by ceramide, galactocerebroside, or sialic acid. In addition to the inhibitory action of gangliosides on ADP-ribosylation, the importance of gangliosides as regulators of NAD+ metabolism is discussed.

Stimulation of haptoglobin synthesis by interleukin-6 and tumor necrosis factor, but not by interleukin-1, in bovine primary cultured hepatocytes.

The hepatic synthesis of acute phase proteins in ruminants has been suggested to be regulated by some mechanisms different from those in other species such as rodents and human. To explore possible regulatory factors unique to ruminants, we examined effects of interleukin (IL)-6, IL-1 and tumor necrosis factor (TNF), on haptoglobin (Hp) synthesis using a primary culture system of bovine hepatocytes. After bovine primary cultured hepatocytes were incubated in the presence of various concentrations of the cytokines, the synthesis and mRNA level of haptoglobin and albumin were measured by labeling with [35S]-methionine and immunoprecipitation, and by Northern blot analysis, respectively. Hp synthesis was dose-dependently increased by recombinant human (rh) IL-6, and also by rhTNF-alpha, but to a less extent, while it was not affected by rhIL-1 beta. The stimulatory effect is mainly pretranslational, because mRNA level of Hp changed in parallel with protein synthesis. In contrast, albumin synthesis was suppressed by these three cytokines similarly. These results are inconsistent with the previously proposed view that TNF and IL-1 overlap in their pathways leading to the transcriptional activation of many acute phase protein genes. In conclusion, there is a species-specific unique signaling system, especially for TNF, in transcriptional activation of bovine Hp gene.

Haptoglobin in Carnivora: a unique molecular structure in bear, cat and dog haptoglobins.

Haptoglobin (Hp), a hemoglobin-binding protein in plasma, consists of alpha and beta subunits and has a tetra-chain arrangement (beta-alpha-alpha-beta) connected by disulfide bridges in most mammals so far examined. Dog Hp has been reported to be unique compared with other Hps in respect that (1) the two alpha beta units are joined by a non-covalent interaction rather than a disulfide bridge and (2) the alpha chain has an oligosaccharide-binding sequence (Asn-X-Ser/Thr) and is glycosylated. To determine whether the unique structures of dog Hp are common in the Carnivora, we purified Hps from sera of bear and cat, and analyzed their subunit structure and partial amino acid sequences. The analyses by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, under both reducing and non-reducing conditions, revealed that bear and cat Hps have similar subunit arrangements to dog Hp, suggesting the absence of a disulfide bridge between two alpha chains. This was confirmed by amino acid sequence analysis of the alpha chains: that is, Cys15 participating in the inter-alpha chain disulfide bridge was replaced by Val in bear or Leu in cat and dog. Thus, the unique subunit arrangement of Hp reported in dog may be common in the Carnivora. In contrast to dog Hp, however, alpha chains of bear and cat Hps were found not to have the typical oligosaccharide binding sequence on their alpha chains and were not glycosylated.

Polyacrylamide gel electrophoretic patterns of chicken serum in acute inflammation induced by intramuscular injection of turpentine.

To develop a method to detect hidden inflammation using serum protein in chickens, changes in serum proteins with acute inflammation were analyzed using a turpentine-induced inflammation model. Inflammation in the pectoral muscle of a 14-wk-old White Leghorn became apparent 3 h after the injection of turpentine and became more severe thereafter. Coincident with the development of inflammation, changes in serum proteins were analyzed by electrophoresis on polyacrylamide gradient gels. The electrophoretic patterns were divided into 21 segments. Two of these segments increased remarkably. These were located near the center of the electrophoretic pattern and were identified as transferrin due to iron staining, correlation of movement against a commercial transferrin sample in SDS-PAGE, and immunoblotting. These results suggest that transferrin may serve as a marker for inflammation in chicken.

Possible involvement of a small G-protein sensitive to exoenzyme C3 of Clostridium botulinum in the regulation of myofilament Ca2+ sensitivity in beta-escin skinned smooth muscle of guinea pig ileum.

The effects of exoenzyme C3 of Clostridium botulinum on Ca(2+)- and drug-induced tension developments were investigated in beta-escin skinned smooth muscle of guinea pig ileum to test the involvement of a small G-protein in the regulation of myofilament Ca2+ sensitivity. C3 is known to ADP-ribosylate the rho p21 family of small G-proteins. Treatment with C3 (0.35 microgram/ml, for 30 min) shifted the pCa-tension curve rightward along the Ca2+ concentration axis, indicating a decrease in Ca2+ sensitivity of the contractile elements. The inhibitory effect of C3 was not preserved after treatment with GDP beta S (1 mM), an antagonist of GTP for the binding to G-proteins. Stimulation of muscarinic receptors with carbachol (CCh, 100 microM) shifted the pCa-tension curve leftward, indicating Ca2+ sensitization of tension development. The Ca(2+)-sensitizing effect of CCh was not observed after C3 treatment. When GTP gamma S (10 microM), an activator of G-proteins, was applied at a plateau of tension development produced by a moderate concentration of Ca2+, further increase in tension was elicited and the effect of GTP gamma S was inhibited by C3 treatment. The results suggest the possible involvement of a rho p21-like small G-protein in the regulation of Ca2+ sensitivity of smooth muscle myofilaments.

Orally administered spermine induces precocious intestinal maturation of macromolecular transport and disaccharidase development in suckling rats.

The effect of orally administered spermine on intestinal cessation in bovine IgG transport and digestive enzymes in the small intestine was examined in the suckling rats. By the repeated oral administration of spermine (0.1 or 0.25 mumol/g bwt) for 5 days, the ratio of protein to DNA was significantly increased. Maltase and lactase activities changed dose dependently in the spermine treated pups. Absorption of bovine IgG transport in the intestine was dose dependently depressed by spermine treatments. Morphological inspection of treated pups showed a decline in the number of epithelial cells that absorb bovine IgG and in their vesicle sizes from basal to upper regions of the villi. The ratio of mitosis in the crypt of treated pups significantly increased in the small intestine and cecum. These results suggest that exogenously administered spermine induces precocious maturation of the macromolecular transmission and disaccharidase activity in the small intestine of the suckling rats.

Precocious cessation of intestinal macromolecular transport and digestive enzymes development by prostaglandin E2 in suckling rats.

The effect of repeated oral administration of prostaglandin analogue (dmPGE2) on intestinal macromolecular transport and digestive enzymes development were investigated in the suckling rats. By the administration of dmPGE2 for 7 days, precocious induction of maltase activity, depression of amylase activity and enhancement of trypsin activity in the pancreas occurred. Absorption of bovine IgG was dose dependently depressed by dmPGE2 treatments. The intestinal cessation was also observed in the adrenalectomized pups, but was not influenced by difluoromethyl ornithine administration. These results suggest that oral administration of PGE2 induces precocious maturation of the small intestine and exocrine pancreas and that the intestinal cessation is not directly related to ornithine decarboxylase activity in the suckling rats.

Isolation and primary culture of bovine hepatocytes: albumin synthesis and adrenergic activation of glycogenolysis.

We describe a technique for isolation and primary culture of bovine hepatocytes, and their metabolic characterization. Hepatocytes were isolated from the caudate lobe of bovine liver by perfusion with 0.25 mM ethylene-glycol tetraacetic acid and 0.05% collagenase. The viability and yield of the cells were 70-92% and 0.1-3.6 x 10(7) cells/g liver, respectively. When the isolated hepatocytes were cultured in Williams' medium E, they began to spread in 3 hr and formed monolayers in 24 hr. These monolayers were retained for at least 6 days. To monitor the metabolic activities specific to liver, synthesis and secretion of albumin were measured by labeling with [35S]-methionine and immunoprecipitation. This activity was low in isolated hepatocytes, but increased after culturing 1-3 days, and decreased again after 6 days. Glycogenolytic activity was also assessed by measuring glucose release to the medium by stimulation with epinephrine. The glycogenolytic response to epinephrine was also enhanced by culturing the hepatocytes 1-3 days, but was decreased after 6 days. Since the isolated bovine hepatocytes retained the liver-specific activities of albumin synthesis and glycogenolysis for several days in culture, these cells are useful for cellular and molecular studies on the functions of bovine liver.