PubMed Health⌕ Search

Biomedical subjects

L A Marshall

Publications and source records attributed to L A Marshall.

At least 37 records · Page 2Linked to original sources
PubMed1998

Selective inhibition of low affinity IgE receptor (CD23) processing.

A series of hydroxamic acids related to the non-selective matrix metalloproteinase inhibitor Batimastat has been prepared, some members of which are potent inhibitors of the processing of the low affinity IgE receptor (CD 23). Increased activity is obtained by appropriate substitution at the alpha-position, whilst selectivity is gained by use of a P1' benzyl group in conjunction with a C-terminal primary amide.

Hydroxamic Acids↗
PubMed1998

Intergenerational gamete donation: ethical and societal implications.

Cases of daughter-to-mother oocyte donation, niece-to-aunt oocyte donation, and father-to-son sperm donation are presented. Comparisons to sibling gamete donation and organ donation, potential ethical conflicts, and societal implications are examined in an attempt to aid decision making when these procedures are requested.

Adult↗
PubMed1998

Beta-lactams SB 212047 and SB 216754 are irreversible, time-dependent inhibitors of coenzyme A-independent transacylase.

The enzyme coenzyme A-independent transacylase (CoA-IT) has been demonstrated to be the key mediator of arachidonate remodeling, a process that moves arachidonate into 1-ether-containing phospholipids. Blockade of CoA-IT by reversible inhibitors has been shown to block the release of arachidonate in stimulated neutrophils and inhibit the production of eicosanoids and platelet-activating factor. We describe novel inhibitors of CoA-IT activity that contain a beta-lactam nucleus. beta-Lactams were investigated as potential mechanism-based inhibitors of CoA-IT on the basis of the expected formation of an acyl-enzyme intermediate complex. Two beta-lactams, SB 212047 and SB 216754, were shown to be specific, time-dependent inhibitors of CoA-IT activity (IC50 = 6 and 20 microM, respectively, with a 10-min pretreatment time). Extensive washing and dilution could not remove the inhibition, suggesting it was irreversible. In stimulated human monocytes, SB 216754 decreased the production of eicosanoids in a time-dependent manner. In an in vivo model of phorbol ester-induced ear inflammation, SB 216754 was able to inhibit indices of both edema and cell infiltration. Taken together, the results support two hypotheses: 1) CoA-IT activity is important for the production of inflammatory lipid mediators in stimulated cells and in vivo and 2) the mechanism by which CoA-IT acts to transfer arachidonate is through an acyl-enzyme intermediate.

Acetyltransferases↗
PubMed1998

Modulation of arachidonic acid metabolism: focus on phospholipase A2(S).

New information has challenged our traditional concepts that the forms and functions of PLA(2) are highly homologous, suggesting now that distinct PLA(2)s may be assigned distinct functions in normal and pathological processes. The nonpancreatic type II 14-kDa PLA(2) and the recently identified type IV "cytosolic" 85-kDa PLA(2) are the two forms most studied in inflammation. Observations in the past suggested that the type II 14-kDa PLA2 is a secreted enzyme that functions extracellularly. Evidence is now emerging that the type II 14-kDa PLA(2) or its recently discovered low-molecular-weight isoforms may be localized and act intracellularly. In view of this, a more complex notion of distinctly functioning PLA(2)s in arachidonic acid release and/or eicosanoid generation can be envisioned. A comparison of the structural and biochemical features of the type II 14-kDa and the 85-kDa PLA(2)s reveals that the enzymes are more distinct than similar. These two enzymes would appear to have distinctly different genetic and biochemical regulatory mechanisms, suggesting that their functions could be quite distinct. Inhibitors of the 14-kDa PLA(2) and to a lesser extent the 85-kDa PLA(2) have been used to obtain a greater understanding of their cellular roles. The concept that the two distinct enzymes might hydrolyze arachidonic acid from different pools and/or supply distinct metabolizing systems in a single cell system has emerged. At this time an intriguing hypothesis can be formed suggesting distinct functional modalities for the two the PLA(2) enzymes in a single cell system. Evidence continues to build implicating the role of the type II 14-kDa PLA(2) in disease, providing a strong rationale for targeting this enzyme in designing novel antiinflammatory therapeutics.

Journal Article↗
PubMed1998

Characteristics of arachidonic acid generation in human basophils: relationship between the effects of inhibitors of secretory phospholipase A2 activity and leukotriene C4 release.

In human basophils, degranulation stimulated by receptor activation or Ca++ ionophores is accompanied by an increase in free arachidonic acid (AA) as determined by gas chromatography negative ion chemical ionization mass spectrometry. Previous studies suggested that there was more than one pool of AA generated during stimulation and indirectly suggested that the leukotriene (LTC4) generated in these reactions was dependent on only one of these pools of AA. Our studies further examined these issues. Preliminary studies demonstrated discordance in the generation of free AA and LTC4 release. Treatment of basophils with triacsin C, a reacylation inhibitor, led to a marked increase in N-formyl-L-methionyl-L-leucyl-L-phenylalanine-(fMLP) stimulated free AA generation with no effect on LTC4 release. Similarly, incubation of basophils with recombinant human secretory phospholipase A2 (sPLA2), before and during fMLP stimulation, led to the generation of extremely high levels of free AA with no effect on LTC4 release. Pretreatment of basophils with anti-14 kDa phospholipase A2 monoclonal antibody (mAb 3F10) inhibited fMLP-induced synthesis of LTC4 but did not attenuate the mass of AA measured nor histamine release. Treating human basophils with zileuton (an inhibitor of 5-lipoxygenase) inhibited the stimulated synthesis of LTC4 and in combination with triacsin C increased the amount of observable AA by an amount approximately equal to the loss in LTC4 mass. Monoclonal antibody 3F10 blocked only the enhanced AA production caused by the combination of zileuton and triacsin C. Monoclonal antibody 3F10 did not inhibit the increases in free AA produced by pretreatment with triacsin C alone. These findings were supported by experiments using another relatively specific inhibitor of sPLA2, SB 203347. In all respects, SB 203347 mimicked the addition of mAb 3F10. Taken together, these data indicate that not all pools of AA are well used for LTC4 synthesis. These experiments also suggest that LTC4 synthesis in human basophils stimulated with fMLP depends on a SB 203347- and monoclonal antibody 3F10-inhibitable deacylation activity, presumably a sPLA2 acting at or near the cell surface. Furthermore, under normal conditions, this pool of AA is not observable because it is efficiently coupled to 5-lipoxygenase. Other deacylating enzymes, which do not supply AA for 5-lipoxygenase metabolism, also appear to be activated by fMLP and these other enzymes appear responsible for the net free AA normally observed after stimulation.

Arachidonate 5-Lipoxygenase↗
PubMed1997

Cytosolic 85-kDa phospholipase A2-mediated release of arachidonic acid is critical for proliferation of vascular smooth muscle cells.

Recent evidence suggests that arachidonic acid (AA) may be involved in regulating cellular proliferation. The predominant mechanism of AA release from cellular phospholipids is via phospholipase A2 (PLA2) hydrolysis. The purpose of this study was to examine the roles of the distinct 14-kDa and 85-kDa PLA2 enzymes in human coronary artery vascular smooth muscle cell (hCAVSMC) proliferation. Cultured hCAVSMCs proliferate in the presence of growth medium with a typical doubling time of 30-40 h, grow at a slower proliferative rate upon reaching confluency (day 8), and eventually undergo contact inhibition of growth (day 10). Neither Type II 14-kDa PLA2 activity nor mass changed over a 10-day culture period. In contrast, 85-kDa PLA2 protein activity and mRNA decreased as time in culture progressed. This reduction in 85-kDa PLA2 correlated with reductions in DNA synthesis and suggested a possible association between 85-kDa PLA2 and proliferation. To directly evaluate the role of the 85-kDa PLA2 in proliferation we examined the effects of an 85-kDa PLA2 inhibitor (AACOCF3) and 85-kDa PLA2 antisense oligonucleotides on proliferation. Both reagents dose dependently inhibited proliferation, whereas a 14-kDa PLA2 inhibitor (SB203347), a calcium-independent PLA2 inhibitor (HELSS), an 85-kDa sense oligonucleotide, and a nonrelevant scrambled control oligonucleotide had no effect. The mechanism by which 85-kDa PLA2 influences cellular proliferation remains unclear. Inhibition of 85-kDa PLA2 activity produced neither phase-specific cell cycle arrest nor apoptosis (fluorescence-activated cell sorter analysis). Addition of AA (20 mu M) attenuated the effects of both AACOCF3 and 85-kDa antisense oligonucleotides implicating AA as a key mediator in cellular proliferation. However, although prostaglandin E2 (PGE2) was present in the culture medium, it peaked early (day 3) in culture, and indomethacin had no effect on cellular proliferation indicating that hCAVSMC proliferation was not mediated through PGE2. These data provide the first direct evidence that PLA2 is involved in control of VSMC proliferation and indicate that 85-kDa PLA2-mediated liberation of AA is critical for cellular proliferation.

Arachidonic Acid↗
PubMed1997

Evidence that 85 kDa phospholipase A2 is not linked to CoA-independent transacylase-mediated production of platelet-activating factor in human monocytes.

Platelet-activating factor (PAF) production is carefully controlled in inflammatory cells. The specific removal of arachidonate (AA) from 1-O-alkyl-2-arachidonoyl-sn-glycero-3-phosphocholine (GPC), thought to be mediated by CoA-independent transacylase (CoA-IT), is required to generate the PAF precursor 1-O-alkyl-2-lyso-GPC in human neutrophils. Exposure of A23187-stimulated human monocytes to the CoA-IT inhibitors SK&F 98625 and SK&F 45905 inhibited PAF formation (IC50s of 10 and 12 microM, respectively), indicating that these cells also need CoA-IT activity for PAF production. Because CoA-IT activity transfers arachidonate to a 2-lyso phospholipid substrate, its activity is obligated to an sn-2 acyl hydrolase to form the 2-lyso phospholipid substrate. SB 203347, an inhibitor of 14 kDa phospholipase A2 (PLA2), and AACOCF3, an inhibitor of 85 kDa PLA2, both inhibited AA release from A23187-stimulated human monocytes. However, AACOCF3 had no effect on A23187-induced PAF formation at concentrations as high as 3 microM. Further, depletion of 85 kDa PLA2 using antisense (SB 7111, 1 microM) had no effect on PAF production, indicating a lack of a role of 85 kDa PLA2 in PAF biosynthesis. Both SB 203347 and the 14 kDa PLA2 inhibitor scalaradial blocked PAF synthesis in monocytes (IC50s of 2 and 0.5 microM, respectively), suggesting a key role of 14 kDa PLA2 in this process. Further, A23187-stimulated monocytes produced two forms of PAF: 80% 1-O-alkyl-2-acetyl-GPC and 20% 1-acyl-2-acetyl-GPC, which were both equally inhibited by SB 203347. In contrast, inhibition of CoA-IT using SK&F 45905 (20 microM) had a greater effect on the production of 1-O-alkyl (-80%) than of 1-acyl (-14%) acetylated material. Finally, treatment of U937 cell membranes with exogenous human recombinant (rh) type II 14 kDa PLA2, but not rh 85 kDa PLA2, induced PAF production. Elimination of membrane CoA-IT activity by heat treatment impaired the ability of 14 kDa PLA2 to induce PAF formation. Taken together, these results suggest that a 14 kDa PLA2-like activity, and not 85 kDa PLA2, is coupled to monocyte CoA-IT-induced PAF production.

Acyltransferases↗
PubMed1997

Depletion of human monocyte 85-kDa phospholipase A2 does not alter leukotriene formation.

Human monocytes possess several acylhydrolase activities and are capable of producing both prostanoids (PG) and leukotriene (LT) products upon acute stimulation with calcium ionophore, A23187 or phagocytosis of zymosan particles. The cytosolic 85-kDa phospholipase (PLA) A2 co-exists with the 14-kDa PLA2 in the human monocyte, but their respective roles in LT production are not well understood. Reduction in 85-kDa PLA2 cellular protein levels by initiation site-directed antisense (SK 7111) or exposure to the 85-kDa PLA2 inhibitor, arachidonyl trifluoromethyl ketone (AACOCF3), prevented A23187 or zymosan-stimulated monocyte prostanoid formation. In contrast, neither treatment altered stimulated LTC4 production. This confirmed the important role of the 85-kDa PLA2 in prostanoid formation but suggests that it has less of a role in LT biosynthesis. Alternatively, treatment of monocytes with the selective, active site-directed 14-kDa PLA2 inhibitor, SB 203347, prior to stimulation had no effect on prostanoid formation at concentrations that totally inhibited LT formation. Addition of 20 microM exogenous arachidonic acid to monocytes exposed to SK 7111 or SB 203347 did not alter A23187-induced PGE2 or LTC4 generation, respectively, indicating that these agents had no effect on downstream arachidonic acid-metabolizing enzymes in this setting. Taken together, these results provide evidence that the 85-kDa PLA2 may play a more significant role in the formation of PG than LT. Further, utilization of SB 203347 provides intriguing data to form the hypothesis that a non-85-kDa PLA2 sn-2 acyl hydrolase, possibly the 14-kDa PLA2, may provide substrate for LT formation.

Arachidonic Acids↗
PubMed1997

IgE secretion is attenuated by an inhibitor of proteolytic processing of CD23 (Fc epsilonRII).

CD23, the low-affinity IgE receptor, is up-regulated on interleukin (IL)-4-stimulated B cells and monocytes, with a concomitant increase in the release of soluble fragments of CD23 (sCD23) into the medium by proteolytic processing of the surface-bound intact CD23. The effect of inhibition of the processing of CD23 on IgE production in human and mouse cells and in a mouse model in vivo was evaluated. CD23 processing to sCD23 from RPMI 8866 (a human Epstein-Barr virus-transformed B cell line) cell membranes was inhibited by a broad-spectrum matrix-metalloprotease inhibitor, batimastat, with an IC50 of 0.15 microM. Batimastat also inhibited CD23 processing in whole RPMI 8866 cells as well as in IL-4-stimulated purified human monocytes with similar IC50. Batimastat inhibited IgE production from IL-4/anti-CD40-stimulated human tonsil B cells as well as mouse splenic B cells in a manner consistent with inhibition of CD23 processing. Release of soluble fragments of CD23 in the cell supernatants of tonsil B cells was inhibited over the concentration range of 1-10 microM batimastat and intact cell surface CD23 was increased on mouse splenic B cells in the presence of these concentrations of batimastat. IgE production of IL-4-stimulated human peripheral blood mononuclear cells was also blocked by 1-10 microM batimastat, again with comparable inhibition of sCD23 release over the same concentration range. Finally, in a mouse model of IgE production, batimastat inhibited IgE production in response to ovalbumin challenge as determined by serum IgE levels. Taken together, the data support a role of CD23 in IgE production and point to CD23 processing to sCD23 as a therapeutically relevant control point in the regulation of IgE synthesis.

Animals↗
PubMed1997

Inhibition of NFkappaB-mediated interleukin-1beta-stimulated prostaglandin E2 formation by the marine natural product hymenialdisine.

Exposure of human rheumatoid synovial fibroblasts (RSF) to interleukin 1beta (IL-1beta) results in the coordinate up-regulation of 85-kDa phospholipase A2 (PLA2) and mitogen-inducible cyclooxygenase (COX II) and subsequent biosynthesis of prostaglandin E2 (PGE2). We have recently demonstrated, through the use of oligonucleotide decoys and antisense, the participation of the proinflammatory transcription factor, nuclear factor kappaB (NFkappaB), in the regulation of the prostanoid-metabolizing enzymes. Hymenialdisine, a marine natural product has recently been characterized as an inhibitor of NFkappaB activation and exposure of IL-1-stimulated RSF-inhibited PGE2 production in a concentration-dependent manner (IC50 approximately 1 microM). Alternatively, both an analog, aldisine, and the protein kinase C inhibitor, RO 32-0432, were without affect. Direct action of hymenialdisine on IL-1-induced NFkappaB activation was demonstrated by a significant reduction (approximately 80%) in NFkappaB binding to the classical kappaB consensus motif (as assessed by electrophoretic mobility shift assay) and inhibition of stimulated p65 migration from the cytosol of treated cells (as assessed by Western analysis). Consistent with the role of NFkappaB in the transcriptional regulation of COX II and 85-kDa PLA2, hymenialdisine-treated RSF did not transcribe the respective mRNAs in response to IL-1. This led to reductions in their respective protein levels and subsequent reductions in the ability to produce PGE2. Specificity of action is suggested as IL-1-stimulated interleukin-8 (IL-8) production, which is known to be an NFkappaB-regulated event, was also inhibited by hymenialdisine, whereas IL-1-induced production of vascular endothelial growth factor, a non-NFkappaB-regulated gene, was not affected by exposure to hymenialdisine. Taken together, hymenialdisine inhibits IL-1-stimulated-RSF PGE2 formation acting predominately through modulation of NFkappaB activation and offers an interesting novel tool to evaluate the role of NFkappaB in inflammatory disease.

Adult↗
PubMed1996

Manipulation of distinct NFkappaB proteins alters interleukin-1beta-induced human rheumatoid synovial fibroblast prostaglandin E2 formation.

Interleukin 1beta (IL-1beta) up-regulates human rheumatoid synovial fibroblast (RSF) 85-kDa phospholipase A2 (PLA2) and mitogen-inducible cyclooxygenase (COX) II. Promoter regions for these genes contain a motif that closely resembles the "classic" NFkappaB consensus site. Immunoblot analysis identified NFkappaB1 (p50), RelA (p65), and c-Rel in RSF. Upon IL-1beta-stimulation, p65 and c-Rel but not p50 protein levels were reduced suggesting nuclear translocation. IL-1beta-induced RSF nuclear extracts contained a p65-containing complex, which bound to the classical NFkappaB consensus motif. An NFkappaB classical oligonucleotide decoy produced a concentration-dependent decrease in IL-1-stimulated PGE2 production (IC50 = approximately 2 microM), indicating a role of NFkappaB. Utilization of antisense technology showed that p65 but not p50 or c-Rel mediated IL-1beta-stimulated PGE2 formation. Treated RSF could not transcribe COX II or 85-kDa PLA2 mRNA, which reduced their respective proteins. Interestingly, stimulated IL-8 production was not inhibited by the classical NFkappaB decoy but was reduced by treatment with antisense to both p65 and c-Rel supporting preferential binding of c-Rel-p65 to the "alternative" IL-8 kappaB motif. Taken together, these data provide the first direct evidence for a role of p65 in COX II and 85-kDa PLA2 gene induction and support the IL-1 activation and participation of distinct NFkappaB protein dimers in RSF prostanoid and IL-8 formation.

Arthritis, Rheumatoid↗
PubMed1996

Urinary N-telopeptides to monitor bone resorption while on GnRH agonist therapy.

OBJECTIVES: To assess the utility of urinary cross-linked N-telopeptides in monitoring bone resorption and predicting bone loss during GnRH agonist administration. METHODS: Ninety patients who were prescribed GnRH agonist therapy for 3-6 months for treatment of endometriosis, leiomyomas or other gynecologic disorders participated in this prospective multicenter study. N-telopeptides, serum estradiol (E2), and bone mineral density were monitored before, during and up to 3 months after the course of GnRH agonist therapy. RESULTS: N-telopeptide levels increased significantly throughout GnRH agonist therapy and returned to baseline levels by 3 months after treatment was completed. A significant negative correlation was seen between N-telopeptide and E2 measurements after 3 months (r=-0.23, P<.05), 4 months (r=-0.32, P < .05), and 5 months (r=-0.41, P<.005) of GnRH agonist therapy. The percent change in bone mineral density at L1-L4 at 6 months of GnRH agonist treatment correlated inversely with the percent change in N-telopeptides from baseline to 2,3,4, and 5 months of treatment; the percent change of bone mineral density at the femoral neck at 6 months correlated inversely with the percent change of N-telopeptides from baseline to month 4. CONCLUSIONS: Urinary N-telopeptide determinations provide a quantitative measure of bone resorption, due to GnRH agonist-induced hypoestrogenism. Increases in resorption as measured by N-telopeptides parallel decreases in in E2 levels. Increases in N-telopeptides on GnRH agonist therapy may provide a tool to predict decreases in bone mineral density.

Adult↗
PubMed1996

Suppression of human synovial fibroblast 85 kDa phospholipase A2 by antisense reduces interleukin-1 beta induced prostaglandin E2.

OBJECTIVE: To evaluate the relative roles of rheumatoid synovial fibroblast phospholipases A2 (PLA2) in interleukin- 1beta (IL-1 beta) stimulated prostaglandin E2 (PGE2) production. METHODS: The role of the cytosolic 85 kDa PLA2 in IL-1beta induced human rheumatoid synovial fibroblast PGE2 formation was directly evaluated using an antisense phosphorothioate oligonucleotide to the initiation site of the 85 kDa PLA2 mRNA. Contribution of the 14 kDa PLA2 was assessed using selective inhibitors or a neutralizing monoclonal antibody (Mab). RESULTS: Antisense, but not sense, decreased IL-1beta upregulation of 85 kDa PLA2 activity and protein levels. The antisense effect was specific, since it did not affect 14 kDa PLA2 activity released into the media or induced cyclooxygenase II protein levels over 24 h. Antisense, but not sense, reduced PGE2 formation in a concentration dependent manner. IL-1 beta significantly upregulated cell associated 14 kDa PLA2 and its subsequent release. Specific inhibition of this enzyme by a neutralizing Mab or selective inhibitors of 14 kDA PLA2 activity did not alter IL-1beta induced PGE2 levels. CONCLUSION: These data directly support a role for the 85 kDa PLA2, but not the 14 kDa PLA2, in IL- 1beta stimulated PGE2 production from human rheumatoid synovial fibroblast.

Antibodies, Monoclonal↗