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Biomedical subjects

H Nakakuma

Publications and source records attributed to H Nakakuma.

At least 55 records · Page 3Linked to original sources
PubMed1992

Ganglioside, adhesion molecule, and HLA antigen expression in basal cell carcinoma lesions.

The antigenic profile of basal cell carcinoma (BCC) cells was analyzed by immunoperoxidase staining of 27 surgically removed BCC lesions with antiganglioside, antiadhesion molecule, and antihistocompatibility locus antigen (HLA) monoclonal antibodies (MoAb). The large majority of BCC lesions were stained by antiganglioside MoAb; among the latter the anti-GD3 ganglioside MoAb R24 displayed the broadest reactivity. The GD3 ganglioside expression by BCC cells, which was corroborated by thin layer chromatography immunostaining with MoAb R24, appears to be a proliferation dependent phenomenon. Among the adhesion molecules tested vitronectin receptor and CDw44 were found in up to 70% of the lesions tested, while intercellular adhesion molecule 1 (ICAM-1) was detected in only a low percentage of BCC cells in one lesion. ICAM-1 was not induced on BCC cells in five and three lesions removed 24 and 48 h, respectively, following the intralesional injection of gamma-interferon. The latter enhanced HLA Class I antigen expression and induced ICAM-1 expression by the surrounding keratinocytes; furthermore gamma-interferon induced HLA Class II antigen expression by a small percentage of BCC cells in three lesions. These results suggest that malignant transformation of keratinocytes is associated with a selective loss of susceptibility to induction by cytokines of ICAM-1 expression. Besides confirming the low HLA Class I and Class II antigen expression by BCC cells, the present investigation has shown a differential expression of distinct monomorphic determinants of HLA Class I antigens and a lower expression of HLA-A antigens than of HLA-B antigens by BCC cells. Furthermore, the present study has shown that HLA Class II antigens can be induced on BCC cells by cytokines.

Aged↗
PubMed1992

Ganglioside-induced inhibition of in vivo immune response in mice.

In vitro immunomodulatory properties of gangliosides have been well characterized such as the ganglioside-induced modulation of CD4 on T lymphocytes and inhibition of lectin-induced proliferative response of lymphocytes. These findings have led to an interesting suggestion that gangliosides play a role as in vivo immune modulators, although this possibility is not clearly defined yet. We then first confirmed in vitro effects of gangliosides on murine immunocytes and examined in vivo effects of gangliosides on immune response in mice. Murine spleen cells that were treated with a ganglioside mixture (GS) purified from bovine brain exhibited a marked decrease in CD4 expression, while CD8 expression was slightly suppressed. Transplantation of GS-untreated control immunocytes that were isolated from syngeneic mice into the immune suppressed mice by X-ray irradiation restored in vivo immune responses, while GS-treated cells could not. Immune response was assayed by the evaluation of footpad swelling which was induced by immunization with sheep erythrocytes as antigens. Moreover, intramuscular administration of gangliosides into mice suppressed both immediate (Arthus)-type and delayed-type allergic reactions. These results suggest that gangliosides would be potential in vivo immune modulators.

Animals↗
PubMed1992

Increased plasma decay-accelerating factor levels in paroxysmal nocturnal hemoglobinuria.

Decay-accelerating factor (DAF) is a complement regulatory membrane protein that is often absent from the cell surface of blood cells in paroxysmal nocturnal hemoglobinuria (PNH). DAF has also recently been found in the body fluids of healthy individuals. However, its precise structure and biological significance are not yet clear. To clarify the clinical and pathological implications of free DAF, we measured plasma DAF levels in PNH patients, using a newly developed quantitative enzyme-linked immunosorbent assay (ELISA), with a measurable range between 0.2-12 ng, for soluble DAF. ELISA assays revealed significantly increased plasma DAF levels in PNH patients (258 +/- 150 ng/ml, mean +/- S.D., n = 9) as compared with healthy controls (80 +/- 41 ng/ml, n = 17) (p less than 0.01). Taken together with the finding that DAF is synthesized in and released extracellularly from affected PNH cells, plasma DAF levels would be useful for clinical diagnosis and for the quantitative evaluation of the clonal expansion of affected cells in PNH.

Adult↗
PubMed1992

Altered expression of protein kinase C in adult T-cell leukemia cells.

Protein kinase C (PKC) has been shown to be involved in the mitogenic response and in oncogenic cell transformation in many experimental models. We analyzed the expression of PKC in both highly purified leukemic T cells freshly isolated from adult T-cell leukemia (ATL) patients and control T lymphocytes obtained from healthy volunteers. PKC activity was decreased in the ATL cells as compared with the control T cells. Cytosolic PKC activity in the ATL cells was remarkably decreased, whereas particulate membrane PKC activity was similar to the control level. The percentage of PKC activity in the particulate fraction was 34% in the ATL cells and 19% in the control cells. Regarding the altered subcellular localization of PKC activity, phorbol ester-induced translocation of cytosolic PKC was inhibited in some ATL cases. Similarly to the decrease in PKC activity, there was a decrease in the expression of the major PKC isozymes II(beta) and III(alpha) in ATL cells. These results suggest impaired regulation of PKC expression in ATL as well as in many experimental cancers.

Adult↗
PubMed1992

Common phenotypic expression of gangliosides GM3 and GD3 in normal human tissues and neoplastic skin lesions.

The expression of gangliosides in non-malignant tissues (epidermis and pigmented nevus) and neoplastic lesions (melanoma, squamous cell carcinoma [SCC] and basal cell carcinoma [BCS]) of the human skin was analyzed immunohistochemically and biochemically to characterize the features associated with malignancy. Immunohistochemical staining with an anti-II3NeuAc-LacCer (GM3) monoclonal antibody (M2590 mAb) and an anti-II3(NeuAc)2-LacCer (GD3) mAb (R24) showed the expression of the gangliosides GM3 and GD3 to vary among the different tissues. M2590 clearly stained epidermal keratinocytes and the tumor cells of BCC and SCC, and strongly stained melanocytes and melanoma cells. In contrast, R24 did not stain epidermal keratinocytes and only faintly stained SCC cells, while it clearly stained BCC cells, and intensely stained melanocytes and melanoma cells. GM3 showed a similar level of staining among the tissue specimens, while the level of GD3 staining was quite variable among the tumor specimens. Biochemical analysis by thin-layer chromatography (TLC) with resorcinol staining and TLC immunostaining with either M2590 or R24 showed both GM3 and GD3 to be commonly expressed by both the normal and malignant skin tissues, including SCC. There was no close correlation between the intensity of immunohistochemical staining and the biochemically detected amounts of these gangliosides. This may have been partly due to the so-called cryptic expression of cell membrane gangliosides. Our results thus suggest that analysis of the tumor-associated expression of gangliosides requires several methods, since the sensitivity of the methods used may have a considerable effect on the diagnostic value of gangliosides as skin cancer markers.

Biomarkers, Tumor↗
PubMed1991

Hemolysis of human erythrocytes is a new bioactivity of gangliosides.

Using sheep erythrocytes and liposomes, an inhibitory effect of gangliosides has been shown on the activation of the alternative pathway of complement. However, in studies using human erythrocytes, we found that gangliosides had hemolytic activity that was possibly mediated through activation of the alternative pathway. Pretreatment of human erythrocytes obtained from healthy volunteers or paroxysmal nocturnal hemoglobinuria (PNH) patients with a ganglioside mixture purified from human erythrocytes enhanced their susceptibility to homologous human complement, and resulted in dose-dependent hemolysis. The enhancement was more marked in PNH erythrocytes than control cells. Protease treatment of the ganglioside mixture did not change its hemolytic activity, but sialidase treatment abolished the activity. Among the major erythrocyte gangliosides, II3NeuAc-LacCer (GM3) was the most potent hemolytic agent. Gangliosides purified from bovine brain were also active, while neither nonsialylated glycosphingolipids, the ceramide moiety, or sialic acid alone were active. Sialic acid residues in the ganglioside molecules were essential to this activity, but the amount of the residue or the source of the gangliosides seemed not to be important. Several treatments inhibiting the alternative but not classical complement pathway markedly reduced the ganglioside hemolytic activity. This novel bioactivity of gangliosides was thus suggested to be mediated partly by activation of the alternative pathway.

Chromatography, Thin Layer↗
PubMed1991

Concanavalin A-stimulated expression of gangliosides with GalNAc beta 1-4(NeuAc alpha 2-3)Gal beta structure in murine thymocytes.

We analysed the glycolipids of mouse thymocytes before and after Concanavalin A (Con A) or recombinant interleukin-2 (rIL-2) stimulation by TLC-immunostaining with carbohydrate-specific antiglycolipid antibodies. The thymocytes were cultured in serum-free medium in the presence of 500 ng ml-1 Con A, 10 U ml-1 rIL-2 or Con A plus rIL-2 for 6, 12, 24, 48, and 72 h, and were found to start proliferating 24 h after cultivation in the presence of Con A or Con A plus rIL-2, the maximum levels being reached at 72 h and 48 h, respectively, in a thymidine uptake experiment. The concentrations of II3Neu-Gg4Cer, Gg4Cer and IV3GalNAc alpha-Gb4Cer after 48 h Con A stimulation were found to be at almost the original levels. Conversely, II3Neu-Gg3Cer, which was not detected in the thymocytes at the start, began to appear after 48 h stimulation with Con A and Con A plus rIL-2, and IV3Neu-Gg5Cer in the cells 48 h after stimulation with Con A and Con A plus rIL-2 has increased to 41 and 44 times higher than in the original cells, respectively, as judged on TLC-immunostaining with monoclonal antibody YHD-06, which detects the GalNAc beta 1-4(NeuAc or NeuGc alpha 2-3)Gal beta-structure. These results indicate that the increased synthesis of both gangliosides, in other words, the activation of N-acetylgalactosaminyltransferase, is associated with the mitogen-induced proliferation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

[Altered metabolism of membrane glycosphingolipids in erythrocytes of paroxysmal nocturnal hemoglobinuria].

Paroxysmal nocturnal hemoglobinuria (PNH) is currently accepted to be a stem-cell disorder of a clonal nature with increased susceptibility to autologous complement attack. Consequent hemolytic feature has been partly explained by lack of complement regulatory membrane proteins such as decay-accelerating factor (DAF) or C8-binding protein that anchored to membrane via glycosyl-phosphatidyl inositol (GPI) lipids. Recent reports suggest essential PNH lesion is the synthetic defect of sugar moiety of the PI-anchor. In PNH, the abnormal expression of C3b/C4b receptor (CRI) glycoproteins, or glycophorin-alpha have been also pointed out. These altered expression of glycoproteins and glyceroglycolipids, especially in the carbohydrate structures, prompted us to analyze biochemically the membrane glycosphingolipids as one of major glycoconjugates in PNH. As results, PNH erythrocytes showed altered metabolism of gangliosides in comparison to control erythrocytes from healthy donors. IV6 NeuAc-nLc4 Cer and highly polar gangliosides variably disappeared in PNH erythrocytes, partly due to impaired sialylation of glycolipids. These results suggest metabolic disorder of carbohydrates of membrane glycoconjugates as a new aspect of PNH.

Adult↗
PubMed1991

[Serum deoxythymidine kinase activity in adult T-cell leukemia].

Serum deoxythymidine kinase activities (s-TK) of the patients with adult T-cell leukemia (ATL), carriers and healthy persons were measured, using a recently developed TK assay with 125I-iodo-deoxyuridine as a substrate. The mean s-TK values were 3.3 +/- 2.7 U/l (n = 21) in normal subjects (HTLV-1(-)), 4.7 +/- 5.0 U/l (n = 35) in carriers, 9.8 U/l (n = 3) in smoldering ATL and 26.7 U/l (n = 6) in chronic ATL. In the patients with acute ATL, the mean s-TK values before and after chemotherapy were 80.9 U/l (n = 2) and 11.6 U/l (n = 4), respectively. In the follow-up studies of the patients with acute ATL, the changes of s-TK levels revealed earlier than those of serum LDH levels. It is suggested that s-TK activity is more useful than LDH as a parameter of monitoring treatment in ATL.

Biomarkers, Tumor↗
PubMed1990

Paroxysmal nocturnal haemoglobinuria with coexisting deficiency of the ninth component of complement: lack of massive haemolytic attack.

A 47-year-old woman with paroxysmal nocturnal haemoglobinuria (PNH) was found to have an inherited deficiency in the ninth complement component (C9). In complement-sensitivity lysis tests, 80% of her erythrocytes were markedly complement-sensitive (PNH-III). Laser cytofluorimetry with a monoclonal antibody against decay-accelerating factor (DAF) revealed that 95% of her erythrocytes were DAF-negative. Surprisingly, she has suffered only mild haemolysis and has never experienced massive spontaneous haemolysis. Gross haemoglobinuria and jaundice occurred only after receiving postoperative transfusion of whole blood. In her serum, C9 was not detectable either by immunological or by functional assays. Both the Ham test and the sugar water test using normal human serum or plasma yielded marked haemolysis of the patient's erythrocytes. When the patient's serum or plasma was used, only a trace of lysis was detected. Addition of purified human C9 to her plasma fully restored haemolysis. These observations indicated that C9 may play a critical role in haemolytic attacks in patients with PNH and that characteristic haemolysis in PNH may be tempered by coexisting C9 deficiency.

Complement C9↗
PubMed1990

Altered expression of gangliosides in erythrocytes of paroxysmal nocturnal hemoglobinuria.

In paroxysmal nocturnal hemoglobinuria (PNH), impaired glycosyl-phosphatidylinositol (PI)-anchoring of membrane proteins such as decay-accelerating factor has been known to lead to increased susceptibility to complement. Moreover, abnormal expression of non-PI-anchoring glycoproteins such as C3b/C4b receptor (CR1) or glycophorin-alpha also has been shown in PNH. Therefore, we biochemically analyzed glycosphingolipids (GSL) as one of the membrane glycoconjugates of PNH erythrocytes. Erythrocytes of all seven PNH patients showed altered expression of sialosyl GSL (gangliosides) as compared with the control erythrocytes of healthy donors. Both a sialosylparagloboside (IV6NeuAc-nLc4Cer) among four major gangliosides and some minor gangliosides in normal erythrocytes variably disappeared in erythrocytes from the peripheral blood of PNH patients. As one of the possible mechanisms of altered expression of gangliosides in PNH erythrocytes, structural analysis suggested impaired sialylation of GSL. These results suggest not only the altered metabolism of gangliosides in PNH erythrocytes, but also a metabolic disorder of membrane glycoconjugates as a new feature of PNH.

Adult↗
PubMed1989

Monoclonal antibody to galactosylceramide: discrimination of structural difference in the ceramide moiety.

A mouse monoclonal antibody (mAb) was developed against monohexaosylceramide. This mAb differentially reacted on thin-layer chromatograms with 3 types of galactosylceramide (GalCer) obtained from bovine brain. Structural analysis of the 3 glycolipids revealed that they consisted of the same galactose and sphingosine but of apparently different fatty acids. Among the GalCers, the mAb reacted with teh two GalCers which contained alpha-hydroxy fatty acids, but not with GalCer composed of nonhydroxy fatty acids. These findings suggest that not only that the mAb discriminated the fatty acid composition in the ceramide moiety of GalCer, but also that the ceramide structure defines the immunological epitope as it is known to do for the carbohydrate moiety of glycosphingolipid.

Animals↗
PubMed1989

Activation of human neutrophil NADPH-oxidase in vitro by the catalytic fragment of protein kinase-C.

Phorbol ester treatment of intact neutrophils both stimulates protein kinase C (PK-C) and causes the rapid proteolytic conversion to a cytosolic, co-factor independent fragment, protein kinase M (PK-M). In intact neutrophils, phorbol ester treatment activates the NADPH-oxidase, the enzyme responsible for the oxidative burst. Addition of purified PK-M to resting neutrophil light density membranes activated the NADPH-oxidase in the presence of PS, ATP and Mg2+. A 3.5-fold greater stimulation of oxidase (ca. 25 nmoles O2-/min/mg membrane protein) was obtained with comparable PK-M concentrations to that observed with the reconstituted PK-C system, and approximately 1/3 that obtained with arachidonic acid (AA) or SDS. In contrast to the reconstituted system using PK-C, PMA and Ca++ were neither required nor affected activity. The effect of PS was unexpected, since PK-M does not require phospholipids for enzymatic activity, and likely represents the action of PS on the oxidase itself or on another component in the plasma membrane fraction. Our studies demonstrate for the first time that purified PK-M permits reconstitution of a physiologic phorbol ester response.

Adenosine Triphosphate↗
PubMed1989

Characteristic mode of action of gangliosides in selective modulation of CD4 on human T lymphocytes.

We have explored the possible mechanisms for selective modulation by gangliosides of CD4 on human T lymphocytes and subsequent re-expression of CD4. Indirect immunofluorescence staining with anti-CD4 antibodies revealed newly internalized CD4 in ganglioside-treated cells after membrane permeabilization with 0.1% saponin. Cycloheximide and other metabolic inhibitors did not alter the modulation but inhibited significantly the re-expression of CD4. These results suggest both selective modulation of CD4 by a process of endocytosis and re-expression of CD4 through de novo protein synthesis.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1989

Inhibition of human immunodeficiency virus infection of human lymphocytes by gangliosides.

As the first step in human immunodeficiency virus (HIV) infection, HIV binds to CD4 molecules on the surface of human T lymphocytes. Expression of CD4 by the cells is remarkably modulated by exogenously added gangliosides, which are normal components of the surface of animal cells. We report here that these physiological molecules also clearly inhibited HIV infection of lymphocytes in vitro in a dose-dependent manner through the selective modulation of CD4 from the cell surface. This raises the possibility of in vivo application of gangliosides as a new strategy for treating acquired immunodeficiency syndrome.

Antibodies, Monoclonal↗
PubMed1989

Effect of microinjected catalytic fragment of protein kinase C on morphological change in Swiss 3T3 cells.

Although phorbol esters can enhance formation of an active, catalytic domain of protein kinase C (PKC) in intact cells, little is known about the actual importance of the proteolytic pathway in mediating cellular responses to the phorbol esters or other PKC activators. To explore this issue, we examined the effect of microinjected catalytic fragment of PKC on Swiss 3T3 cell morphology. In contrast to the dramatic, rapid response upon phorbol ester treatment, catalytic fragment microinjected in the presence of bovine serum albumin or normal goat immunoglobulin G as carrier protein had no effect. A morphological response similar but not identical to the effect of phorbol ester treatment was obtained, however, if catalytic fragment was microinjected in the presence of normal rabbit immunoglobulin G rather than the usual carrier proteins. The normal rabbit immunoglobulin by itself was inactive. Although the mechanism remains undefined, normal rabbit immunoglobulin but not other carrier proteins modulated PKC activity in vitro. We conclude that the generation of free catalytic fragment of PKC cannot account for the morphological response of Swiss 3T3 cells to the phorbol esters; secondary factors may, however, potentiate its action.

Animals↗