Kohji Egawa

Solubilization of the enzyme catalyzing CDP-diglyceride-independent incorporation of myo-inositol into phosphatidyl inositol and its comparison to CDP-diglyceride:Inositol transferase

Abstract A solubilized preparation with activity for catalyzing the incorporation of free myo-inositol into phosphatidyl inositol was obtained from a rat liver microsomal fraction. The incorporation took place both in the presence and in the absence of cytidine diphosphodiglyceride (CDP-DG). The pH optimum of the incorporation in the absence of CDP-DG was 7.4–7.5, while that of the incorporation in its presence was 8.5–8.6. The incorporation in the absence of CDP-DG was activated by Mn2+ but not by Mg2+, while that in the presence of CDP-DG was activated by either Mn2+ or Mg2+. These results indicated that the incorporation in the absence of CDP-DG and the incorporation in its presence were catalyzed by different enzymes. Before Solubilization, the CDP-DG-independent enzyme was bound to endoplasmic reticulum. The CDP-DG-dependent enzyme also was bound mainly to endoplasmic reticulum and, to a minor extent, to plasma membrane. The CDP-DG-independent enzyme was more easily solubilized by sodium cholate than the CDP-DG-dependent enzyme. There were also differences between these two enzyme activities of the solubilized preparation with respect to their sensitivity to various detergents and their dependence on exogenous lipids. The CDP-DG-independent incorporation was inhibited by CDP-DG, by some nucleotides, and by phosphatidyl serine, while the CDP-DG-dependent incorporation was not inhibited by these substances. Both activities were both inhibited by thiol-reactive compounds.

Stimulation of human butyrophilin 3 molecules results in negative regulation of cellular immunity

The BTN molecule consists of three subfamilies, BTN1, BTN2. and BTN3, and possesses interesting properties for biological regulation. Although the biological significance of BTN1 and BTN2 has been progressively clarified, the receptor function of BTN3 remains to be elucidated as a result of the absence of appropriate agonists. To clarify the participation of BTN3 in immune regulation, BTN3‐specific mAb, referred to as 34‐7 and 232‐5, were generated from BTN3 gene‐immunized mice. The 232‐5 mAb, specific to the extracellular domain of the BTN3 molecule, stained almost all populations of human PBMCs, including T, NK, NKT, and B cells. Notably, treatment with the 232‐5 mAb resulted in phosphorylation of BTN3A3 molecules, leading to attenuated proliferation and cytokine secretion by CD4+ and CD8+ T cells in a CD4+ CD25+ Treg cell‐independent manner, demonstrating the agonistic property of the 232‐5 mAb in BTN3‐mediated negative signal transduction. The magnitude of the cell surface expression of BTN3 molecules correlated inversely with lymphocyte activity, suggesting that BTN3 molecules contribute to the maintenance of the immune system. Taken together, our findings provide new insights for the role of BTN3 as an inhibitor of excessive cellular immune responses.

Phosphatidyl inositol: myo-Inositol exchange enzyme from rat liver: Partial purification and characterization

Abstract The enzyme which catalyzes CDP-diglyceride-independent incorporation of myo -inositol into phosphatidyl inositol was solubilized from rat liver microsomes by sodium cholate and was partially purified by ammonium sulfate fractionation and sucrose density gradient centrifugation. Addition of phospholipids during purification and assay procedures prevented irreversible loss of the enzyme activity to some extent. The resulting preparation contained about 3.7% of the protein and 35% of the original activity of the microsomal fraction. The activity of the enzyme preparation was strongly enhanced by addition of phosphatidyl inositol. The enzyme required Mn 2+ for activity. The K m for myo -inositol was 4 × 10 −5 m . The pH optimum was 7.4. The activity was inhibited by thiol-reactive reagents and also to some extent by inosose-2 but not by scyllitol. Phosphorus-containing acidic substances such as acidic phospholipids and nucleotides were generally inhibitory. It was found that the preparation catalyzed liberation of inositol moiety from phosphatidyl inositol in a manner dependent on the concentration of free myo -inositol and also on Mn 2 . The K m of this reaction for free myo -inositol was estimated to be 7 × 10 −5 m . This result indicates that CDP-diglyceride-independent incorporation, which has been assumed to show inositol exchange reaction, actually represents an exchange reaction between the myo -inositol moiety of phosphatidyl inositol and free myo -inositol. Phosphatidyl choline and phosphatidyl ethanolamine did not play a role as acceptor of the exchange reaction.

Suppression of cytotoxic T lymphocyte activity by γ/δ T cells in tumor-bearing mice

Spleen cells derived from tumor-bearing mice prove useful for the elucidation of the mechanism determining how tumor cells evade cytotoxic T lymphocytes (CTL) in tumor-bearing hosts. Our data indicate that inactive CTL or precursor CTL specific for tumor antigens are present among lymphocytes of tumor-bearing mice. However, their activity is inhibited by a soluble factor produced by other cells present in the same source. Inhibition of the cytolytic reaction was also detected in the culture supernatant of spleen cells obtained from normal mice, precultured in the presence of tumor cell culture supernatant and interleukin-2 (IL-2). Cell-depletion and cell-purification studies let us conclude that cells that produced the CTL-inhibitory factor (CTL-IF) were γ/δ T cells. The γ/δ T cells that were activated in vivo in tumor bearers were able to produce CTL-IF after isolation and in vitro culture. Maximum activation of γ/δ T cells was achieved by antigenic stimulation and by suppression of cells that interfered with the activation of γ/δ T cells. CTL-IF, which was assayed by use of CTL clones, did not show antigen specificity. Inhibition depended on a relatively heat- and acidstable, but alkali-labile molecule with a molecular mass of less than 10 kDa. The latter characteristics imply that CTL-IF does not resemble any of the known lymphokines produced by γ/δ T cells. These observations emphasize the crucial role of the γ/δ T cells in the escape of tumor cells from the attack of tumorspecific CTL.

Revised sequence of the nusA gene of Escherichia coli and identification of nusA11 (ts) and nusA1 mutations which cause changes in a hydrophobic amino acid cluster.

SummaryThe mutant nusA DNAs (nusA11 and nusA1) were sequenced. Single base substitutions caused by these mutations were found in the coding region of nusA. The nusA11 mutation, which is conditionally lethal, substituted Thr for the 181st Ala. Also, nusA1, which restricts λ growth, substituted Ala for the 183rd Ser. These two positions were located in the same hydrophobic amino acid cluster. This cluster seemed to be an essential region in the functional domain of NusA. In the course of these experiments, several mistakes in the published nusA nucleotide sequence were found. These errors are revised in this article. The molecular weight of NusA is accordingly revised to 54,430.

Effect of retinoic acid and 12-O-tetradecanoyl phorbol-13-acetate on the binding of epidermal growth factor to its cellular receptors

Binding of 125I-labelled epidermal growth factor (EGF) to C3H/2K cells and the effect of a tumor promotor, 12-O-tetradecanoyl phorbol-13-acetate (TPA) and of a tumor promotor antagonist, retinoic acid, on the binding was studied. Scatchard plot analysis of the binding showed the presence of two types of binding sites with different affinity to EGF. Treatment of the cells with retinoic acid for 1 h resulted in elevation of the affinity of both sites without changing their number per cell. Prolonged exposure to retinoic acid abrogated this elevation of the affinity and caused cycloheximide-sensitive increase of the number of the binding sites of both types. TPA inhibited binding of EGF to the cells by abolishing the binding to the high affinity sites, whereas retinoic acid, in the presence of TPA, enhanced it by increasing the number of the low affinity sites.

An N-acetylneuraminic acid-specific lectin from the body surface mucus of African giant snail

Abstract 1. 1. A novel lectin (Achatinin) was isolated from the body-surface mucus of African giant snail, Achatina fulica Ferussac. 2. 2. The lectin recognizes N-acetylneuraminic acid preferentially and also other N-acetyl aminosugars to some extent, while it does not bind to N-glycolylneuraminic acid. 3. 3. The molecular weight of the lectin estimated by SDS polyacrylamide gel electrophoresis was 78,000. 4. 4. The lectin requires Ca2+ for its haemagglutinating activity. 5. 5. The isolated lectin was free of antibacterial activity which has been detected in the body-surface mucus.

Detection of allogeneic Qa/Tl and Ly specificities on murine tumor cells with IgD in tumor-regressor serum

SummarySerum from C3H/He mice, which show regression of MM2 tumor cells after transplantation and removal (regressor serum, RS) contains non-gammaglobulin components that cross-react with various tumor cells of mice [22, 23]. In addition to tumor cells, various allogeneic lymphocytes are also susceptible to an RS-dependent lymphocyte-mediated cytotoxic reaction. To identify tumor cell surface antigens that cause the cross-reactive host response, the serum components were analyzed by absorption of RS with allogeneic lymphocytes. RS components were found to recognize allogeneic lymphocyte antigens including Qa-2 and Ly6.2. Specificity for the Qa-2 antigen was further tested using Qa-2-congenic mice. The expression of Qa-2 antigen was detected on the surfaces of MM2 and other tumor cells derived from H-2k mice (seven among nine cell lines tested) by a membrane immunofluorescence method using a Qa-2-specific mAb. Physical characteristics of the Qa-2-specific component in RS were determined and found to differ from those of regular IgGs but to be similar to those of IgDs. Using an enzyme-linked immunosorbent assay with an IgD-specific mAb and Qa-2-lacZ fusion protein, the existence of IgD in RS with specificity for Qa-2 was confirmed. These results suggest that the RS component with Qa-2 specificity is an IgD, the specificity and physiological role of which are unknown.

Recognition of the Qa-2k tumor antigen by T cell receptor γ/δ of an immunopotentiator-induced tumoricidal T cell of mice

Tumor-specific expression of Qa-2k antigen coded by the Q5k gene on various mouse tumor cells and immunological response of the host mice to the antigen have been demonstrated [Seo et al. (1992) J Exp Med 175: 547; Tanino et al. (1992) Cancer Immunol Immunother 35: 230]. The possibility was examined that Qa-2 antigen is one of the recognition target molecules of immunopotentiator-induced, H-2-nonrestricted tumoricidal lymphocytes of Qa-2− mice. Lymphocytes stimulated in vivo withP. acnes or culture-induced anomalous killers of B6.K1 mice did not exhibit significant in vitro cytotoxicity against B6.K1 lymphoblasts but lysed their Qa-2,3-congenic counterpart B6 lymphoblasts. To demonstrate the Qa-2 specificity of such cytotoxic cells more precisely, an L cell transformant clone (LQ7b/Kb), which expressed the α1 and α2 domains of the Qa-2 antigen (Q7b gene product), was generated by transfecting a cloned plasmid DNA containing a hybrid gene constructed from the 5′ half of the Q7b gene and the 3′ half of the H-2Kb gene (pQ7b/Kb). Using LQ7b/Kb cells as the target cells and the nylon-wool-nonadherent fraction of lymphocytes fromP. acnes-stimulated (C3H/He × B6.K1)F1 mice (H-2k, Qa-2−) as the effector cells of the in vitro cytotoxicity reaction, the presence of cytotoxic cells that recognize the α1/α2 region of the Q7b gene product was demonstrated. The cytotoxic activity was dependent on T cells bearing T cell receptors of the γ/δ type (TCRγ/δ). The (C3H/He × B6.K1)F1 effector cells, as well as the B6.K1 effector cells also lysed BW5147 lymphoma cells (Qa-2k+) derived from AKR mice (Qa-2−, H-2k). By target-competition experiments it was shown that some of the effector cells lytic to BW5147 were identical to those that lysed LQ7b/Kb. Therefore some of the tumoricidal cells induced by the immunopotentiator interact with the target tumor cells through recognition of the α1/α2 region of the Qa-2k tumor antigen by TCRγ/δ.

Conjugation to octa‐arginine via disulfide bonds confers solubility to denatured proteins in physiological solution and enables efficient cell internalization

Some protein transduction methods have already been developed for regenerative medicine application. These methods can be applied to soluble proteins but not to insoluble proteins, such as those that originate from inclusion bodies, for example, Escherichia coli. We have developed a method that allows the in vitro solubilization of denatured proteins without refolding and their efficient cellular internalization through conjugation to the peptide, octa‐arginine (R8), via disulfide bonds with cysteine residues. Ovalbumin (OVA), denatured in urea solution containing dithiothreitol, was used as a model protein. The R8 peptide was conjugated with OVA in urea solution. Denatured OVA was recovered in the insoluble fraction after dialysis against phosphate‐buffered saline. However, almost all the R8‐conjugated OVA was recovered in the soluble fraction and used for translocation experiments in HeLa, Chinese hamster ovary‐K1, Cos‐7, and matured dendritic cells, where efficient internalization of the protein conjugate was observed. Furthermore, we formulated R8‐conjugated β‐galactosidase and R8‐conjugated luciferase using a similar procedure, and investigated how the conjugated proteins are processed after cell internalization. We also observed that only a small fraction of these proteins refolded and almost all underwent intracellular degradation. These results suggest that this method is suitable for the transduction of antigen‐presenting cells and will benefit research and innovation in vaccine design and discovery.