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Biomedical subjects

M J O'Donnell

Publications and source records attributed to M J O'Donnell.

At least 19 recordsLinked to original sources

Effective control of dental chair unit waterline biofilm and marked reduction of bacterial contamination of output water using two peroxide-based disinfectants.

Bacterial biofilm in dental unit waterlines (DUWs) is a widespread problem, and poses a potentially significant risk of infection to dental staff and patients, particularly those who are medically compromised or immunocompromised. The purpose of the present study was to investigate the level of bacterial contamination of dental chair unit output water in the Dublin Dental Hospital, and to investigate the efficacy of two hydrogen peroxide-based disinfectants in reducing bacterial loads to < or =200 cfu/mL as recommended by the American Dental Association. The chemical quality of dental chair unit input and output water was well within the limits recommended for potable water. Water supplied to the units yielded an average aerobic heterotrophic bacterial cell density of 184 cfu/mL. However, the corresponding density in output water was considerably higher; the average cell density in water from the three-in-one air/water syringes and cup fillers in 12 chairs was 8200 and 4300 cfu/mL, respectively. Dental unit water obtained from 18 separate reservoir-supplied units in general practices in the Dublin area yielded an average of 66000 cfu/mL. The bacterial species found were predominantly environmental organisms, which were also present at low levels in the input water. Some of the species identified (e.g., Burkholderia cepacia and Pseudomonas fluorescens) are known opportunistic pathogens. The capacity of two disinfectants, Sterilex Ultra and Sanosil, to reduce bacterial contamination to safe levels was compared. In a controlled study, once weekly overnight (15 h) disinfection using either agent reduced the bacterial density to below the American Dental Association recommended level of 200 cfu/mL. However, once disinfection ceased the bacterial loads increased to unacceptably high levels within three weeks. Electron microscopic analysis showed that both disinfectants markedly reduced biofilm in the DUWs, but the biofilm rapidly became extensive again when once weekly disinfection ceased. While both disinfectants were equally effective in lowering the bacterial counts to acceptable levels, Sterilex Ultra was associated with clogging of DUWs in some dental chair units after repeated usage, suggesting that Sanosil is a more suitable agent for routine use.

Biofilms↗

Transepithelial potential in Malpighian tubules of Rhodnius prolixus: lumen-negative voltages and the triphasic response to serotonin.

Previous studies of the Malpighian tubules of Rhodnius reported lumen-negative values of transepithelial potential (TEP), and a characteristic triphasic change in TEP in response to stimulation of tubule fluid secretion by serotonin. TEP was measured using the Ramsay technique, in which electrodes are positioned in bathing and secreted fluid droplets for tubules isolated under paraffin oil. The validity of this method of TEP measurement has been questioned on the grounds that, in tubules of some species, it may permit shunting of current from lumen to bath through the cells or through the thin layer of fluid adherent to the surface of that portion of the tubule in the oil. The triphasic response of TEP to serotonin has been confirmed in this study of tubules of fifth instar Rhodnius prolixus using two different techniques that eliminate the possibility of shunting artefacts. From an initially negative value in unstimulated tubules ( approximately -25 mV, lumen-negative), TEP shifted to approximately -33 mV in phase 1, approximately +30 mV in phase 2 and approximately -32 mV in phase 3. TEP during each phase was similar irrespective of the measurement technique. Ion substitution experiments and the effects of specific pharmacological reagents support the proposal that the three phases of the response of TEP to serotonin correspond to sequential activation of an apical Cl(-) channel, an apical V-type H(+) ATPase and a basolateral Na(+):K(+):2Cl(-) cotransporter.

Journal Article↗

A maxi Cl- channel in cultured pavement cells from the gills of the freshwater rainbow trout Oncorhynchus mykiss.

Primary cultures of pavement cells from the gills of a freshwater fish, the rainbow trout Oncorhynchus mykiss, have been studied for the first time using the patch-clamp technique. Gigaohm seals were obtained in approximately 95% of cells studied, and channel activity was evident in a high proportion (>90%). A large-conductance Cl- channel was evident in 6 % of cell-attached and in 31% of inside-out patches. Single-channel conductance in inside-out patches was 372 pS, and current/voltage relationships were linear over the range -60 to +60 mV. The channel was activated by patch excision, and activation was often associated with polarization of the patch. The mean number of channels per patch was 1.9, and there were several subconductance states. The relationship between channel activity (NP(o)) and voltage was in the form of an inverted U, and channel activity was highest between 0 and +20 mV. Large-conductance Cl- channels showed a progressive time-dependent reduction in current in response to sustained polarization to voltages outside the range -20 mV to +20 mV. Permeability ratios (P) of Cl- to other anions were P(HCO3)/P(Cl)=0.81, P(SO4)/P(Cl)=0.31 and P(isethionate)/P(Cl)=0.53. The channel was blocked by Zn2+, SITS, DIDS and diphenylamine carboxylate. This is the first description of a large-conductance Cl- channel in gill cells from freshwater or marine species. Possible functions of the channel are discussed.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

Analysis of epithelial K(+) transport in Malpighian tubules of Drosophila melanogaster: evidence for spatial and temporal heterogeneity.

Transport of K(+) by the lower, main and distal segments of the Malpighian tubules of Drosophila melanogaster was analyzed using self-referencing K(+)-selective microelectrodes. Transport properties of the Malpighian tubules of Drosophila melanogaster change along their length. Self-referencing ion-selective (SeRIS) microelectrode measurements (relative to the bath concentration of 20 mmoll(-1)) showed a 1% reduction (P<0.05) of [K(+)] in the unstirred layer adjacent to the main segment of the Malpighian tubules, confirming secretion of K(+) from the bath to the tubule lumen. Conversely, SeRIS measurements showed a 0.7% increase (P<0.05) in [K(+)] in the unstirred layer adjacent to the lower segment of Malpighian tubules, confirming reabsorption of K(+) from the luminal fluid to the bath. Measurements using SeRIS also showed that the distal segment neither secreted nor reabsorbed K(+). There was pronounced spatial heterogeneity in K(+) transport by the lower segment and the main segment; not all morphologically similar cells participated equally in K(+) transport, nor did all main segment cells respond equally to stimulation of K(+) transport by cyclic AMP. Pronounced temporal heterogeneity in K(+) reabsorption by the lower Malpighian tubules was also observed. We suggest that this reflects periodic reduction in K(+) reabsorption due to retention of fluid within the lower segment when the ureter contracts.

Absorption↗

Na(+) and Cl(-) transport by the urinary bladder of the freshwater rainbow trout (Oncorhynchus mykiss).

Freshwater (FW) rainbow trout (Oncorhynchus mykiss) urinary bladders mounted in vitro under symmetrical saline conditions displayed electroneutral active absorption of Na(+) and Cl(-) from the mucosal side; the transepithelial potential (V(t)) was 0.1 mV, and the short-circuit current was less than 1 microA cm(-2). Removal of Na(+) from mucosal saline decreased Cl(-) absorption by 56% and removal of Cl(-) decreased Na(+) absorption by 69%. However, active net absorption of both Na(+) and Cl(-) was not abolished when Cl(-) or Na(+) was replaced with an impermeant ion (gluconate or choline, respectively). Under physiological conditions with artificial urine (¿Na(+) = 2.12 mM, ¿Cl(-) = 3.51 mM) bathing the mucosal surface and saline bathing the serosal surface, transepithelial potential (V(t)) increased to a serosal positive approximately +7.6 mV. Unidirectional influx rates of both Na(+) and Cl(-) were 10-20-fold lower but active absorption of both ions still occurred according to the Ussing flux ratio criterion. Replacement of Na(+) with choline, or Cl(-) with gluconate, in the mucosal artificial urine yielded no change in unidirectional influx of Cl(-) or Na(+), respectively. However, kinetic analyses indicated a decrease in maximum Na(+) transport rate (J(max)) of 66% with no change in affinity (K(m)) in the low Cl(-) mucosal solution relative to the control solution. Similarly, there was a 79% decrease in J(max) values for Cl(-), again with no change in K(m), in the low-Na(+) mucosal bathing. The mucosal addition of DIDS, amiloride or bumetanide (10(-4) M) had no effect on either Na(+) or Cl(-) transport, under either symmetrical saline or artificial urine/saline conditions. Addition of the three drugs simultaneously (10(-4) M), or chlorothiazide (10(-3) M), under symmetrical saline conditions also had no effect on Na(+) or Cl(-) transport rates. Cyanide (10(-3) M) addition to mucosal artificial urine caused a slowly developing decrease of Na(+) influx to 59% and Cl(-) influx to 50% in the period after drug addition. Na(+) and Cl(-) reabsorption appears to be a partially coupled process in the urinary bladder of O. mykiss; transport mechanisms are both dependent upon and independent of the other ion.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

Calcium homeostasis in larval and adult Drosophila melanogaster.

Calcium homeostasis in Drosophila melanogaster was examined in response to the challenges imposed by growth, reproduction and variations in dietary calcium content. Turnover time for calcium, calculated as the time for (45)Ca(2+)to accumulate to half the steady state value of 3.46 nmol/fly, was 3.3 days. Although larvae weighed 2x as much as adults, they contained 3-4x as much calcium. Anterior Malpighian tubules (Mts) contain much more calcium than posterior Mts, accounting for 25-30% of the calcium content of the whole fly. In response to a 6.2-fold increase in dietary calcium level, calcium content of whole flies increased only 10%. Hemolymph calcium concentration ( approximately 0.5 mM) was similar in males and females and in animals raised on diets differing in calcium content. Fluid secretion rate, secreted fluid calcium concentration, and transepithelial calcium flux in tubules isolated from flies raised on high and low calcium diets did not differ significantly. Malpighian tubules secrete calcium at rates sufficient to eliminate whole body calcium content in 0.5 and 3 days for tubules secreting fluid at basal and maximal rates, respectively. It is suggested that flies absorb high quantities of calcium from the diet and maintain homeostasis through the combined effects of elimination of calcium in fluid secreted by the Malpighian tubules and the sequestration of calcium in granules, especially within the distal segment of the anterior pair of Malpighian tubules.

Animals↗

Directional coronary atherectomy in intermediate sized vessels: final results of the intermediate vessel atherectomy trial (IVAT).

Revascularization options for intermediate sized vessels (2.5-2.9 mm) have historically been limited. IVAT is a pilot study to assess the efficacy and safety of debulking intermediate sized vessels using directional coronary atherectomy (DCA). Between March 1996 and June 1997, 50 patients were enrolled at seven hospitals in the United States. Of those patients, 70% presented with unstable angina and 52% had single vessel disease. Of the lesions treated, 96% were de novo. Adjunctive PTCA after DCA was performed in 90% of cases at the discretion of the investigator to maximize luminal diameter. The GTO DCA device was used in 90% of cases. Procedural success (residual stenosis <50% without major complications) was 94%. Stents were placed in 12% of patients. The only complications were three non-Q wave MIs. Mean reference vessel diameter increased from 2.49 mm pre-procedure to 2.57 mm after DCA and 2.61 post-procedure; mean MLD increased from 0.76 mm to 2.03 mm to 2.31 mm; and mean stenosis decreased from 70% to 21% post DCA and to 11% post procedure. At six months follow-up, 18.0% of target lesions required revascularization. Total revascularization, including non-target vessels, was 32%. These results suggest that DCA has a high procedural success rate and a low target lesion revascularization rate in intermediate sized vessels.

Adult↗

Pulsed-dye laser versus conventional therapy in the treatment of warts: a prospective randomized trial.

BACKGROUND: The clinical management of verrucae vulgaris is often challenging, and no ideal treatment currently exists. Early studies suggested that pulsed-dye laser (PDL) therapy might be highly effective in the treatment of verrucae, although more recent reports have been less optimistic. Previous studies have been retrospective or have consisted of a series of patients treated with PDL alone. OBJECTIVE: Our objective was to prospectively evaluate the efficacy of PDL therapy versus conventional therapy with liquid nitrogen cryotherapy or cantharidin in the treatment of warts. METHODS: Forty healthy adult patients with verrucae were randomized to receive either PDL (585 nm) therapy or conventional therapy. All enrollees also performed home therapy. Patients were eligible for up to 4 treatment sessions at 1-month intervals. Warts were individually counted and measured at the time of each treatment session. Complete response was defined as complete absence of verrucae with the presence of dermatoglyphics, and partial response was defined as a 50% or greater reduction in wart size. RESULTS: A total of 194 warts were evaluated by the conclusion of the study. Complete response was noted in an average of 70% of the warts treated with conventional therapy and in 66% of those in the PDL group; this was statistically insignificant. Partial response was observed in an average of 82% of the warts in patients treated with conventional therapy and 87% in the PDL group. Verruca vulgaris responded more readily than verruca plantaris in both treatment groups. The mean number of treatments to achieve success was similar in both groups. CONCLUSION: The PDL is an efficacious form of therapy for verrucae, as is conventional therapy. PDL therapy should be considered among the better established approaches in the treatment of warts, although data from this trial suggest that this approach is probably not superior.

Adult↗

Hepatic versus gallbladder bile composition: in vivo transport physiology of the gallbladder in rainbow trout.

Ion and water transport across the teleost Oncorhynchus mykiss gallbladder were studied in vivo by comparing flow and composition of hepatic bile, collected by chronic catheter, to volume and composition of terminally collected gallbladder bile. Differences in composition were comparable with those of other vertebrates, whereas bile flow (75 microl. kg(-1). h(-1)) was below values reported for endothermic vertebrates. The gallbladder concentrates bile acids five- to sevenfold and exhibits higher net Cl(-) than Na(+) transport in vivo, in contrast to the 1:1 transport ratio from gallbladders under saline/saline conditions. Transepithelial potential (TEP) in the presence of bile, at the apical surface, was -13 mV (bile side negative) but +1.5 mV in the presence of saline. Bile acid in the apical saline reversed the TEP, presumably by a Donnan effect. We propose that ion transport across the gallbladder in vivo involves backflux of Na(+) from blood to bile resulting in higher net Cl(-) than Na(+) flux. This Na(+) backflux is driven by a bile side negative TEP and low Na(+) activity in bile due to the complexing effects of bile acids.

Animals↗

Transport properties of cultured branchial epithelia from freshwater rainbow trout: a novel preparation with mitochondria-rich cells.

A new double-seeded insert (DSI) technique is described for culture of branchial epithelial preparations from freshwater rainbow trout on filter supports. DSI epithelia contain both pavement cells and mitochondria-rich (MR) cells (15.7+/-2.5 % of total cell numbers). MR cells occur singly or in clusters, are voluminous, open apically to the 'external environment' and exhibit ultrastructural characteristics similar to those found in the 'chloride cells' of freshwater fish gills. After 6-9 days in culture with Leibovitz's L-15 medium on both surfaces (symmetrical conditions), transepithelial resistance (TER) stabilized at values as high as 34 k capomega cm(2), indicative of electrically 'tight' epithelia. The density of MR cells, the surface area of their clusters and transepithelial potential (TEP; up to +8 mV basolateral positive, mean +1.9+/-0.2 mV) were all positively correlated with TER. In contrast, preparations cultured using an earlier single-seeded insert (SSI) technique contained only pavement cells and exhibited a negligible TEP under symmetrical conditions. Na(+)/K(+)-ATPase activities of DSI preparations were comparable with those in gill filaments, but did not differ from those of SSI epithelia. Replacement of the apical medium with fresh water to mimic the in vivo situation (asymmetrical conditions) induced a negative TEP (-6 to -15 mV) and increased permeability to the paracellular marker PEG-4000. Under symmetrical conditions, unidirectional Na(+) and Cl(-) fluxes were in balance, and there was no active transport by the Ussing flux ratio criterion. Under asymmetrical conditions, there were large effluxes, small influxes and evidence for active Cl(-) uptake and Na(+) extrusion. Unidirectional Ca(2+) fluxes were only 0.5-1.0 % of Na(+) and Cl(-) fluxes; active net Ca(2+) uptake occurred under symmetrical conditions and active net extrusion under asymmetrical conditions. Thus, DSI epithelia exhibit some of the features of the intact gill, but improvements in culture conditions are needed before the MR cells will function as true freshwater 'chloride cells'.

Animals↗

Novel aspects of the transport of organic anions by the malpighian tubules of Drosophila melanogaster.

Para-aminohippuric acid (PAH) is a negatively charged organic ion that can pass across the epithelium of Malpighian tubules. Its mode of transport was studied in Malpighian tubules of Drosophila melanogaster. PAH transport was an active process, with a K(m) of 2. 74 mmol l(-)(1) and a V(max) of 88.8 pmol min(-)(1). Tubules had a low passive permeability to PAH, but PAH transport rates (832 nmol min(-)(1 )mm(2)) and concentrative ability ([PAH](secreted fluid):[PAH](bath)=81.2) were the highest measured to date for insects. Competition experiments indicated that there were two organic anion transporters, one that transports carboxylate compounds, such as PAH and fluorescein, and another that transports sulphonates, such as amaranth and Indigo Carmine. PAH transport appears to be maximal in vivo because the rate of transport by isolated tubules is not increased when these are challenged with cyclic AMP, cyclic GMP, leucokinin I or staurosporine. Basolateral PAH transport was inhibited by ouabain and dependent on the Na(+) gradient. The Malpighian tubules appeared not to possess an organic acid/ &agr; -keto acid exchanger because PAH accumulation was not affected by low concentrations (100 micromol l(-)(1)) of &agr; -keto acids ( &agr; -ketoglutarate, glutarate, citrate and succinate) or the activity of phosphokinase C. PAH transport may be directly coupled to the Na(+) gradient, perhaps via Na(+)/organic acid cotransport. Fluorescence microscopy showed that transport of the carboxylate fluorescein was confined to the principal cells of the main (secretory) segment and all the cells of the lower (reabsorptive) segment. Organic anions were transported across the cytoplasm of the principal cells both by diffusion and in vesicles. The accumulation of punctate fluorescence in the lumen is consistent with exocytosis of the cytoplasmic vesicles. Apical PAH transport was independent of the apical membrane potential and may not occur by an electrodiffusive mechanism.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

Contributions of K+:Cl- cotransport and Na+/K+-ATPase to basolateral ion transport in malpighian tubules of Drosophila melanogaster.

Mechanisms of Na+ and K+ transport across the basolateral membrane of isolated Malpighian tubules of Drosophila melanogaster were studied by examining the effects of ion substitution and putative inhibitors of specific ion transporters on fluid secretion rates, basolateral membrane potential and secreted fluid cation composition. Inhibition of fluid secretion by [(dihydroindenyl)oxy]alkanoic acid (DIOA) and bumetanide (10(-)4 mol l-1) suggested that a K+:Cl- cotransporter is the main route for K+ entry into the principal cells of the tubules. Differences in the effects of bumetanide on fluxes of K+ and Na+ are inconsistent with effects upon a basolateral Na+:K+:2Cl- cotransporter. Large differences in electrical potential across apical (>100 mV, lumen positive) and basolateral (<60 mV, cell negative) cell membranes suggest that a favourable electrochemical gradient for Cl- entry into the cell may be used to drive K+ into the cell against its electrochemical gradient, via a DIOA-sensitive K+:Cl- cotransporter. A Na+/K+-ATPase was also present in the basolateral membrane of the Malpighian tubules. Addition of 10(-)5 to 10(-)3 mol l-1 ouabain to unstimulated tubules depolarized the basolateral potential, increased the Na+ concentration of the secreted fluid by 50-73 % and increased the fluid secretion rate by 10-19 %, consistent with an increased availability of intracellular Na+. We suggest that an apical vacuolar-type H+-ATPase and a basolateral Na+/K+-ATPase are both stimulated by cyclic AMP. In cyclic-AMP-stimulated tubules, K+ entry is stimulated by the increase in the apical membrane potential, which drives K+:Cl- cotransport at a faster rate, and by the stimulation of the Na+/K+-ATPase. Fluid secretion by cyclic-AMP-stimulated tubules was reduced by 26 % in the presence of ouabain, suggesting that the Na+/K+-ATPase plays a minor role in K+ entry into the tubule cells. Malpighian tubules secreted a Na+-rich (150 mmol l-1) fluid at high rates when bathed in K+-free amino-acid-replete saline (AARS). Secretion in K+-free AARS was inhibited by amiloride and bafilomycin A1, but not by bumetanide or hydrochlorothiazide, which inhibit Na+:Cl- cotransport. There was no evidence for a Na+ conductance in the basolateral membrane of unstimulated or cyclic-AMP-stimulated tubules. Possible mechanisms of Na+ entry into the tubule cells include cotransport with organic solutes such as amino acids and glucose.

Animals↗

Advances in DNA diagnostics.

The key advances in DNA diagnostics during the past year are techniques which will lead to advanced throughput without sacrificing sensitivity: miniaturization of samples to reduce material cost and preparation time, and parallelization through use of measurement arrays. The most promising gains have come in the areas of DNA arrays and mass spectrometry, where differential sequencing measurements are now possible.

Diagnostic Techniques and Procedures↗

Sequencing exons 5 to 8 of the p53 gene by MALDI-TOF mass spectrometry.

Matrix-assisted laser desorption ionization time of flight mass spectrometry was used to sequence exons 5 to 8 of the human p53 gene. A single tube procedure was established for target amplification and mass spectrometric (MS) sequencing. The MS sequencing scheme is designed for high throughput and parallel sample processing, and is amenable to full automation. Reliable sequencing data were obtained using fmol sample amounts. The high resolution and accuracy of MS sequencing was demonstrated by direct sequencing of a heterozygous template.

Base Sequence↗