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G D Johnson

Publications and source records attributed to G D Johnson.

At least 37 records · Page 2Linked to original sources
PubMed1996

Protein kinase C-delta associates with vimentin intermediate filaments in differentiated HL60 cells.

The subcellular localization of protein kinase C (PKC)-delta was determined in HL60 cells differentiated toward monocytes/macrophages by treatment with TPA. PKC-delta was detected in the nucleus and cytoplasm of differentiated HL60 cells and, more specifically, associated with structures resembling intermediate filaments. Indirect immunostaining revealed that PKC-delta colocalized with vimentin in the cytosol and perinuclear region of these cells. Immunoprecipitation studies showed that PKC-delta was in an active (autophosphorylated) state in differentiated HL60 cells and that vimentin immunoprecipitated from these cells was also phosphorylated. Treatment of HL60 cells with the PKC-specific inhibitor chelerythrine decreased the phosphorylation of vimentin. These data suggest that vimentin is a substrate for PKC-delta and that this PKC isoenzyme may play a specific role in the regulation of shape change and cell adhesion during HL60 differentiation.

Antibody Specificity↗
PubMed1996

Bcl-2 delays macrophage engulfment of human B cells induced to undergo apoptosis.

The capacity to be recognized and engulfed by phagocytes is an important characteristic of cells dying by apoptosis. Phagocytosis of apoptotic cells occurs rapidly in vivo, probably prior to plasma membrane breakdown. While the molecular mechanisms mediating phagocytosis of apoptotic cells are beginning to be defined, little is yet known of the relationship between the cell-death program itself and the surface changes on the dying cells that signal for engulfment. Here, we investigate to what extent the apoptosis repressor Bcl-2 can modulate the recognition and phagocytosis of human B cells exposed to triggers of apoptosis. Burkitt lymphoma (BL)-derived, Bcl-2- B cells were induced into apoptosis either by the Ca(2+)-ionophore ionomycin or by the inhibitor of protein synthesis cycloheximide. Apoptotic BL cells, but not viable BL cells, were recognized and phagocytosed by monocyte-derived macrophages. bcl-2-transfected BL populations showed a reduced capacity both to undergo apoptosis in response to these inducing agents and to interact with macrophages. Like their Bcl-2- counterparts, Bcl-2+ BL cells interacted with macrophages only after activation of their apoptotic program as assessed by changes in nuclear morphology. These results demonstrate not only that continued protein synthesis in B cells undergoing apoptosis is not essential for their recognition by macrophages, but also that macrophage recognition of apoptotic B cells cannot be uncoupled from the cell-death program that is controlled by Bcl-2. In this respect, the behavior of B cells contrasts markedly with that of neutrophils in which Bcl-2 has been reported to inhibit apoptosis without affecting phagocytic clearance.

Apoptosis↗
PubMed1996

S-adenosyl-L-methionine prevents disruption of canalicular function and pericanalicular cytoskeleton integrity caused by cyclosporin A in isolated rat hepatocyte couplets.

Isolated rat hepatocyte couplets were used to study the effect of S-adenosyl-L methionine (SAMe) treatment on disruption of canalicular function caused by cyclosporin A (CyA). Canalicular function was assessed by counting the percentage of couplets that were able to accumulate the fluorescent cholephile choly-lysyl-fluorescein (CLF) into the canalicular vacuole between the two cells, i.e., canalicular vacuole accumulation (CVA). Cotreatment with 1 mmol/L SAMe prevented the inhibition of canalicular vacuole accumulation caused by CyA (75 nmol/L and 100 nmol/L), whereas treatment with it after CyA was unsuccessful. SAMe prevented the dose dependent reduction caused by CyA (5 nmol/L-1 mumol/L) both on CVA and on retention of CLF preaccumulated within the canaliculus, the effect on retention being complete. No difference in intracellular content of reduced glutathione (GSH) between the control and any dose level of the immunosuppressor, with or without SAMe treatment was observed, suggesting that changes in intracellular reduced GSH levels are not involved in the effects of SAMe. F-actin was stained with fluorescein-isothiocyanate phalloidin and fluorescence measurements were performed by confocal microscopy. The ratio of the percanalicular area fluorescence/total couplet fluorescence, indicative of F-actin distribution, significantly decreased with CyA. However, cotreatment of CyA with SAMe protected the integrity of the pericanalicular cytoskeleton, suggesting that this beneficial effect on canalicular function may maintain canalicular contractions and/or preserve tight junction function. Results are discussed in relation to possible involvement of the transmethylation pathway, modifications in membrane fluidity, effects on bile acid transport, and of inhibition of uptake of CyA. They suggest that SAMe could be a good candidate for protecting against CyA-induced membrane dysfunction.

Actins↗
PubMed1996

Doppa induces cell death but not differentiation of U937 cells: evidence for the involvement of PKC-beta 1 in the regulation of apoptosis.

Recent reports have claimed that activation of protein kinase C (PKC)-beta is sufficient for both differentiation and apoptosis in promyeloid HL60 cells. Phorbol esters which differentially activate PKC isoenzymes in vitro were used to induce differentiation and apoptosis in U937 cells; TPA and Dopp activate all U937 PKC isoenzymes, except PKC-zeta and Doppa activate only PKC-beta l. At concentrations of Doppa below 50 nM, only PKC-beta l was activated by 2 min and apoptosis was induced, but there was no differentiation of cells towards monocytes. TPA (1-25 nM) and Dopp (5-100 nM) activated PKC-alpha, -beta l and-gamma within 2 min and induced differentiation, but only increased apoptosis at the highest concentrations used. Thus, initial activation of PKC-beta l is insufficient for differentiation of U937 cells, but may lead to the induction of apoptosis.

Apoptosis↗
PubMed1996

Expression of subunits of the metalloendopeptidase meprin in renal cortex in experimental hydronephrosis.

Meprin A is a metalloendopeptidase in the proximal tubular epithelium of rodents that is capable of hydrolyzing a great variety of peptides and proteins. The aim of the present investigation was to investigate effects of ureteral ligation on the expression of meprin subunits. Ureteral ligation resulted in marked decreases in the expression of both alpha- and beta-meprin subunits within 12 h of ureteral obstruction. Even greater downregulation of expression of meprin alpha- and beta-mRNA was noted at 24, 48, and 96 h after ureteral ligation. The greatest decrease in meprin mRNA expression in obstructed kidneys over contralateral unobstructed control kidneys (CUK) occurred at 24 h postunilateral ureteral obstruction (post-UUO) for the meprin alpha-subunit (20-fold decrease compared with controls) and at 48 h for the meprin beta-subunit (90-fold decrease). On immunolabeling, the intensity for the two meprin subunits at the corticomedullary junction was dramatically decreased at 24 to 96 h after ureteral ligation in contrast to the CUK specimens. Results of in situ hybridization indicated that the CUK specimens expressed meprin beta-mRNA at the corticomedullary junction, whereas the obstructed kidneys exhibited a decrease in mRNA signal for meprin beta-subunit. There was a steady increase in the interstitial macrophage number in UUO rat kidneys over the 96 h of evaluation post-UUO. ED-1-positive macrophages were observed almost exclusively in the peritubular cortical interstitial space in a ringlike pattern with a preponderance of macrophage clusters around glomeruli. Unexpectedly, after reversal of UUO, the interstitial macrophage number remained higher than controls, despite the demonstrable decompression of the renal pelvis and caliceal system. In summary, this investigation demonstrates downregulation of meprin alpha and beta within hours of UUO and indicates a novel tubular response to ureteral obstruction.

Animals↗
PubMed1996

Differences in the germinal centres of palatine tonsils and lymph nodes.

The germinal centres of human palatine tonsils typically have four clearly recognizable compartments. The dark zone is identified by the presence of centroblasts and a thin follicular dendritic cell (FDC) network. The dense FDC network is divided into a CD23(low/-) portion adjacent to the dark zone (the basal light zone) and a CD23(high) portion, the apical light zone). The outer zone, which lies between the apical light zone and the follicular mantle, has only fine CD23- FDC processes. While these compartments were seen in 48 follicles from eight tonsils, the compartmental structure of 54 germinal centres in lymph nodes from 11 individuals was markedly different. The CD23+ FDC network in lymph node follicles extended into part of the dark zone and the inner part of the follicular mantle, and consequently no outer zone or basal light zone was identified. In both the lymph nodes and tonsils most T cells were CD4+ and located outside the dark zone; the concentration of these cells at the edge of germinal centres was typical of tonsil centres but was noted only occasionally in lymph nodes. The substantial minority of T cells that were CD4,CD57+ were located mainly in the dense FDC network.

Adolescent↗
PubMed1995

IL-4 promotes macrophage development by rapidly stimulating lineage restriction of bipotent granulocyte-macrophage colony-forming cells.

Granulocyte macrophage colony-forming cells (GM-CFC) are bipotential progenitor cells that can proliferate and develop into macrophages in response to macrophage CSF or into neutrophils in response to stem cell factor or granulocyte CSF. These cytokines promoted growth and development in highly enriched GM-CFC. In [3H]thymidine suicide assays, IL-4 was shown to stimulate proliferation of GM-CFC to the same degree as IL-3 and other potent mitogens for GM-CFC. IL-4 also maintained the clonogenic potential of enriched GM-CFC over a 2-day period. However, after several days in the presence of IL-4, the GM-CFC began to die and retained blast cell morphology characteristic of the isolated GM-CFC. When a high concentration of IL-4 was added to GM-CFC with neutrophilic stimuli, the response of these cells was altered because macrophages were formed. This effect was achieved by a 4-h preincubation with IL-4, suggesting that an early signal produced by IL-4 promotes lineage restriction, although IL-4 itself cannot promote development. IL-4, like macrophage CSF, translocates PKC-alpha to the nucleus in GM-CFC, this redistribution of protein kinase C alpha (PKC-alpha) being inhibited by calphostin C (a PKC inhibitor). Calphostin C also blocked IL-4-mediated development of macrophages in stem cell factor- and granulocyte-CSF-treated cells. This is further evidence that PKC-alpha translocation is involved in the commitment of GM-CFC to macrophage development. This data also suggests that agonist-stimulated lineage commitment can be uncoupled from development in normal hematopoietic cells.

Animals↗
PubMed1995

COOH-terminal proteolytic processing of secreted and membrane forms of the alpha subunit of the metalloprotease meprin A. Requirement of the I domain for processing in the endoplasmic reticulum.

Cell surface isoforms of meprin A (EC 3.4.24.18) from mice and rats contain beta subunits that are type I integral membrane proteins and alpha subunits that are disulfide-linked to or noncovalently associated with membrane-anchored meprin subunits. Both alpha and beta subunits are synthesized with COOH-terminal domains predicted to be cytoplasmic, transmembrane, and epidermal growth factor-like; these domains are retained in beta subunits but are removed from alpha during maturation. The present studies establish that an inserted 56-amino acid domain (the "I" domain), present in alpha but not in beta, is necessary and sufficient for COOH-terminal proteolytic processing of the alpha subunit. This was demonstrated by expression of mutant meprin subunits (deletion mutants, chimeric alpha beta subunits, and beta mutants containing the I domain) in COS-1 cells. Mutations of two common processing sites present in the I domain (a dibasic site and a furin site) did not prevent COOH-terminal proteolytic processing, indicating that the proteases responsible for cleavage are distinct from those having these specificities. Deletion of the I domain from the alpha subunit resulted in accumulation of unprocessed subunits in a preGolgi compartment. Furthermore, COOH-terminal proteolytic processing of wild-type alpha subunits occurred before acquisition of endoglycosidase H resistance. Pulse-chase experiments and expression of an alpha subunit transcript containing a c-myc epitope tag, confirmed that proteolytic processing at the COOH terminus occurs in the endoplasmic reticulum. This work identifies the region of the alpha subunit that is essential for COOH-terminal processing and demonstrates that the differential processing of the evolutionarily-related subunits of meprin A that results in a structurally unique tetrameric protease begins in the endoplasmic reticulum.

Amino Acid Sequence↗
PubMed1995

Subcellular localization of pyruvate dehydrogenase dihydrolipoamide acetyltransferase in human intrahepatic biliary epithelial cells.

In previous histological studies, biliary epithelial cells (BEC) in the liver of patients with primary biliary cirrhosis (PBC), but not controls, reacted strongly with antibodies specific for the major autoantigen associated with PBC, the E2 component of pyruvate dehydrogenase complex (PDC-E2). In this study we have used transmission electron microscopy (TEM) to document the precise subcellular localization of PDC-E2 in BEC. Two antibodies which recognize PDC-E2 were used: affinity-purified anti-PDC-E2 raised in rabbits; and human antibody from the serum of patients with PBC, affinity-purified against human heart PDC. The intracellular localization of antibody binding was determined by laser scanning confocal microscopy and TEM. Both antibodies bound to the inner membrane of mitochondria in BEC isolated from both patients with PBC and controls, but binding to the external aspect of the plasma membrane was observed only in BEC from patients with PBC. Surface antigen expression in PBC may make BEC immunological targets.

Adult↗
PubMed1995

Changes in protein kinase C isoenzyme expression associated with apoptosis in U937 myelomonocytic cells.

Spontaneously apoptotic U937 cells from exponentially growing cell cultures were enriched on discontinuous Percoll density gradients. Increased PKC-beta and reduced PKC-zeta expression were detected in apoptotic cells by Western blotting. Using confocal microscopy, changes in the level of PKC isoenzymes were confirmed and in addition alterations in the subcellular location of PKC isoenzymes were detected in apoptotic cells compared with nonapoptotic cells. The data indicate that the expression of specific PKC isoenzymes is modulated during apoptosis and that PKC-beta and PKC-zeta may play specific roles in the regulation of the apoptotic program.

Apoptosis↗
PubMed1995

A novel protein expressed in mammalian cells undergoing apoptosis.

Human and rodent cells undergoing apoptosis were observed to express high levels of a novel 45,000 M(r) protein. The protein, which we have termed apoptosis specific protein (ASP), was found in Burkitt lymphoma (BL) cells and in adenovirus-transformed human and rat embryo cells induced into apoptosis by a variety of stimuli, including serum deprivation, exposure to the Ca2+ ionophore, ionomycin, treatment with inhibitors of macromolecular synthesis (cycloheximide and actinomycin D), and cold shock. In BL cells treated with apoptotic stimuli, expression of the oncoprotein Bcl-2 was found to both protect from apoptosis and prevent expression of ASP. ASP was not detected either in viable cells or in cells dying passively by necrosis. Laser scanning confocal microscopy showed high levels of ASP in the cytoplasm of cells displaying the chromatin condensation and fragmentation patterns typical of apoptosis. Retention of ASP was observed even when DNA was no longer detectable, and two-color immunofluorescence staining indicated that the protein primarily colocalized with, but was clearly distinct from, non-muscle actin. These findings, together with the observation that biochemical extraction of ASP was only possible under conditions which caused solubilization of the cytoskeleton, leads us to conclude that ASP forms part of, or at least strongly associates with, a modified cytoskeleton unique to cells undergoing apoptosis. While elucidation of its function will require further work, ASP constitutes a powerful marker for the diagnosis and quantitation of apoptosis in vivo and in vitro.

Animals↗
PubMed1995

Isolation and characteristics of tonsil centroblasts with reference to Ig class switching.

Most tonsil B cells have high levels of surface CD44 but this molecule is either expressed at low levels or is absent from germinal centre B cells (GCB). On average 62% of isolated GCB were found to be CD44- and the remainder CD44low. Most CD44- GCB were in cell cycle, indicating that they were centroblasts, while centrocytes, non-dividing GCB, were mainly CD44low. Immunohistological analysis confirms that centrocytes, which are located in the light zone of germinal centres, express low levels of CD44, while centroblasts, cells of the dark zone, are CD44-. While most CD77high GCB are centroblasts and CD77low GCB centrocytes, many centroblasts and centrocytes express intermediate levels of CD77, making this less reliable than CD44 for discriminating between these cells. Most CD44low and CD44- GCB were shown to have undergone Ig switch recombination in vivo. This indicates that switch recombination is independent of the maturation of centroblasts to centrocytes and precedes the signals that induce GCB to differentiate to plasma cells or memory B cells. The average rate of entry of the CD44- GCB fraction to apoptosis on culture at 37 degrees C was faster than that of the total GCB preparation. It is suggested that this may reflect strict stromal-dependence of centroblasts while centrocytes have to survive for long enough to have the chance of receiving antigen-specific selection signals. Inhibition of apoptosis by CD40 mAb with IL-4 or phorbol myristate acetate with ionomycin was similar in the CD44- and CD44low preparations.

ADP-ribosyl Cyclase↗
PubMed1995

Expression of fibroblast growth factors in thyroid cancer.

We have examined immunoreactive fibroblast growth factor-1 (FGF-1) and FGF-2 in thyroid sections from normal tissue, follicular adenoma, differentiated follicular and papillary carcinoma, and anaplastic carcinoma. Polyclonal primary antibodies (Dr. A. Baird, Whittier Institute, La Jolla, CA) to FGF-1 and FGF-2 and fluorescein-conjugated secondary antibodies were used with confocal microscopy to allow quantitation and subcellular localization of the antigens. Staining for FGF-1 and FGF-2 was intense in the differentiated malignant tumor specimens, whereas staining in the normal thyroid tissue controls was not detectable above background fluorescence. Staining for FGF-1 and FGF-2 was intracellular and was not found in the nucleus. Staining using either antibody was enhanced in follicular adenomas, but was less intense than that in the malignant tumors. Sections from anaplastic carcinomas also stained positively. In primary cultures of thyroid cells derived from a papillary carcinoma, staining for FGF-2 was 10-fold greater than that from normal thyroid cells from the same patient. The data suggest a possible role for FGFs in the etiology of thyroid carcinoma.

Adenocarcinoma, Follicular↗
PubMed1994

Expression of meprin subunit precursors. Membrane anchoring through the beta subunit and mechanism of zymogen activation.

The biosynthesis of the membrane-bound metalloendopeptidase meprin was studied after the introduction of cDNAs encoding the rat meprin alpha and beta subunits into human 293 cells. When expressed individually the meprin alpha subunit was found to be primarily secreted into the culture medium, while the beta subunit was determined to be an integral membrane protein. Coexpression of the alpha and beta subunits results in the localization of both subunits to the plasma membrane. In this case the alpha subunit is specifically released from the cell surface by dithiothreitol, indicating the alpha subunit is associated with the membrane via disulfide bond(s) to the beta subunit. Meprin expressed in 293 cells is similar to the rat kidney enzyme in that it forms disulfide-linked dimers and has a similar pattern of glycosylation. However, the expressed protein displayed no detectable peptidase activity against four meprin substrates. Processing of the alpha subunit was followed after the introduction of sequences coding for the human c-myc peptide epitope EQKLISEEDL into its cDNA in the region of its prosequence and at the COOH terminus. Detection of the resulting proteins using a monoclonal antibody specific for the c-myc epitope indicates the alpha subunit is processed at its COOH terminus but retains the prosequence which is absent from the enzyme purified from rat kidney. Limited trypsin digestion of meprin precursors expressed in 293 cells results in both the activation of the enzyme and the removal of the prosequence. This result supports the hypothesis of Bode et al. (Bode W., Gomis-Ruth, F. X., Huber, R., Zwilling, R., and Stocker, W. (1992) Nature 358, 164-167) that meprin and other astacin family proteases require removal of NH2-terminal prosequences at the junction of the astacin protease domain for zymogen activation. Trypsin-activated meprin was assayed with the protein substrate azocasein and three synthetic peptide substrates. The membrane-bound beta subunit was found to be more active than the secreted alpha subunit against azocasein but much less active toward the synthetic peptide substrates.

Amino Acid Sequence↗
PubMed1994

Effect of oxidative stress and disruption of Ca2+ homeostasis on hepatocyte canalicular function in vitro.

Isolated rat hepatocyte couplets were used to study the effects of menadione and a rise in the intracellular concentration of calcium on biliary canalicular function. Canalicular function was assessed by counting the percentage of couplets which were able to accumulate the fluorescent cholephile, cholyl lysyl fluorescein (CLF) into the canalicular vacuole between the two cells. Menadione induced a concentration-dependent inhibition of the canalicular vacuole accumulation (CVA) of CLF reaching 7.6 +/- 1.8% of control at 100 microM menadione. This disruption was not prevented by blocking receptor-operated calcium channels with Ni2+ (300 microM). The concentration range of menadione used did not deplete cellular ATP content. In contrast glutathione content was reduced to 52% of its control value by 100 microM menadione. A rise in cytosolic calcium induced by the calcium ionophore, A23187 (up to 30 microM) also disrupted CVA in a concentration-dependent manner. Release of endoplasmic reticulum calcium stores by thapsigargin (50 nM) affected the retention of canalicular contents to a much lesser extent, although it was able to stimulate a reduction in canalicular area to 40% of its original value, assumed to be due to canalicular contraction. Menadione (30 and 100 microM) reduced the fluorescence of phalloidin-FITC-labelled F-actin in both the total and pericanalicular cytoskeleton. Canalicular function was therefore disrupted by non-lethal concentrations of menadione via a mechanism which does not appear to involve ATP depletion or the entry of extracellular calcium, but is associated with a depletion of both cellular glutathione and F-actin. An increase in the concentration of intracellular calcium can stimulate canalicular contraction, and at relatively high concentrations calcium can also disrupt canalicular function.

Actins↗
PubMed1994

Classification of CEA-related positivity in primary and metastatic malignant melanoma.

Using a panel of antibodies to carcinoembryonic antigen (CEA), in paraffin-processed biopsy material patchy, predominantly membranous positivity was seen on tumour cells in 70 per cent of cases of superficial spreading melanoma, 60 per cent of nodular melanomas, and 75 per cent of secondary deposits studied with unabsorbed polyclonal anti-CEA only. No staining was seen using monoclonal anti-CEAs. Localization of CEA to the cell membrane was confirmed with confocal microscopy. Immunoblotting of fresh frozen material detected CEA of around 180 kD in both primary and metastatic melanomas migrating with an apparent molecular weight of between 150 and 200 kD, indicating variable glycosylation of the protein. Recognition of an adhesive role for CEA with roles in immunolocalization and immunotherapy emphasizes the importance of more precise classification of CEA-related positivity in human tumours.

Carcinoembryonic Antigen↗