Increased binding and chemotactic capacities of PDGF-BB on fibroblasts in radiation pneumonitis

Although pulmonary fibrosis is a frequent and serious consequence of radiotherapy for thoracic malignant diseases such as lung cancer, the pathogenesis of this radiation-induced lung disorder remains unclear. To clarify the mechanisms underlying radiation pneumonitis and pulmonary fibrosis, we investigated the expression of platelet-derived growth factor receptor (PDGFR) on fibroblasts obtained from irradiated rat lungs and on control fibroblasts. Whole lungs of male Wistar rats were irradiated with a single dose of 15 Gy, and lung fibroblasts were isolated at 4 weeks after the irradiation. The chemotactic response of irradiated lung fibroblasts to PDGF-BB was significantly higher than that of control lung fibroblasts, whereas there was no significant difference between irradiated lung fibroblasts and control lung fibroblasts in the response to PDGF-AA. Receptor binding assay showed more specific binding sites for PDGF-BB on irradiated lung fibroblasts than on control lung fibroblasts, and the displacement of (125)I-labeled PDGF binding to fibroblasts by unlabeled PDGF showed that (125)I-labeled PDGF-BB was displaced by PDGF-BB but not by PDGF-AA. These results suggest that the increased binding sites for PDGF-BB on irradiated lung fibroblasts correspond mainly to PDGFRB. Scatchard analysis of the saturation data demonstrated an approximately twofold increase both in the number of PDGF-BB binding sites and in the binding affinity in irradiated lung fibroblasts compared to that in control lung fibroblasts. Those results suggest that the increased chemotactic response of irradiated lung fibroblasts to PDGF-BB is related to the overexpression of PDGFRB, which may have an important role in the pathogenesis of radiation-induced pneumonitis and pulmonary fibrosis.     

Induction of protein kinase Czeta-related protein kinase by growth suppression in carcinogen-initiated epidermal cell-line WYF31 cells

In primary cultured mouse epidermal cells, protein kinase C isozyme zeta (PKCzeta) consists of multiple forms, for example, low-salt eluted PKCzeta (1-PKCzeta; 79 and 85 kDa) and high-salt eluted PKCzeta (h-PKCzeta; 79 and 85 kDa) on anion-exchange column chromatography. In this study, biochemical and biophysical differences between 1-PKCzeta and h-PKCzeta were examined by using carcinogen-initiated mouse epidermal cell-line WYF31 cells, whose growth is stimulated by tumour promoter phorbol 12-myristate 13-acetate (PMA). The binding efficiency of h-PKCzeta to anti-PKCzeta antibody-affinity column was 10 times higher than that of 1-PKCzeta. T7-tagged rat PKCzeta overexpressed in WYF31 cells was recovered only in the high-salt eluted area on the anion-exchange column. Furthermore, when rat PKCzeta was stably overexpressed in WYF31 cells, the content of h-PKCzeta increased 4 to 5 times compared to that of parental cells, but the content of 1-PKCzeta was not altered. All of these results indicate that h-PKCzeta is the product of the PKCzeta gene (referred to as PKCzeta) and that 1-PKCzeta is closely related but different from PKCzeta (referred to as PKCzeta-related kinase). Interestingly, serum starvation of WYF31 cells caused a marked increase of the content of PKCzeta-related kinase with a concomitant decrease of PKCzeta content. These changes were reversed by stimulating the cell growth with 10% foetal calf serum. Prolonged treatment of starved cells with PMA, which induces the proliferation of WYF31 cells, also caused the downregulation of PKCzeta-related kinase. These results suggest that the expression levels of PKCzeta-related kinase and PKCzeta are differently regulated, and that the increased expression of PKCzeta-related kinase might play a significant role in the growth-suppression processes of WYF31 cells.     

Inactivation of glycogen synthase kinase-3 by protein kinase C delta: implications for regulation of tau phosphorylation

The role of the phosphatidylinositol 3-kinase (PI3K) pathway in the hyperphosphorylation of tau was investigated in SY5Y human neuroblastoma cells. Wortmannin, an inhibitor of PI3K, induced transient (after 1 h) activation of glycogen synthase kinase-3 (GSK-3), hyperphosphorylation of tau and dose-dependent cytotoxicity. However, continuous inactivation of protein kinase (PK) B was observed from 1 to 24 h, suggesting the involvement of protein kinase(s) other than PKB in the phosphorylation and inactivation of GSK-3 after 3 h. In cells treated with wortmannin, PKC delta fragments were observed, and the PKC activity increased after 3 h, whereas treatment of cells with z-DEVD-fmk, an inhibitor of caspase 3, also inhibited fragmentation of PKC delta and induced continuous activation of GSK-3. It is suggested that fragmentation of PKC delta during the process of apoptosis results in the phosphorylation and inactivation of GSK-3 and consequently inhibition of the phosphorylation of tau.     

Redundant and segregated functions of granule-associated heparin-binding group II subfamily of secretory phospholipases A2 in the regulation of degranulation and prostaglandin D2 synthesis in mast cells

We herein demonstrate that mast cells express all known members of the group II subfamily of secretory phospholipase A2 (sPLA2) isozymes, and those having heparin affinity markedly enhance the exocytotic response. Rat mastocytoma RBL-2H3 cells transfected with heparin-binding (sPLA2-IIA, -V, and -IID), but not heparin-nonbinding (sPLA2-IIC), enzymes released more granule-associated markers (beta-hexosaminidase and histamine) than mock- or cytosolic PLA2alpha (cPLA2alpha)-transfected cells after stimulation with IgE and Ag. Site-directed mutagenesis of sPLA2-IIA and -V revealed that both the catalytic and heparin-binding domains are essential for this function. Confocal laser and electron microscopic analyses revealed that sPLA2-IIA, which was stored in secretory granules in unstimulated cells, accumulated on the membranous sites where fusion between the plasma membrane and granule membranes occurred in activated cells. These results suggest that the heparin-binding sPLA2s bind to the perigranular membranes through their heparin-binding domain, and lysophospholipids produced in situ by their enzymatic action may facilitate the ongoing membrane fusion. In contrast to the redundant role of sPLA2-IIA, -IID, and -V in the regulation of degranulation, only sPLA2-V had the ability to markedly augment IgE/Ag-stimulated immediate PGD2 production, which reached a level comparable to that elicited by cPLA2alpha. The latter observation reveals an unexplored functional segregation among the three related isozymes expressed in the same cell population.     

HMB-45 staining for cytology of primary melanoma of the vagina. A case report

BACKGROUND: Malignant melanoma in the vagina is very rare, but its diagnosis is usually easy if a melanin pigment is present. With cytodiagnosis, however, it is difficult to differentiate amelanotic melanoma or scantily pigmented melanoma from other conditions. In the present case, monoclonal antibody HMB-45, the efficacy of which has been established in histologic studies, was used in the cytodiagnosis of amelanotic melanoma in the vagina. CASE: A woman, aged 78 years, presented with a brownish, nodular tumor, diameter 3 cm, in the vagina. Scraping smears with Papanicolaou staining showed nonepithelial malignant cells without granules suggesting melanin. Smears stained with HMB-45 showed positive immunoreactivity. The diagnosis underwent histologic confirmation of amelanotic melanoma on the initial biopsy. CONCLUSION: Cytodiagnosis was made with HMB-45, which proved very effective in the differential cytodiagnosis of amelanotic melanoma and scantily pigmented melanoma, particularly because it obviated the need for tissue invasion.     

Serine 727 phosphorylation and activation of cytosolic phospholipase A2 by MNK1-related protein kinases

We have previously reported that in thrombin-stimulated human platelets, cytosolic phospholipase A(2) (cPLA2) is phosphorylated on Ser-505 by p38 protein kinase and on Ser-727 by an unknown kinase. Pharmacological inhibition of p38 leads to inhibition of cPLA2 phosphorylation at both Ser-505 and Ser-727 suggesting that the kinase responsible for phosphorylation on Ser-727 is activated in a p38-dependent pathway. By using Chinese hamster ovary, HeLa, and HEK293 cells stably transfected with wild type and phosphorylation site mutant forms of cPLA2, we show that phosphorylation of cPLA2 at both Ser-505 and Ser-727 and elevation of Ca(2+) leads to its activation in agonist-stimulated cells. The p38-activated protein kinases MNK1, MSK1, and PRAK1 phosphorylate cPLA2 in vitro uniquely on Ser-727 as shown by mass spectrometry. Furthermore, MNK1 and PRAK1, but not MSK1, is present in platelets and undergo modest activation in response to thrombin. Expression of a dominant negative form of MNK1 in HEK293 cells leads to significant inhibition of cPLA2-mediated arachidonate release. The results suggest that MNK1 or a closely related kinase is responsible for in vivo phosphorylation of cPLA2 on Ser-727.     

Three-dimensional solution structure of oryzacystatin-I, a cysteine proteinase inhibitor of the rice, Oryza sativa L. japonica

The three-dimensional structure of oryzacystatin-I, a cysteine proteinase inhibitor of the rice, Oryza sativa L. japonica, has been determined in solution at pH 6.8 and 25 degrees C by (1)H and (15)N NMR spectroscopy. The main body (Glu13-Asp97) of oryzacystatin-I is well-defined and consists of an alpha-helix and a five-stranded antiparallel beta-sheet, while the N- and C-terminal regions (Ser2-Val12 and Ala98-Ala102) are less defined. The helix-sheet architechture of oryzacystatin-I is stabilized by a hydrophobic cluster formed between the alpha-helix and the beta-sheet and is considerably similar to that of monellin, a sweet-tasting protein from an African berry, as well as those of the animal cystatins studied, e.g., chicken egg white cystatin and human stefins A and B (also referred to as human cystatins A and B). Detailed structural comparison indicates that oryzacystatin-I is more similar to chicken cystatin, which belongs to the type-2 animal cystatins, than to human stefins A and B, which belong to the type-1 animal cystatins, despite different loop length.