Structural comparison of the cleavage-activation site of the fusion glycoprotein between virulent and avirulent strains of Newcastle disease virus

The nucleotide sequence of the mRNA encoding the fusion (F0) protein of a virulent strain of Newcastle disease virus was determined. A single open reading frame in the sequence encodes a protein of 553 amino acids with a calculated molecular weight of 59058. The amino acid sequence predicted several structural features involving the fusion-inducing hydrophobic stretch (residues 117-142) and the cleavage-activation site (residues 112-116) to generate the disulfide-linked F1 and F2 subunits. The cleavage-activation site as well as a part of the fusion-inducing sequence were compared among a series of virulent and avirulent strains by the chain-termination method using a synthetic oligonucleotide primer. It was found that without exception, the cleavage-activation site of virulent strains consisted of two dibasic residues with an intervening glutamine, Arg-Arg-Gln-Arg-Arg, whereas the corresponding region of avirulent strains was made of a sequence with single basic residues scattered among uncharged residues, Gly-LysArg-Gln-GlySer-Arg. On the basis of these observations and the previous results showing a strict correlation between the pathogenicity and the cleavability of the fusion protein of NDV (Y. Nagai, H-D. Klenk, and R. Rott, Virology, 72, 494-508, 1976), we propose the importance of the dibasic residues for efficient proteolytic activation of the fusion protein and for the pantropic property of NDV. Some strains were found to have Leu-Ile-Gly as the N-terminus of F1, whereas others contained Phe-Ile-Gly, indicating that Phe-X-Gly is not always conserved at F1 N-terminus of paramyxovirus.     

Activities of Aeromonas hydrophila hemolysins and their interaction with erythrocyte membranes.

The activity of hemolysin produced by Aeromonas hydrophila CA-11, isolated from an environmental source, was more sensitive to temperature than that of hemolysin produced by strain AH-1, isolated from a diarrheal case. CA-11 hemolysin failed to elicit hemolysis below 10 degrees C. Immunoblotting analyses showed that both hemolysins formed into oligomers in rabbit erythrocyte membrane even when no hemolysis occurred. suggesting that the binding and the subsequent oligomerization are temperature independent. Sodium salicylate inhibited lysis of rabbit erythrocytes by both hemolysins, but selected monosaccharides and oligosaccharides did not. Thin-layer immunostaining indicated that both hemolysins bound to phosphoglycerides with net negative charge but weakly to the ones with no net negative charge. Neither sphingomyelin nor lysophosphoglyceride reacted with the hemolysins, whereas the hemolysins bound to free fatty acids. These results suggest that the binding of either hemolysin to the membrane component, probably phospholipid, requires both negative charge of the polar head group and suitable hydrophobicity of the nonpolar tails.     

Some in vivo electrophysiological properties of locus coeruleus neurones in fetal rats

Summary The electrical activity of locus coeruleus (LC) neurones was recorded extracellularly in fetal rats still in contact with their dams by an intact umbilical cord. Pregnant rats, at gestation days 18 to 22, were anesthetized with urethane. The head of a fetal rat was exposed from the uterus and fixed to a conventional stereotaxic apparatus by means of a simple device. The location of the LC in the fetal rats was determined by the appearance of field responses evoked by stimulation of the dorsal noradrenergic bundle. Antidromic spikes of single LC neurones were evoked superimposed upon the field responses. The mean conduction velocity of LC axons was calculated to be 0.25 m/s. Some fetal LC units were activated antidromically by stimulation of the frontal cortex (FC) with latencies ranging from 21 to 67 ms, values nearly the same as those obtained in neonates and adults. Although the majority of fetal LC neurones recorded were not spontaneously active, a small number of them revealed epochs of sporadic firing, which appeared to occur synchronously in many or all of the LC neurones. Sensory stimuli (e.g., air puffs to the skin) were effective in activating LC neurones. These results indicate that LC neurones have already developed projections to the FC, and are functionally active in prenatal periods.     

Approaches to immunotherapies for Japanese cedar pollinosis

Japanese cedar (Cryptomeria japonica; CJ) pollinosis is a typical type I allergy induced by CJ pollen and one of the most common allergic diseases in Japan. New immunotherapies have been developed for treatment of CJ pollinosis. We focus here on new immunotherapies for CJ pollinosis including sublingual immunotherapy with crude extract of CJ antigen, oral immunotherapy with transgenic rice expressing CJ allergens, a peptide vaccine using T cell epitopes of CJ allergens, DNA vaccines encoding either the CJ allergen gene or T cell epitope gene, and adjuvant-conjugated vaccines using CJ allergen conjugated with adjuvants such as CpG oligodeoxynucleotide or pullulan.     

Targeting Treg cells in cancer immunotherapy

Foxp3‐expressing regulatory T (Treg) cells, which are indispensable for preventing autoimmunity, also suppress effective tumor immunity. Treg cells abundantly infiltrate into tumor tissues, which is often associated with poor prognosis in cancer patients. Removal of Treg cells enhances anti‐tumor immune responses but may also elicit autoimmunity. A key issue in devising Treg‐targeting cancer immunotherapy is, therefore, how to specifically deplete Treg cells infiltrating into tumor tissues without affecting tumor‐reactive effector T cells, while suppressing autoimmunity. This can be achieved by differentially controlling Treg and effector T cells by various ways. In this review, we discuss how tumor‐infiltrating Foxp3⁺ Treg cells hamper effective anti‐tumor immune responses especially in tumor tissues and how they can be specifically targeted for augmenting tumor immunity but not autoimmunity.     

In silico analysis‐based identification of the target residue of integrin α6 for metastasis inhibition of basal‐like breast cancer

Metastasis causes death in breast cancer patients. To inhibit breast cancer metastasis, we focused on integrin α6, a membrane protein that contributes to cell migration and metastasis. According to in silico analysis, we identified Asp‐358 as an integrin α6‐specific vertebrate‐conserved residue and consequently as a potential therapeutic target. Because Asp‐358 is located on the surface of the β propeller domain that interacts with other molecules for integrin α6 function, we hypothesized that a peptide with the sequence around Asp‐358 competitively inhibits integrin α6 complex formation. We treated basal‐like breast cancer cells with the peptide and observed reductions in cell migration and metastasis. The result of the immunoprecipitation assay showed that the peptide inhibited integrin α6 complex formation. Our immunofluorescence for phosphorylated paxillin, a marker of integrin‐regulated focal adhesion, showed that the peptide reduced the number of focal adhesions. These results indicate that the peptide inhibits integrin α6 function. This study identified the functional residue of integrin α6 and designed the inhibitory peptide. For breast cancer patients, metastasis inhibition therapy may be developed in the future based on this study.     

Adhesion polypeptides are useful for the prevention of peritoneal dissemination of gastric cancer

We examined the effect of adhesion polypeptides on the adhesion and invasiveness of gastric cancer cell lines. We previously reported the establishment of an extensively peritoneal-seeding cell line, OCUM-2MD3, from a poorly seeding human scirrhous gastric carcinoma cell line, OCUM-2M. Both 21 and 31 integrin expression was markedly increased on OCUM-2MD3 cells compared with OCUM-2M cells, and the ability of OCUM-2MD3 cells to bind to the extracellular matrix (ECM) was also significantly higher than that of OCUM-2M cells. The adhesion polypeptides, YIGSR and RGD, and two RGD derivatives significantly inhibited the adhesion of OCUM-2MD3 cells to the submesothelial ECM, while not inhibiting the adhesiveness of OCUM-2M cells and two well differentiated human gastric cell lines, MKN-28 and MKN-74. The YIGSR and RGD peptides also significantly inhibited the invasiveness of OCUM-2MD3 cells. The survival of nude mice with peritoneal dissemination given YIGSR sequenc e intraperitoneally was obviously longer than that of untreated mice. The survival of mice treated with RGD was also improved, and this effect was increased using the RGD derivatives, poly(CEMA-RGDS) and CM-chitin RGDS. These polypeptides appear to block the binding of integrins, which are expressed on OCUM-2MD3 cells, to the submesothelial ECM, and consequently inhibit peritoneal implantation. The peritoneal injection of adhe-sion polypeptides may be a new therapy against the dissemination of scirrhous gastric cancer, and may be useful for the prevention of dissemination in high-risk patients. © Rapid Science Ltd.     

Immunologic self-tolerance maintained by CD25+CD4+ naturally anergic and suppressive T cells: induction of autoimmune disease by breaking their anergic/suppressive state

Elimination of CD25+ T cells, which constitute 5-10% of peripheral CD4+ T cells in normal naive mice, leads to spontaneous development of various autoimmune diseases. These immunoregulatory CD25+CD4+ T cells are naturally unresponsive (anergic) in vitro to TCR stimulation, and, upon stimulation, suppress proliferation of CD25-CD4+ T cells and CD8+ T cells. The antigen concentration required for stimulating CD25+CD4+ T cells to exert suppression is much lower than that required for stimulating CD25-CD4+ T cells to proliferate. The suppression, which results in reduced IL-2 production by CD25-CD4+ T cells, is dependent on cellular interactions on antigen-presenting cells (and not mediated by far-reaching or long-lasting humoral factors or apoptosis-inducing signals) and antigen non-specific in its effector phase. Addition of high doses of IL-2 or anti-CD28 antibody to the in vitro T cell stimulation culture not only breaks the anergic state of CD25+CD4+ T cells, but also abrogates their suppressive activity simultaneously. Importantly, the anergic/suppressive state of CD25+CD4+ T cells appeared to be their basal default condition, since removal of IL-2 or anti-CD28 antibody from the culture milieu allows them to revert to the original anergic/suppressive state. Furthermore, transfer of such anergy/suppression-broken T cells from normal mice produces various autoimmune diseases in syngeneic athymic nude mice. These results taken together indicate that one aspect of immunologic self-tolerance is maintained by this unique CD25+CD4+ naturally anergic/suppressive T cell population and its functional abnormality directly leads to the development of autoimmune disease.      

Bradykinin Stimulates Type II Alveolar Cells to Release Neutrophil and Monocyte Chemotactic Activity and Inflammatory Cytokines

In the present study, we evaluated the potential of bradykinin (BK) to induce the release of neutrophil and monocyte chemotactic activity (NCA and MCA) and cytokines from an alveolar type II epithelial cell line, A549 cells. BK stimulated A549 cells to release NCA and MCA in a dose- and time-dependent manner (P < 0.001). Checkerboard analysis revealed that both NCA and MCA involved chemotactic and chemokinetic activity. Molecular sieve column chromatography showed three molecular weight masses (near 19 kd, 8 kd, and 400 d) for NCA and several molecular weight peaks (near 66 kd, 25 kd, 19 kd, 16 kd, and 400 d) for MCA. The release of NCA and MCA was inhibited by cycloheximide and lipoxygenase inhibitors (P < 0.01). The NCA and MCA were inhibited by leukotriene B4 (LTB4) receptor antagonist (P < 0.01), and the concentration of LTB4 was high enough for NCA and MCA. Antibodies to interleukin (IL)-8 and granulocyte colony-stimulating factor (G-CSF) attenuated NCA (P < 0.01), and antibodies to monocyte chemotactic protein-1 (MCP-1), G-CSF, and transforming growth factor (TGF)-β attenuated MCA (P < 0.01). The levels of IL-8, G-CSF, MCP-1, and TGF-β increased time dependently (P < 0.01). BK also stimulated the release of ILeukin-6 from A549 cells (P < 0.001). The receptors responsible for the release of NCA, MCA, and individual chemokines involved both BKB1 and BKB2 receptors. These data suggest that BK may stimulate alveolar type II pneumocytes to release inflammatory cytokines, which then may modulate the lung inflammation.