Potent inhibitors of amyloid β fibrillization, 4,5-dianilinophthalimide and staurosporine aglycone, enhance degradation of preformed aggregates of mutant Notch3

Cytotoxic T lymphocytes (CTL) are critical effector cells in tumor immunity. Adoptive transfer therapy with in vitro-expanded tumor-specific CTL is a promising approach for preventing cancer metastasis and recurrence. Transferred CTL are not effective in clinical trials, however, due to inadequate tumor-infiltration. Therefore, the development of functionally modified CTL, such as tumor-targeting CTL, is widely desired. Here, we designed the tumor-targeting CTL expressing a single-chain antibody fragment (scFv-CTL) specific for vascular endothelial growth factor receptor 2 (VEGFR2/flk1) by transducing the CTL with a retroviral vector. The scFv-CTL bound to VEGFR2/flk1-expressing cells and retained their cytotoxic activity against tumor cells. In addition, adoptive transfer of scFv-CTL into tumor-bearing mice effectively suppressed tumor growth due to the augmented accumulation of the transferred CTL in the tumor tissue. These findings indicate that the creation of CTL capable of targeting tumor vascular endothelial cells by scFv-expression technique is considerably promising for improvement of efficacy in adoptive immunotherapy.     

Resequencing and Association Analysis of PTPRA,a Possible Susceptibility Gene for Schizophrenia and Autism Spectrum Disorders

Background

The PTPRA gene, which encodes the protein RPTP-α, is critical to neurodevelopment. Previous linkage studies, genome-wide association studies, controlled expression analyses and animal models support an association with both schizophrenia and autism spectrum disorders, both of which share a substantial portion of genetic risks.

Methods

We sequenced the protein-encoding areas of the PTPRA gene for single nucleotide polymorphisms or small insertions/deletions (InDel) in 382 schizophrenia patients. To validate their association with the disorders, rare (minor allele frequency <1%), missense mutations as well as one InDel in the 3′UTR region were then genotyped in another independent sample set comprising 944 schizophrenia patients, 336 autism spectrum disorders patients, and 912 healthy controls.

Results

Eight rare mutations, including 3 novel variants, were identified during the mutation-screening phase. In the following association analysis, L59P, one of the two missense mutations, was only observed among patients of schizophrenia. Additionally, a novel duplication in the 3′UTR region, 174620_174623dupTGAT, was predicted to be located within a Musashi Binding Element.

Major Conclusions

No evidence was seen for the association of rare, missense mutations in the PTPRA gene with schizophrenia or autism spectrum disorders; however, we did find some rare variants with possibly damaging effects that may increase the susceptibility of carriers to the disorders.     

Evaluation of the process for superoxide production by NADPH oxidase in human neutrophils: evidence for cytoplasmic origin of superoxide

We present an up-to-date insight into the function of NADPH oxidase in human neutrophils, the signalling pathways involved in activation of this enzyme and the process of association of its components with the cytoskeleton. We also discuss the functional implications of morphological studies revealing localization of the sites of NADPH oxidase activity. An original model of the process of superoxide (O2*-) production in human neutrophils is shown. Organization of NADPH oxidase is associated with several components. Upon stimulation, tri-phox cytosolic components of NADPH oxidase (p40-phox, p47-phox and p67-phox) bind to actin filaments. This process involves other actin-binding proteins, such as cofilin and coronin. Activated protein kinase C, translocated from the plasma membrane, phosphorylates cytosolic components at a scaffold of cytoskeleton. Subsequently, p40-phox, responsible for maintaining the resting state of NADPH oxidase, is separated from other two cytosolic phox proteins following an attachment of the active form of small GTP-binding protein Rac to p67-phox. Cytosolic duo-phox proteins (p47-phox and p67-phox) conjugate with membrane components (gp91-phox, p22-phox and Rapla) of NADPH oxidase residing within membranes of intracellular compartments. This chain of events triggers production of O2*-. Then, oxidant-producing intracellular compartments associate with the plasma membrane. Eventually, intracellularly produced O2*- is released to the extracellular environment through the orifice formed by fusion of oxidant-producing compartments with the plasma membrane. Intracellular movement of the oxidant-producing compartments may be regulated by myosin light chain kinase. The review emphasizes that functional assembly of NADPH oxidase and, therefore, generation of O2*- is accomplished essentially within the intracellular compartments. Upon neutrophil stimulation, intracellularly generated O2*- is transported to the plasma membrane to be released and to ensure host defense against infection.     

Cholesteryl glucoside-induced protection against gastric ulcer

The cytoprotective effect of heat shock proteins (HSPs) promises new therapeutic modalities for medical treatment. We examined the anti-ulcer effect of cholesteryl glucoside (1-O-cholesteryl-beta-D-glucopyranoside, CG) on cold-restraint stress-induced gastric ulcer in rats, in terms of its correlative ability to activate heat shock factor (HSF) and to induce HSP70. Rapid induction of CG occurred in animal tissues, especially in stomach, after exposure to stress, indicating that this glycolipid might act as an anti-stress, lipid mediator involved in the very early stages of stress-induced signal transduction. Orally administered CG apparently showed anti-ulcer activity in rats via HSF activation and HSP70 induction. When compared with geranylgeranylacetone (GGA), the well known as an effective, synthetic anti-ulcer agent, CG proved to have the same level of strength on ulcer inhibition. GGA caused CG and HSP70 induction in gastric mucosa, indicating that GGA induced HSP70 via CG production. CG thus might be useful for medical treatment of stress-induced diseases, and as an anti-stress supplement for daily diet.     

Relationships of p40(phox) with p67(phox) in the activation and expression of the human respiratory burst NADPH oxidase

p40(phox) of the phagocyte NADPH oxidase forms a complex with p67(phox) in cytosol, and coincidentally decreases in patients who lack p67(phox). Here we investigated the mode of translocation of p40(phox) to the membrane, its cytoskeletal localization on activation of the NADPH oxidase, and the dependency of its expression relative to that of p67(phox). When human polymorphonuclear leukocytes (PMNs) were stimulated with phorbol myristate acetate (PMA), p40(phox) was translocated to the membrane along with p67(phox), and not was released into the cytosol. Studies with resting PMNs using Triton X-100 revealed the exclusive localization of p67(phox) in the cytoskeletal fraction. Unexpectedly, however, about half of p40(phox), which is deemed to be fully associated with p67(phox), was recovered in the non-cytoskeletal fraction. Unlike p47(phox), the association of p40(phox) with cytoskeleton was not induced by the PMA-stimulation. These results indicate not only that p40(phox) associates with cytoskeleton via a molecule of p67(phox), but also that there are distinct states of p40(phox) that can be manipulated with Triton X-100. Lastly, Western-blot analysis of hematopoietic cells revealed no correlation between p40(phox) and p67(phox) in their protein expressions during cell differentiation, and also that p40(phox) can be stably present alone in cells, unless in the case of mature PMNs. In this regard, definitive proof was obtained with Epstein-Barr virus-transformed B cells of a p67(phox)-deficient patient, in which p40(phox) was normally expressed.     

Comparison of transforming growth factor-beta and a macrophage- deactivating polypeptide from tumor cells. Differences in antigenicity and mechanism of action

A factor in medium conditioned by mouse tumor cells was shown previously to suppress the capacity of mouse peritoneal macrophages to undergo a respiratory burst and to kill protozoal pathogens (macrophage deactivation factor, MDF). Recently, pure transforming growth factor-beta (TGF-beta) proved to be a potent macrophage deactivator as well. Two lines of evidence suggest that MDF is not identical with TGF-beta. First, rabbit anti-TGF-beta IgG neutralized the respiratory burst-suppressing activity of TGF-beta without affecting the bioactivity of MDF, even when the latter was treated with acid to activate potentially latent TGF-beta. Second, in contrast to MDF, which decreases the affinity of the NADPH oxidase for NADPH, permeabilized macrophages that had been deactivated with TGF-beta displayed the same Km and Vmax of the oxidase as activated macrophages. As with MDF, TGF-beta had no effect on two other potential control points over the secretion of respiratory burst products, namely, hydrogen peroxide catabolism or glucose uptake. Finally, neither MDF nor TGF-beta affected the extent or affinity of binding of phorbol diesters to macrophages, the activity or cofactor requirements for protein kinase C, or the ability of protein kinase C to translocate quantitatively from cytosol to membrane fractions in response to phorbol diesters. Thus, 1) MDF is not identical with TGF-beta, and 2) in contrast to the activation or deactivation of macrophages by numerous other agents, TGF-beta regulates macrophage respiratory burst capacity at a level other than the apparent affinity of the oxidase for its substrate.     

Activation of guinea pig polymorphonuclear leukocytes with soluble stimulators leads to nonrandom distribution of NADPH oxidase in the plasma membrane

Guinea pig polymorphonuclear leukocytes (PMN) were briefly activated with soluble stimulators such as sodium myristate (SM) or phorbol myristate acetate (PMA) and then disrupted by the nitrogen cavitation method to study the subcellular distribution of NADPH oxidase, which is responsible for O2 - generation. Fc-receptor and 5'-nucleotidase activities were measured as plasma membrane markers. 1) The homogenate was first fractionated by differential centrifugation. The O2- -generating activity of PMN activated either by SM or PMA was recovered in a 2 X 10(4) g pellet which contained a large amount of granules and about 50% of the plasma membrane markers, but not in a 1 X 10(5) g pellet which consisted of plasma membranes and few granules. 2) Further separation of the 2 X 10(4) g pellet from PMA-activated PMN was attempted by an iso-osmotic Percoll density gradient centrifugation. The O2- -generating activity was recovered in light fractions in which plasma membrane markers were found, but neither in specific nor in azurophil granules. The 1 X 10(5) g pellet showed a similar distribution of the plasma membrane markers to that of the 2 X 10(4) g pellet, except that the peak of the O2- -generating activity was much smaller on an identical density gradient. The results showed that NADPH oxidase is located in the plasma membranes precipitated by centrifugation at 2 X 10(4) X g but not in the ones precipitated at 1 X 10(5) X g. The results suggest that the plasma membrane of activated PMN has a mosaic distribution of NADPH oxidase.     

Direct interaction of actin with p47(phox) of neutrophil NADPH oxidase

The cell-free activation of human neutrophil NADPH oxidase is enhanced by actin, and actin filaments formed during activation are suggested to stabilize the oxidase. In an attempt to elucidate the mechanism, we examined the protein-protein interactions between actin and cytosolic components of the oxidase. Far-Western blotting using recombinant phox proteins showed that both alpha- and beta-actin interacted with p47(phox) and rac1, and weakly with rac2. A deletion mutant of p47(phox) proved that its C-terminal region was essential for the interaction. The dissociation constant (K(d)) for interaction between actin and p47(phox) was estimated to be 0.45 microM by surface plasmon resonance, and that between actin and rac1 or rac2 was 1.7 or 4.6 microM, respectively. Far-Western blotting using cytosol as a target showed an interaction between actin and endogenous p47(phox) and rac proteins. These results suggest that actin can directly interact with p47(phox) and possibly with rac in the cells.     

Suppressive effect of rebamipide, an antiulcer agent, against activation of human neutrophils exposed to formyl-methionyl-leucyl-phenylalanine

Rebamipide, an antiulcer agent, has been shown to be able to prevent gastric mucosal injury resulting in part from activation of neutrophils. The mechanism of its suppressive action, however, remains to be established. The present study aimed to determine the effect of rebamipide on activation of isolated human neutrophils and to identify the signal transduction pathway involved in its regulation. In unstimulated cells, alkaline phosphatase activity was found residing in short rod-shaped intracellular granules. Upon stimulation with a chemotactic peptide formyl-methionyl-leucyl-phenylalanine, the granules fused to form elongated tubular structures and spherical vacuoles. Rebamipide inhibited reorganization of alkaline phosphatase-containing granules along with upregulation of alkaline phosphatase activity and CD16, a marker of the granules. It also suppressed chemotaxis, an increase in intracellular calcium ion concentration, and NADPH oxidase activation in cells stimulated with formyl-methionyl-leucyl-phenylalanine. In contrast, the drug showed no inhibitory action toward upregulation of alkaline phosphatase activity and CD16, and activation of NADPH oxidase in cells stimulated with phorbol myristate acetate, an activator of protein kinase C. These findings demonstrate that rebamipide exerts a broad spectrum of suppressive actions toward biological functions of human neutrophils stimulated with formyl-methionyl-leucyl-phenylalanine, but not with phorbol myristate acetate, and suggest that the upstream point of protein kinase C is the signal transduction pathway involved in its regulation.