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P R Walker

Publications and source records attributed to P R Walker.

At least 55 records · Page 3Linked to original sources
PubMed1996

Degradation of nuclear matrix and DNA cleavage in apoptotic thymocytes.

In dexamethasone-treated thymocyte cultures an increase in nuclear proteolytic activity paralleled chromatin fragmentation and the appearance of small apoptotic cells. The elevation of nuclear proteolytic activity was accompanied by site-specific degradation of nuclear mitotic apparatus protein and lamin B, two essential components of the nuclear matrix. Nuclear mitotic apparatus protein phosphorylation and cleavage into 200 and 48 kDa fragments occurred within 30 minutes of dexamethasone treatment. Cleavage of lamin B, which generated a fragment of 46 kDa consistent with the central rod domain of the protein, was also detected after 30 minutes of exposure to the steroid hormone. The level of lamin B phosphorylation did not change as a result of the dexamethasone treatment and the lamina did not solubilize until the later stages of apoptosis. Initial DNA breaks, detected by the terminal transferase-mediated dUTP-biotin nick end labeling assay, occurred throughout the nuclei and solubilization of lamina was not required for this process to commence. The data presented in this paper support a model of apoptotic nuclear destruction brought about by the site-specific proteolysis of key structural proteins. Both the nuclear mitotic apparatus protein and lamin B were specifically targeted by protease(s) at early stages of the cell death pathway, which possibly initiate the cascade of degradative events in apoptosis.

Animals↗
PubMed1995

Distinct phenotypes of antigen-selected CD8 T cells emerge at different stages of an in vivo immune response.

We have previously described a unique system for identifying Ag-selected CD8 T cells during an in vivo response in normal mice. In this system, lymphocytes isolated from DBA/2 mice injected i.p. with HLA-CW3 transfected syngeneic (H-2d) P815 cells show a remarkable expansion of CD8 cells that utilize TCR expressing the V beta 10 gene segment and additional structural features characteristic of Kd-restricted CW3-specific CTL clones. We have now taken advantage of this system to characterize the surface phenotype of CD8 cells selected by Ag in vivo. We observed several distinct phenotypes at different stages of the response. At the peak of the response, Ag-selected cells were low in CD62L and CD45RB expression but displayed high levels of CD44. In addition, there was a partial down-regulation of CD8 and TCR. Cells of this phenotype were present in lymphoid tissues for several mo after immunization. Much later in the response, Ag-selected cells expressed higher levels of CD8 and TCR. Moreover, a distinct subset of these long-term immune cells emerged that now expressed CD62L and CD45RB. Analysis of CD8 cells from different tissues also revealed certain differences, particularly in TCR and co-receptor levels from liver-derived cells compared with circulating cells at the peak of the response. Our findings suggest that the function of Ag-selected CD8 cells may be regulated over time and according to location by subtle changes in cell-surface phenotype.

Animals↗
PubMed1995

Reversal of atropine-resistant atrioventricular block with intravenous aminophylline in the early phase of inferior wall acute myocardial infarction following treatment with streptokinase.

We report three patients with acute inferior myocardial infarction treated with aspirin (150 mg) and streptokinase (1.5 MU over 60 min), who developed atropine-resistant bradyarrhythmias during or immediately following streptokinase. The bradyarrhythmias responded to aminophylline, thus avoiding the need for temporary pacing.

Aged↗
PubMed1995

Degradation of chromatin in apoptotic cells.

We present here a model for the degradation of chromatin in cells undergoing apoptosis. This model rationalises all aspects of the fragmentation process that have been described to date, explaining not only the patterns of degradation seen within individual cells, but also the variability in extent of degradation seen in different cells. Although DNA fragmentation in apoptosis was initially considered to be solely internucleosomal, it is now apparent that the process is much more complex and most, if not all, cells also produce much larger DNA fragments. However, in the same way that internucleosomal DNA fragmentation is a reflection of chromatin structure, the generation of these larger fragments is a reflection of chromatin structure, too. By comparing the ionic requirements for the complete pattern of chromatin degradation in nuclei with those required for apoptosis, it is apparent that the whole process may be catalysed by two pools of Mg-activated\Ca-modulated DNase I-like enzyme activities.

Journal Article↗
PubMed1994

Endonuclease activities associated with high molecular weight and internucleosomal DNA fragmentation in apoptosis.

We have used pulsed-field gel electrophoresis and electron microscopy to correlate stages of DNA fragmentation with alterations of nuclear structure during apoptosis. DNA fragmentation occurs in two stages. The first is initiated by a previously undescribed endonucleolytic activity that cleaves DNA into 50- to 300-kb fragments. Electron microscopy showed that this degree of cleavage was sufficient to cause the chromatin to undergo condensation. The second stage of fragmentation is catalyzed by the previously described calcium-magnesium endonuclease. The enzyme activity responsible for the initial fragmentation of DNA was found to be distinct from that causing subsequent internucleosomal DNA cleavage based upon its cation requirements, independence of proteolysis and lack of inhibition by zinc. Both activities were found to preexist in nuclei from thymocytes, liver, HL60, and IL2-dependent CTLL cells. Thus, in apoptosis DNA degradation involves two distinct endonucleolytic activities, with only the first activity being essential for cell death.

Animals↗
PubMed1994

Role of topoisomerase II in the structural and functional evolution of mitogen-stimulated lymphocyte nuclei.

To examine the role of DNA topoisomerase II (Topo II) in the mitogenic activation of mouse lymphocytes, we applied the Topo II inhibitor VM26 throughout the stimulation period and monitored morphological and functional parameters of lymphocyte activation. Cell viability and the usual increase in cell size were little affected at doses between 0.05 and 0.5 microM. DNA synthesis, however, was already significantly inhibited at 0.05 microM, with RNA synthesis inhibited to a lesser extent. Light microscope autoradiography showed that a smaller proportion of cells entered S phase, with each S phase cell incorporating less [3H]thymidine. In immunofluorescence studies, the nucleolar antigen fibrillarin was reduced, although only minor effects on the snRNP Sm antigen and the internal component labeled by antibody PI1 were observed. At the electron microscope level, nucleoli were remodeled and chromatin became aggregated. At a high dose of VM26 (5 microM), cells showed the expected high levels of apoptosis and strong inhibition in all activation parameters assayed. The results support the hypothesis that the Topo II beta isoform is involved in the very early phases of lymphocyte activation, with function of the Topo II alpha isoform, which is more sensitive to VM26, being required for progression through S phase.

Animals↗
PubMed1994

Evidence for trans regulation of apoptosis in intertypic somatic cell hybrids.

The genetic components required for glucocorticoid induction of apoptosis were studied by using somatic cell hybridization. Intertypic whole-cell hybrids were generated by crossing the glucocorticoid-resistant rat liver cell line Fado-2 with the glucocorticoid-sensitive mouse thymoma cell line BW5147.3. Morphological and biochemical criteria were used to assess sensitivity or resistance to glucocorticoid-induced cell death. Both phenotypes were observed, and all of the hybrids retained a functional glucocorticoid receptor as judged by their abilities to induce the metallothionein gene in response to dexamethasone (Dex). Sensitivity to apoptosis did not correlate with morphological phenotype in that not all suspension cells were sensitive. The effect of glucocorticoids on the expression of apoptosis-linked genes was analyzed in a subset of Dex-sensitive and Dex-resistant hybrids. p53 and c-myc mRNAs were present in parental cells as well as sensitive and resistant hybrid cells, and their levels were not affected by glucocorticoid treatment. bcl-2 expression was restricted to the thymoma cell line and was also not affected by glucocorticoids. We did not detect any bcl-2 mRNA in the hepatoma cell line and the hybrids, suggesting that, as with most tissue-specific genes, bcl-2 is regulated in trans. Furthermore, while the majority of hybrids analyzed retained a full complement of mouse chromosomes, sensitive hybrids were missing some rat chromosomes (preferentially chromosomes 16 and 19), indicating that apoptosis is subject to trans repression. Resistant cells thus appear to repress the activity or synthesis of a nuclear factor that interacts with a glucocorticoid-dependent gene(s) to activate the cell death pathway.

Animals↗
PubMed1993

Identification of a new cAMP response element-binding factor by southwestern blotting.

We have identified in mammalian cells a novel cyclic AMP response element (CRE)-binding protein of molecular mass 47 kDa. This protein was not recognized by either the CREB-327/341 or c-Jun antisera, and its tissue distribution did not overlap with those of the CREB and Jun families. For example, hepatoma and placental tissue did not contain the 47-kDa DNA-binding protein, but did contain the CREB isoforms. On the other hand, S49 lymphoma cells contained a high level of the 47-kDa DNA-binding protein but did not contain a 47-kDa Jun-related protein which was found in normal liver and hepatoma. This new 47-kDa factor bound to the CRE in the dephosphorylated form, and phosphorylation of the protein by the catalytic subunit of protein kinase A completely abolished its DNA-binding activity. The isoforms of the CREB-327/341 family, on the other hand, bound to DNA in the phosphorylated form, and alkaline phosphatase treatment reduced significantly their interaction with CRE sequence. This reverse effect of phosphorylation/dephosphorylation on the DNA-binding property of this new 47-kDa protein in particular distinguishes it from other known CREB factors and suggests that the protein might play a unique role in the regulation of cAMP-mediated transcription.

Animals↗
PubMed1993

Apoptotic death in epithelial cells: cleavage of DNA to 300 and/or 50 kb fragments prior to or in the absence of internucleosomal fragmentation.

To date, apoptosis has been characterized biochemically by the production of 180-200 bp internucleosomal DNA fragments resulting from the activation of an endonuclease(s). The principal morphological feature of apoptosis is the condensation of chromatin and it has been assumed that this may reflect the oligonucleosomal fragmentation pattern. We have re-examined this dogma by comparing the biochemical and morphological features of cell death in several epithelial cell types (HT-29-I1 colon adenocarcinoma, CC164 mink lung, DU-145 human prostatic carcinoma and MCF-7 human breast adenocarcinoma) and one mesenchymal cell line (H11ras-R3 ras-transformed rat fibroblasts). Cell death was induced either by serum deprivation, TGF-beta 1 or etoposide, or by leaving cells to reach confluence. Cell death was assessed with respect to detachment from monolayers, morphological changes and DNA integrity. The DNA-binding fluorophore Hoechst 33258 revealed chromatin condensation patterns consistent with apoptotic cell death in all cell types except MCF-7 cells. Using field inversion gel electrophoresis in conjunction with conventional 2% agarose gel electrophoresis, cleavage of DNA to 50 kbp fragments was observed in all cases except MCF-7 cells. This preceded the appearance of oligonucleosomal fragments in HT-29-I1, CC164 and H11ras-R3 cells. Although the DNA of DU-145 cells fragmented into 50 kbp units, and although the cells exhibited classical apoptotic morphology, no subsequent internucleosomal cleavage was observed. These results suggest that changes in the integrity of DNA indicative of the release of chromatin loop domains occur before cleavage at internucleosomal sites is initiated and that the latter is not an essential step in the apoptotic process.

Animals↗
PubMed1993

Relationship between apoptosis and the cell cycle in lymphocytes: roles of protein kinase C, tyrosine phosphorylation, and AP1.

The mechanism of switching between the cell cycle and active cell death (apoptosis) was investigated in cytokine-dependent CTLL cells. These cells proliferate in the presence of interleukin 2 (IL2), but accumulate in early G1 and undergo apoptosis in its absence. In the absence of IL2 the cells also become sensitive to glucocorticoid-induced apoptosis. Using specific inhibitors of protein kinase C and tyrosine kinases we established that two signals are required to fully repress cell death and stimulate G1 progression. One of these signals activates protein kinase C (PKC) which represses cell death and the other activates a tyrosine kinase which confers glucocorticoid resistance and permits cell cycle progression. Thus, phorbol esters can activate PKC and maintain cell viability in the absence of IL2, but the cells cannot proliferate. Moreover, the cells remain sensitive to glucocorticoid-induced apoptosis unless the tyrosine kinase-mediated signal is also given. There is a correlation between the presence of AP1 DNA-binding activity and the repression of the cell death pathway. The c-jun gene is expressed constitutively and both IL2 and phorbol esters induce the expression of c-fos to generate a functional AP1 capable of repressing cell death. However, only interleukin 2 can initiate the tyrosine kinase-mediated modification that confers dexamethasone resistance and permits G1 progression. In the absence of IL2 glucocorticoids stimulate AP1 degradation and induce apoptosis.

Adaptor Protein Complex 1↗
PubMed1993

Two-stage cell shrinkage and the OER for radiation-induced apoptosis of rat thymocytes.

Suspensions of rat thymocytes were given 0.09-100 Gy using 60Co gamma-rays. The radiation-induced changes in the thymocytes were examined from minutes to hours post-irradiation using electron microscopy, agarose gel electrophoresis, staining and Coulter Counter sizing. Sizing by Coulter Counter showed, for the first time, that thymocytes which undergo apoptosis shrink in two distinct stages, first by a sudden decrease from an original volume of 99 microns 3 to a volume of 76 microns 3, followed by a gradual decrease to 57 microns 3 over the space of a few hours. The oxygen enhancement ratio for apoptosis was measured to be about 3.5, similar to the value for reproductive death for many mammalian cells.

Animals↗
PubMed1993

Mapping major and minor T-cell epitopes in vitro and their immunogenic or tolerogenic effect in vivo in non-human primates.

The immunogenicity of synthetic peptides of in vitro mapped T- and B-cell epitopes from a Streptococcus mutans cell-surface antigen were investigated in non-human primates. Peptide (1-15) contains T-cell (7-15) and B-cell (8-13) epitopes, but is only immunogenic if dimerized (1-15)2 or linked to the carrier tetanus toxoid (1-15)TT. Monomers and dimers of T- and B-cell epitopes were prepared and used to immunize macaques. Immunogenicity was assayed in lymphocytes by the uptake of [3H]thymidine and serum antibodies by a solid-phase radioimmunoassay. Macaques immunized with the dimerized (1-15)2 or carrier-linked peptide (1-15)TT exhibited in vitro T-cell proliferative responses to peptides (1-15) and (7-15). T cells from animals immunized with peptides (1-15), (7-15) or (7-15)2 failed to elicit an immune response. In order to establish if these non-immunogenic peptides might induce tolerance, the same macaques were challenged with the immunogenic peptide (1-15)TT. The results suggest that T-cell responses to peptide (1-15) were reestablished, but instead of responding to peptide (7-15) they were stimulated by a hitherto silent epitope (1-7). Tolerance to the major T-cell epitope (7-15) and the expression of a minor (silent) T-cell epitope (1-7) was associated with B-cell tolerance, suggesting that T-cell help for antibodies resides in the major T-cell epitope (7-15). However, short-term T-cell lines revealed T-cell responses to peptides (1-7) and (7-15) in both tolerized and immunized macaques, but the relative frequency of the minor epitope (1-7)-reactive lines was significantly higher in tolerized animals, whilst that for the major epitope (7-15) was higher in immunized animals. These findings suggest that the silent epitope (1-7) is really cryptic, in that it can be detected if the cell lines are first expanded in vitro with the whole peptide (1-15) and then stimulated with the truncated peptides (1-7) or (7-15). The results are consistent with the concept of a hierarchy of major and minor T-cell epitopes, now demonstrated in non-human primates, in which tolerance to the major T-cell epitope is associated with tolerance to antibody formation and the emergence of a minor T-cell epitope.

Animals↗
PubMed1993

Evidence that an additional conserved element with the consensus C/GAGA/C is essential for maximal responsiveness of the cyclic AMP enhancer.

We analyzed the ability of cyclic AMP-response element binding proteins (CREBs) to interact with the CRE sequences derived from different genes and examined the role of sequences flanking the core CRE element in rendering cAMP-responsiveness to the enhancer. We were able to detect reproducibly, sing the Southwestern blotting technique, five major CREB factors of molecular weights 56, 47, 40, and 36-34 kDa which were present in various rat tissues and cultured cells. The 34-40 kDa proteins (CREB-327/341) were able to bind to the CRE of cAMP-inducible genes (somatostatin, c-fos, E2A), but not to genes whose expression is not controlled by cAMP (glucagon, parathyroid hormone). The novel 47 kDa CREB had a high specificity for the core octameric CRE sequence and it bound equally well to the consensus CRE of cAMP-inducible and noninducible genes. On the other hand, the 47 kDa CREB did not bind at all to the phorbol ester response element (TRE), whereas the 56 kDa protein, reminiscent of the CRE-BP1 protein, could bind to both elements. A computer aided sequence analysis of cAMP-inducible gene promoters revealed the presence of an additional conserved element starting 4-6 nucleotides 3' to the octomer with the consensus C/GAGA/C. We have shown this element to be essential for maximal cAMP-responsiveness of the enhancer in transient expression assays of CRE-CAT plasmid constructs indicating that the functional interaction of CREB proteins with the cAMP-inducible enhancer involves an additional 8-10 base pairs immediately downstream from the CRE core element.

Animals↗
PubMed1993

Detection of the initial stages of DNA fragmentation in apoptosis.

DNA fragmentation during apoptosis proceeds through an ordered series of stages commencing with the production of DNA fragments of 300 kbp, which are then degraded to fragments of 50 kbp. The 50-kbp fragments are further degraded, in some but not all cells, to smaller fragments (10-40 kbp) and release the small oligonucleosome fragments that are recognized as the characteristic DNA ladder on conventional agarose gels. Methodology is presented for the detection of the initial stages of DNA fragmentation using pulsed-field gel electrophoresis or a combination of pulsed-field gel electrophoresis and conventional agarose gel electrophoresis that allows detection of the DNA ladder in the same sample. A new method for the detection of high molecular weight DNA fragments on conventional agarose gels is presented, together with a rationale for the analysis of DNA fragmentation during apoptosis.

Animals↗
PubMed1992

The topoisomerase II inhibitor teniposide (VM-26) induces apoptosis in unstimulated mature murine lymphocytes.

This study shows that not only concanavalin A-stimulated proliferating lymphocytes but also unstimulated mouse splenic lymphocytes are sensitive to the topoisomerase II (topo II) inhibitor teniposide (VM-26). When unstimulated lymphocytes are pretreated with VM-26 for a 2-h period and are then incubated in drug-free medium, cell viability, as determined by trypan blue exclusion, decreases to 40% of the control by 6 h. The drug-treated cultures show two to three times the level of detergent soluble DNA than the control cultures and agarose gel electrophoresis of the soluble DNA shows the presence of oligonucleosomal-sized fragments, a feature considered to be a hallmark of apoptosis. Phase contrast microscopy, Hoechst staining for DNA, and immunofluorescence microscopy of various nuclear and cytoplasmic antigens (nucleolar fibrillarin, snRNP, ubiquitin, vimentin, tubulin) in the VM-26-treated cells characterize the morphological changes during apoptosis of these cells. The role of topo II as the mediator of the VM-26 effects is supported by pulsed field gel electrophoresis, which shows the typical topo II-induced cleavage of supercoiled DNA into loop-sized 300- and 50-kbp fragments. We conclude that the cancer chemotherapeutic agent VM-26 interacts with topo II and induces apoptosis in unstimulated lymphocytes.

Animals↗
PubMed1991

Characterization of streptococcal antigen-specific CD8+, MHC class I-restricted, T cell clones that down-regulate in vitro antibody synthesis.

T cell clones were generated from the peripheral blood of rhesus monkeys that had been immunized with a soluble Mr 185,000 Ag (SAI/II) derived from Streptococcus mutans. The clones were CD3+ CD8+ CD4- alpha beta TCR+ and were specifically stimulated to proliferate by SAI/II. The proliferative responses of the cloned cells were class I restricted, as demonstrated by reconstitution of the cloned T cells with APC matched at various MHC class I and II loci, as well as by inhibition with anti-class I and not anti-class II mAb. The function of the CD8+ cloned cells was examined in vitro for their effect on antibody synthesis by Ag-stimulated CD4+ cells and B cells from immunized animals. Indeed, four of the five clones suppressed SAI/II-specific IgG antibody synthesis when activated with SAI/II and the appropriate MHC-matched APC. Although activation of the suppressor clones was Ag specific, the effector function of the suppression of antibody synthesis was Ag nonspecific. The latter was probably mediated by lymphokines and, indeed, the culture supernatant generated by stimulating the cloned CD8+ cells with anti-CD3 mAb suppressed both the specific and nonspecific antibody synthesis. Cytotoxicity studies showed that all five CD8+ clones showed a low level of lectin-dependent cytotoxicity. However, because four of the five clones expressed significant suppression of antibody synthesis, the suppressor activity was unlikely to be a function of the weak cytotoxicity. The results suggest that immunization of rhesus monkeys with a soluble streptococcal Ag induced CD8+ alpha beta TCR+ T cell clones that show SAI/II-specific, MHC class I-restricted proliferative responses and nonspecific down-regulatory function of in vitro antibody synthesis.

Animals↗