[Neutrophil adherence-inhibiting factor, AIF, is released from lysosomes of activated platelets].
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Biological Factors↗
Biomedical subjects
K Iwabuchi
Publications and source records attributed to K Iwabuchi.
[Neuropathology of familial spastic paraplegia].
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Axons↗
Separation of "glycosphingolipid signaling domain" from caveolin-containing membrane fraction in mouse melanoma B16 cells and its role in cell adhesion coupled with signaling.
Two membrane subfractions, one enriched in GM3 ganglioside and the other containing caveolin, were separated from low density detergent-insoluble membrane fraction prepared by sucrose density gradient centrifugation of postnuclear fraction of mouse melanoma B16 cells. The GM3-enriched subfraction, separated by anti-GM3 monoclonal antibody DH2, contained sphingomyelin, cholesterol, c-Src, and Rho A but not caveolin. In contrast, the caveolin-containing subfraction, separated by anti-caveolin antibody, contained neither GM3, c-Src, nor Rho A but did contain glucosylceramide, Ras, a very small quantity of sphingomyelin, and a very large quantity of cholesterol. The GM3/c-Src-enriched membrane subfraction was characterized by (i) maintenance of GM3-dependent adhesion and (ii) susceptibility to being activated for signal transduction through GM3. 32P-Phosphorylation of c-Src (Mr 60,000) together with two other components (Mr 45,000 and 29,000) was enhanced in the fraction bound to dishes coated with asialo-GM2 (Gg3) or with anti-GM3 monoclonal antibody DH2, detected by incubation with [gamma-32P]ATP at 37 degreesC for 5 min. GM3-dependent adhesion of B16 cells to Gg3-coated dishes and associated signaling were not reduced or abolished in the presence of either filipin or nystatin, which are cholesterol-binding reagents known to abolish caveolae structure and function. B16 melanoma cells incubated with filipin (0.16-0.3 micrograms/ml) or with nystatin (25 micrograms/ml) for 30 min showed depletion of cholesterol in detergent-insoluble membrane fraction but were still capable of binding to Gg3-coated plate and capable of the associated signaling. Thus, the GM3-enriched subfraction, involved in cell adhesion and capable of sending signals through GM3, represents a membrane domain distinguishable from caveolin-containing subfraction or caveolae. This microdomain is hereby termed the "glycosphingolipid signaling domain" or "glycosignaling domain".
Animals↗
Chemotactic activity of soluble Fas ligand against phagocytes.
A recombinant soluble form of human Fas ligand (sFasL) was tested for its chemotactic activity against human and mouse polymorphonuclear neutrophils (PMN) by the Boyden chamber method. sFasL exhibited a potent chemotactic activity against both human and mouse PMN and HL-60 cells when differentiated into neutrophils or monocytes. A neutralizing anti-FasL mAb abolished the chemotactic activity, while control mAb did not. Ligation of Fas by either IgM- or IgG-type anti-Fas mAb also induced PMN migration. PMN derived from lpr mice that express few Fas molecules did not respond to sFasL. In contrast, those derived from lpr(cg) mice that express Fas molecules with a mutated death domain normally responded to sFasL chemotaxis. These results directly indicated a chemotactic activity of sFasL against PMN and suggest a novel signaling function of Fas, which appears to be independent of the death domain-mediated apoptosis.
Animals↗
Stimulation of p53-mediated transcriptional activation by the p53-binding proteins, 53BP1 and 53BP2.
p53 is a tumor suppressor protein that controls cell proliferation by regulating the expression of growth control genes. In a previous study, we identified two proteins, 53BP1 and 53BP2, that are able to bind to wild type but not to mutant p53 via the DNA-binding domain of p53. We isolated cDNAs expressing a full-length human 53BP1 clone, which predicts a protein of 1972 residues that can be detected in the H358 human lung carcinoma cell line. The 53BP1 and 53BP2 genes were mapped to chromosomes 15q15-21 and 1q41-42, respectively. Immunofluorescence studies showed three types of staining patterns for 53BP1 as follows: both cytoplasmic and nuclear, homogeneous nuclear, and a nuclear dot pattern. In contrast, 53BP2 localized exclusively to the cytoplasm, and this pattern did not change upon coexpression of wild type p53. Although our previous study revealed that p53 is not able to bind simultaneously to either 53BP1 or 53BP2 and to DNA carrying a consensus binding site, both 53BP1 and 53BP2 enhanced p53-mediated transcriptional activation and induced the expression of a p53-dependent protein, suggesting that these proteins might function in signal transduction pathways to promote p53 activity.
Amino Acid Sequence↗
Phenotype variation correlates with CAG repeat length in SCA2--a study of 28 Japanese patients.
Spinocerebellar ataxia-2 (SCA2) is an autosomal dominant ataxia caused by an abnormal CAG repeat expansion in a novel gene on chromosome 12q24.1. The size of the mutant allele is unstable during transmission, and correlates inversely with age at onset. We studied eight Japanese SCA2 families, including 28 patients, to assess the effect of repeat length on the phenotype features of SCA2. Frequencies of slow eye movements (SEM), reflex activity, dementia, choreiform movements, and axial tremor correlated significantly with CAG repeat size. Parkinsonism was seen in a man homozygote for SCA2 mutation. The clinical variety of SCA2 is apparently influenced by the size of the mutant allele, as is the case in other CAG repeat disorders.
Adolescent↗
Intrathymic selection of NK1.1(+)alpha/beta T cell antigen receptor (TCR)+ cells in transgenic mice bearing TCR specific for chicken ovalbumin and restricted to I-Ad.
Generation and negative selection of NK1.1(+)alpha/beta T cell receptor (TCR)+ thymocytes were analyzed using TCR-transgenic (B10. D2 x DO10)F1 and (C57BL/6 x DO10)F1 mice and Rag-1(-/-)/DO10 mice, which had been established by breeding and backcrossing between Rag-1(-/-) and DO10 mice. Almost all T cells from these mice were shown to bear Valpha13/Vbeta8.2 that is specific for chicken ovalbumin (cOVA) and restricted to I-Ad. A normal proportion of the NK1.1(+) Valpha13/Vbeta8.2(+) thymocytes was generated in these mice. However, the actual cell number of both NK1.1(+) and NK1.1(-) thymocytes in I-Ad/d mice (positive selecting background) was larger than that in I-Ab/d mice (negative selecting background). Markedly low but significant proportions of NK1.1(+) Valpha13/Vbeta8.2(+) cells were detected in the spleens from I-Ad/d and I-Ab/d mice. It was shown that the splenic NK1.1(+) T cells of the I-Ab/d mice were anergized against stimulation through TCR. When (B10.D2 x DO10)F1 and (C57BL/6 x DO10)F1 mice were given cOVA, extensive or intermediate elimination of NK1.1(+)alpha/betaTCR+ thymocytes was induced in I-Ad/d or I-Ab/d mice, respectively. However, the clonal elimination was not as complete as that seen in the major NK1.1(-) thymocyte population. The present findings indicate that normal generation of NK1.1(+)alpha/betaTCR+ thymocytes occurs in the absence of Valpha14-Jalpha281 and that substantial negative selection operates on the NK1.1(+)alpha/betaTCR+ cells.
Animals↗
GM3-enriched microdomain involved in cell adhesion and signal transduction through carbohydrate-carbohydrate interaction in mouse melanoma B16 cells.
Mouse melanoma B16 cells are characterized by the predominant presence of ganglioside GM3 and adhere to lactosylceramide- or Gg3-coated plates through interaction of GM3 with lactosylceramide or Gg3, whereby not only adhesion but also spreading and enhancement of cell motility occur (Kojima, N., Hakomori, S. (1991) J. Biol. Chem. 266, 17552-17558). We now report that the adhesion process is based essentially on a glycosphingolipid-enriched microdomain (GEM) at the B16 cell surface, since >90% of GM3 present in the original cells is found in GEM, and GEM is also enriched in several signal transducer molecules, e.g. c-Src, Ras, Rho, and focal adhesion kinase (FAK). GEM was isolated as a low density membranous fraction by homogenization of B16 cells in lysis buffer under two different conditions (i.e. buffer containing 1% Triton X-100, or hypertonic sodium carbonate without detergent), followed by sucrose density gradient centrifugation. A close association of GM3 with c-Src, Rho, and FAK was indicated by co-immunoprecipitation of GM3 present in GEM by anti-GM3 monoclonal antibody DH2, followed by Western blotting with antibodies directed to these transducer molecules. The following data indicate that GEM is a structural and functional unit for initiation of GM3-dependent cell adhesion coupled with signal transduction. 1) Tyrosine phosphorylation in FAK was greatly enhanced in B16 cells adhered to Gg3-coated plates but was minimal in cells adhered to GM3-coated, GlcCer-coated, or noncoated plates. 2) GTP loading on Ras and Rho increased significantly when cells were adhered to Gg3-coated plates, compared with GM3-coated, GlcCer-coated, or noncoated plates. Since Ras and Rho are closely associated with GM3 in GEM, cell adhesion/stimulation through GM3 in GEM may induce activation of Ras and Rho through enhanced GTP binding.
Animals↗
Modulation of neutrophil adherence to endothelial cells by platelet-derived adherence-inhibiting factor through interactions with selectin molecules.
Platelet-derived adherence-inhibiting factor (AIF) has been demonstrated to regulate the neutrophil binding to type IV collagen. In this study, we have examined the effect of AIF on neutrophil adherence to confluently cultured human umbilical vein endothelial cells (EC). AIF inhibited neutrophil adherence to thrombin- or tumor necrosis factor alpha (TNF-alpha)-stimulated EC by 75 or 43%, respectively, but hardly affected neutrophil adherence to resting EC. It is interesting to note that the inhibitory activity of AIF was reduced by N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulation of neutrophils. Pretreatment of neutrophils or EC with AIF inhibited neutrophil adherence to thrombin- or TNF-alpha-stimulated EC, suggesting that neutrophils and EC have AIF-binding proteins. Using AIF-Sepharose affinity chromatography, AIF-binding proteins containing L-selectin were isolated from 125I-labeled resting neutrophils. However, L-selectin was markedly decreased in the AIF-binding fraction from fMLP-stimulated neutrophils. With the use of AIF-affinity chromatography, P- and E-selectins were obtained in the AIF-binding fractions from resting, thrombin-, and TNF-alpha-stimulated EC. It is important to note that P- and E-selectin were greatly increased in the AIF-binding fractions from thrombin- and TNF-alpha-stimulated EC, respectively. Furthermore, AIF was able to bind to L-selectin-IgG chimeric protein and inhibit the binding of chimeric protein to thrombin or TNF-alpha-stimulated EC. In addition, AIF inhibited the binding of anti-P- or anti-E-selectin monoclonal antibody to the lysates of thrombin- or TNF-alpha-stimulated EC. Together these observations indicate that AIF could recognize L-, P-, and E-selectins, and modulate neutrophil adherence to EC through interactions with selectin molecules.
Blotting, Western↗
Heat shock protein expression in hearts hypertrophied by genetic and nongenetic hypertension.
Genetically hypertensive animals are characterized by greater thermosensitivity and overexpression of heat shock proteins (HSP) upon thermal stimulation. We examined HSP72 expression under conditions of brief coronary occlusion or thermal stimulation, and the effects of the severity of these stimuli and of myocardial hypertrophy on the expression in hearts of spontaneously hypertensive rat (SHR) and Wistar Kyoto rat (WKY) groups, A snare was created around the left coronary artery in the SHR (n = 16) and WKY (n = 19) groups. In 7 WKY rats, the ascending aorta was banded and a snare was created simultaneously (WKY-AoB). By tying the snare, 4 weeks later, we applied 5- or 10-min coronary occlusion without opening the chest. For thermal stimulation, the SHR (n = 13) and WKY (n = 11) rats were placed in a 42 degrees C chamber for 15 or 40 min. The mRNA or protein level was estimated 1 or 24h after stimulation. In the SHR vs WKY groups, the mRNA and protein levels were higher after 5-min occlusion or 15-min thermal stimulation. After 10-min occlusion or 40-min thermal stimulation the difference was no longer observed. The overexpression was not observed in the WKY-AoB group despite the presence of hypertrophy similar to that seen in the SHR group (3.11+/-0.11 vs 3.20+/-0.06 mg/g in left ventricular weight/body weight). The HSP72 was overexpressed in hearts of genetically hypertensive animals after brief ischemia. Differential expression between the two groups was observed after mild stimuli, but not after more severe stimuli. Cardiac hypertrophy was not a major factor for determining the overexpression of HSP72.
Animals↗
Evaluation of neutrophil functions after experimental abdominal surgical trauma.
OBJECTIVE: In order to determine the effect of surgical trauma on neutrophil functions, we set up an experimental abdominal surgical model using rats and analyzed neutrophil functions. In addition, we measured tumor necrosis factor-alpha (TNF-alpha), cytokine-induced neutrophil chemoattractant/growth-regulated oncogene (CINC/GRO) and nitric oxide (NO) production. MATERIALS AND METHODS: Male Sprague-Dawley rats, 8 weeks old and weighing 250-270 g, underwent laparotomy (4 rats for each experiment). After the operation, neutrophil chemotaxis was assayed using a modified Boyden chamber, and phagocytosis, active oxygen production and adhesion molecule expression were analyzed by flow cytometry. TNF-alpha and CINC/GRO levels were quantified by an immuno-dot-blot assay, and NO levels were measured by the Griess method. At the operation, NO inhibitor, N(G)-monomethyl-L-arginine acetate (L-NMMA, 40 mg) was intraperitoneally administered, and the effect of L-NMMA was studied. RESULTS: After the surgical trauma (24-48 h), blood neutrophil counts significantly increased (p < 0.001), and neutrophil chemotaxis, phagocytosis and active oxygen production were markedly enhanced (p < 0.01). Moreover, up-regulation of Mac-1 and down-regulation of L-selectin on neutrophils were observed (p < 0.05). The levels of TNF-alpha, CINC/GRO and NO increased remarkably in both blood and ascites at 8-48 h after the surgical trauma (p < 0.01): TNF-alpha increased from 194 +/- 9 (the mean +/- SD, n = 4) and 183 +/- 12 pg/ml (preoperation) to 797 +/- 28 and 1045 +/- 137 pg/ml at 24 h in blood and ascites, respectively; CINC/GRO increased from 0.1 +/- 0 and 0.1 +/- 0 ng/ml (pre-operation) to 66.4 +/- 4.5 and 60.3 +/- 17.9 ng/ml at 8 h in blood and ascites, respectively; NO increased from 2.4 +/- 1.0 and 4.2 +/- 1.1 microM (pre-operation) to 11.9 +/- 0.7 and 36.9 +/- 2.1 microM in blood and ascites at 24 h and 48 h in blood and ascites, respectively. Interestingly, L-NMMA treatment significantly reduced the increased levels of TNF-alpha and CINC/GRO and altered the enhanced neutrophil functions (p < 0.05). CONCLUSION: These observations indicate that abdominal surgical trauma induces the production of NO, TNF-alpha and CINC/GRO, and enhances neutrophil functions such as chemotaxis, phagocytosis and active oxygen production. Furthermore, L-NMMA likely modulates the neutrophil functions and the production of TNF-alpha and CINC/GRO after the surgical trauma.
Abdomen↗
Development of peptide vaccines inducing production of neutralizing antibodies against HIV-1 viruses in HLA-DQ6 mice.
Peptide vaccines against HIV-1 were prepared according to the cassette theory that we had proposed previously. An amino acid sequence of B subtype consensus of the HIV-1 V3 region was introduced into the MHC binding component with a supermotif for various MHC class II. The peptide vaccines induced T-cell responses in the DQ6 mice in which only DQ6 molecules were expressed as MHC class II. By contrast, an original V3 peptide including the consensus sequence was non-immunogenic in the DQ6 mice. Antibodies obtained from the DQ6 mice immunized with the peptide vaccines neutralized laboratory B subtype strains of HIV-1 in vitro. It may be anticipated that these peptide vaccines protect infection of HIV-1 in DQ6 positive individuals.
AIDS Vaccines↗
Identification of a peptide inducing experimental autoimmune uveoretinitis (EAU) in H-2Ak-carrying mice.
When certain strains of mice bearing H-2Ak are immunized with the interphotoreceptor retinoid-binding protein (IRBP), EAU is induced. Thus far uveitogenic determinant(s) has not been determined in the H-2Ak mouse system. In addition it is hard to prepare purified IRBP. In the present study, to circumvent these problems we attempted to identify uveitogenic peptides derived from bovine IRBP in H-2Ak haplotype mice. Six peptides which had been selected according to the H-2Ak binding motif (Dxxxxxxxx[A, R, T]) were synthesized. We report here that all the peptides are immunogenic but only one peptide, K2, which consisted of IRBP201-216 residues, induces EAU in various mice carrying H-2Ak. Amino acid substitution of K2 revealed that the core region interacted with both H-2Ak and T cell antigen receptor (TCR). The amino acid sequence of the core region derived from bovine IRBP was identical to the corresponding region of mouse IRBP. In addition, K2 appeared to be a natural peptide antigen processed from bovine IRBP. Altogether, we concluded that K2 is one of the natural autoantigens involved in induction of EAU in H-2Ak mice.
Amino Acid Sequence↗
An increase in basal glucocorticoid concentration with age induces suppressor macrophages with high-density Fc gamma RII/III.
Ageing is usually accompanied by a decline in immune and neuroendocrine functions. To elucidate the mechanisms underlying age-related immunosuppression, the functions and surface phenotypes of peritoneal cells in the monocyte/macrophage lineage from old mice were investigated. The role of glucocorticoids (GC) in the immunomodulation was also examined. Proliferative responses of spleen cells from control mice stimulated with concanavalin A (Con A) were significantly suppressed by adding peritoneal exudate cells from old mice. Flow cytometry analysis revealed that the proportion of MAC-1+ cells with a high density of type II or type III receptor for the Fc portion of IgG (Fc gamma RII/IIIbright cells) was increased markedly in the periotoneal exudate cells from old mice. The prominent suppressor activity for Con A responses of control spleen cells was found in the Fc gamma RII/IIIbright cells, whereas MAC-1+ cells with a low density of Fc gamma RII/III (Fc gamma RII/IIIdull cells) did not suppress the Con A responses. On the other hand, both the basal corticosterone concentrations in serum and the mRNA expression for GC receptor in peritoneal exudate cells increased significantly in old mice. Furthermore, the proportion of Fc gamma RII/IIIbright cells in peritoneal exudate cells from old mice was normalized on administration of the GC antagonist RU 38,486 (mifepristone). These results suggest that the increase in basal corticosterone concentrations in old mice induces the generation of Fc gamma RII/IIIbright suppressor cells, possibly leading to the immune-suppressive state.
Aging↗
Immunohistochemical analysis of a case of gastritis cystica profunda associated with carcinoma development.
We report a rare case of gastritis cystica profunda (GCP) accompanied by carcinoma that developed in a 51-year-old Japanese man without antecedent gastric surgery. The polypoid tumor was located in the upper body of the resected stomach. Histologically, it was characterized by herniation of surface epithelium and cystic glands in the submucosa, muscularis propria, and subserosa. Marked chronic atrophic gastritis was found throughout the stomach, and dysplastic epithelia and a few adenocarcinoma cells were found in the deeper parts of the GCP. The Ki-67, p53, and p21WAF1/CIP1 labeling indices for the deeper part of the GCP were higher than those for the superficial parts or the surrounding mucosa, suggesting that both epithelial cell proliferation and p53-dependent p21WAF1/CIP1 expression in DNA-damaged cells, which might be associated with gastritis, are enhanced in line with penetration of glands. The underlying mechanisms might be linked in a chain of factors leading to malignancy.
Adenocarcinoma↗
Japanese Creutzfeldt-Jakob disease patients exhibiting high incidence of the E200K PRNP mutation and located in the basin of a river.
Seven cases with Creutzfeldt-Jakob disease (CJD) located in the basin of the Fuji river (Fuji area) in Japan were examined genetically and clinicopathologically. The onset of the disease was between 1989 and 1995. All cases were from different families, although 3 cases were family members of previously reported CJD patients. They had clinical and/or neuropathological features, corresponding to subacute spongiform encephalopathy. Five of the 7 cases, including the 3 familial cases, had the E200K mutation in the gene encoding prion protein (PRNP). It is suggested that there is a small cluster of CJD patients with a founder effect of the E200K mutation in the Fuji area, because the incidence of CJD with the E200K mutation appears to be much higher in this area than other areas in Japan. The disease penetrance of the 5 cases with the E200K mutation seems to be low, and they may have an age-related incidence in the Fuji area. These findings support the hypothesis that the phenotypes of CJD patients with the PRNP mutations are linked to the position of the mutation, but not related to ethnic or environmental factors.
Aged↗
Sarcomatoid carcinoma of the descending colon--a histological, immunohistochemical and ultrastructural analysis.
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Aged↗
New insights in glycosphingolipid function: "glycosignaling domain," a cell surface assembly of glycosphingolipids with signal transducer molecules,involved in cell adhesion coupled with signaling.
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Animals↗