Biomedical subjects

M K Owada

Publications and source records attributed to M K Owada.

At least 19 recordsLinked to original sources

PubMed2000

Scraped-wounding causes activation and association of C-Src tyrosine kinase with microtubules in cultured keratinocytes.

In order to elucidate the function of c-Src in keratinocytes, we studied the intracellular distribution of its active and inactive form in cultured normal human keratinocyte, using anti-c-Src monoclonal antibody clone 28, which recognizes the active form of c-Src (dephosphorylated at COOH-terminal residue Tyr 530), and monoclonal antibody clone 327 which recognizes both active and inactive forms. Since c-Src has been suggested to be involved in the control of cell adhesion in other cells, we produced a dynamic condition of cell migration by cutting culture cell colonies into squares to form a mesh pattern with a blade (culture wound model). Before cutting, the active form was expressed in cells located only at the periphery of colonies or isolated migrating cells, and was associated with microtubules. Wounding the colony generated a dramatic and rapid activation of c-Src in a few rows of cells along the cut edges, which were made even at the middle of colony, resulting in the association of the active form with microtubules. This increase of the active form was also detected by immunoblotting of cell extracts. These reactions were inhibited by 1 mM sodium orthovanadate, a protein-tyrosine phosphatase inhibitor. ST 638, a potent Src family tyrosine kinase inhibitor, inhibited the migration of keratinocytes in the culture wound healing model. These results suggest that wounding the culture causes activation of c-Src in keratinocytes, and thus activated c-Src may play a role in the function of microtubules during cell migration, especially at an early stage of wound healing.

CSK Tyrosine-Protein Kinase↗

PubMed1999

A pathogenic autoantibody, pemphigus vulgaris-IgG, induces phosphorylation of desmoglein 3, and its dissociation from plakoglobin in cultured keratinocytes.

Pemphigus vulgaris (PV) is an autoimmune blistering skin disease, which is characterized by autoantibodies to a specific desmosomal constituent, i.e. desmoglein 3 (Dsg3). In this study, we analyzed phosphorylation of desmosomal proteins and their molecular interactions after PV-IgG binding to Dsg3 using DJM-1 cells, a squamous cell carcinoma cell line, and normal human keratinocytes. Cells were metabolically labeled with 32P inorganic phosphate, followed by stimulation with the IgG fractions from five PV patients or normal individuals for 20 min. Phosphorylation of specific desmosomal components and their molecular interactions were studied in immunoprecipitates using PV-IgG and anti-plakoglobin (PG) antibodies. PV-IgG binding alone induced the phosphorylation of Dsg3 at serine residues. Although Dsg3 and PG were coprecipitated by PV-IgG-immunoprecipitation when treated with normal IgG, PG was not coprecipitated with Dsg3 when stimulated with PV-IgG, suggesting that PV-IgG binding to Dsg3 caused the dissociation of Dsg3 from PG. These results demonstrate that the binding of pathogenic PV autoantibodies to the cell surface antigen Dsg3, which is an adhesion molecule categorized into desmosomal cadherins, caused particular phosphorylation of Dsg3 and its dissociation from PG.

Autoantibodies↗

PubMed1998

An epitope localized in c-Src negative regulatory domain is a potential marker in early stage of colonic neoplasms.

In previous work, we established a new monoclonal antibody that specifically recognizes the active form of c-Src tyrosine kinase (Kawakatsu et al, 1996). To determine whether c-Src is active in colorectal tumorigenesis, we examined the expression of an active form of c-Src in human normal mucosa, hyperplastic polyps, adenomas, and adenocarcinomas. The tissue distribution of the active form of c-Src was studied by immunohistochemistry using this antibody, termed Clone 28. Among 66 cases of adenoma tested, 61 (92%) showed positive staining (adenoma with mild atypia, 3 of 3; adenoma with moderate atypia, 38 of 42; adenoma with severe atypia, 20 of 21). In contrast to the frequent and intense staining in adenomas, adenocarcinoma showed weak staining with less frequency in 4 of 16 (25%) cases. The number of specimens with positive staining in well- and moderately differentiated adenocarcinomas was limited to an early stage. The active form of c-Src mainly localized to the nuclear membrane and the perinuclear region. These results provide the first direct evidence that the activation of c-Src appears to be an early event in colonic carcinogenesis in situ. The findings of the present study thus allow us to propose a molecular mechanism involving c-Src activation in the process of malignant transformation of the human colonic neoplastic cells.

Antibodies, Monoclonal↗

PubMed1997

Inflammatory cytokines cause coronary arteriosclerosis-like changes and alterations in the smooth-muscle phenotypes in pigs.

We recently developed a porcine model in which chronic, local treatment with interleukin-1 beta (IL-1 beta) causes coronary arteriosclerosis-like changes and hyperconstrictive responses. This study was designed to examine whether or not other major inflammatory cytokines [tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 alpha (IL-1 alpha) might also cause similar coronary responses and whether those responses are associated with alterations in the smooth-muscle phenotypes. A segment of the porcine coronary artery was aseptically wrapped with cotton mesh, absorbing IL-1 beta, TNF-alpha, and IL-1 alpha. Two weeks after the operation, coronary arteriography showed the development of mild stenotic lesions at the cytokine-treated sites, where hyperconstrictive responses were repeatedly induced by intracoronary serotonin or histamine. Histologically mild intimal thickening was noted at those cytokine-treated sites. Immunostaining and immunoblotting demonstrated that all three myosin heavy chain isoforms, SM1, SM2 (smooth-muscle type), and SMemb (nonmuscle type), were noted in the normal coronary segments, whereas in the segments treated with inflammatory cytokines, SM1 and SM2 were markedly reduced, and only SMemb was noted. These results indicate that inflammatory cytokines all have a similar ability to induce coronary arteriosclerosis-like changes and hyperconstrictive responses, which are associated with alterations in smooth-muscle phenotypes toward dedifferentiation.

Animals↗

PubMed1997

Tyrosine kinase inhibitor markedly suppresses the development of coronary lesions induced by long-term treatment with platelet-derived growth factor in pigs in vivo.

Platelet-derived growth factor (PDGF) plays an important role in the development of coronary atherosclerosis. However, it remains to be examined what morphologic and functional changes are induced in vivo by the long-term treatment with PDGF itself or what pharmacologic interventions could suppress those changes in vivo. Our study was designed to address these points. We examined the effects of long-term treatment with PDGF on the porcine coronary artery in vivo. Under aseptic conditions, the proximal segments of the left porcine coronary artery were gently wrapped with cotton mesh absorbing sepharose beads either with or without recombinant human PDGF-AA or -BB. Two weeks after the operation, coronary hyperconstrictions to intracoronary serotonin or histamine were noted at the sites treated with PDGF-AA or -BB. Histologically, neointimal formation and geometric remodeling (reduction of the total vessel area) were noted at the PDGF-treated sites. These functional and histologic changes of the coronary artery induced by PDGF were markedly inhibited by cotreatment with ST 638, a specific inhibitor of tyrosine kinases. A Western blot analysis showed that ST 638 markedly suppressed the PDGF-induced tyrosine phosphorylations in the coronary segment. These results indicate that long-term treatment with PDGF induces neointimal formation, geometric remodeling, and vasospastic responses in vivo, for all of which, activation of tyrosine kinases is substantially involved.

Absorption↗

PubMed1996

A new monoclonal antibody which selectively recognizes the active form of Src tyrosine kinase.

Phosphorylation and dephosphorylation of Tyr-530 in human c-Src (Tyr-527 in avian c-Src) is critical in regulating c-Src kinase activity. So far, it has not been possible to distinguish the active and inactive forms in vivo. We now report a new monoclonal antibody that selectively recognizes the active form of c-Src. This antibody, termed clone 28, recognized a region adjacent to Tyr-530 (Q529YQP532) in the C-terminal regulatory domain of c-Src, and its binding was hindered by phosphorylation of this tyrosine as determined by peptide competition assay. Combined immunoprecipitation/Western blotting revealed that clone 28 reacted with a 60-kDa protein that was precipitated by mAb 327, a well known monoclonal antibody against v-Src and c-Src. Cyanogen bromide cleavage and two-dimensional tryptic maps confirmed that clone 28 was specific for the active form (Tyr-530 not phosphorylated), whereas mAb 327 recognized the inactive form (Tyr-530 phosphorylated) as well as the active form. Clone 28 selectively immunoprecipitated the active form and augmented its kinase activity. Preabsorption experiments revealed that clone 28 could not completely immunoprecipitate the mAb 327 binding 60-kDa protein in either an in vitro or an in vivo phosphorylation system. These observations, taken together, strongly suggest the existence of multiple forms of c-Src as proposed by Cooper and Howell (1993) (Cooper, J. A., and Howell, B. (1993) Cell 73, 1051-1054). Using clone 28, we demonstrated a distinct localization of the active form of c-Src within cultured normal fibroblast cells. In liver tissue sections, we also examined the distribution of the active form in embryonic mice. Megakaryocytes were strongly stained, in contrast to completely negative immunoreactivity in hepatocytes, reticulocytes, and granulocytes. This result provides the first direct evidence that c-Src is highly activated in platelets.

Amino Acid Sequence↗

PubMed1996

Tyrosine kinase inhibitor suppresses the (re) stenotic changes of the coronary artery after balloon injury in pigs.

OBJECTIVE: Restenosis after percutaneous transluminal coronary angioplasty (PTCA) still remains a serious late complication. Many growth factors induced in restenotic lesions may be responsible for restenosis after PTCA. Most of the receptors for such growth factors possess tyrosine kinase activity. This study was designed to determine whether or not a specific tyrosine kinase inhibitor, ST 638, can prevent (re)stenotic changes of the coronary artery after balloon injury. METHODS: A segment of the porcine coronary artery was aseptically wrapped with cotton mesh absorbing either ST 638 or vehicle, followed by balloon injury. Two weeks after the procedure, coronary stenosis and vasoconstricting responses were examined by coronary arteriography and (re)stenotic changes of the coronary artery were histologically examined. Antiphosphotyrosine immunoblotting was also performed to examine the inhibitory effects of ST 638. RESULTS: Coronary arteriography showed the development of mild stenotic lesions at the balloon-injured sites, where hyperconstrictive responses were repeatedly induced by intracoronary serotonin and histamine. Histologically, neointimal formation was noted at the balloon-injured site, where the total vessel area also tended to decrease (geometric remodeling). The treatment with ST 638 suppressed all the hyperconstrictive responses, the neointimal formation, and the geometric remodeling induced by balloon injury. Immunoblotting for phosphotyrosine proteins demonstrated the elevation of proteins at the balloon-injured site, which was suppressed by ST 638. CONCLUSIONS: These results indicate that tyrosine kinases are activated at the balloon-injured site and the inhibition of such kinase activities is effective in reducing both the (re)stenotic changes (neointimal formation and geometric remodeling) and the hyperconstrictive responses of the coronary artery after balloon injury.

Angioplasty, Balloon, Coronary↗

PubMed1996

The importance of the hyperpolarizing mechanism increases as the vessel size decreases in endothelium-dependent relaxations in rat mesenteric circulation.

Endothelium-dependent relaxations are achieved by a combination of endothelium-derived prostacyclin (PGI2), nitric oxide (NO), and endothelium-derived hyperpolarizing factor (EDHF). However, it remains to be fully clarified whether the relative contribution of these three mechanisms to endothelium-dependent relaxations varies as a function of the vessel size. This study was designed to clarify this point. Acetylcholine (ACh)-induced endothelium-dependent relaxations were examined in isolated blood vessels taken from the aorta and the proximal and distal mesenteric arteries of the rat. The contributions of PGI2, NO, and EDHF were evaluated by the inhibitory effects of indomethacin, N omega-nitro-L-arginine methyl ester (L-NAME) in the presence of indomethacin, and KCl in the presence of indomethacin and L-NAME, respectively. The membrane potentials were recorded with microelectrodes. The expression of endothelial No synthase (eNOS) was examined by both immunostaining and immunoblotting. The contribution of PGI2 was negligible in three different-sized blood vessels. The contribution of NO was most prominent in the aorta, whereas that of EDHF was most prominent in the distal mesenteric arteries. The resting membrane potential was significantly deeper and the ACh-induced hyperpolarization was greater in the distal mesenteric arteries than those in the aorta. The expression of eNOS was the highest in the aorta and the lowest in the distal mesenteric arteries. These results indicate that the importance of EDHF increases as the vessel size decreases in endothelium-dependent relaxations in the rat mesenteric circulation.

Acetylcholine↗

PubMed1996

Vanadate causes synthesis of endothelium-derived NO via pertussis toxin-sensitive G protein in pigs.

The effects of sodium orthovanadate, an inhibitor of protein tyrosine phosphatases, on the endothelial nitric oxide (NO) pathway were studied in vitro. Vanadate caused endothelium-dependent relaxations in isolated porcine coronary arteries, which were abolished by N omega-nitro-L-arginine methyl ester. The relaxations were also abolished by pertussis toxin, an inhibitor of certain G proteins. Tyrosine kinase inhibitors, genistein and alpha-cyano-3-ethoxy-4-hydroxy-5-phenyl-methylcinnamamide (ST-638), significantly attenuated the vanadate-induced relaxations. Vanadate also caused pertussis toxin-sensitive, endothelium-dependent relaxations in isolated porcine renal and femoral arteries and jugular veins. Immunoblots, using an antibody to phosphotyrosines and to c-Src in native porcine aortic endothelial cells, respectively, showed that vanadate induced an elevation of phosphotyrosine proteins and a decrease in the amount of the active form of c-Src family kinases; both changes were markedly suppressed by cotreatment with ST-638. These results indicate that in porcine blood vessels, vanadate causes a synthesis of endothelium-derived NO for which endothelial tyrosine kinases and pertussis toxin-sensitive G protein are considered to be closely involved.

Animals↗

PubMed1995

Tyrosine kinase inhibitor suppresses coronary arteriosclerotic changes and vasospastic responses induced by chronic treatment with interleukin-1 beta in pigs in vivo.

We recently demonstrated that chronic treatment with IL-1 beta induces coronary arteriosclerotic changes and vasospastic responses to autacoids in pigs in vivo and that those responses are importantly mediated by PDGF. The receptors for PDGF and other major growth factors are known to have tyrosine kinase activity. We therefore investigated the effects of a selective tyrosine kinase inhibitor, ST 638, on those responses induced by IL-1 beta in our swine model. Intimal thickening and coronary vasospastic responses to serotonin and histamine were induced at the site of the coronary artery where IL-1 beta was chronically and locally applied. These responses were significantly suppressed in a dose-dependent manner by cotreatment with ST 638. In addition, ST 494, which is an inactive form of ST 638, did not inhibit those responses. The treatment with ST 638 alone did not affect the coronary vasoconstricting responses to the autacoids. Immunoblotting using an antibody to phosphotyrosines confirmed the inhibitory effects of ST 638 on the tyrosine phosphorylations induced by IL-1 beta. These results thus suggest that tyrosine kinase activation may play an important role in mediating the effects of IL-1 beta, while also suggesting that ST 638 has an inhibitory effect on the arteriosclerotic changes and vasospastic responses to autacoids in our swine model in vivo.

Animals↗

PubMed1995

A hemidesmosomal transmembrane collagenous molecule, the 180-kDa bullous pemphigoid antigen (BPA II), is phosphorylated with 12-O-tetradecanoylphorbol-13-acetate in a human squamous cell carcinoma cell line (DJM-1).

We have previously shown that the 180-kD bullous pemphigoid antigen (BPAII), which is a transmembrane collagenous protein of hemidesmosomes, is distributed at adhesion sites on glass coverslips on the basal membrane forming a concentric ring, or arch pattern, in a human squamous cell carcinoma cell line (DJM-1), when studied by immunofluorescence microscopy using monoclonal antibodies to BPA II. This concentric ring/arch pattern of "footsteps" of BPA II has been shown to be collapsed in association with a transient activation of protein kinase C by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA). In the present study, therefore, the effects of TPA on the phosphorylation of BPA II was examined. DJM-1 cells, which were metabolically labelled with [32Pi], were lysed and the extracts were subjected to immunoprecipitation with anti-BPAII and anti-230 kDa bullous pemphigoid antigen (BPAI) monoclonal antibodies. The results showed that only BPA II, but not BPA I, was phosphorylated at serine residues before TPA treatment. After TPA treatment phosphorylation was prominently increased so as to generate a 190 kDa-phosphorylated peptide. This 190-kDa peptide was reacted with anti-BPA II monoclonal antibodies by immunoblotting, and it was not detected when cells were pretreated with a specific protein kinase C inhibitor (H7) before TPA treatment, suggesting that the 190 kDa peptide is phosphorylated BPAII with TPA. Prolonged treatment with TPA abolished both of 180- and 190-kDa BPA II from Triton X-100-soluble fractions. These findings suggest that the BPA II, but not BPA I, is a substrate of protein kinase C, and the generation of 190-kDa-phosphorylated BPA II has a key role in the TPA-induced collapse of the assembly of BPA II on the basal plasma membrane, probably, at hemidesmosomes.

Amino Acids↗

PubMed1994

Specificity of the high affinity interaction of protein kinase C with a physiological substrate, myristoylated alanine-rich protein kinase C substrate.

Although myristoylated alanine-rich C kinase substrate (MARCKS), has been employed as an indicator for the activation of protein kinase C (PKC) in intact cells, little is known about its specificity for PKC family members. To address this question, we partially purified human MARCKS from baculovirus-infected cells and compared the kinetic parameters for phosphorylation by PKC isozymes, conventional PKC alpha (cPKC alpha), novel PKC delta (nPKC delta), nPKC epsilon, and atypical PKC zeta (apKC zeta), all of which are distributed in a wide variety of cells. cPKC alpha, nPKC delta, and nPKC epsilon efficiently phosphorylated intact MARCKS protein in vitro. The affinity of MARCKS for cPKC alpha, nPKC delta, and nPKC epsilon was extremely high and decreased in the order alpha > delta > epsilon with Km values of 10.7, 20.7, and 29.8 nM, respectively. The rate of phosphorylation also decreased in the same order. In contrast, a PKC zeta did not phosphorylate MARCKS efficiently, and we were unable to estimate the kinetic parameters. These results suggest that cPKC alpha, nPKC delta, and nPKC epsilon but not a PKC zeta are enzymes that phosphorylate MARCKS in response to PKC activators in intact cells. The structural requirements of MARCKS for efficient phosphorylation by these PKC members were then examined using a peptide that surrounds the phosphorylation site of MARCKS (peptide MARCKS). Interestingly, intact MARCKS showed a 90-150 times lower rate of phosphorylation by PKCs compared with peptide MARCKS, whereas the former showed a 40-180 times higher affinity for these PKC members. This implies that intact MARCKS protein retains a very high affinity for PKC with the sacrifice of its phospho-accepting activity. The structural requirements of PKC were then examined using a calpain-cleaved active fragment of nPKC delta. MARCKS was phosphorylated by the active catalytic fragment as efficiently as by intact nPKC delta, indicating that the kinase domain is sufficient for the high affinity interaction with intact MARCKS. However, gel overlay assay revealed that both intact nPKC delta and its regulatory domain bind to MARCKS, suggesting that both the kinase and regulatory domains of nPKC delta are involved in the high affinity interaction with intact MARCKS protein.

Amino Acid Sequence↗

PubMed1994

High-level expression of human lipocortin I in the fission yeast Schizosaccharomyces pombe using a novel expression vector.

We have developed a novel expression system that allows the fission yeast, Schizosaccharomyces pombe, to be used for the efficient overproduction of heterologous proteins. As an example of the utility of this system, human lipocortin I was expressed to 50 percent of soluble protein, and 150 mg of highly purified material was obtained from 10 grams of wet cell paste. Expression of lipocortin I was driven by the human cytomegalovirus (hCMV) promoter in a vector that also contains a neomycin resistance gene (neo) under the control of the SV40 early promoter, permitting selection for increasing copy-number with increasing concentrations of the antibiotic G418. The purified protein was equivalent to its native counterpart with respect to antigenicity and biochemical properties such as phospholipase A2 inhibition, actin binding and N-terminal acetylation. We have also used this system to produce comparable amounts of other proteins including rat arginase, rat NDP-kinase and human interleukin-6.

Animals↗

PubMed1992

Expression of platelet-derived growth factor-independent phenotypes in BALB/c 3T3 cell variant with high susceptibility to chemically or physically induced neoplastic cell transformation: dissociation from activation of protein kinase C.

A31-I-13, a clonal cell variant of nontransformed BALB/c 3T3 that is highly susceptible to chemically or physically induced malignant cell transformation but is not sensitive to cell killing or susceptible to induced somatic cell mutation compared with another less transformation-susceptible A31-I-1 cell variant, was previously found to be constitutively competent [platelet-derived growth factor (PDGF)-independent] to synthesize DNA (M. Tatsuka et al., J. Cell. Physiol., 139: 18-23, 1989). The present study has demonstrated that density-arrested, quiescent A31-I-13 cells autonomously exhibit disruption of actin filamentous bundles and perturbations of dynamic morphology. PDGF induced these cytoskeletal modulations in quiescent A31-I-1 cells, which require PDGF for the induction of DNA synthesis. Furthermore, the cytoskeletal modulations of quiescent A31-I-13 cells were not accompanied by an increased production of plasminogen activators, activation of protein kinase C, or phosphorylation of a Triton X-100-soluble protein (molecular weight, 90,000) known as 80K, a major substrate for protein kinase C. However, these modulations were accompanied by the tyrosine phosphorylation of Triton X-100-insoluble (cytoskeletal) proteins with molecular weights of 24,000, 32,000-33,000, and 36,000. These Triton X-100-insoluble proteins, as well as the 80K protein, were phosphorylated by the exposure of quiescent A31-I-1 cells to PDGF. Thus the pathway for producing the transformation-susceptible phenotype in A31-I-13 appears to coincide with the PDGF signaling pathway but does not involve the protein kinase C pathway.

3T3 Cells↗

PubMed1991

Calcium-induced intracellular cross-linking of lipocortin I by tissue transglutaminase in A431 cells. Augmentation by membrane phospholipids.

Covalently cross-linked multimers of lipocortin I are shown to be present in human epidermoid carcinoma A431 cells treated with epidermal growth factor or the calcium ionophore A23187. This intracellular cross-linking of lipocortin I is suggested to be mediated by the action of tissue transglutaminase, a Ca2(+)-dependent protein cross-linking enzyme. Cross-linking of lipocortin I competes with proteolytic digestion of the protein, and pretreatment of the cells with inhibitors for calpain (Ca2(+)-dependent intracellular protease) markedly enhanced the cross-linking of lipocortin I. Cross-linked lipocortin I is shown to be present in the soluble fraction of A431 cells as well as in the particulate fraction; a 34-kDa fragment of lipocortin I was solubilized successfully by plasmin digestion of the latter fraction. Immunofluorescence microscopy using specific antilipocortin-I antibody showed that cross-linked lipocortin I forms an envelope-like structure, which is not extracted with [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) or Triton X-100. In vitro incubation of purified lipocortin I with tissue transglutaminase resulted in the formation of covalently cross-linked lipocortin I dimer, tetramer, and so on. Amine incorporation and cross-linking studies using lipocortin I and its N-terminal truncated derivatives indicated that the cross-linking site is localized within the plasmin-susceptible N-terminal 29 amino acids of lipocortin I. The cross-linking of lipocortin I is shown to be accelerated more than 10 times by the addition of phosphatidylserine vesicles, on which lipocortin I molecules are most likely aligned in a conformation suitable for cross-linking. Collectively, these findings suggest that an increase of intracellular calcium concentration results in the attachment of lipocortin I onto the plasma membrane phospholipids through the C-terminal domain of the molecule where the membrane-bound lipocortin I is cross-linked by the action of tissue transglutaminase through the N-terminal domain.

Annexins↗

PubMed1991

Lipocortin I (annexin I) is preferentially localized on the plasma membrane in keratinocytes of psoriatic lesional epidermis as shown by immunofluorescence microscopy.

Lipocortin I (LPC-I, also called annexin I) is a 35-kD protein that binds phospholipids and actin in a Ca(++)-dependent manner. It is also a major substrate for EGF receptor/kinase and protein kinase C, and a putative inhibitor of phospholipase A2, which produces chemical mediators to cause inflammation. Psoriasis (PS) is an inflammatory skin disease characterized by a rapid turnover of keratinocytes and a defect in keratinization with increased activities of phospholipase C and A2, and EGF receptor. To understand the mechanism of the PS lesion formation and the function of LPC-I, its distribution was studied in the epidermis of PS, subacute eczema and normal skin, and in tumor cells of seborrheic keratosis and Bowen's disease. This study involved immunofluorescence and immunoblotting using affinity-purified polyclonal and monoclonal antibodies specific to LPC-I and to its Ca(++)-bound form. In normal, nonlesional PS and subacute eczema epidermis, LPC-I was detected mainly in the cytoplasm of the suprabasal cells, although it was on the inner aspects of the plasma membrane in some parts of the granular layer. In lesional epidermis of PS, it was localized mainly on the inner aspects of the plasma membrane, but not in the cytoplasm of the whole suprabasal cells as the Ca(++)-bound form, indicating a preferential localization on the plasma membrane. This membrane-binding of LPC-I was also observed in seborrheic keratosis, but not in Bowen's disease. These results suggest that the binding of LPC-I to the plasma membrane occurs actually in living cells, plays a role, not necessarily disease specific, in the PS lesion formation, and has some relevance to normal or abnormal differentiation of keratinocytes.

Annexins↗

PubMed1990

Monoclonal antibodies specific to a Ca2(+)-bound form of lipocortin I distinguish its Ca2(+)-dependent phospholipid-binding ability from its ability to inhibit phospholipase A2.

Lipocortin I, a Ca2(+)-and phospholipid-binding protein without EF-hand structures, has many biological effects in vitro. Its actual role in vivo, however is unknown. We obtained and characterized five monoclonal antibodies to lipocortin I. Two of these monoclonal antibodies (L2 and L4-MAbs) reacted with the Ca(+)-bound form of lipocortin I, but not with the Ca2(+)-free form, both in vivo and in vitro. Lipocortin I required greater than or equal to 10 microM-Ca2+ to bind the two antibodies, and this Ca2+ requirement was not affected by phosphatidylserine. L2-MAb abolished the phospholipase A2 inhibitory activity of lipocortin I and inhibited its binding to Escherichia coli membranes and to phosphatidylserine in vitro. L4-MAb abolished the phospholipase A2 inhibitory activity of lipocortin I, but did not affect its binding to E. coli membranes or to phosphatidylserine. These findings indicated that the inhibition of phospholipase A2 by lipocortin I was not simply due to removal or capping of the substrates in E. coli membranes. Furthermore, an immunofluorescence study using L2-MAb showed the actual existence of Ca2(+)-bound form of lipocortin I in vivo.

Annexins↗

PubMed1990

Association of protein tyrosine phosphorylation with B cell differentiation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA).

We have assessed whether tyrosine protein kinase (TPK) is involved in B cell differentiation. In vitro phosphorylation of an endogenous substrate in B cell leukemias showed that leukemic B cells at different stages of differentiation had specific endogenous substrates in tyrosine phosphorylation as well as distinct TPK activity. To clarify the relationship between TPK and the process of B cell differentiation, we studied protein tyrosine phosphorylation in two kinds of leukemic B cells, which showed distinct responses to TPA (12-O-tetradecanoylphorbol-13-acetate) in B cell differentiation. TPA-treated leukemic B cells from patients with B cell chronic lymphocytic leukemia (B-CLL) differentiated into cytoplasmic immunoglobulin (clg)+ plasmacytoid cells, while TPA-treated leukemic B cells from patients with hairy cell leukemia (HCL) did not differentiate into clg+ cells, but showed a peculiar morphological change, spreading. Untreated B-CLL cells and HCL cells showed similar TPK activities and tyrosine protein phosphorylation. When treated with TPA, enhanced phosphorylation was seen in B-CLL cells, while a clear reduction in phosphorylation was found in HCL cells. However, using 4-hydroxycinnamide derivatives which reduce TPK activity, we found that only the reduction of TPK activity did not lead HCL cells to spreading. These data suggest that protein tyrosine phosphorylation and/or dephosphorylation might be involved in B cell differentiation, but only the change of TPK activity in HCL cells is not sufficient to induce effects.

B-Lymphocytes↗