Separation of toxic activity and ADP-ribosylation activity of botulinum neurotoxin D

Neurotoxin from Clostridium botulinum type D strain South African (neurotoxin D) has shown ADP-ribosylation activity as well as toxic activity (Matsuoka, I., Sakuma, H., Syuto, B., Moriishi, K., Kubo, S., and Kurihara, K. (1989) J. Biol. Chem. 264, 706-712). Separation of these activities from each other was attempted by means of gel filtration, hydroxylapatite column chromatography, or immunoaffinity chromatography. Approximately 90% of toxic activity was recovered in each chromatography. Although ADP-ribosylation activity was incompletely separated from neurotoxin D by gel filtration, it was separated by hydroxylapatite column chromatography. In immunoaffinity chromatography with a column of Sepharose 4B coupling antibodies against botulinum ADP-ribosyltransferase, no ADP-ribosylation activity was detected by autoradiography in the unabsorbed toxic fraction. These results indicate that neurotoxin D does not have ADP-ribosylation activity.     

Evidence that PI3K, Rac, Rho, and Rho kinase are involved in basic fibroblast growth factor-stimulated fibroblast-Collagen matrix contraction

Fibroblast-collagen matrix contraction has been used as a model system to study how cells organize connective tissue. Previous work showed that lysophosphatidic acid (LPA)-stimulated floating collagen matrix contraction is independent of Rho kinase while platelet-derived growth factor (PDGF)-stimulated contraction is Rho kinase-dependent. The current studies were carried out to determine the signaling mechanisms of basic fibroblast growth factor (bFGF)-stimulated fibroblast-collagen matrix contraction. Both bFGF and LPA promoted equally collagen matrix contraction well. Three different inhibitors, LY294002 for phosphatidylinositol-3-kinase (PI3K), C3 exotransferase for Rho and Y27632 for Rho kinase, suppressed the bFGF-stimulated fibroblast-collagen matrix contraction. With bFGF stimulation, fibroblasts spread with prominent stress fiber network formation and focal adhesions. In the presence of Rho kinase inhibitor, focal adhesions and stress fibers were mostly lost. We demonstrated that bFGF stimulation for fibroblast caused transient Rac and Rho activation but did not activate Cdc42. In addition, bFGF enhanced fibroblast migration in wound healing assay. The present study implicates PI3K, Rac, Rho, and Rho kinase as being involved in bFGF-stimulated collagen matrix contraction. The elucidation of bFGF-triggered signal transduction may be an important clue to understand the roles of bFGF in wound healing.     

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.     

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.     

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

Haptoglobin (Hp), a hemoglobin-binding protein in plasma, consists of α and β subunits and has a tetra-chain arrangement (β-α-α-β) 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 αβ units are joined by a non-covalent interaction rather than a disulfide bridge and (2) the α 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 α chains. This was confirmed by amino acid sequence analysis of the α chains: that is, Cys₁₅ participating in the inter-α 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, α chains of bear and cat Hps were found not to have the typical oligosaccharide binding sequence on their α chains and were not glycosylated.     

A new purification procedure for bovine C-reactive protein and serum amyloid P component.

1. A new purification procedure was started with salting-out fractionation of serum proteins at 45-75% saturated ammonium sulfate concentration, followed by HE agarose affinity chromatography by which calcium-dependently bound CRP and SAP were purely eluted with EDTA-containing buffer. 2. Pure CRP and SAP were finally separated by DEAE-5PW HPLC. 3. This procedure gave recovery of 15 and 26%, and fold purification of 2650 and 2400 for CRP and SAP, respectively. 4. Each subunit of CRP and SAP had one intrasubunit disulfide bond, determined by reduction and carboxymethylation.     

The complete nucleotide sequence of the gene coding for botulinum type C1 toxin in the C-ST phage genome

Two DNA fragments, 3 kbp and 7.8kbp, which encode the type C1 botulinum neurotoxin gene, were obtained from toxigenic bacteriophage DNA by treatment with a restriction enzyme. They were cloned into the plasmid vectors for nucleotide sequence determination. The nucleotide sequence contained a single open reading frame coding for 1,291 amino acids corresponding to a polypeptide with a molecular weight of 149,000. The amino acid sequence of the C1 toxin has a few regions highly homologous with tetanus toxin.     

Isolation and molecular size of Clostridium botulinum type C toxin.

A procedure is described for the purification of hemagglutinin-free Clostridium botulinum type C toxin. The toxin was purified approximately 1,000-fold from the original culture supernatant in an overall yield of 60% to a final specific toxicity of 4.4 x 10(7) minimal lethal doses/mg of protein. The toxin had a molecular weight of 141,000 and consisted of a heavy and a light chain. The molecular weights of the subunits were approximately 98,000 and 53,000. When comparing the molecular size and composition of type C toxin to that of botulinum toxins of different types, some common features may be suggested; i.e., the toxin has a molecular weight between 141,000 to 160,000 and is comprised of a heavy and a light chain linked by disulfide bonds (or bond).     

Hepatic bile and pancreatic exocrine secretions evoked by gastrointestinal peptides in sheep

The secretory response of hepatic bile and exocrine pancreas to gastrointestinal peptides has been studied in chronically cannulated sheep. Pancreatic juice flow and protein output were evoked dose dependently by intraportal injection of secretin, CCK-8, caerulein, VIP and neurotensin. However, biliary secretion was evoked by only secretin. Biliary and pancreatic exocrine secretions were enhanced by delivered gastric juice into the duodenum as followed by the increased plasma concentration of immunoreactive secretin (IRS). Results suggest that secretin is the major peptide that regulates pancreatic exocrine secretion and hepatic bile production in the sheep.