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.

ADP-ribosylation of specific membrane proteins in pheochromocytoma and primary-cultured brain cells by botulinum neurotoxins type C and D.

Type C1 and D toxins produced by Clostridium botulinum caused ADP-ribosylation of a protein of 24 kDa in membrane preparations of rat clonal pheochromocytoma cells (PC12) and of proteins of 25 and 26 kDa in neuron-rich culture of fetal rat brain cells. The ADP-ribosylation reaction was dependent on the presence of MgCl2, GTP and GTP gamma S. The results obtained suggested that the ADP-ribosylation reaction is responsible for the development of the biological activity of the botulinum neurotoxins and that the target of this reaction may be novel GTP-binding proteins localized on cell membranes.

Binding of Clostridium botulinum neurotoxin to gangliosides.

The binding characteristics of Clostridium botulinum neurotoxins of types B, C1, and F to gangliosides was studied by thin layer chromatography plate and microtiter plate methods at low (10 mM NaCl in 10 mM Tris-HCl buffer, pH 7.2) or high (150 mM NaCl in 10 mM Tris-HCl buffer, pH 7.2) ionic strengths and at 0 or 37 degrees C. The three types of toxins bound exclusively to three kinds of gangliosides, GD1a, GD1b, and GT1b, in both the thin layer chromatography plate and the microtiter plate methods. Type C1 toxin bound to the three gangliosides under all the conditions, while type B and F toxins bound only at low ionic strength and 37 degrees C. At low ionic strength, the binding kinetics for the three toxins was monophasic in Scatchard plots, and the association constants obtained in the microtiter plate system were 2-4 X 10(8) M-1. In contrast, the binding kinetics of type C1 toxin in high ionic strength was biphasic in the Scatchard plot, and two association constants were obtained in the microtiter plate system. The heavy chain facilitated the binding of the toxin to the gangliosides. These results indicate that different types of botulinum toxins bind to the gangliosides under different optimal conditions and that gangliosides may not be the common receptor for all types of botulinum toxins. The gangliosides may bind to type C1 toxin together with other potential receptor(s) on synaptosomal membranes.

The structural relation between the antigenic determinants to monoclonal antibodies and binding sites to rat brain synaptosomes and GT1b ganglioside in Clostridium botulinum type C neurotoxin.

The inhibition of the binding of 125I-labeled Clostridium botulinum type C neurotoxin to synaptosomes by unlabeled toxin indicated that there were two kinds of receptors on the synaptosomal membrane. The dissociation constants (Kd) were calculated as 79 pM and 35 nM from the concentration of unlabeled toxin that induced half-displacement of bound 125I-toxin. These values agree satisfactorily with the values obtained from direct binding experiments (Agui, T, Syuto, B., Oguma, K., Iida, H., & Kubo, S. (1983) J. Biochem. 94, 521-527). The inhibition of the binding of 125I-toxin to synaptosomes and N-acetylneuraminyl(alpha 2-3)galactosyl(beta 1-3)N-acetylgalactosaminyl(beta 1-4) [N-acetylneuraminyl(alpha 2-8) N-acetylneuraminyl(alpha 2-3)]galactosyl(beta 1-4)glucosyl(beta 1-1)ceramide (GT1b) by unlabeled heavy chain indicated that heavy chain facilitates the binding of toxin to synaptosomes and GT1b. The synaptosomal and heavy chain complex Kd values were estimated as 12 nM and 24 microM. Monoclonal antibodies C-9 and CA-12 recognized the binding sites to GT1b and synaptosomes, respectively. Antigenic determinants against the two antibodies are presumably partially overlapping, and the overlapping area seems to be essential to the reaction between toxin and C-9 antibody.

Binding of Clostridium botulinum type C neurotoxin to rat brain synaptosomes.

The binding of Clostridium botulinum type C neurotoxin to rat brain synaptosomes was determined by the use of 125I-neurotoxin. The binding was independent of the incubation temperature (0 degrees C and 37 degrees C) and was equilibrated in 10 min. The dose dependent of 125I-toxin binding to synaptosomes at 0 degrees C showed that there were two kinds of toxin receptors on the synaptosomal membrane; the association constants and maximum binding values were 1.05 x 10(10 M-1, 5.25 x 10(-13) mol/mg of synaptosomal protein and 5.00 x 10(6) M-1, 5.00 x 10(-12) mol/mg of synaptosomal protein, respectively. When the incubation of toxin with synaptosomes was continued at 37 degrees C after 125I-toxin had been pre-incubated with synaptosomes at 0 degrees C for 10 min, the displacement of labeled toxin by the addition of excess amounts of unlabeled toxin decreased slightly with increasing incubation time, and finally 0.4% of the bound 125I-toxin was not displaced from synaptosomes. The binding of 125I-toxin to synaptosomes was inhibited by anti-heavy chain IgG and a monoclonal antibody which neutralized toxin and recognized heavy chain. These results suggest that the binding sites of toxin to synaptosomes are localized on heavy chain and a small amount of the bound toxin is incorporated into the synaptosomal membrane or synaptosomes.

Action of botulinum neurotoxin on acetylcholine release from rat brain synaptosomes: putative internalization of the toxin into synaptosomes.

The action of botulinum neurotoxin type C1 on the release of acetylcholine from rat brain synaptosomes was studied by using anti-toxin heavy chain Fab and anti-toxin light chain Fab. The toxin was bound to synaptosomes at 0 degrees C for 10 min, in which [14C]acetylcholine had been accumulated previously. The toxin-binding synaptosomes were pre-incubated at 37 degrees C, and the release of acetylcholine was determined after the synaptosomes had been incubated in 25 mM KCl-incubation medium for 20 min at 37 degrees C. Inhibition of [14C]acetylcholine release from the synaptosomes was observed with increasing pre-incubation time and toxin concentration, and the maximum inhibition was seen after pre-incubation for at least 15 min, which was called the "lag time." The toxin-binding synaptosomes were reacted with anti-toxin heavy chain and anti-toxin light chain Fabs at 0 degrees C for 1.5 min before pre-incubation of the synaptosomes at 37 degrees C. Both Fabs reversed the acetylcholine release inhibition by the toxin. However, when the Fabs were added during the pre-incubation time at 37 degrees C, they showed less restoration with increasing pre-incubation time. The restoration was completely abolished if the Fabs were added to the synaptosomes after the first half of the "lag time." On the other hand, when 125I-labeled toxin-binding synaptosomes were reacted with the Fabs at 0 degrees C for 1.5 min before pre-incubation of the synaptosomes at 37 degrees C, anti-heavy chain Fab removed 125I-toxin from the synaptosomes, but anti-light chain Fab did not. However, if the Fabs were added to toxin-binding synaptosomes during the pre-incubation time at 37 degrees C, the Fabs could not remove 125I-toxin from the synaptosomes, and the synaptosomes retained more labeled toxin with increasing pre-incubation time. These results suggest that there are three distinct steps in the inhibition of acetylcholine release from synaptosomes by botulinum neurotoxin. The first is binding, which is reversible, temperature-independent, and mediated by the heavy chain of the toxin. The second is temperature-dependent internalization, that takes place in the first half of the "lag time," in which both the chains are internalized into synaptosomes. The third is the development of toxicity, which requires the latter half of the "lag time."

Low-molecular-weight GTP-binding proteins serving as ADP-ribosylation substrate for ADP-ribosyltransferase from Clostridium botulinum and their relation to phosphoinositides metabolism in thymocytes.

ADP-ribosyltransferase from Clostridium botulinum type C strain was found to induce an increase of inositol phosphates (IPs) formation in murine thymocytes membranes. Incubation of electropermeabilized murine thymocytes with the enzyme also caused an increase of IPs formation in the cells. This increase of IPs formation in the enzyme-treated membranes and electropermeabilized cells was dependent on the amount of both NAD and the enzyme, suggesting that the stimulation of phosphoinositide-specific phospholipase C (PLC) was related to ADP-ribosylation of membrane proteins by the enzyme. On the other hand, in calf and murine thymocytes two proteins with the same molecular weight of 21,000 were found to be ADP-ribosylated by the botulinum ADP-ribosyltransferase. A minor ADP-ribosylation substrate was shown by two-dimensional polyacrylamide gel electrophoresis to be G21k, a low-molecular-weight GTP-binding protein (G protein) suggested previously by us to be involved in PLC regulation [Wang, P. et al. (1987) J. Biochem. 102, 1275-1287; (1988) 103, 137-142; and (1989) 105, 461-466], and the other major ADP-ribosylation substrate was identified as a rho A protein. Under the experimental conditions of the IPs formation study, ADP-ribosylation of both G21k and rho A proteins by botulinum ADP-ribosyltransferase in membranes and permeabilized cells was observed. These results suggest that botulinum ADP-ribosyltransferase-induced PLC stimulation in thymocytes is closely correlated with ADP-ribosylation of the low-molecular-weight G proteins.

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.

Binding of botulinum type Cl, D and E neurotoxins to neuronal cell lines and synaptosomes.

Clostridium botulinum 125I-labeled Cl neurotoxin bound to NG108 hybridoma cell line. Unlabeled type Cl neurotoxin inhibited the binding of the labeled Cl toxin but neither types D nor E toxin. 125I-labeled type D neurotoxin bound to rat brain synaptosomes but did not bind to NG108 cells. It is suggested that receptors for types C and D or E toxins on neuronal cell membranes are different.

Inhibition of norepinephrine secretion from digitonin permeabilized PC12 cells by botulinum type D toxin.

Botulinum type D neurotoxin inhibited Ca(2+)-evoked norepinephrine secretion in digitonin permeabilized PC12 cells. Inhibition by the toxin required prior incubation with dithiothreitol (DTT). The inhibition was dependent on both concentration and incubation times of the toxin, and was affected by Ca2+ concentration. With less than 0.7 microM Ca2+ almost complete inhibition was observed; however, above 0.7 microM, Ca2+ stimulated additional norepinephrine release in a dose-dependent manner.

Bovine haptoglobin: single radial immunodiffusion assay of its polymeric forms and dramatic rise in acute-phase sera.

Using purified bovine haptoglobin (Hp) and specific antisera, a single radial immunodiffusion (SRID) assay method has been developed to measure the serum Hp level in cattle. Bovine Hp is a highly polymerized protein showing heterogeneous molecular forms in serum. After treatment with cysteine or glutathione, Hp was partially reduced to a homogeneous form, suitable for SRID assay. This method gives values comparable to those obtained by hemoglobin-binding capacity assay, and has the advantage of being simple and convenient. Although serum Hp was not detectable in healthy cattle, it was found more than 50-fold after invasive surgery, indicating that Hp is a characteristic acute-phase protein in cattle.

Chemiluminescence of neutrophils from patients with Behçet's disease and its correlation with an increased proportion of uncommon serotypes of Streptococcus sanguis in the oral flora.

Zymosan-induced chemiluminescence was investigated in whole blood and in neutrophils: in both, the peak count was frequently elevated in Behçet's disease, and was significantly higher than in healthy controls; similarly the peak time was shorter. There were more uncommon serotypes of Streptococcus sanguis in the oral flora of patients with Behçet's disease. Common serotypes were present in the flora of healthy controls, but not in patients with the disease. The percentage of Strep. sanguis in the oral flora was significantly correlated with the level of chemiluminescence response. Thus infection with uncommon serotypes of Strep. sanguis may play a role in the aetiology of Behçet's disease.

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.

Soluble fractions of Pasteurella multocida: their protective qualities against fowl cholera in turkeys.

Soluble fractions of Pasteurella multocida strain P1059 were extracted from a single source by four methods, and their immunogenicity was evaluated by challenge exposure in turkeys. The fractions were extracted by 1) heating in 2.5% NaCl, 2) 0.5M potassium thiocyanate, 3) 1.0M sodium salicylate, and 4) prolonged stirring in formalin solution followed by pelleting (LPS-protein antigen). Eighty percent to 90% of infected turkeys were protected in two trials by vaccination with the saline extract or LPS-protein antigen, whereas less consistent protection was associated with the other two preparations. Endotoxin content was the highest in LPS-protein antigen, followed by KSCN, Na salicylate, and saline extract in that order. The four fractions contained at least one common antigen, which had previously been shown to be a surface-protective antigen.

Comparison of pancreatic exocrine secretion via endogenous secretin by intestinal infusion of hydrochloric acid and monocarboxylic acid in anesthetized piglets.

The secretory response of the exocrine pancreas via endogenous secretin (IRS) by intraduodenal instillation of hydrochloric acid (HCl) and various monocarboxylic acid solutions was studied in anesthetized piglets. The secretion induced by HCl solutions of various concentrations containing 250 mM NaCl occurred when pH of the solutions was lower than 1.5. After instillation of the HCl solution of pH 1.0, juice flow and protein output increased 26 times and 9 times, respectively, as compared with basal levels. Such pancreatic responses paralleled an increase in plasma IRS concentration in the portal vein. The pancreatic response induced by a lactic acid solution occurred when pH of the solutions was lower than 3.8. The juice flow and protein output stimulated by a lactic acid solution of 250 mM and pH 2.0 were 16 and 8 times higher than the basal levels. The responses to the lactic acid solution of pH 2.0 increased concentration dependently, and were followed by an increase in IRS concentration in the portal vein. The pancreatic exocrine responses induced by other monocarboxylic acid solutions (250 mM) of pH 2.0 were in the following order: formic acid greater than lactic acid greater than pyruvic acid much greater than acetic acid greater than butyric acid greater than propionic acid. Lactamide, an analogous substance of lactic acid, did not evoke any pancreatic secretion. The results indicate the possibility that pancreatic exocrine response induced by HCl is dependent upon hydrogen ion, while the response induced by monocarboxylic acid is not always dependent on dissociation constant of acid.

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.

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.

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.

Purification and identification of a serum protein increased by anthelmintic drugs for Dirofilaria immitis in dogs.

Polyacrylamide gel electrophoretic analysis of canine serum protein has revealed that the administration of anthelmintics elicits an increase in a certain serum protein. This protein, named PT60, was partially purified by ammonium sulfate fractionation and preparative electrophoresis. The purified PT60 gave a single band with the molecular size of 53 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under non-reducing conditions. After reduction with 2-mercaptoethanol, two bands appeared at 35 kDa and 17 kDa, indicating that PT60 consists of two subunits which are linked with each other by disulfide bonds. PT60 had the capacity to bind to hemoglobin. In an immunodiffusion test, an antiserum against PT60 cross-reacted with canine haptoglobin (Hp). N-terminal amino acid sequences of two PT60 subunits were identical to those of alpha and beta subunits of canine Hp, respectively. Thus, PT60 was identified as Hp.

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.