A novel 96-kDa aminopeptidase localized on epithelial cell membranes of Bombyx mori midgut, which binds to Cry1Ac toxin of Bacillus thuringiensis

Proteins in the brush border membrane (BBM) of the midgut binding to the insecticidal Cry1Ac toxin from Bacillus thuringiensis were investigated to examine the lower sensitivity of Bombyx mori to Cry1Ac, and new aminopeptidase N that bound to Cry1Ac was discovered. DEAE chromatography of Triton X-100-soluble BBM proteins from the midgut revealed 96-kDa aminopeptidase that bound to Cry1Ac. The enzyme was purified to homogeneity and estimated to be a 96.4-kDa molecule on a silver-stained SDS-PAGE gel. However, the native protein was eluted as a single peak corresponding to approximately 190-kDa on gel filtration and gave a single band on native PAGE. The enzyme was determined to be an aminopeptidase N (APN96) from its substrate specificity. Antiserum to class 3 B. mori APN (BmAPN3) recognized APN96, but peptide mass fingerprinting revealed that 54% of the amino acids of matched peptides were identical to those of BmAPN3, suggesting that APN96 was a novel isoform of the APN3 family. On ligand blots, APN96 bound to Cry1Ac but not Cry1Aa or Cry1Ab, and the interaction was inhibited by GalNAc. K(D) of the APN96-Cry1Ac interaction was determined to be 1.83 +/- 0.95 microM. The lectin binding assay suggested that APN96 had an N-linked bi-antennal oligosaccharide or an O-linked mucin type one. The role of APN96 was discussed in relation to the insensitivity of B. mori to Cry1Ac.     

Subacute thyroiditis associated with systemic multi-organ disorders

Subacute thyroiditis is generally thought to be a self-limited inflammatory disease of the thyroid gland. This paper describes serial observations on the clinical course of a typical patient with subacute thyroiditis. This patient showed specific features of destructive thyrotoxicosis with increases in the serum levels of acute phase reactants and in the erythrocyte sedimentation rate. She also showed signs of liver dysfunction [slightly increased alanine aminotransferase (ALT), alkaline phosphatase (ALP), gamma-glutamyl transpeptidase (gamma-GTP), and leucine aminopeptidase (LAP)], slight anemia, glucose intolerance, increased pancreatic enzymes, splenomegaly, and an increase in peripheral Leu 7 positive (NK/K) cells. These abnormalities all improved with recovery from disease. These findings indicate that in this patient with subacute thyroiditis inflammation is not limited to the thyroid gland but also involves the liver, pancreas and spleen. Thus the subacute thyroiditis in this patient may be a systemic multi-organ disease.     

Dual role of vitamin C in an oxygen-sensitive system: discrepancy between DNA damage and cell death

Although vitamin C is considered to act both as pro-oxidant and antioxidant, the mechanisms underlying these actions are still unclear. Using the oxygen-sensitive system of a strict anaerobe, Prevotella melaninogenica, we investigated both the pro-oxidant and antioxidant mechanisms of vitamin C. In the presence of vitamin C, the 8-hydroxydeoxyguanosine (8OHdG) formation induced by oxygen exposure was enhanced, probably due to the action of vitamin C on hydrogen peroxide generated during oxygen exposure: while catalase almost completely suppressed the enhancing effect of vitamin C, 8OHdG formation induced by hydrogen peroxide was enhanced by vitamin C. By contrast, the presence of vitamin C inhibited bacterial cell death, membrane damage, and lipid peroxidation induced by oxygen exposure. Sodium azide showed similar effects to vitamin C, thus the antioxidant action of vitamin C may be due to its quenching of the singlet oxygen generated in this system. Both the pro-oxidant and antioxidant effects of vitamin C were observed only in acidic conditions.     

A simple culture protocol to detect peptide-specific cytotoxic T lymphocyte precursors in the circulation

The detection and monitoring of peptide-specific cytotoxic T lymphocyte (CTL) precursors is essential for successful peptide-based immunotherapy against cancers. In contrast to the development of effective methods of detecting antigen-specific CTL, such as ELISpot and HLA-class I tetramer assay, stimulation with peptide-pulsed antigen-presenting cells (APC) has for some time been conventionally employed to induce peptide-specific CTL from peripheral blood mononuclear cells (PBMC). This culture protocol, however, needs a substantial number of PBMC to test the reactivity against a panel of peptides. In the present study, we established a simple culture protocol which has no need of additional APC. Addition of a corresponding peptide every 3 days was found to induce not only Epstein-Barr virus (EBV)-specific CTL from healthy donors, but also tumor antigen-derived peptide-specific CTL from cancer patients. A 10-ml blood sample was almost sufficient to test the presence of CTL precursors against 20 different peptides in triplicate assays. Overall, this culture protocol can be useful in detecting and monitoring peptide-specific CTL precursors in the circulation in peptide-based immunotherapy against cancer.     

Design,synthesis and structure-activity relationships of dual inhibitors of acetylcholinesterase and serotonin transporter as potential agents for Alzheimer's disease

We have designed and synthesized a dual inhibitor of acetylcholinesterase (AChE) and serotonin transporter (SERT) as a novel class of treatment drugs for Alzheimer's disease on the basis of a hypothetical model of the AChE active site. Dual inhibitions of AChE and SERT would bring about greater therapeutic effects than AChE inhibition alone and avoid adverse peripheral effects caused by excessive AChE inhibition. Compound (S)-6j exhibited potent inhibitory activities against AChE (IC(50)=101 nM) and SERT (IC(50)=42 nM). Furthermore, (S)-6j showed inhibitory activities of both AChE and SERT in mice brain following oral administration.     

Thrombomodulin enhances the invasive activity of mouse mammary tumor cells

Thrombomodulin (TM) is a thrombin receptor on the surface of endothelial cells that converts thrombin from a procoagulant to an anticoagulant. Thrombin promotes invasion by various tumor cells, and positive or negative correlations are found between the expression of TM and tumorigenesis in some patients. In this study, we used an invasion assay to investigate the effect of TM on the invasive activity of a mouse mammary tumor cell line, MMT cells, and the effects of TM were compared with those of thrombin as a positive control. In the presence of 1% fetal calf serum (FCS), TM significantly stimulated MMT cell invasion in a dose-dependent manner, resulting in an approximately 3-fold increase at 1-10 pg/ml over the untreated control. Thrombin also caused a similar degree of stimulation at 50 ng/ml. Since thrombin activity was detected in the components of the assay system, an invasion assay was also performed in a thrombin-activity-depleted assay system constructed to eliminate the effect of thrombin activity; TM (10 pg/ml) plus thrombin (1 pg/ml) stimulated invasion by approximately 3.5-fold in this assay system. Hirudin, a specific thrombin inhibitor, inhibited stimulation by TM as well as by thrombin in both the presence and absence of 1% FCS. Investigations of the effects of TM on proliferation, adhesion and chemotaxis to clarify the mechanism of stimulation by TM revealed that TM does not affect proliferation or adhesion in the presence of 1% FCS, but stimulates chemotaxis by approximately 2.3-fold. Similar results were obtained in experiments using thrombin. TM (10 pg/ml) plus thrombin (1 pg/ml), on the other hand, stimulated chemotaxis by approximately 2.3-fold in the thrombin-activity-depleted assay system. Binding studies using [125I]-thrombin revealed that the cells have specific saturable binding sites for thrombin. These results show that TM stimulates the invasive activity of MMT cells, probably by acting as a cofactor for the thrombin-stimulated invasion of the cells via its receptor and lowering the effective concentration of thrombin. The findings also indicate that the stimulation of invasive activity in the presence of 1% FCS and in the thrombin-activity-depleted assay system may mainly be mediated by the stimulation of chemotaxis.