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J D Walters

Publications and source records attributed to J D Walters.

At least 37 records · Page 2Linked to original sources
PubMed1995

Gangliosides inhibit PDGF-induced signal transduction events in U-1242 MG human glioma cells.

In this study we investigated the responses of intracellular calcium ([Ca2+]i) and protein kinase C (PKC) to PDGF in U-1242 MG cells. PDGF-BB stimulated [3H]PDBu binding approximately 2-3 fold. This response was inhibited by preincubating the cells with an inhibitor of phospholipase C (PLC), U73122, suggesting that PLC mediates the induction of PKC translocation by PDGF. PDGF also increased the concentration of [Ca2+]i that was attenuated in a calcium-free medium. This indicates that PDGF-induced elevation of [Ca2+]i is mainly due to influx of extracellular calcium. PDGF-stimulated translocation of PKC was inhibited by the intracellular calcium buffer BAPTA/AM. All gangliosides studied except GM3 inhibited these responses with similar efficacy. Collectively, these results indicate that the signal transduction pathway initiated by PDGF leading to PKC translocation in U-1242 MG cells is intact, and this pathway is inhibited by several gangliosides.

Calcium↗
PubMed1995

Polyamines found in gingival fluid enhance the secretory and oxidative function of human polymorphonuclear leukocytes in vitro.

Many bacterial and host cells contain large amounts of polyamines that can be released at infection sites as a result of cell lysis. Consequently, the putrescine and spermidine content of gingival fluid from inflamed periodontal pockets (0.1 to 1 mM) is sharply elevated in comparison to peripheral blood. At these levels, polyamines potentiated fMet-Leu-Phe-induced Ca2+ signaling in polymorphonuclear leukocytes (PMNs) in vitro. Consistent with the essential role of Ca2+ signaling in PMN activation, secondary granule release and superoxide anion production by fMet-Leu-Phe-stimulated PMNs was enhanced in the presence of polyamines. Thus, polyamines may play a local role in modulating the antimicrobial activity of PMNs in periodontal disease.

Calcium↗
PubMed1995

Polyamines found in gingival fluid inhibit chemotaxis by human polymorphonuclear leukocytes in vitro.

Putrescine and spermidine occur at concentrations approaching 1 mM in gingival fluid at diseased periodontal sites. Previous work demonstrates that these polyamines potentiate Ca2+ signaling in polymorphonuclear leukocytes (PMNs), resulting in enhanced degranulation and superoxide generation. The present study extends this work by characterizing the effects of polyamines on PMN chemotaxis and phagocytosis, in which Ca2+ signaling plays a less defined regulatory role. Putrescine (1 mM) and spermidine (0.1 to 0.5 mM) significantly inhibited chemotaxis to fMet-Leu-Phe and C5a (P < 0.05). This inhibition was not strongly related to any effect polyamines have on PMN adhesion, actin polymerization, or formyl peptide receptor expression. Neither putrescine nor spermidine had a significant impact on phagocytosis of opsonized bacteria by PMNs. Thus, at concentrations similar to those found in gingival fluid, polyamines could potentially inhibit recruitment of PMNs to diseased pockets without impairing their ability to engulf invading bacteria.

Analysis of Variance↗
PubMed1994

Ethics decision-making in Australian hospitals.

OBJECTIVE: To describe the methods used to resolve ethical issues in hospitals throughout Australia. DESIGN: A descriptive study from data collected by a questionnaire survey of Australian public and private hospitals. PARTICIPANTS: Hospital administrators and employees nominated by hospital administrators. RESULTS: Completed questionnaires were received from 739 (74%) hospitals. Methods used to resolve ethical issues included personal reflection, discussion between health professionals and discussion between health professionals and patients. A variety of committees were identified as having some role in the discussion of ethical issues, but only a small number of hospitals had established ethics committees. CONCLUSION: Doctor-patient interaction and discussion between practitioners are the most common methods used to resolve ethical issues. Various committees are also used and, of these, ethics committees are not the most commonly consulted.

Australia↗
PubMed1994

Ethical issues in Australian hospitals.

OBJECTIVE: To identify the most common ethical issues of concern in Australian hospitals. DESIGN: A descriptive study using data collected by means of a questionnaire. PARTICIPANTS: Seven hundred and thirty-nine (74%) hospital administrators and employees nominated by hospital administrators in Australian public and private hospitals. RESULTS: Over half the respondents reported that ethical concerns had been raised in relation to: making "not for resuscitation orders"; the treatment of patients with HIV and AIDS; interprofessional conflict; and the allocation of resources. CONCLUSION: "End of life" concerns, patient autonomy issues, questions of resource distribution, and communication difficulties commonly raise ethical concerns in Australian hospitals.

Australia↗
PubMed1994

Polyamine transport in human promyelocytic leukemia cells and polymorphonuclear leukocytes.

We examined the kinetics of polyamine uptake by human myeloid cells at different stages of maturity. The Km values of putrescine, spermidine and spermine transport by HL-60 cells were 52, 7.9 and 8.1 microM, respectively. These values decreased to 5.1, 1.7 and 0.77 microM, respectively, in HL-60 cells induced to mature past the promyelocyte stage by DMSO. In human PMNs, the respective Km values were 501, 479 and 381 microM. Transport by HL-60 cells was enhanced when intracellular polyamine levels were reduced with difluoromethylornithine. Thus, HL-60 cell maturation is accompanied by an increase in the affinity of their polyamine transport system. This system is much more efficient than that found in end-stage PMNs, suggesting that it plays a more important role in supporting the metabolic requirements of HL-60 cells. Alternatively, the low affinity of the PMN polyamine transport system could represent an adaptation to the high polyamine concentrations found at infection sites.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1994

Signal transduction mechanisms in mesenchymal cells.

Mesenchymal cells are continually stimulated by a wide spectrum of biological mediators. These mediators bind to receptors on the cell surface and initiate a cascade of signaling events. The initial signal transduction pathways known to be stimulated in mesenchymal cells included phospholipase C, phospholipase D, phospholipase A2, adenylate cyclase, receptor tyrosine kinases, and receptor serine/threonine kinases. These pathways are reviewed and specific applications for therapeutic intervention in wound healing and regenerative therapy in the periodontium are discussed.

Adenylyl Cyclases↗
PubMed1992

Polyamines enhance calcium mobilization in fMet-Leu-Phe-stimulated phagocytes.

Spermidine and putrescine (50 microM-1 mM), found in exudates from infection sites, significantly enhance fMet-Leu-Phe-induced Ca2+ mobilization in differentiated HL-60 cells and polymorphonuclear leukocytes (PMNs) by delaying the return to basal cytosolic Ca2+ levels. This enhancement by polyamines is associated with inhibition of Ca2+ efflux across the plasma membrane. In parallel with their effects on Ca2+ signaling, polyamines also significantly prolong the kinetics of fMet-Leu-Phe-induced protein kinase C translocation. Thus, polyamines may play a novel role in modulating regulatory events in phagocytes.

Calcium↗
PubMed1992

The relationship of gingival fluid leukocyte elastase activity to gingival fluid flow rate.

Recent studies have suggested that leukocyte elastase activity (EA) in tissue exudates is an indicator of inflammatory disease. We assayed gingival fluid (GF) EA with a selective peptide substrate and compared it to GF flow rate with regard to its ability to detect differences in the clinical status of existing inflammatory periodontal disease in 56 human subjects. Compared to healthy sites (Gingival Index = 0, 1 to 3 mm) and mild gingivitis sites (GI = 1, 2 to 5 mm), mean GF EA was significantly (P < 0.05) higher at periodontitis sites with deep probing depths (GI = 2, 6 to 9 mm depth), but not at periodontitis sites with intermediate probing depths (GI = 2, 4 to 5 mm). When expressed as specific EA (i.e., normalized to GF protein content), mean EA was also significantly higher at deep periodontitis sites compared to healthy sites and mild gingivitis sites. In addition, specific EA was significantly higher at periodontitis sites with intermediate probing depths than at healthy sites. As predicted by previous studies, these significant increases in specific EA were associated with significant increases in mean GF flow rate. In contrast to specific EA, however, mean GF flow rate was significantly higher at gingivitis sites than at healthy sites. A strong correlation was observed between GF flow rate and specific EA (rs = 0.737, P = 0.0006). Thus, GF flow rate and GF EA appear to be related indicators of inflammation, but GF flow rate may be more sensitive to early inflammatory changes leading to mild gingivitis.

Alveolar Bone Loss↗
PubMed1991

A continuous fluorescence assay for protein kinase C.

A 6-acryloyl-2-dimethylaminonapthalene (acrylodan)-labeled 25-amino acid peptide (acrylodan-CKK-KKRFSFKKSFKLSGFSFKKNKK-COO-), containing the protein kinase C (PKC) phosphorylation sites of brain myristoylated alanine-rich kinase C substrate protein, undergoes a 20% fluorescence decrease when it is phosphorylated by phospholipid/calcium-dependent protein kinase (PKC). This fluorescence decrease is dependent on the presence of PKC, calcium (half-maximal stimulation at pCa = 6.2), phosphatidylserine, diacylglycerol, or phorbol-12-myristate-13-acetate (half-maximal stimulation at 2 nM) and ATP, and correlates well (r = 0.997) with [32P]phosphate incorporation into the peptide. This fluorescence assay allows detection of 0.02 nM PKC, while similar concentrations of cyclic AMP-dependent or type II calmodulin-dependent protein kinases produced no change in peptide fluorescence. The method can be used to assay purified PKC as well as activity in crude brain homogenates. Incubation of PKC with staurosporine inhibits the fluorescence decrease with an IC50 of 2 nM. Thus the fluorescence decrease that occurs in the acrylodan-peptide provides a continuous fluorescence assay for PKC activity.

2-Naphthylamine↗
PubMed1991

Phosphorylation-dependent binding of a synthetic MARCKS peptide to calmodulin.

A 25-amino acid peptide, containing the four protein kinase C (PKC) phosphorylation sites and the calmodulin (CaM) binding domain of the myristoylated alanine-rich C kinase substrate (MARCKS) protein, has been synthesized and used to determine the effects of phosphorylation on its binding and regulation of CaM. PKC phosphorylation of this peptide (3.0 mol of Pi/mol of peptide) produced a 200-fold decrease in its affinity for CaM. PKC phosphorylation of the peptide resulted in its dissociation from CaM over a time course that paralleled the phosphorylation of 1 mol of serine/mol of peptide. The peptide inhibited CaM's binding to myosin light chain kinase and CaM's stimulation of phosphodiesterase and calcineurin. PKC phosphorylation of the peptide resulted in a rapid release of bound CaM, allowing its subsequent binding to myosin light chain kinase (t1/2 = 1.6 min), stimulation of phosphodiesterase (t1/2 = 1.2 min) and calcineurin (t1/2 = 1.7 min). Partially purified MARCKS protein produced a similar inhibition of CaM-phosphodiesterase which was reversed by PKC phosphorylation. PKC phosphorylation of the peptide occurred primarily at serine 8 and serine 12, and phosphorylation of serine 12 regulated peptide affinity for CaM. Thus, PKC phosphorylation of the peptide and the MARCKS protein results in the rapid release of CaM and the subsequent activation of CaM-dependent enzymes. This process might allow for interplay between PKC and CaM-dependent signal transduction pathways.

2-Naphthylamine↗
PubMed1991

Signal transduction in human B cells during aging: alterations in stimulus-induced phosphorylations of tyrosine and serine/threonine substrates and in cytosolic calcium responsiveness.

Protein phosphorylation is considered an early cellular mechanism of signal transduction by surface immunoglobulins (sIg) and other receptors of B cells. Using intact human peripheral blood B cells of young subjects labeled with orthophosphate, increased phosphorylation levels of serine/threonine and tyrosine substrates were demonstrated on indicator phosphoproteins corresponding to the CD20 isoforms and microtubule-associated protein 2 kinase after cross-linking sIg and costimulation with phorbol diesters. By contrast, stimulated B cells from certain elderly subjects displayed substantial alterations in the phosphorylation patterns of serine/threonine or tyrosine indicator phosphoproteins. Also, age-related impairments in sIg stimulated mobilization of cytosolic protein kinase C (PKC) enzymatic activity and in cytosolic calcium [Ca2+]i responses of B cells were observed with the altered phosphorylation reactions. Comparison of the substrate phosphorylation profiles to the proliferative responses of stimulated B cells from individual elderly subjects suggested a model of signal transduction in which differing stimuli have different dependencies on phosphorylation reactions. Diminished proliferative responses after sIg ligation coincided with decreased phosphorylations of either tyrosine or serine/threonine indicator substrates. However, the decreased proliferative responses of B cells from elderly subjects with substantial reductions of tyrosine phosphorylation after sIg ligation were enhanced by the direct stimulation of serine/threonine kinase activity with phorbol diesters or CD40 ligation. Experiments with kinase inhibitors evaluated the relative dependency of different B cell stimuli on tyrosine and serine/threonine phosphorylation reactions. The proliferative responses of normal B cells to sIg ligation were quite sensitive to the tyrosine kinase inhibitor genistein whereas those observed following costimulations with phorbol diesters or CD40 ligation were more resistant. However, treatment of B cells with H7, an inhibitor of PKC activity, led to a more uniform reduction of B-cell responses after different stimuli. Results from RNase protection assays of c-myc expression also suggested that different B-cell stimuli might utilize distinct intracellular signaling pathways. Both the type of stimuli and mode of sIg ligation were important in determining the stimulated levels of c-myc mRNA expression. Thus, the current findings suggest that age-related defects are present in human B cell signaling pathways as reflected by tyrosine and serine/threonine phosphorylation reactions. Also, these age-related defects can coexist with altered mobilization of PKC enzymatic activity and with alterations in [Ca2+]i and proliferative responses.

Aging↗
PubMed1990

Terbium as a luminescent probe of metal-binding sites in protein kinase C.

In the present report, we demonstrate that Tb3+ binds to protein kinase C and serves as a luminescent reporter of certain cationic metal-binding sites. Tb3+ titration of 50 nM protein kinase C results in a 20-fold enhancement of Tb3+ luminescence which is half-maximal at 12 microM Tb3+. A Kd of approximately 145 nM was determined for Tb3+ binding to the enzyme. The excitation spectrum of bound Tb3+ exhibits a peak at 280 nm characteristic of energy transfer from protein tryptophan or tyrosine residues. The luminescence of this complex can be markedly decreased by other metals, including Pb2+ (IC50 = 25 microM), La3+ (IC50 = 50 microM), Hg2+ (IC50 = 300 microM), Ca2+ (IC50 = 6 mM), and Zn2+ (IC50 greater than 10 mM), and chelation of Tb3+ by 2 mM EGTA. Tb3+ binding to protein kinase C is correlated with its inhibition of protein kinase activity (IC50 = 8 microM), r = 0.99) and phorbol ester binding (IC50 = 15 microM, r = 0.98). Tb3+ inhibition of protein kinase C activity cannot be overcome by excess Ca2+, but can be partially overcome with excess phosphatidylserine or by chelation of Tb3+ with EGTA. Tb3+ noncompetitively inhibits phorbol ester binding by decreasing the maximal extent of binding without significantly altering binding affinity. The results suggest that the Tb3(+)-binding site is at or allosterically related to the enzyme's phosphatidylserine-binding site, but is distinct from the phorbol ester-binding domain and the Ca2(+)-binding site that regulates enzyme activity.

Binding Sites↗
PubMed1989

Relationship of human gingival crevicular fluid polyamine concentration to the percentage of spirochaetes in subgingival dental plaque.

This relationship was examined at 45 inflamed periodontitis sites in 28 patients. A statistically significant positive correlation between polyamine concentration and spirochaetes was observed overall (r = 0.551, p = 0.0001). Within subgroups of this population (n = 15 each), there was no such relationship among midly inflamed sites with 5-6 mm probing depths (r = 0.0003, p = 0.999), but significant relationships among moderately inflamed sites with 5-6 mm depths (r = 0.567, p = 0.027) and 7-11 mm depths (r = 0.639, p = 0.010). Thus polyamines may be involved in subgingival plaque homeostasis at moderately inflamed periodontal sites.

Dental Plaque↗
PubMed1989

Ganglioside modulation of cyclic AMP-dependent protein kinase and cyclic nucleotide phosphodiesterase in vitro.

Purified cyclic AMP-dependent protein kinase (cAK) catalytic subunit phosphorylated 180-, 49-, 31-, 19-, and 14-kilodalton (kDa) proteins of rabbit sciatic nerve membranes. The ability of cAK to phosphorylate these membrane substrate proteins was inhibited by gangliosides GM1, GD1a, and GT1b with half-maximal inhibitory concentration (I50) = 7-25 microM. Neutral glycolipids and lysophosphatidylcholine were much less effective. Cyclic AMP (cAMP) kinase phosphorylation of histone IIA was inhibited by GM1, GD1a, and GT1b (I50 = 115 microM, 75 microM, and 75 microM, respectively). Inhibition by GM1 was competitive with respect to histone (Ki = 108 microM). Autophosphorylation of cAMP kinase was inhibited by GM1 (I50 = 15 microM). GT1b, GD1a, and GM1 half-maximally stimulated calmodulin-dependent cyclic nucleotide phosphodiesterase at 0.1 microM, 0.2 microM, and 0.3 microM, respectively. Although GT1b stimulated phosphodiesterase by increasing Vmax and decreasing Km (similar to calmodulin), GD1a and GM1 produced only an increase in Vmax. These results suggest that ganglioside can modulate the activity of cAMP kinase by both direct inhibition of the enzyme and indirect reduction of cAMP levels through activation of phosphodiesterase. Through these mechanisms, gangliosides may alter cAMP-dependent protein phosphorylation and cell function within the nervous system.

Animals↗
PubMed1988

Inhibition of cyclic nucleotide phosphodiesterase and calcineurin by spermine, a calcium-independent calmodulin antagonist.

Spermine binding to calmodulin and its effects on two calmodulin-dependent enzymes were studied. Spermine bound to dansylated calmodulin with an apparent Ki of 0.7 mM, and to native calmodulin with a Kd of 1.1 mM in equilibrium dialysis experiments. Its binding was found to be independent of calcium. Spermine inhibited calmodulin-activated cyclic nucleotide phosphodiesterase noncompetitively with respect to calcium (Ki = 1.1 mM). Calmodulin activation of calcineurin was inhibited at similar concentrations (Ki = 1.2 mM). Spermine had little effect on basal phosphodiesterase activity or nickel-activated calcineurin activity. Inhibition of both enzymes correlated well with spermine binding to dansylcalmodulin. These findings suggest that spermine might modulate calcium-dependent events in the cell by inactivation of calmodulin via a novel calcium-independent mechanism.

Animals↗
PubMed1988

Activation of cyclic nucleotide phosphodiesterase by a monosaccharide precursor of Escherichia coli lipid A.

The E. coli lipid A precursor lipid X (N2,O3-diacylglucosamine 1-phosphate) activates calmodulin-dependent cyclic nucleotide phosphodiesterase in a noncooperative, calcium-independent manner by increasing its Vmax and decreasing its Km for substrate. The glycolipid produces half-maximal activation at 11 micrograms/ml and does not further enhance activation by calcium-calmodulin. Lipid X activation of phosphodiesterase requires the presence of the O3-linked hydroxymyristoyl residue. These findings suggest that lipid X could produce some of its biological effects by modulating intracellular cAMP levels.

Calcineurin↗