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Biomedical subjects

M P Ryan

Publications and source records attributed to M P Ryan.

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

Purinoceptor activation of chloride transport in cystic fibrosis and CFTR-transfected pancreatic cell lines.

The regulation of chloride efflux from cystic fibrosis pancreatic adenocarcinoma cells (CFPAC-1) and wild-type CFTR-transfected CFPAC-1 cells (TPAC) was compared. Forskolin (10 microM) stimulated chloride efflux from the corrected TPAC cells but not from CFPAC-1 cells. Chloride efflux from both cell types was activated by thapsigargin (0.5 microM). The nucleotides ATP and UTP and the non-hydrolyzable ATP analogue, adenosine 5'-O-(3-thio) triphosphate (ATPgammaS), stimulated chloride efflux from both cell types. None of the other P2 purinoceptor agonists investigated elicited a response. The order of potency was ATP > or = UTP > or = ATPgammaS. Adenosine (10-100 microM) activated choride efflux from the TPAC but not the CFPAC cell line with no increase in intracellular cyclic AMP. Small but statistically significant inhibitions of the adenosine-(50 microM)-stimulated increase in chloride efflux were elicited by the A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (CPX, 100 nM) and the A2 receptor antagonist 3,7-dimethyl-1-propylargylxanthine (DMPX, 10 microM). The A2A receptor antagonist 8-(3-chlorostyryl)caffeine (CSC, 100 nM) had no significant effect. These results provide evidence for the regulation of chloride efflux by P2Y2 purinoceptors in genetically-corrected and CF pancreatic cell lines. Studies with adenosine receptor antagonists indicate some possible involvement of A1 and A2 (but not A2A) receptors in the adenosine stimulation of chloride efflux, but the relatively small effects of the inhibitors coupled with lack of increase in cyclic AMP and a response only in the CFTR-transfected cells also suggests a possible direct effect of adenosine on CFTR.

Adenosine Triphosphate↗
PubMed1998

Apoptosis and necrosis: mechanisms of cell death induced by cyclosporine A in a renal proximal tubular cell line.

BACKGROUND: The mechanisms of cyclosporine (CsA)-induced nephrotoxicity are not fully understood. While hemodynamic changes may be involved in vivo, there is also some evidence for tubular involvement. We previously showed direct toxicity of CsA in the LLC-PK1 renal tubular cell line. In the current study we examined mechanisms (apoptosis or necrosis) of cell death induced by CsA in the LLC-PK1 renal proximal tubular cell line. The possible role of the Fas (APO-1/CD95) antigen-Fas ligand system in the mediation of CsA-induced cell death was also investigated. METHODS: Cells were treated with CsA (0.42 nM to 83 microM) for 24 hours and alterations in DNA and protein synthesis and membrane integrity were examined. Flow cytometry was used to investigate: (i) alterations in the DNA content and cell cycle; (ii) the forward (FSC) and side (SSC) light scattering properties (indicators of cell size and granularity, respectively); (iii) the externalization of phosphatidylserine (PS) as a marker of early apoptosis using FITC-annexin V binding; and (iv) expression of the apoptotic Fas protein. DNA fragmentation in apoptotic cells was also determined by the TUNEL assay. RESULTS: CsA (all doses) caused a block in the G0/G1 phase of the cell cycle as indicated by a decrease in DNA synthesis and supported by an increase in the % of cells in the G0/G1 phase with concurrent decreases of those in the S and G2/M phases. The effect on protein synthesis appeared to be much less. Lower doses of CsA (4.2 nM) caused the appearance of a "sub-G0/G1" peak, indicative of reduced DNA content, on the DNA histogram that was paralleled by a reduction in cell size and an increased cell granularity and an increase in FITC-annexin V binding. DNA fragmentation was evident in these cells as assessed using the TUNEL assay. Higher doses of CsA increased cell size and decreased cell granularity and reduced membrane integrity. Expression of Fas, the cell surface molecule that stimulates apoptosis, was increased following low dose CsA exposure. CONCLUSIONS: These results indicate that CsA is directly toxic to LLC-PK1 cells with reduced DNA synthesis and cell cycle blockade. The mode of cell death, namely apoptosis or necrosis, is dose dependent. Fas may be an important mediator of CsA induced apoptosis in renal proximal tubular cells.

Animals↗
PubMed1998

Lacticin 3147, a broad-spectrum bacteriocin which selectively dissipates the membrane potential.

Lacticin 3147 is a broad-spectrum bacteriocin produced by Lactococcus lactis subsp. lactis DPC3147 (M. P. Ryan, M. C. Rea, C. Hill, and R. P. Ross, Appl. Environ. Microbiol. 62:612-619, 1996). Partial purification of the bacteriocin by hydrophobic interaction chromatography and reverse-phase fast protein liquid chromatography revealed that two components are required for full activity. Lacticin 3147 is bactericidal against L. lactis, Listeria monocytogenes, and Bacillus subtilis; at low concentrations of the bacteriocin, bactericidal activity is enhanced when target cells are energized. This finding suggests that the presence of a proton motive force promotes the interaction of the bacteriocin with the cytoplasmic membrane, leading to the formation of pores at these low lacticin 3147 concentrations. These pores were shown to be selective for K+ ions and inorganic phosphate. The loss of these ions resulted in immediate dissipation of the membrane potential and hydrolysis of internal ATP, leading to an eventual collapse of the pH gradient at the membrane and ultimately to cell death. Our results suggest that lacticin 3147 is a pore-forming bacteriocin which acts on a broad range of gram-positive bacteria.

Adenosine Triphosphate↗
PubMed1998

Evaluation of lacticin 3147 and a teat seal containing this bacteriocin for inhibition of mastitis pathogens.

Lacticin 3147 is a broad-spectrum bacteriocin produced by Lactococcus lactis subsp. lactis DPC3147 which is bactericidal against a range of mastitis-causing streptococci and staphylococci. In this study, both lacticin 3147 and the lantibiotic nisin were separately incorporated into an intramammary teat seal product. The seal containing lacticin 3147 exhibited excellent antimicrobial activity and might form the basis of an improved treatment for the prevention of mastitis in dry cows.

Animals↗
PubMed1998

SWI-SNF complex participation in transcriptional activation at a step subsequent to activator binding.

The SWI-SNF complex in yeast and related complexes in higher eukaryotes have been implicated in assisting gene activation by overcoming the repressive effects of chromatin. We show that the ability of the transcriptional activator GAL4 to bind to a site in a positioned nucleosome is not appreciably impaired in swi mutant yeast cells. However, chromatin remodeling that depends on a transcriptional activation domain shows a considerable, although not complete, SWI-SNF dependence, suggesting that the SWI-SNF complex exerts its major effect at a step subsequent to activator binding. We tested this idea further by comparing the SWI-SNF dependence of a reporter gene based on the GAL10 promoter, which has an accessible upstream activating sequence and a nucleosomal TATA element, with that of a CYC1-lacZ reporter, which has a relatively accessible TATA element. We found that the GAL10-based reporter gene showed a much stronger SWI-SNF dependence than did the CYC1-lacZ reporter with several different activators. Remarkably, transcription of the GAL10-based reporter by a GAL4-GAL11 fusion protein showed a nearly complete requirement for the SWI-SNF complex, strongly suggesting that SWI-SNF is needed to allow access of TFIID or the RNA polymerase II holoenzyme. Taken together, our results demonstrate that chromatin remodeling in vivo can occur by both SWI-SNF-dependent and -independent avenues and suggest that the SWI-SNF complex exerts its major effect in transcriptional activation at a step subsequent to transcriptional activator-promoter recognition.

Chromatin↗
PubMed1997

Assessment of viability and mitochondrial function of equine spermatozoa using double staining and flow cytometry.

An objective double-staining method was developed to evaluate viability and mitochondrial function of stallion spermatozoa using flow cytometry. Sperm viability was assessed by propidium iodide (PI) exclusion, and mitochondrial function was measured by the intensity of rhodamine 123 (R123) fluorescence. Flow cytometry estimates of sperm viability measured by PI were equivalent (P > 0.05) to estimates made using Hoechst 33258 stain and fluorescent microscopy (% dead: 25 +/- 2.4 vs 21.5 +/- 3.5). The use of both PI and R123 was validated by addition of various proportions of freeze-shocked (membrane damaged) cells to viable spermatozoa. There was a high correlation (r(2) = 0.996) between increased PI positivestained (dead) cells and the number of membrane-damaged spermatozoa added (% dead: 29 +/- 0.4, 44 +/- 1.4, 58 +/- 0.9, 75 +/- 0.7 and 91+/- 0.25 vs 0, 25, 50, 75 and 100% damaged cells, respectively). Optimal mitochondrial activity (OMA), as assessed by R123 uptake, was also reduced proportionally (r(2) = 0.976) by the percentage of membrane-damaged cells added (% OMA: 48 +/- 0.6, 37 +/- 1.7, 29 +/- 0.5, 16 +/- 1, 3.8 +/- 1.3 vs 0, 25, 50, 75 and 100% damaged cells, respectively). The mitochondrial inhibitors rotenone and monensin significantly depressed optimal mitochondrial activity (P < 0.001), and there was a significant positive correlation (r(2) = 0.959) between the dose of inhibitors added and the population of sperm cells exhibiting minimal R123 staining (4 -/+ 0.9, 12 -/+ 1.6, 14 -/+ 0.1 and 28 -/+ 2% for treatments with 0, 0.5, 1 and 2 x 10(-5) M rotenone and 0, 0.5, 1, and 2 x 10(-4) M monensin, respectively). Finally, it was shown that treatments containing identical proportions of membrane-damaged cells yielded similar results in terms of viability and mitochondrial activity, irrespective of whether the staining procedure was single or double (P > 0.05). The results of the double-staining method revealed that the percentage of spermatozoa with optimally functioning mitochondria was significantly correlated with the percentage of viable (PI negative) sperm cells (r(2) = 0.998). Flow cytometric analyses using this staining procedure provides reliable and rapid (10,000 cells/min) qualitative assessment of stallion semen.

Journal Article↗
PubMed1997

P52PAI-1 gene expression in butyrate-induced flat revertants of v-ras-transformed rat kidney cells: mechanism of induction and involvement in the morphological response.

Sodium n-butyrate-induced flat reversion in v-K-ras oncogene-transformed rat kidney (KNRK) cells is associated with transcriptional activation of the p52PAI-1 gene (which encodes the type-1 inhibitor of plasminogen activator). Butyrate-initiated expression of p52PAI-1 mRNA and protein correlated with induced cell spreading and preceded development of cell-to-substrate focal adhesions. Such undersurface matrix contact structures, which are absent from parental KNRK cells, require proximal PAI-1 deposition for their stabilization. Stimulated p52PAI-1 expression by flat revertants (approximating 25-fold that of control cells) and the accompanying cytoarchitectural reorganization appeared to be programmed responses to butyrate as both events required de novo RNA and protein synthesis, metabolic characteristics consistent with a secondary pathway of gene regulation. To assess the relevance of p52PAI-1 induction to the process of flat reversion more critically, a molecular genetic approach was designed to maintain high-level constitutive p52PAI-1 synthesis in the absence of butyrate. KNRK cells transfected with a Rc/CMVPAI plasmid construct, in which expression of a p52PAI-1 cDNA insert was driven by enhancer-promoter sequences from the immediate-early gene of human cytomegalovirus, formed colonies comprised of flat-revertant-like cells with a greater frequency than did cells transfected with the Rc/CMV vector alone (24.8% and 1.7% respectively). Comparative analysis of randomly selected Rc/ CMVPAI clones indicated that a 10-fold increase in immunoreactive p52PAI-1 protein, relative to Rc/CMV isolates, correlated with generation of the flat phenotype. These data suggest that induced p52PAI-1 expression probably functions in the development of morphological revertants in the KNRK cell system.

Animals↗
PubMed1997

Alpha-glutathione s-transferase (alpha-GST) release, an early indicator of carbon tetrachloride hepatotoxicity in the rat.

1. The use of the cytoplasmic enzyme, alpha glutathione s-transferase (alpha-GST) as an early index of carbon tetrachloride (CCl4) toxicity in the rat was investigated and compared with a standard enzyme, marker, aspartate aminotransferase (AST). The hepatotoxic effects of CCl4 in the rat were determined in a time and dose-response study. 2. Following CCl4 exposure, alpha-GST release was shown to be an earlier and more sensitive biomarker of hepatotoxicity than AST. 3. Significant increases in alpha-GST were detected 2 h after CCl4 exposure. Using the enzyme marker AST, this early hepatotoxic injury went undetected. At 6 and 16 h, alpha-GST was also a more sensitive indicator of hepatotoxicity than AST. 4. alpha-GST release was significantly increased at a dose of 5 microliters/kg, the lowest concentration of CCl4 administered and clearly responded in a dose-dependent manner with increasing doses of CCl4. In contrast, release of AST did not reach statistical significance until a dose of 25 microliters/kg. 5. Thus, these findings indicate that alpha-GST is a more sensitive and more accurate reflector of CCl4 induced hepatotoxicity than AST.

Administration, Oral↗
PubMed1996

Complex regulation of plasminogen activator inhibitor type-1 (PAI-1) gene expression by serum and substrate adhesion.

Expression of plasminogen activator inhibitor type-1 (PAI-1), a member of the serine protease inhibitor (SERPIN) superfamily that functions to negatively regulate the plasmin-based pericellular proteolytic cascade, was induced early after exposure of growth-arrested normal rat kidney (NRK) cells to serum-containing medium. Increased PAI-1 transcription was rapid (evident within 10 min of serum addition) and involved immediate-early response kinetics. [3H]Thymidine autoradiography was used to map the time frame of PAI-1 expression during a synchronous growth cycle. PAI-1 transcript accumulation peaked in mid-G1 phase (approx. 4-6 h post-stimulation) and declined prior to, or concomitant with, the onset of DNA synthetic phase. Serum increased PAI-1 expression in NRK cells in agarose suspension, as well as monolayer, culture; induction in suspended cells (similar to monolayer culture conditions) also occurred in the presence of cyclohexamide or puromycin. The serum-inductive pathway leading to PAI-1 gene activation is thus functional regardless of adhesive conditions or capacity for de novo protein synthesis. The amplitude of induction and maintenance of expression in later stages of G1, however, were subject to adhesive influences. PAI-1 transcript accumulation at 4 and 8 h post-stimulation in newly adherent cells, moreover, was blocked by puromycin, indicating that both immediate-early and secondary mechanisms regulate PAI-1 mRNA levels during progression of NRK cells through an 'activated' G1 growth phase.

Animals↗
PubMed1996

Regulation of changes in cytosolic Ca2+ and Na+ concentrations in rat submandibular gland acini exposed to carbachol and ATP.

The relationship between cytosolic concentrations of Ca2+ (Ca2i) and Na+ (Na+i) were studied in preparations of rat submandibular and pancreatic acini loaded with the Ca(2+)-sensitive dye Fura-2 or the Na(+)-sensitive dye SBFI. Pancreatic acini showed no changes in Na+i during either transient or persistent changes in Ca2+i. Increases in Ca2+i produced by exposure of submandibular gland acini to carbachol, a muscarinic cholinergic agonist, were followed by an increase in Na+i after a delay of 5-10 s. When Ca2+ stores were mobilized without Ca2+ influx Na+i also increased, but in acini loaded with BAPTA, a nonfluorescent Ca2+ chelator, the transient increase in Ca2+ caused by mobilization of stored Ca2+ was virtually abolished, as was the increase in Na+i. In the presence of inomycin, increases in Ca2+i were followed by increases in Na+i. Ca(2+)-dependent increases in Na+i were abolished in Na(+)-free buffer and by the presence of furosemide, a blocker of Na(+)-K(+)-2Cl- cotransport. In other studies, extracellular ATP (ATPo) produced an increase in Ca2+i and Na+i. The steady-state increase in Ca(i)2+ was reduced by increasing extracellular Na+ concentrations (Na+o in dose-dependent fashion (IC50 = 16.4 +/- 4.7 mM Na+). Likewise, increasing Na+o reduced ATPo-stimulated 45Ca2+ uptake at steady state (IC50 = 15.8 +/- 9.2 mM Na+). Changing Na+o had no effect on carbachol-stimulated increases in Ca2+i. We conclude that, in rat submandibular gland acini, ATPo promotes an increase in Ca2+i and Na+i via a common influx pathway and that, under physiologic conditions, Na+ significantly limits the ATPo-stimulated increase in Ca2+i. In the presence of carbachol, however, Na+i rises in Ca2+i-dependent fashion in submandibular gland acini via stimulation of Na(+)-K(+)-2Cl- cotransport.

Adenosine Triphosphate↗
PubMed1996

An application in cheddar cheese manufacture for a strain of Lactococcus lactis producing a novel broad-spectrum bacteriocin, lacticin 3147.

Lactococcus lactis DPC3147, a strain isolated from an Irish kefir grain, produces a bacteriocin with a broad spectrum of inhibition. The bacteriocin produced is heat stable, particularly at a low pH, and inhibits nisin-producing (Nip+) lactococci. On the basis of the observation that the nisin structural gene (nisA) does not hybridize to DPC3147 genomic DNA, the bacteriocin produced was considered novel and designated lacticin 3147. The genetic determinants which encode lacticin 3147 are contained on a 63-kb plasmid, which was conjugally mobilized to a commercial cheese starter, L. lactis subsp. cremoris DPC4268. The resultant transconjugant, DPC4275, both produces and is immune to lacticin 3147. The ability of lacticin 3147-producing lactococci to perform as cheddar cheese starters was subsequently investigated in cheesemaking trials. Bacteriocin-producing starters (which included the transconjugant strain DPC4275) produced acid at rates similar to those of commercial strains. The level of lacticin 3147 produced in cheese remained constant over 6 months of ripening and correlated with a significant reduction in the levels of nonstarter lactic acid bacteria. Such results suggest that these starters provide a means of controlling developing microflora in ripened fermented products.

Bacteriocins↗
PubMed1995

The standard electrocardiogram as a screening test for hypertrophic cardiomyopathy.

Phenotypic heterogeneity in hypertrophic cardiomyopathy (HC) makes definitive diagnosis difficult, particularly during family screening. We studied the electrocardiogram (ECG) as a potential initial screening test in patients with HC. Using accepted diagnostic criteria, we examined the ECGs and echocardiograms of 159 patients with a confirmed clinical or genetic diagnosis of HC. An abnormal ECG was found in 154 patients (97%) while only 146 (92%) showed an abnormal echocardiogram. Of the former, 9 patients (6%) had normal echocardiograms and had been diagnosed on the basis of identification of a mutation in the beta myosin heavy chain gene (n = 8) or obligate carrier status (n = 1). Only 1 of these 9 patients was under age 20, the time at which hypertrophy is normally expressed on the echocardiogram. The remaining 5 patients (3%) without ECG abnormality consisted of 1 patient with an echocardiogram clearly diagnostic of HC and 4 clinically normal patients (aged 13, 24, 29, and 33 years) with normal echocardiograms who had been diagnosed by mutation identification (n = 3) or obligate carrier status (n = 1). Thus only these latter 4 patients (3%) would not have been diagnosed as having HC based on an abnormal ECG and/or abnormal echocardiogram. Screening relatives for HC by ECG criteria alone detects all those whom an echocardiogram will diagnose. While echocardiography aids in the specificity of HC diagnosis, the ECG, within the context of a family with a proven case of HC, is a more sensitive marker of the disease. It is therefore both a cost-effective and useful tool for screening those to proceed to echocardiography.

Adolescent↗
PubMed1995

Cell-shape-dependent modulation of p52(PAI-1) gene expression involves a secondary response pathway.

Expression of the rat p52(PAI-1) gene is positively regulated by agents that influence cellular microfilament organization and/or cell-to-substrate adhesion [e.g. cytochalasin D (CD) and sodium n-butyrate (NaB)] [Higgins, Chaudhari and Ryan (1991) Biochem. J. 273, 651-658; Higgins, Ryan and Providence (1994) J. Cell. Physiol. 159, 187-195]. As shape-responsive genes may be subject to inducer-specific controls, the biochemical mechanisms underlying the shape-dependent pathway of p52(PAI-1) gene regulation were examined in v-ras-transformed rat kidney (KNRK) cells. NaB and/or CD effectively stimulated p52(PAI-1) run-off transcription and augmented de novo p52(PAI-1) mRNA and protein synthesis in KNRK cells; induction at both the mRNA and protein levels was inhibited by actinomycin D. Pretreatment with cycloheximide (CX) markedly attenuated NaB- and/or CD-stimulated p52(PAI-1) expression. CX alone, however, induced low levels of p52(PAI-1) mRNA; increased p52(PAI-1) protein synthesis was evident after release of KNRK cells from CX blockade. Such CX-mediated induction was also sensitive to actinomycin D. Full stimulation of p52(PAI-1) expression in KNRK cells in response to the shape modulators NaB and/or CD involves transcriptional activation of the p52(PAI-1) gene, requires de novo RNA synthesis and occurs through a secondary-response (i.e. protein-synthesis-dependent) pathway.

Butyrates↗
PubMed1995

Evidence for location of the CFTR in human placental apical membrane vesicles.

Ion transport (36Cl uptake) and immunochemical studies were undertaken to detect the cystic fibrosis transmembrane conductance regulator (CFTR) in apical membrane vesicles prepared from human placenta. 36Cl uptake into membrane vesicles was studied in the absence and presence of inwardly directed potassium gradients and valinomycin (Ko = Ki and Ko > Ki, where Ko is potassium concentration outside and Ki is potassium concentration inside the vesicles). The sensitivities of 36Cl uptake to the inhibitors 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), bumetanide, and diphenylamine-2-carboxylate were investigated. Each compound significantly inhibited uptake under both sets of conditions. Additional inhibition of 36Cl uptake was found when the compounds were added together, indicating that they were acting at least partly on different components of the 36Cl uptake. The DIDS- and bumetanide-insensitive component of transport was more selective for Cl than I. These findings suggested that this component may, at least in part, represent Cl transport via CFTR. Addition of adenosine 5'-O-(3-thiotriphosphate) (0.8 mM) led to a decrease in total 36Cl uptake but masked in the overall decrease was an increase in the DIDS- and bumetanide-insensitive component of 36Cl uptake. Western blot analysis of the apical membrane proteins with an antibody specific for a region of human CFTR detected a protein band of approximately 190 kDa. These ion transport and immunochemical studies provide evidence that CFTR is located in human placental apical membrane vesicles.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1995

Protection from cisplatin nephrotoxicity by A68828, an atrial natriuretic peptide.

The ability of a 13 amino-acid analog of atrial natriuretic peptide (ANP), A68828, to prevent development of cisplatin toxicity was evaluated in a rat model. ANP (1 microgram/kg/min), A68828 (10 micrograms/kg/min), A68828 (50 micrograms/kg/min), or peptide buffer was given as an intravenous infusion over 1 h beginning 15 min prior to an infusion of 5 mg/kg cisplatin. Animals receiving cisplatin plus peptide buffer vehicle developed predictable renal failure, with mean plasma creatinine and blood urea nitrogen concentrations of 1.09 +/- 0.09 mg/dL and 50.13 +/- 5.96 mg/dL, 72 h after treatment. ANP and A68828 (10 micrograms/kg/min) attenuated the increase in these indices of nephrotoxicity (mean plasma creatinine 0.86 +/- .06 mg/dL and 0.76 +/- 0.11 mg/dL, respectively). Surprisingly, the higher dose of A68828 (50 micrograms/kg/min) did not reduce cisplatin nephrotoxicity (72-h plasma creatinine 1.61 +/- 0.34 mg/dL). These results indicate that a short-term infusion of ANP or the analog A68828 can reduce the severity of cisplatin toxicity. At high doses of A68828 the beneficial effects of treatment may be lost.

Animals↗