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M P Ryan

Publications and source records attributed to M P Ryan.

At least 19 recordsLinked to original sources
PubMed1992

Cell-shape-associated transcriptional activation of the p52(PAI-1) gene in rat kidney cells.

The microfilament-disrupting agent cytochalasin D (CD) increased (by 10-22-fold) the synthesis de novo and extracellular matrix deposition of plasminogen-activator inhibitor type-1 [p52(PAI-1)] in normal rat kidney (NRK) cells. Transition from a flat to a round phenotype occurred concomitantly with, and may actually precede, p52(PAI-1) induction; both the morphological and p52(PAI-1) responses were dose-dependent. Augmented synthesis became evident between 4 and 5 h of treatment of NRK cells with 100 microM-CD, correlating with a transition from 25 to more than 60% rounded cells. CD-associated increases in p52(PAI-1) mRNA abundance and protein biosynthesis were maximal between 6 and 8 h of continuous CD exposure, declined by 50% thereafter, but remained elevated (by at least 6-21-fold respectively over control values) for 24 h. Changes in p52(PAI-1) mRNA abundance at this 24 h point reflected an approx. 5-fold increase in p52(PAI-1)-gene transcription. These data confirm previous suggestions, based on actinomycin D-sensitivity of the inductive response [Higgins & Ryan (1992) Biochem. J. 284, 433-439], that CD-mediated increases in p52(PAI-1) expression are at least partly due to transcription-level events. Since CD also augments specific cellular responses to growth factors or cytokines, the potential effectiveness of this inducer was evaluated both in the presence and absence of serum growth factors using quiescent NRK cells [a growth state in which p52(PAI-1) is not expressed] as a model system. Induction of p52(PAI-1) synthesis and matrix deposition in CD-stimulated quiescent NRK cells was as efficient under growth-factor-deficient conditions as when CD was added simultaneously with serum. CD alone is thus a complete inducer of p52(PAI-1) expression in NRK cells, an observation that supports the contention that cell shape is an important regulatory element in p52(PAI-1)-gene control.

Animals↗
PubMed1992

Identification of the 52 kDa cytoskeletal-like protein of cytochalasin D-stimulated normal rat kidney (NRK/CD) cells as substrate-associated glycoprotein p52 [plasminogen-activator inhibitor type-1 (PAI-1)]. Expression of p52 (PAI-1) in NRK/CD cells is regulated at the level of mRNA abundance.

Cell shape profoundly affects cellular metabolic activity, protein and nucleic acid synthesis, and cytoskeletal organization. To examine the influence of cell shape on protein expression, normal rat kidney (NRK) cells were exposed to the microfilament-disrupting drug cytochalasin D (CD), labelled with [35S]methionine, and newly synthesized cellular and cytoskeletal proteins examined by two-dimensional gel electrophoresis. CD produced dramatic changes in cell shape (from a flat to round phenotype) with concomitant 3-7-fold increases in the cellular content and cytoskeletal deposition of the microfilament-associated proteins actin, alpha-actinin, and tropomyosin isoform 1. Augmented actin protein content in NRK/CD cells was paralleled by a corresponding increase in actin mRNA abundance and was inhibited by prior addition of actinomycin D. A detergent-insoluble protein of 52 kDa was also detected at high levels in the cytoskeletal fraction of NRK/CD cells. Two-dimensional electrophoretic mapping of total cellular and cytoskeletal proteins revealed this 52 kDa protein to be the previously described glycoprotein p52 [Higgins & Ryan (1989) Biochem. J. 257, 173-182]. By using electrophoretic and immunochemical criteria, p52 was identified as plasminogen-activator inhibitor type-1 (PAI-1). Like actin, CD-induced p52(PAI-1) synthesis, cellular content, and partitioning to the detergent-insoluble cytoskeletal compartment reflected a corresponding increase in p52(PAI-1) mRNA. Such induction was similarly inhibited by actinomycin D. p52(PAI-1) expression in the NRK-cell system is thus responsive to CD-mediated shape changes and requires ongoing RNA synthesis for its induction. Differential extraction of detached cell bodies and the substrate-adherent 'remnant' fraction of NRK/CD cultures, furthermore, indicated that p52(PAI-1) was not an intrinsic internal cytoskeletal element but, rather, selectively localized to the extracellular residue. p52(PAI-1) retained its detergent-insoluble characteristics even in this isolated 'remnant' fraction, where it was also the predominant protein species resolved.

Animals↗
PubMed1992

Factors affecting chloride conductance in apical membrane vesicles from human placenta.

Apical membrane vesicles from human term placenta were isolated using a magnesium precipitation technique, and the purity of the vesicles was assessed morphologically using scanning and transmission electron microscopy, and biochemically, using marker enzymes. The vesicles were found to be morphologically intact and significantly enriched in enzymes associated with apical membranes. 36Cl- uptake into these vesicles was studied in the presence of an outwardly directed Cl- gradient. This uptake was found to be time dependent, with an initial rapid uptake tending to peak between 10 and 20 min and thereafter decline. Uptake was found to be voltage dependent since 5 microM valinomycin caused a decrease in uptake. The effects of N-phenylanthranilic acid (NPA) and 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS) and bumetanide on the initial rate of Cl- were examined in the presence and absence of 5 microM valinomycin. NPA and DIDS inhibited isotope uptake strongly with IC50 values of 0.83 +/- 0.35 microM and 3.43 +/- 0.37 microM, respectively, in the absence of valinomycin. Although valinomycin reduced 36Cl- uptake by about 80% when added before the isotope, DIDS reduced the uptake which remained in a concentration-dependent fashion with an IC50 of 5.6 +/- 2.1 microM. Under these conditions, NPA was without effect at concentrations below 100 microM. Bumetanide was without effect at the concentrations used in the absence of valinomycin. However, following valinomycin pretreatment, bumetanide reduced 36Cl- uptake significantly at 100 microM concentration. Vesicle diameter, as assessed by flow cytometry, did not change under the conditions employed. The effects of some fatty acids were also investigated. Arachidonic acid and linoleic acid inhibited Cl- uptake with IC50 values of 37.6 +/- 14.9 microM and 4.59 +/- 0.51 microM, respectively. Arachidonyl alcohol and elaidic acid were found to be without effect. These studies show that human placental brush border membrane vesicles possess a chloride conductance channel, the activity of which can be measured in the presence of an outwardly directed Cl- gradient and this channel is sensitive to Cl- channel inhibitors, especially N-phenylanthranilic acid, and can be inhibited by unsaturated fatty acids such as arachidonic acid and linoleic acid.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1992

Changes of cytosolic Ca2+ interfere with measurements of cytosolic Mg2+ using mag-fura-2.

In rat pancreatic and submandibular gland acini during exposure to carbachol, changes in the fluorescence emission intensity ratio (R) of acini loaded with mag-fura-2 resemble changes in cytosolic Ca2+ concentration (Ca2+i) in acini loaded with fura-2. Furthermore, changes of R depend on the presence of extracellular Ca2+ (Ca2+o) but are much less influenced by changes in extracellular Mg2+ (Mg2+o). To evaluate interference with measurement of cytosolic Mg2+ (Mg2+i) by changes in Ca2+i, we determined the dissociation constant (Kd) and Hill coefficient (NH) of the Ca(2+)-mag-fura-2 and Mg(2+)-mag-fura-2 complexes in standard solutions, in intact acini after loading with the acetoxymethyl ester of mag-fura-2 (mag-fura-2/AM), and in lysates derived from acini loaded with mag-fura-2/AM. The Kd of the Ca(2+)-mag-fura-2 complex in acini (determined with ionomycin) was 20.49 +/- 5.20 microM, and NH was 1.44 +/- 0.16 (n = 24). Kd of the Mg(2+)-mag-fura-2 complex in acini was 2.25 +/- 0.98 mM, and NH was 1.20 +/- 0.20 (n = 25). Mean Kd values were slightly lower in acinar lysates and in solutions of standard mag-fura-2. In acini from either gland, 1,2-bis(2-aminophenoxy)ethan-N,N,N',N'-tetraacetic acid (BAPTA) suppressed carbachol-induced Ca2+i transients. The R value in stimulated acini loaded with BAPTA and mag-fura-2 increased slightly when Mg2+o was increased from less than 10 nM to 1.2 mM, suggesting that Mg2+ influx contributes to the maintenance of Mg2+i during exposure to carbachol. Under these conditions, pancreatic acinar Mg2+i is 0.53 +/- 0.14 mM (n = 5).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Refractory potassium repletion. A consequence of magnesium deficiency.

Experimental and clinical observations support the view that uncorrected magnesium (Mg) deficiency impairs repletion of cellular potassium (K). This is consistent with the observed close association between K and Mg depletion. Concomitant Mg deficiency in K-depleted patients ranges from 38% to 42%. Refractory K repletion due to unrecognized concurrent Mg deficiency can be clinically perplexing. Refractory K repletion as a consequence of Mg deficiency may be operative in patients with congestive failure, digitalis toxicity, cisplatin therapy, and in patients receiving potent loop diuretics. Therefore, we recommend that: (1) serum Mg be routinely assessed in any patients in whom serum electrolytes are necessary for clinical management and (2) until serum Mg is routinely performed consideration should be given to treating hypokalemic patients with both Mg as well as K to avoid the problem of refractory K repletion due to coexisting Mg deficiency.

Electrolytes↗
PubMed1991

p52(PAI-1) and actin expression in butyrate-induced flat revertants of v-ras-transformed rat kidney cells.

Flat revertants of v-ras-transformed (KNRK) rat kidney cells, which express elevated levels of p21ras protein, were generated to high efficiencies with sodium butyrate (NaB). Overall protein synthesis in revertants was not different from parental cells, although changes were evident in expression and distribution of specific microfilament-associated cytoskeletal proteins. Quantitative two-dimensional electrophoresis revealed revertant-associated 3-4-fold increases in cytoskeletal deposition of the microfilament-associated proteins gelsolin and vinculin correlating with microfilament reorganization and focal-contact formation respectively. Similar increases in actin content were evident at both the total-cellular- and cytoskeletal-associated-protein levels. In contrast, intermediate-filament family elements (vimentin, lamins) remained unaltered. The only unique protein resolved in flat revertants was p52, a 52 kDa extracellular-matrix-associated protein previously identified as plasminogen-activator inhibitor type 1 (PAI-1). p52(PAI-1) expression was induced early during generation of the revertant phenotype and preceded development of focal-contact structures. NaB-induced p52(PAI-1) synthesis and generation of early morphological reversion in KNRK cells required ongoing RNA synthesis, since exposure to actinomycin D before addition of NaB inhibited both events. p52(PAI-1) induction by NaB was regulated at the level of mRNA abundance; in contrast, actin mRNA levels were the same in parental and revertant cells, suggesting that the increased actin content which typified the revertant phenotype was due to augmented actin microfilament stability.

Animals↗
PubMed1991

AIDS in Thailand.

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Acquired Immunodeficiency Syndrome↗
PubMed1991

Cell-shape regulation and matrix protein p52 content in phenotypic variants of ras-transformed rat kidney fibroblasts. Functional analysis and biochemical comparison of p52 with proteins implicated in cell-shape determination.

The 52 kDa transformation-sensitive protein p52 was previously identified as a major substrate-associated component of normal rat kidney (NRK) fibroblasts [Higgins & Ryan (1989) Biochem. J. 257, 173-182]. p52 selectively localized to cellular fractions enriched in substrate focal-contact sites and associated ventral undersurface elements. Rapid attachment/spreading of NRK cells on to prepared p52 matrices and inhibition of fibroblast spreading by antibodies to p52 indicated that this protein participates in shape determination or cell-to-substrate adhesion. NRK cells transformed with Kirsten murine sarcoma virus (KiMSV), with a temperature-sensitive mutant (ts-371 KiMSV) and maintained at the permissive temperature, or with the cloned EJrasval.12 oncogene, exhibited down-regulated accumulation of p52 in the ventral undersurface region. Immunochemical, lectin-affinity and electrophoretic analyses indicated that p52 shares considerable sequence similarity with plasminogen-activator inhibitor type-1, which is consistent with its subcellular localization and likely morphoregulatory activity. The marked down-regulation of p52 expression seen in four different ras-mediated transformation systems, its induction prior to butyrate-induced morphological reorganization in KiMSV-transformed cells, and the morphological consequences of exogenously added p52 or p52 antibodies on NRK fibroblasts suggest that this protein probably functions in cell-shape regulation. Abrogation of p52 matrix accumulation typically seen in ras transformants may contribute, therefore, to the aberrant cytoarchitecture characteristic of malignant fibroblasts.

Animals↗
PubMed1991

Myocardial contractility and ischaemia in the isolated perfused rat heart with propofol and thiopentone.

The effects of propofol and thiopentone on myocardial contractility and global ischaemia were evaluated using an isolated non-working perfused rat heart preparation. Contractility was assessed using a tension transducer linked to the cardiac apex, and the contractility was expressed as a ratio of the deflection size before and after infusion of the drug. Ischaemia-induced leakage of myocardial proteins and ions (potassium and magnesium) was assessed by comparing the concentrations in the effluent perfusate immediately before and after 60 min of isothermic ischaemia, in the presence of propofol, thiopentone or plain Krebs' buffer solution (control). Mean contractility ratios of 1.15 and 1.3 were obtained with control and propofol groups respectively (NS), but were reduced to 0.5 in the thiopentone group (P less than 0.001). The magnitude of the post-ischaemic leakage of proteins and potassium was similar in each group; however, the post-ischaemic leakage of magnesium was greater in the thiopentone group than in the propofol or control groups. These data suggest that, compared with thiopentone, propofol is not a potent negative inotrope, and that it may cause less disturbance of myocardial magnesium homeostasis during myocardial ischaemia.

Animals↗
PubMed1990

Human red blood cell insulin-degrading enzyme and rat skeletal muscle insulin protease share antigenic sites and generate identical products from insulin.

The mechanisms of cellular insulin degradation remain uncertain. Considerable evidence now exists that the primary cellular insulin-degrading activity is a metallothiol proteinase. Two similar degrading activities have been purified and characterized. Insulin protease has been purified from rat skeletal muscle and insulin-degrading enzyme from human red blood cells. Whereas the two degrading activities share a number of similar properties, significant differences have also been reported; and it is not at all established that they are the same enzyme. To examine this, we have compared antigenic and catalytic properties of the two enzymatic activities. Monoclonal antibodies against the red blood cell enzyme adsorb the skeletal muscle enzyme; and on Western blots, the antibodies react with an identical 110-kDa protein. Immunoaffinity-purified enzymes from both red blood cells and skeletal muscle degrade [125I]iodo(B26)insulin to the same products as seen with purified insulin protease and with intact liver and kidney. Chelator-treated muscle and red blood cell enzymes can be reactivated with either Mn2+ or Ca2+. Thus, insulin-degrading enzyme and insulin protease have similar properties. These results support the hypothesis that these activities reside in the same enzyme.

Animals↗
PubMed1990

General practice computing in Scotland.

OBJECTIVE: To investigate a method of assessing the extent of routine patient data held on computer by Scottish general practitioners. DESIGN: An "electronic questionnaire" in the form of an interrogation questionnaire was used to extract a subset of data from practice computers running a standard software package (the general practice administrative system for Scotland, GPASS). The data were retained by each practice and also collected and analysed centrally to produce regional and national data. SUBJECTS: All 257 general practices in Scotland using GPASS software were sent the electronic questionnaire; data from 154 practices, including 759 general practitioners and covering 1,010,452 patients, were collected. RESULTS: Ninety three practices had all their patient records on computer; others had selectively entered data on, for example, only those patients receiving repeat prescriptions. The number of computerised patient records per practitioner ranged from 46 to 2373. Altogether 194,261 patients had repeat prescribing data and 204,005 morbidity or clinical data. CONCLUSION: An electronic questionnaire is a simple and effective way of investigating the information held on practice computers, allowing analysis and feedback of information to practitioners. Development of this system will provide a cumulative information system for Scottish general practitioners.

Computers↗
PubMed1990

p52 induction by cytochalasin D in rat kidney fibroblasts: homologies between p52 and plasminogen activator inhibitor type-1.

Normal rat kidney (NRK) fibroblasts respond to the cell shape-modulating chemical agent cytochalasin D (CD) with augmented synthesis of the 52-kDa substrate-associated protein p52. p52 is a complex glycoprotein, existing as 12 different isoforms, which include a 43-kDa "core" protein (p43), four 50-kDa species (p50-0,1,2,3), and at least seven distinct pI variants of the mature 52-kDa protein. A threshold of 2-4 microM CD was found to be necessary to augment p52 deposition into both the secreted protein- and saponin-resistant cytomatrix (SAP) fractions of NRK cells. This concentration of CD was also necessary to initiate significant cell rounding. Augmented p52 production in CD-treated NRK (NRK/CD) cells provided a means to assess the identity of this protein. p52 was found to be identical to rat plasminogen activator inhibitor type-1 (rPAI-1) and to PAI-1-like proteins of other species by comparative immunoprecipitation, 2-D electrophoretic profile, V8 protease digest mapping, and subcellular fractionation criteria. Quantitation of rPAI-1 cytoplasmic mRNA abundance, using the rPAI-1 cDNA probe pSS1-3, revealed an induction of rPAI-1 mRNA in NRK/CD cells which paralleled the increased protein production. CD-augmented p52(rPAI-1) synthesis and SAP deposition was blocked by actinomycin D, implicating a need for RNA synthesis during the period of CD exposure to effect induction. Augmentation of p52 expression in NRK/CD fibroblasts, thus, appears to involve both cell shape-associated metabolic processes and concomitant RNA synthesis.

Animals↗
PubMed1990

GPASS.

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Computer Systems↗