Biomedical subjects

P D Allen

Publications and source records attributed to P D Allen.

At least 37 records · Page 2Linked to original sources

PubMed2001

8-Cl-adenosine mediated cytotoxicity and sensitization of T-lymphoblastic leukemia cells to TNFalpha-induced apoptosis is via inactivation of NF-kappaB.

These data show that 8-Cl-cAMP is cytotoxic to the lymphoblastic leukemia cell line CEM and its vinblastine selected multidrug resistant derivative, CEM/VLB100 although PKA was not involved in these effects. The cytotoxic effects of 8-Cl-cAMP was abrogated by cotreatment with either ADA or IBMX which indicated a degradation form of 8-Cl-cAMP was needed for this cytotoxicity. CEM and CEM/VLB100 cells displayed a notable sensitivity to 8-Cl-adenosine-induced growth inhibition and apoptosis. 8-Cl-adenosine increased the cytosolic levels of IkappaBalpha which prevented NF-kappaB nuclear translocation. 8-Cl-adenosine also prevented TNFalpha-induced IkB decay and NF-kappaB activation in CEM and CEM/VLB100 cells.

2-Chloroadenosine↗

PubMed2001

Effect of size and disease on estimated deposition of drugs administered using jet nebulization in children with cystic fibrosis.

STUDY OBJECTIVES: To develop a model that quantified the nebulizer output that was inhaled by subjects with cystic fibrosis (CF) in order to predict the amount of drug likely to enter the upper airway contained in particles small enough to be deposited in the lower respiratory tract of individual patients. DESIGN: Forty-three patients (age, 6 to 18 years) with CF, with FEV(1) of 26 to 124% of predicted, breathed through a nebulizer circuit with a pneumotachograph in place at the distal end. Algorithms were developed from the measured flows through the pneumotachograph, allowing partitioning of inspiration into undiluted aerosol and fresh gas. In order to validate the algorithms, argon was added to the nebulizing gas flow and then its concentration was analyzed at the mouth by mass spectrometry. RESULTS: Predictions of the concentration of argon at the mouth were concordant with that measured by mass spectrometry, thus validating the model. Combining data from the model with in vitro nebulizer performance data, predictions for estimates for lung deposition for individuals were possible. Total estimate was independent of patient size or FEV(1). The respiratory duty cycle was 0.44 +/- 0.05 (mean +/- SD) and correlated (r = 0.91, p < 0.001) with estimated deposition and minute ventilation (r = 0.60, p < 0.01). However, when expressed in milligrams per kilogram of body weight, the estimated deposition in smaller children was fourfold higher than in larger children. CONCLUSIONS: If the effect of patient size and pattern of breathing on estimated drug deposition are not considered when prescribing drugs given by nebulization, the result may be overdosing younger children, underdosing older children, or both.

Administration, Inhalation↗

PubMed2001

Expression of functional Na,K-ATPase isozymes in normal human cardiac biopsies.

In human heart failure, disturbances in Ca2+ homeostasis are well known but the fate of the Na,K-ATPase isoforms (alpha1beta1, alpha2beta1 and alpha3beta1), the receptors for cardiac glycosides, still remains under study. Microsomes have been purified from non-failing human hearts. As judged by the sensitivities of Na,K-ATPase activity to ouabain (IC50 values: 7.0 +/- 2.5 and 81 +/- 11 nM), 3H-ouabain-binding measurements at equilibrium with and without 10 mM K+ and by a biphasic ouabain dissociation process, at least two finctionally active Na,K-ATPase isozymes coexist in normal human hearts. These are demonstrated as a very high- and a high affinity ouabain-binding site. The KD values are 3.6 +/- 1.6 nM and 17 +/- 6 nM, respectively. The two dissociation rate constants are 42 x 10(4) min(-1) and 360 x 10(-4) min(-1). Addition of 10 mM K+ ions shifted the respective KD values for ouabain from 3.6 +/- 1.6 to 20 +/- 5 nM and from 17 +/- 6 nM to 125 +/- 25 nM, respectively. The isozymes involved are identified by comparing these three pharmacological parameters to those of each alpha/beta-isozyme separately expressed in Xenopus oocytes (9). In human heart, the very high affinity site for ouabain is the alpha1beta1 dimer and the high affinity site is alpha2beta1.

Animals↗

PubMed2000

Transmembrane redox sensor of ryanodine receptor complex.

Inositol 1,4,5-trisphosphate receptors (IP(3)R) and ryanodine receptors (RyR) mediate the release of endoplasmic and sarcoplasmic reticulum (ER/SR) Ca(2+) stores and regulate Ca(2+) entry through voltage-dependent or ligand-gated channels of the plasma membrane. A prominent property of ER/SR Ca(2+) channels is exquisite sensitivity to sulfhydryl-modifying reagents. A plausible role for sulfhydryl chemistry in physiologic regulation of Ca(2+) release channels and the fidelity of Ca(2+) release from ER/SR is lacking. This study reveals the existence of a transmembrane redox sensor within the RyR1 channel complex that confers tight regulation of channel activity in response to changes in transmembrane redox potential produced by cytoplasmic and luminal glutathione. A transporter selective for glutathione is co-localized with RyR1 within the SR membrane to maintain local redox potential gradients consistent with redox regulation of ER/SR Ca(2+) release. Hyperreactive sulfhydryls previously shown to reside within the RyR1 complex (Liu, G., and Pessah, I. N. (1994) J. Biol. Chem. 269, 33028-33034) are an essential biochemical component of a transmembrane redox sensor. Transmembrane redox sensing may represent a fundamental mechanism by which ER/SR Ca(2+) channels respond to localized changes in transmembrane glutathione redox potential produced by physiologic and pathophysiologic modulators of Ca(2+) release from stores.

Animals↗

PubMed2000

Expression of ryanodine receptor RyR3 produces Ca2+ sparks in dyspedic myotubes.

Discrete, localized elevations of myoplasmic [Ca2+], Ca2+ 'sparks', were readily detected using the fluorescent Ca2+ indicator fluo-3 and laser scanning confocal microscopy in 'dyspedic' 1B5 myotubes, i.e. myotubes which do not express ryanodine receptors (RyRs), transduced with virions containing cDNA for RyR type 3 that were saponin permeabilized to allow dye entry. Ca2+ sparks were never observed in non-transduced RyR null myotubes. The spatial locations of sparks observed in permeabilized myotubes roughly corresponded to regions of RyR protein expression in the same myotube as detected after subsequent fixation and antibody staining. Permeabilized RyR3-transduced myotubes exhibited similar punctate peripheral RyR3 protein immunohistochemical patterns as myotubes fixed before permeabilization indicating that permeabilization did not affect the structural organization of the triad. Ca2+ sparks, recorded in line scan mode, in permeabilized myotubes expressing RyR3 exhibited mean amplitudes (change in fluorescence/mean fluorescence, DeltaF/F: 1.20 +/- 0.04) and temporal rise times (10-90%; 6.31 +/- 0.12 ms) similar to those of sparks recorded in permeabilized frog skeletal muscle fibres (0.98 +/- 0.01; 6.11 +/- 0.07, respectively) using the same confocal system. Spatial extent and temporal duration of the Ca2+ sparks were approximately 40% larger in the RyR3-expressing myotube cultures than in frog fibres. Ca2+ sparks recorded in line scan mode often occurred repetitively at the same spatial location in RyR3-expressing myotubes. Such repetitive events were highly reproducible in amplitude and spatio-temporal properties, as previously observed for repetitive mode sparks in frog skeletal muscle. Ca2+ sparks recorded in xy mode were frequently compressed in the y (slower scan) direction compared to the x direction. This asymmetry was reproduced assuming spatially symmetric events having the time course of Ca2+ sparks recorded in line scan (xt) mode. These expression studies demonstrate that the presence of RyR3 is sufficient for the production of Ca2+ sparks in a skeletal muscle system lacking the expression of any other RyR isoform.

Aniline Compounds↗

PubMed2000

Chemical synthesis and characterization of maurocalcine, a scorpion toxin that activates Ca(2+) release channel/ryanodine receptors.

Maurocalcine is a novel toxin isolated from the venom of the chactid scorpion Scorpio maurus palmatus. It is a 33-mer basic peptide cross-linked by three disulfide bridges, which shares 82% sequence identity with imperatoxin A, a scorpion toxin from the venom of Pandinus imperator. Maurocalcine is peculiar in terms of structural properties since it does not possess any consensus motif reported so far in other scorpion toxins. Due to its low concentration in venom (0.5% of the proteins), maurocalcine was chemically synthesized by means of an optimized solid-phase method, and purified after folding/oxidation by using both C18 reversed-phase and ion exchange high-pressure liquid chromatographies. The synthetic product (sMCa) was characterized. The half-cystine pairing pattern of sMCa was identified by enzyme-based cleavage and Edman sequencing. The pairings were Cys3-Cys17, Cys10-Cys21, and Cys16-Cys32. In vivo, the sMCa was lethal to mice following intracerebroventricular inoculation (LD(50), 20 microg/mouse). In vitro, electrophysiological experiments based on recordings of single channels incorporated into planar lipid bilayers showed that sMCa potently and reversibly modifies channel gating behavior of the type 1 ryanodine receptor by inducing prominent subconductance behavior.

Amino Acid Sequence↗

PubMed2000

Low-frequency low-field magnetic susceptibility of ferritin and hemosiderin.

Low-frequency low-field magnetic susceptibility measurements were made on four samples of mammalian tissue iron oxide deposits. The samples comprised: (1) horse spleen ferritin; (2) dugong liver hemosiderin; (3) thalassemic human spleen ferritin; and (4) crude thalassemic human spleen hemosiderin. These samples were chosen because Mössbauer spectroscopic measurements on the samples indicated that they exemplified the variation in magnetic and mineral structure found in mammalian tissue iron oxide deposits. The AC-magnetic susceptometry yielded information on the magnetization kinetics of the four samples indicating samples 1, 2, and 3 to be superparamagnetic with values of around 10(11) s(-1) for the pre-exponential frequency factor in the Néel-Arrhenius equation and values for characteristic magnetic anisotropy energy barriers in the range 250-400 K. Sample 4 was indicated to be paramagnetic at all temperatures above 1.3 K. The AC-magnetic susceptometry data also indicated a larger magnetic anisotropy energy distribution in the dugong liver sample compared with samples 1 and 3 in agreement with previous Mössbauer spectroscopic data on these samples. At temperatures below 200 K, samples 1-3 exhibited Curie-Weiss law behavior, indicating weak particle-particle interactions tending to favor antiparallel alignment of the particle magnetic moments. These interactions were strongest for the dugong liver hemosiderin, possibly reflecting the smaller separation between mineral particles in this sample. This is the first magnetic susceptometry study of hemosiderin iron deposits and demonstrates that the AC-magnetic susceptometry technique is a fast and informative method of studying such tissue iron oxide deposits.

Animals↗

PubMed2000

Cyclic ADP-ribose induces a larger than normal calcium release in malignant hyperthermia-susceptible skeletal muscle fibers.

Malignant hyperthermia (MH) is associated with abnormal regulation of intracellular calcium in skeletal muscle fibers. Cyclic adenosine diphosphate-ribose (cADPR) is an endogenous metabolite of beta-NAD+ that induces Ca2+ release from intracellular stores in many tissues. Microinjection of cADPR (0.5 or 1 microM) increased the intracellular resting Ca2+ concentration ([Ca2+]i) in intact swine skeletal muscle in a dose-dependent manner. However, the increase in [Ca2+]i was greater in malignant-hyperthermia-susceptible (MHS) fibers than in non-susceptible (MHN) fibers. Incubation of muscle fibers in low external Ca2+ solution or in the presence of L-type Ca2+ channel entry blockers, or intracellular microinjection of heparin or ruthenium red did not modify the effect of cADPR on [Ca2+]i. Dantrolene (50 microM), a known inhibitor of intracellular Ca2+ release, decreased resting [Ca2+]i and prevented the cADPR-induced increase in [Ca2+]i. These results provide evidence: (1) for the existence of Ca2+ release mechanisms occurring via non-ryanodine or inositol 1,4,5-trisphosphate (InsP3) receptor mechanisms; (2) that MHS skeletal muscles exhibit a higher responsiveness to cADP-ribose-induced release of Ca2+ and (3) that the ability of dantrolene to block cADP-ribose-induced release of Ca2+ could be related to its pharmacologic effect on resting [Ca2+]i.

Adenosine Diphosphate Ribose↗

PubMed2000

RYR1 and RYR3 have different roles in the assembly of calcium release units of skeletal muscle.

Calcium release units (CRUs) are junctions between the sarcoplasmic reticulum (SR) and exterior membranes that mediates excitation contraction (e-c) coupling in muscle cells. In skeletal muscle CRUs contain two isoforms of the sarcoplasmic reticulum Ca(2+)release channel: ryanodine receptors type 1 and type 3 (RyR1 and RyR3). 1B5s are a mouse skeletal muscle cell line that carries a null mutation for RyR1 and does not express either RyR1 or RyR3. These cells develop dyspedic SR/exterior membrane junctions (i.e., dyspedic calcium release units, dCRUs) that contain dihydropyridine receptors (DHPRs) and triadin, two essential components of CRUs, but no RyRs (or feet). Lack of RyRs in turn affects the disposition of DHPRs, which is normally dictated by a linkage to RyR subunits. In the dCRUs of 1B5 cells, DHPRs are neither grouped into tetrads nor aligned in two orthogonal directions. We have explored the structural role of RyR3 in the assembly of CRUs in 1B5 cells independently expressing either RyR1 or RyR3. Either isoform colocalizes with DHPRs and triadin at the cell periphery. Electron microscopy shows that expression of either isoform results in CRUs containing arrays of feet, indicating the ability of both isoforms to be targeted to dCRUs and to assemble in ordered arrays in the absence of the other. However, a significant difference between RyR1- and RyR3-rescued junctions is revealed by freeze fracture. While cells transfected with RyR1 show restoration of DHPR tetrads and DHPR orthogonal alignment indicative of a link to RyRs, those transfected with RyR3 do not. This indicates that RyR3 fails to link to DHPRs in a specific manner. This morphological evidence supports the hypothesis that activation of RyR3 in skeletal muscle cells must be indirect and provides the basis for failure of e-c coupling in muscle cells containing RyR3 but lacking RyR1 (see the accompanying report, ).

Amino Acid Sequence↗

PubMed2000

Divergent functional properties of ryanodine receptor types 1 and 3 expressed in a myogenic cell line.

Of the three known ryanodine receptor (RyR) isoforms expressed in muscle, RyR1 and RyR2 have well-defined roles in contraction. However, studies on mammalian RyR3 have been difficult because of low expression levels relative to RyR1 or RyR2. Using the herpes simplex virus 1 (HSV-1) helper-free amplicon system, we expressed either RyR1 or RyR3 in 1B5 RyR-deficient myotubes. Western blot analysis revealed that RyR1- or RyR3-transduced cells expressed the appropriate RyR isoform of the correct molecular mass. Although RyR1 channels exhibited the expected unitary conductance for Cs(+) in bilayer lipid membranes, 74 of 88 RyR3 channels exhibited pronounced subconductance behavior. Western blot analysis with an FKBP12/12.6-selective antibody reveals that differences in gating behavior exhibited by RyR1 and RyR3 may be, in part, the result of lower affinity of RyR3 for FKBP12. In calcium imaging studies, RyR1 restored skeletal-type excitation-contraction coupling, whereas RyR3 did not. Although RyR3-expressing myotubes were more sensitive to caffeine than those expressing RyR1, they were much less sensitive to 4-chloro-m-cresol (CMC). In RyR1-expressing cells, regenerative calcium oscillations were observed in response to caffeine and CMC but were never seen in RyR3-expressing 1B5 cells. In [(3)H]ryanodine binding studies, only RyR1 exhibited sensitivity to CMC, but both RyR isoforms responded to caffeine. These functional differences between RyR1 and RyR3 expressed in a mammalian muscle context may reflect differences in association with accessory proteins, especially FKBP12, as well as structural differences in modulator binding sites.

Animals↗

PubMed2000

Gating of store-operated channels by conformational coupling to ryanodine receptors.

We report here that RyRs interact with and gate the store-operated hTrp3 and Icrac channels. This gating contributes to activation of hTrp3 and Icrac by agonists. Coupling of hTrp3 to IP3Rs or RyRs in the same cells was found to be mutually exclusive. Biochemical and functional evidence suggest that mutually exclusive coupling reflects clustering and segregation of hTrp3-IP3R and hTrp3-RyR complexes in plasma membrane microdomains. Gating of CCE by RyRs indicates that gating by conformational coupling is not unique to skeletal muscle but is a general mechanism for communication between events in the plasma and endoplasmic reticulum membranes.

Barium↗

PubMed2000

Mechanisms of resistance to apoptosis in human AML blasts: the role of differentiation-induced perturbations of cell-cycle checkpoints.

Alterations in the response of leukaemic cells to apoptosis-inducing stimuli may account for resistance to chemotherapy and treatment failure, either by disruption of the apoptotic pathway itself or by altered DNA repair; quiescent cells and those with disrupted cell-cycle checkpoints may also display decreased apoptosis. Quiescence can be induced by the differentiation of myeloid cells, and this led us to investigate whether the modulation of drug-induced apoptosis associated with differentiation might be a model for quiescence-associated resistance generally. We have demonstrated that resistance to idarubicin-induced apoptosis increased with greater duration of incubation of HL60 and U937 cells with ATRA and 1,25(OH)2 D3 and that this protective effect correlated with the degree of G0/G1 accumulation. In addition, the cytoprotective effects held for other classes of cytotoxic drugs with different mechanisms of action to idarubicin. Prolonged exposure to idarubicin or vinblastine was associated with diminution of the protective effect and re-entry of cells into cycle. The full cytoprotective effect was restored by resupplementation with ATRA or 1,25(OH)2 D3 during exposure to idarubicin, with concomitant persistence of G0/G1 accumulation. Differentiating agents prevented the accumulation of leukaemic cells at the G2/M checkpoint in response to low concentrations of idarubicin. Understanding how differentiating agents modulate these cell-cycle checkpoints, and how quiescent cells evade apoptosis, may allow the development of therapeutic strategies to limit such apoptosis-inhibiting effects and maximise cell kill from chemotherapy.

Acute Disease↗

PubMed2000

Constitutive levels of cAMP-dependent protein kinase activity determine sensitivity of human multidrug-resistant leukaemic cell lines to growth inhibition and apoptosis by forskolin and tumour necrosis factor alpha.

The cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) signal pathway regulates cell proliferation, differentiation and cell death. It may also regulate the multidrug resistance (MDR) phenotype in leukaemic cells. These data showed that MDR1+ K/Dau600 cells exhibited a higher basal level of PKA activity than MDR- parental cells. The significance of this on tumour necrosis factor alpha (TNFalpha)-induced apoptosis and cytostasis was investigated further. In comparison with MDR1- parental cells, K/Dau600 cells had a higher expression of PKA regulatory subunit RIalpha and nuclear catalytic subunit PKAcalpha. They were also more susceptible to inhibition of proliferation and induction of apoptosis by TNFalpha and/or forskolin, but this could be attenuated by H89. An increase in cAMP was associated with the apoptosis in the K/Dau600 cell line. Forskolin inactivated NF-kappaB in K/Dau600 cells but not in K562 cl. 6 cells, whereas TNF activated NF-kappaB in K562 cl.6 cells but not in K/Dau600 cells. 8-Cl-cAMP exhibited similar inhibitory effects on the proliferation of all of the cell lines used via its metabolite 8-Cl-adenosine, which indicates that these effects were independent of residual PKA or cAMP. Therefore, the differential sensitivity to apoptosis and/or growth inhibition could be mediated via cAMP, partly through PKA via NF-kappaB and partly by PKA-independent pathways.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed2000

Hypersensitivity of malignant hyperthermia-susceptible swine skeletal muscle to caffeine is mediated by high resting myoplasmic [Ca2+ ].

BACKGROUND: Malignant hyperthermia (MH) is an inherited pharmacogenetic syndrome that is triggered by halogenated anesthetics and/or depolarizing muscle relaxants. MH-susceptible (MHS) skeletal muscle has been shown to be more sensitive to caffeine-induced contracture than muscle from nonsusceptible (MHN) subjects and is the basis for the most commonly used clinical diagnostic test to determine MH susceptibility. METHODS: We studied the effects of caffeine on myoplasmic free calcium concentration ([Ca2+]i) in MHN and MHS swine muscle fibers by means of Ca2+-selective microelectrodes before and after K+-induced partial depolarization. RESULTS: [Ca2+]i in untreated MHN fibers was 123 +/- 8 nm versus 342 +/- 33 nm in MHS fibers. Caffeine (2 mm) caused an increase in [Ca2+]i in both groups (296 +/- 41 nm MHN vs. 1,159 +/- 235 nm MHS) with no change in resting membrane potential. When either MHN or MHS, muscle fibers were incubated in 10 mm K+ [Ca2+]i transiently increased to 272 +/- 22 nm in MHN and 967 +/- 38 nm in MHS for 6-8 min. Exposure of MHN fibers to 2 mm caffeine while resting [Ca2+]i was elevated induced an increment in [Ca2+]i to 940 +/- 37 nm. After 6-8 min of exposure to 10 mm K+, [Ca2+]i returned to control levels in all fibers, and the effect of 2 mm caffeine on resting [Ca2+]i returned to control, despite continued partial membrane depolarization. CONCLUSIONS: These results suggest that the increased "sensitivity" to caffeine of MHS swine muscle fibers is a nonspecific response related, at least in part, to the high resting [Ca2+]i and not an increased caffeine sensitivity of the sarcoplasmic reticulum Ca2+ release channel per se.

Animals↗

PubMed2000

HSV-1 amplicon vectors are a highly efficient gene delivery system for skeletal muscle myoblasts and myotubes.

Analysis of RyR1 structure function in muscle cells is made difficult by the low (<5%) transfection efficiencies of myoblasts or myotubes using calcium phosphate or cationic lipid techniques. We inserted the full-length 15.3-kb RyR1 cDNA into a herpes simplex virus type 1 (HSV-1) amplicon vector, pHSVPrPUC between the ori/IE 4/5 promoter sequence and the HSV-1 DNA cleavage/packaging signal (pac). pHSVGN and pHSVGRyR1, two amplicons that expressed green fluorescent protein, were used for fluorescence-activated cell sorter analysis of transduction efficiency. All amplicons were packaged into HSV-1 virus particles using a helper virus-free packaging system and yielded 10(6) transducing vector units/ml. HSVRyR1, HSVGRyR1, and HSVGN virions efficiently transduced mouse myoblasts and myotubes, expressing the desired product in 70-90% of the cells at multiplicity of infection 5. The transduced cells appeared healthy and RyR1 produced by this method was targeted properly and restored skeletal excitation-contraction coupling in dyspedic myotubes. The myotubes produced sufficient protein to allow single-channel analyses from as few as 10 100-mm dishes. In most cases this method could preclude the need for permanent transfectants for the study of RyR1 structure function.

Animals↗

PubMed1999

Inhibition of FAS/FAS-ligand does not block chemotherapy-induced apoptosis in drug sensitive and resistant cells.

It has been suggested that one means by which chemotherapeutic agents exert their effect on leukaemic cells, is via autocrine induction of fas-ligand which then binds to fas (CD95), activates the caspase pathway and results ultimately in apoptotic death. In order to test this hypothesis, we have treated leukaemic cell lines with various chemotherapeutic agents (idarubicin, etoposide, fludarabine and 2-CdA) with and without pre-treatment with fas (ZB4) and fas-ligand (NOK-1) blocking monoclonal antibodies. Cell cycle analysis and quantitation of apoptosis were performed by flow cytometry following propidium iodide staining. HL-60 cells were found to be sensitive to the induction of apoptosis with all drugs tested but were highly resistant to treatment with a fas-ligating antibody (CH11). Apoptosis was neither inhibited in parental CEM cells nor their mdr-expressing drug resistant counterpart, CEM/VLB100 by pre-treatment with either ZB4 or NOK1. In addition, CEM/VLB100 were slightly more sensitive to treatment with CH11 (100 ng/ml) than parental CEM cells (% age apoptosis = 30.35 and 23.675, p = 0.024) and at least as sensitive to recombinant fas-ligand (50 ng/ml) (% age apoptosis = 26.6 and 20.2, p = NS). We conclude that it is unlikely that fas/fas-ligand interactions play a significant role in the induction of apoptosis by these chemotherapeutic agents in the leukaemic cell lines tested.

Antibodies, Monoclonal↗

PubMed1999

Effects of PARP inhibition on drug and Fas-induced apoptosis in leukaemic cells.

Poly (ADP-ribose) polymerase (PARP) is activated following binding to DNA strand breaks and is cleaved in cells undergoing apoptosis. Work predominantly in murine systems has suggested that inhibitors of PARP might potentiate the effects of chemotherapeutic agents and be used as adjuncts to cancer therapy. Therefore, we studied the role of PARP in drug-induced apoptosis in HL-60, myeloid leukaemia cells and found that pre-treatment with 3-aminobenzamide (3AB) or 6(5H)-phenanthridinone, inhibitors of PARP, resulted in resistance to, rather than potentiation of apoptotic death induced by DNA-damaging agents, idarubicin, etoposide and fludarabine, as determined by flow cytometry, following propidium iodide staining. 3AB treated CEM/VLB100, mdr-expressing human lymphoblastic leukaemia cells were also found to be more resistant to idarubicin compared to cells treated with idarubicin alone, however, apoptosis was not reduced in parental CCRF-CEM cells under the same conditions. Similar results were obtained using agents with primary modes of action which do not involve DNA damage, vinblastine and a fas-ligating antibody (CH11). The precise role of PARP has yet to be defined but might involve effects on cell cycle progression. We conclude that PARP activation appears to be involved in apoptosis in certain leukaemic cell lines and that these effects are independent of lineage or p-glycoprotein. Constitutive failure to activate PARP might be responsible for conferring resistance to apoptosis.

Antineoplastic Agents↗

PubMed1999

Do sinusoidal models of respiration accurately reflect the respiratory events of patients breathing on nebulizers?

The amount of drug that is delivered by nebulization is a combination of the physical properties of the agent being nebulized, the performance of the nebulizer, and the pattern of breathing of the patient. To avoid biological variation, mechanical models of breathing are frequently employed during the evaluation of the performance of a device. For simplicity, many investigators use sinusoidal models of breathing to calculate the expected inhaled mass, although some use square waves and other more complex models. Most assume that the duration of inspiration (Ti) is half of the total respiratory time (Ttot). This study compared the calculated inhaled mass from which the expected pulmonary deposition was estimated from the actual pattern of breathing of 43 children with cystic fibrosis (CF) breathing from an unvented nebulizer with a low dead volume and appropriate particle size distribution with that from a sinusoidal pattern of breathing using the same tidal volume (VT) and respiratory rate. The respiratory duty cycle (Ti/Ttot) was 0.45 +/- 0.05, which meant that less time was spent during inspiration than that found in a pure sinusoidal pattern. The difference between the predicted deposition from the actual pattern of breathing and that calculated from the sinusoidal model was 12 +/- 7%, which correlated with the respiratory rate (r = 0.67, P < 0.001). The degree of lung disease did not influence the discrepancy between the two values. In general, the actual VTs and respiratory rates were less in the patients than those employed in mechanical models of pediatric breathing. Although some patients had respiratory patterns that could be represented accurately with a sinusoidal model, most did not, and there were wide variations from child to child. These results suggest that there are both systematic and random errors arising from the use of a sinusoidal waveform to mimic respiratory events in patients.

Adolescent↗