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Biomedical subjects

R C Franson

Publications and source records attributed to R C Franson.

67 records · Page 4Linked to original sources
PubMed1980

Lipids of myocardial membranes: susceptibility of a fraction enriched in sarcolemma to hydrolysis by an exogenous mammalian phospholipase A2.

A myocardial membrane fraction enriched in sarcolemma was used to determine the susceptibility of the lipids to hydrolysis by a phospholipase A2 from granulocytes. After incubation (37 C, pH 7.0, 5 mM Ca2+) with the phospholipase A2 for 30 min, a more than 3-fold increase in unesterified fatty acids was found (up to 47 nmol/mg protein; P < 0.001) relative to a control incubated without phospholipase A2 or Ca2+. This included a 10-fold increase in the arachidonic acid content (up to 42 mol%) and at least a 7-fold increase in lysophosphatidylethanolamine (up to 7.4 mol% total phospholipid-P). However, the exogenous phospholipase did not augment the quantity of lysophosphatidylcholine produced by endogenous phospholipases in the presence of Ca2+ (5 mM). These results demonstrate the in vitro susceptibility of phospholipids of myocardial membranes, particularly phosphatidylethanolamine, to the neutral-active, Ca2+-dependent phospholipase A2 from granulocytes. This work may be relevant to myocardial inflammation and tissue damage during ischemia, where heterolytic injury of the myocardium may occur subsequent to the accumulation of granulocytes.

Animals↗
PubMed1979

Modulation of purified phospholipase A2 activity from human platelets by calcium and indomethacin.

A membrane bound phospholipase A2 (phosphatide 2-acylhydrolase, EC 3.1.1.4) from human platelets has been purified 3500-fold, and partially characterized. Phospholipase A2 activity was assayed using [1(-14)C] oleate-labeled Escherichia coli or sonicated dispersions of synthetic phospholipids. The 2-acyl specificity of the phospholipase activity was confirmed using phosphatidylethanolamine labeled in the C-1 position as substrate. The purified enzyme was maximally active between pH 8.0 and 10.5, and had an absolute requirement for low concentrations of Ca2+. Indomethacin, but not aspirin, inhibited phospholipase A2 activity.

Blood Platelets↗
PubMed1979

Separation and purification of a potent bactericidal/permeability-increasing protein and a closely associated phospholipase A2 from rabbit polymorphonuclear leukocytes. Observations on their relationship.

Two antibacterial proteins from rabbit polymorphonuclear leukocytes, a potent bactericidal cationic protein that increases the envelope permeability of susceptible gram-negative bacteria and a phospholipase A2, have been purified to near homogeneity by ion exchange, gel filtration, and hydrophobic interaction chromatography. The apparently noncatalytic bactericidal/permeability-increasing protein has an approximate molecular weight of 50,000 and is isoelectric at pH 9.5 to 10.0. The molecular properties, including amino acid composition, and the antibacterial potency and specificity of this rabbit leukocyte protein and of the bactericidal/permeability-increasing protein from human granulocytes that we have recently purified (J. Biol. Chem. 253, 2664-2672, 1978) are closely similar. Both proteins kill several strains of Escherichia coli and Salmonella typhimurium. Rough strains are more sensitive than smooth strains. All gram-positive bacterial species tested are insensitive to high concentrations of either rabbit or human protein. The phospholipase A2, purified by hydrophobic interaction chromatography on phenyl-Sepharose, ran as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 14,000 and had a specific enzymatic activity comparable to that of purified phospholipases A2 from other sources. Separation of the phospholipase A2 from the bactericidal/permeability-increasing protein has no noticeable effect on the bactericidal and permeability-increasing activities of the purified bactericidal protein, but removes the ability of the phospholipase A2 to hydrolyze the phospholipids of intact Escherichia coli. Upon recombination of the phospholipase A2 with the bactericidal/permeability-increasing protein, the phospholipase A2 regains its activity toward the phospholipids of intact E. coli suggesting that these two antibacterial leukocyte proteins act in concert.

Amino Acids↗
PubMed1977

Identification and partial characterization of phospholipases in isolated adrenocortical cells. The effects of synacthen [corticotropin-(1--24)-tetracosapeptide] and calcium ions.

Phospholipase A activity was determined in homogenates and subcellular fractions of trypsin-dispersed cat adrenocortical cells. At pH 7.4 homogenate phospholipid hydrolysis was activated by added Ca2+ and inhibited by EGTA. Phospholipid degradation in the presence and absence of Synacthen was completely blocked by EGTA. Ca2+-dependent activation of a membrane-bound phospholipase may be a critical control mechanism for regulating the molecular changes taking place during stimulation by Synacthen.

Adrenal Cortex↗
PubMed1975

Partial characterization and purification of a rabbit granulocyte factor that increases permeability of Escherichia coli.

Recently we reported that rapid killing of Escherichia coli by granulocytes or granulocyte fractions is accompanied by an equally rapid and discrete increase in permeability of the microbial envelope (Beckerdite, Mooney, Weiss, Franson, and Elsbach. 1974. J. Exp. Med. 140: 396-409). Most of this permeability-increasing activity (PI) is found in a crude granule preparation. PI is quantitatively recovered in a 23,000-g supernatant fraction (Sup II) after sulfuric acid extraction of granulocyte homogenates prepared in water. PI is nondialyzable, destroyed by pronase and trypsin, stable at 4degreesC for at least 2 mo, and destroyed by heating at 94degreesC. Anionic substances, such as heparin sulfate and isolated E. coli lipopolysaccharide, bind to and inhibit PI. PI has been purified up to 1,000-fold from homogenate in a yield of 50percent by acid extraction and carboxymethyl-Sephadex chromatography. Such purified fractions have bactericidal activity that equals that of disrupted granulocytes and Sup II, are similarly enriched with respect to granule-associated phospholipase, and protease activities. Whereas E. coli, sensitive to PI, binds or inactivates solubilized PI, a resistant strain of Serratia marcescens does not. Binding of PI to sensitive microorganisms seems to be necessary for expression of its biological activity since both the apparent binding to and the biological effect of PI on E. coli are completely blocked by 10-20 mM Mg2+ or Ca2+. Mg2+ or Ca2+ can reverse the effect on E. coli permeability produced by Sup II or the carboxymethyl-Sephadex fraction but not that produced by granulocyte homogenate. The close association of bactericidal, phospholipase A2, and permeability-increasing activities towards several gram-negative bacterial species suggests that they may be related.

Animals↗
PubMed1973

Lysosomal phospholipase A 1 and A 2 of normal and bacillus calmette guerin-induced alveolar macrophages.

A single intravenous injection of 0.1 mg of heat-killed Bacillus Calmette Guérin (BCG) in 0.1 ml of Bayol F produced an accumulation of activated alveolar macrophages (BCG induced). Cells were collected 3.5-4.0 wk after injection. Phospholipases A and three lysosomal marker enzymes (acid phosphatase, beta-glucuronidase, and lysozyme) were measured in homogenates, and the distribution of the phospholipases A and lysosomal, mitochondrial, and microsomal marker enzymes were examined after sucrose gradient centrifugation of a postnuclear (1,000 g) supernatant. Homogenates of normal and BCG-induced macrophages contained phospholipases A(1) and A(2) which had optimal activity at pH 4.0 in the presence of 2.0 mM ethylenediaminetetraacetate (EDTA). These activities were inhibited 50-70% by 2.0 mM CaCl(2). Homogenates of BCG-induced macrophages had specific activities of beta-glucuronidase, acid phosphatase, and lysozyme, which were increased 1.5- to 3.0-fold over the controls, whether expressed as activity per mg protein or activity per 10(7) cells. The specific activities of the phospholipases A, on the other hand, were consistently lower than those of the control. Distribution of the phospholipases A and the lysosomal marker enzymes after sucrose gradient centrifugation suggested that the phospholipases A active at pH 4.0 in the presence of EDTA are of lysosomal origin since: (a) BCG treatment caused a selective increase in the density of particles which contained both the phospholipases A and three lysosomal marker enzymes; and (b) since the density of mitochondria and microsomes were not affected by BCG treatment. The increase in the density of lysosomes seen here may be related to previously described morphologic changes of BCG-induced alveolar macrophages.

Acid Phosphatase↗
PubMed1972

Effect of methylprednisolone on some BCG-mediated changes in rabbit alveolar macrophage.

The effect of methylprednisolone sodium succinate treatment on Bacillus Calmette-Guérin (BCG) immunization was examined in alveolar macrophages of New Zealand White rabbits. Intravenous BCG injection caused a marked increase in cell number and in the hexosemonophosphate shunt activity. There was also a selective augmentation in the activity of acid phosphatase and beta-glucuronidase and an increase in lysosomal density. Activity of the lysosomal phospholipases was decreased. Steroid treatment suppressed the BCG-induced increase in cell number, hexosemonophosphate shunt activity, acid phosphatase, and beta-glucuronidase, and prevented the increase in lysosomal density. On the other hand, steroid treatment increased the activity of the phospholipases to levels higher than either control values or those obtained with cells from BCG-treated rabbits.

Acid Phosphatase↗
PubMedDate not supplied

Fatty acid oxidation and myocardial phospholipase A2 activity.

Cis-unsaturated fatty acids, but not saturated fatty acids, inhibited phospholipase A2 activity (PLA2) in vitro, and may function as endogenous suppressors of lipolysis. To probe the possible role of lipid peroxidation in the regulation of myocardial lipid catabolism, a neutral-active and Ca2+-dependent PLA2 was extracted from rat heart and was partially purified by sulfopropyl cation exchange chromatography. Myocardial PLA2 activity was inhibited in a dose-dependent manner by oleic, linoleic, linolenic, and arachidonic acids; the IC50 for arachidonic acid was approx 65 microM. Palmitic acid was not inhibitory. When arachidonic acid was incubated at 37 degrees C, exposed to air, there was a time- and pH-dependent peroxidation of the arachidonic acid as monitored by turbidity, thiobarbituric acid reactivity, and thin layer chromatography. Peroxidation was increased as the pH was lowered from 7.5 to 4.5, and was accompanied by a decrease in PLA2 inhibitory potency. Thus, arachidonate incubated for 24 hours at pH's 4.5, 6.0 and 7.5 lost 84%, 32%, and 20% respectively, of its inhibitory potency. Therefore, in vitro acidosis promotes the oxidation of cis-unsaturated fatty acids and relieves their inhibitory or suppressive activity toward PLA2s. Increased lipid peroxidation of unesterified unsaturated fatty acids during acidosis may therefore promote lipolysis observed during myocardial ischemia and reperfusion injury.

Animals↗
PubMedDate not supplied

Cellular regulation of arachidonate mobilization and metabolism.

Synthesis of eicosanoids is initiated by signal transduction cascades which result in the hydrolysis of free arachidonic acid from membrane phospholipids. Both a cytosolic 85 kDa and a nonpancreatic 14 kDa PLA2 may contribute to cellular arachidonate mobilization. In many cells, agonist-stimulated fatty acid release is dependent upon increases in intracellular free calcium and is enhanced by pretreatment with agents such as phorbol esters and soluble diglycerides. The response is specific for arachidonate and structurally similar polyunsaturated fatty acids containing a cis 5, 6 double bond. DMSO-differentiation of U937 cells markedly enhances the A23187-stimulated release of [3H]arachidonate, which appears to be coupled to differentiation-induced enhancement of capacitance calcium entry. Although both phorbol esters and soluble diglycerides enhance subsequent fMLP or A23187-stimulated arachidonate release in human neutrophils, several lines of evidence indicate that the effects of oleoylacetylglycerol and 1,2-dioctanoylglycerol are protein kinase C-independent. Soluble diglycerides, but not phorbol esters, enhance the coupling of arachidonate mobilization to subsequent leukotriene B4 synthesis. Further studies will be required to elucidate the mechanisms which regulate activation of cellular phospholipases and subsequent synthesis.

Arachidonic Acid↗
PubMedDate not supplied

Biochemical alterations in rat Thiry-Vella fistulas.

In order to obtain baseline information on the secretory function of normal rat bowel for our work on intestinal graft ischemia, we studied several biochemical parameters in rat Thiry-Vella fistulas (TVF). TVFs were created in 200-g male Lewis rats (n = 11) using the 25-cm segment of jejunum normally used as a graft in our intestinal transplant model. The stomas were matured primarily and the animals were allowed to recover. The TVFs were flushed at 0, 6, and 24 h and then daily for up to 21 days with 12 mL normal saline solution. The effluent was collected and analyzed for total protein (TP), secretory phospholipase A2 (sPLA2), intestinal fatty acid binding protein (I-FABP), lactate dehydrogenase (LDH), and N-acetylglucosamine (NAGA). TP content was 0.12 +/- 0.01 mg/mL up to 48 h, then gradually increased and stabilized at 0.39 +/- 0.05 mg/mL at day 21. By sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), one major protein band was identified in the low-molecular-mass range (15 kD), consistent with I-FABP and sPLA2. Secretory PLA2 levels decreased over the first 4 days to a low of 115 +/- 24.8% hydrolysis/min/fraction, then gradually rose to a plateau at approximately 529.76 +/- 88.36% hydrolysis/min/fraction by day 18. I-FABP levels rose rapidly from 0 ng/mL at 2 h to 900 +/- 250.0 ng/mL at 6 h and approximately 3000 +/- 304.9 ng/mL by day 14. LDH levels at 2 h and 48 h did not differ, with 0.03 +/- 0.004 and 0.03 +/- 0.005 optical density units (OD)/min/mL, respectively. NAGA levels were 0.07 +/- 0.05 OD/h/mL at 2 h and rose to 0.14 +/- 0.04 OD/h/mL at 48 h. These data suggest that after an early equilibration period, biochemical secretion into the lumen of normal rat bowel reaches a state of equilibrium, and therefore appears to reflect the baseline biochemical status of the bowel. Some of these levels are not negligible as one would expect in "normal" bowel. This information should prove extremely helpful as a baseline study of abnormal conditions of the intestine, such as ischemia or rejection.

Acetylglucosamine↗
PubMedDate not supplied

Secretory phospholipase A2 levels in rat small bowel.

Preliminary studies on ischemia/reperfusion injury in transplanted small bowel grafts showed that secretory phospholipase A2 (sPLA2) may play a substantial role by breaking down membrane phospholipids. This study sought to determine the normal values of sPLA2 in the rat small bowel as a function of site and length as a baseline for future studies. The entire small bowel of male Lewis rats (200 g) was flushed with normal saline to eliminate solid contents. In group 1, the entire small bowel was divided into 5-cm segments (numbered 1-9), which were snap frozen and processed the same day for sPLA2. In group 2, a 25-cm segment of bowel (corresponding to segments 2-6 in group 1) was harvested from each animal, snap frozen, and immediately processed for sPLA2. To assess the effect of bowel storage on enzyme content, group 3 and group 4 grafts were stored for 7 and 14 days, respectively, at -85 degrees C prior to processing. All samples were homogenized in buffer, extracted with H2SO4 and assayed for sPLA2 activity using [1-14C]oleate-labeled autoclaved Escherichia coli as substrate. Results were analyzed statistically by ANOVA. sPLA2 activity rose from 85.46 +/- 14.46% hydrolysis/min fraction-1 in segment 1, to 476.38 +/- 176.75% hydrolysis/min fraction-1 in segment 9. The increase was linear and statistically significant (p < .0001). There was no significant difference in enzymatic activity between groups 2, 3, and 4. Group 2 activity was 263.02 +/- 43.74% hydrolysis/min fraction-1. This value was not statistically different from the mathematically calculated mean of segments 2-6 in group 1 (237.75). The results show that (1) sPLA2 activity increases predictably with distance from the ligament of Treitz (2) storage at -85 degrees C does not affect sPLA2, activity, and (3) 25-cm grafts may be evaluated in toto with reproducible baseline enzyme activity. Given the variability of enzyme activity along the course of the rat small bowel, it is imperative that exact location be identified in any studies evaluating sPLA2 activity.

Animals↗