An RCT on the effects of topical CGP on surgical wound appearance and residual scarring in bilateral total-knee arthroplasty patients.

OBJECTIVE: To test the hypothesis that topically applied calcium glycerophosphate (CGP) would improve the appearance of the wound following bilateral knee replacement. METHOD: Healthy patients, aged 45-75 years, scheduled for bilateral total-knee replacement surgery were recruited into the study. One knee was randomly assigned to the treatment group, while the contralateral knee was designated the control (standard care). Subjects were instructed to apply a preparation of 10% CGP in an aqueous lotion to the treated knee once daily for 42 days, starting at the third postoperative day. Functional sealing and cosmetic appearance of the incision were evaluated by two surgeons by direct examination of the patient and then by two experienced assessors from photographs. The investigators qualitatively scored the intensity and extent of erythema along the incision and over the entire knee, the appearance of visible oedema along the incision and over the knee, and the overall clinical impression of wound healing. All four assessors were blinded to the subjects' allocation and the latter two assessors to the initial investigators' assessments. Subjects were also followed up for an additional 46 weeks, giving a total study duration of 12 months. RESULTS: Twenty patients completed the study. Statistical analysis showed that both the area and intensity of erythema along the incision were significantly reduced in the treated vs untreated knee over the entire study period. The analysis further showed that treatment significantly reduced oedema, both along the incision and across the entire knee. The differences were most marked at the seventh postoperative day and diminished with time. No adverse effects were observed for any patient, in either treated or untreated knees. CONCLUSION: These data demonstrate that postoperative application of 10% CGP could improve the appearance of the wound following total knee arthroplasty.

Altered expression of the RON receptor tyrosine kinase in various epithelial cancers and its contribution to tumourigenic phenotypes in thyroid cancer cells.

Aberrant expression of the RON receptor tyrosine kinase has been implicated in the pathogenesis of epithelial tumours. The aim of this study was to determine RON expression in various normal epithelial cells and their corresponding tumours by immunohistochemistry. The role of RON in regulating tumourigenic phenotypes was also studied using thyroid cancer cells as a model. RON was almost exclusively expressed at variable levels in normal epithelial cells from the digestive track, lung, kidney, pancreas, liver, breast, bladder, skin, and others. Among 15 types of cancer studied, RON was overexpressed in significant numbers in cancers derived from breast (56%), colon (51%), lung (48), thyroid (42%), skin (37%), bladder (36%), and pancreas (33%). In contrast, limited RON overexpression was observed in cancers from stomach, kidney, brain, liver, ovary, and prostate. Detailed analysis of thyroid tissues showed that RON was hardly detected in normal thyroid cells, moderately expressed in adenoma samples, but overexpressed in about half of papillary and follicular cancer specimens. Overexpression correlated with advanced clinical stage and was associated with lymph node metastasis. In cultured thyroid cancer cells, RON was highly expressed, with constitutive phosphorylation. Activation of RON increased cell growth and migration via the MAP kinase and AKT pathways. Silencing RON expression significantly prevented cell growth and increased cell apoptotic death. These findings show that RON overexpression occurs in a particular group of epithelial cancers. The requirement for RON in sustaining tumourigenic phenotypes suggests that it is a potential target for therapeutic intervention.

Comparison of long-chain fatty acyl-CoA synthetases from rabbit heart and liver: substrate preferences and effects of Mg2+.

Rabbit heart has a single, non-specific, fatty acyl-CoA synthetase (HP1) which is dependent on Mg2+, apart from the requirement for MgATP2-. Two long-chain fatty acyl-CoA synthetase activities (LP1 and LP2) can be resolved by hydroxyapatite chromatography of liver preparations; the Mg2+ requirement for these enzymes is undefined. These experiments were done to define the Mg2+ requirements of the liver enzymes and to compare them with the heart enzyme. For all three sources of enzyme and for arachidonic, oleic and palmitic acid substrates, the overall velocity of the reaction increased as [Mg2+] increased. Depending on the substrate and the source of enzyme, the increase in overall velocity could be attributed to changes in affinity or maximal velocity or both. The substrate preference of the HP1 enzyme for arachidonic acid (AA) was fifth or sixth of eight substrates regardless of the concentration of Mg2+. In contrast, increasing [Mg2+] shifted the relative substrate preference of both liver enzymes for AA. At low [Mg2+], AA was ranked seventh or eighth (least preferred) of eight substrates, whereas at high [Mg2+], AA was ranked as fifth or sixth. Hill plots of competition studies were consistent with Mg2+-induced positive co-operativity in LP1, but not in HP1 or LP2. Although enzymes from the three sources exhibit substantial kinetic differences, it is uncertain whether they are three different enzymes.

Evidence for a single non-arachidonic acid-specific fatty acyl-CoA synthetase in heart which is regulated by Mg2+.

Previous reports indicated that arachidonic acid is incorporated into the isolated perfused rabbit heart in preference to other fatty acids, and that incorporation of arachidonic acid, but not other fatty acids, is inhibited during Mg2+ depletion. In this study, we have not been able to demonstrate an arachidonic acid-specific fatty acyl-CoA synthetase in rat or rabbit heart by hydroxyapatite chromatography. Kinetic evidence was consistent with a single enzyme, as the slopes of pseudo-Hill plots were not significantly different from -1. The single fatty acyl-CoA synthetase present appears to prefer C18:0 unsaturated fatty acids to arachidonate, and had about the same affinity for C10:0 -C14:0 saturated fatty acids as for arachidonate. At 35 microM arachidonate, enzyme velocity increased as the total Mg2+ was increased from 3 to 80 mM. Calculated [MgATP] indicated that the MgATP complex was not rate-limiting. At low concentrations, Mn2+ and Ni2+ supported activity, but Cu2+ and Zn2+ did not. Low Ca2+ concentrations activated only oleic acid conversion. Kinetic analysis indicated that the Vmax of the enzyme was increased with increasing concentrations of ionized Mg2+ for both oleic acid and arachidonic acid. The data are constant with the hypothesis that Mg2+ has a direct effect on fatty acyl-CoA synthetase activity, and suggest that preference for oleic acid and arachidonic acid can be influenced by the ionic milieu.

Magnesium influences incorporation of [3H]arachidonic acid in perfused rabbits hearts.

Ca(2+)-free perfusion of rabbit hearts produces a 50% loss of tissue Ca2+ and Mg2+ and results in reduced incorporation of arachidonic acid into tissue lipids. This work was undertaken to determine whether the reduced incorporation was specific for arachidonic acid and whether it was correlated to the loss of either (or both) cation(s). Single 170-pmol bolus doses of [3H]arachidonic or [3H]oleic acid were given to perfused hearts with or without Ca2+ in the perfusate. The experiment was terminated, and the lipids were extracted 1-15 min after administration of label. At all time points, significantly less arachidonate was incorporated into total tissue lipids in the absence of than in the presence of Ca2+. The presence or absence of Ca2+ had no effect on the incorporation of oleate. When [3H]arachidonate was administered to hearts perfused with buffer containing 104 mM Li+ and 35 mM Na+ (which reduces tissue Mg2+ by approximately 25% and increases tissue Ca2+ by approximately 35%), the incorporation into lipids was reduced from 0.882 +/- 0.0739 to 0.598 +/- 0.0722 pmol/mg lipid (P < 0.05; n = 6). The incorporation of [3H]oleate during 104 mM Li+ and 35 mM Na+ was not different from control. These data suggest that the incorporation of arachidonic, but not oleic, acid into tissue lipids is dependent on the tissue Mg2+ content.

Magnesium and arachidonic acid metabolism.

The arachidonic acid content of plasma lipoproteins is altered during dietary magnesium deficiency, although the tissue arachidonic acid content seems to be unchanged. The primary event triggering these changes is probably the loss of extracellular Mg2+, as it is not clear whether dietary magnesium deficiency produces loss of intracellular Mg2+. In the isolated rabbit heart, in vitro perfusion conditions which produce loss of intracellular Mg2+ also result in disturbances of arachidonic acid metabolism. The metabolism of exogenous arachidonic acid to prostaglandins is increased without changing the Km or Vmax of cyclo-oxygenase. The incorporation of arachidonic acid into tissue phospholipids is significantly reduced, although the incorporation of oleate, stearate, and linolenate is either increased or unchanged. These data indicate that the activity of the enzymes (CoA synthetases and acyl transferases) which mediate arachidonate incorporation is reduced during Mg2+ depletion. Since protein-kinase-C-mediated phosphorylation of both CoA synthetase and acyl transferase reduces their activity, and since protein kinase C has an Mg2+ binding site, it is possible to speculate that loss of intracellular Mg2+ may lead to the activation of protein kinase C, with the consequent reduction of arachidonic acid reacylation enzyme activity.

Vasopressin stimulates the mobilization and metabolism of triacylglycerol in perfused rabbit hearts.

Vasopressin stimulates several metabolic processes, including glycogenolysis, gluconeogenesis, and fatty acid oxidation, and promotes lipolysis in rabbit and hamster suprarenal adipose tissue. This study was conducted to determine whether arginine vasopressin (AVP) stimulates the metabolism of triacylglycerols in the isolated perfused rabbit heart. Since the basal output of glycerol in the rabbit heart is very low, the triacylglycerol pool was labeled with [3H]triolein, and triacylglycerol metabolism was followed by analysis of the radioactive products in the perfusate. Administration of AVP (100 ng, 92 pmol) produced a 10-fold increase in the perfusate radioactivity associated with free fatty acids and mono- and diglycerides, as well as an 8-fold increase in the effluent radioactivity associated with triacylglycerol. The V1-receptor antagonist d[(CH2)5Tyr(Me)]AVP blocked the AVP-induced increase in the output of radioactivity in a dose-related manner. The V2-receptor agonist desmopressin (DDAVP) did not increase the outflow of radioactivity. Likewise, AVP-induced release of radioactivity was inhibited when Ca2+ was omitted from the perfusion buffer. Analysis of total lipid extracts of hearts labeled with [3H]triolein showed that the residual radioactivity was associated almost exclusively with authentic triolein both before and after AVP treatment. These data suggest that AVP promotes triacylglycerol mobilization and utilization and that these processes are Ca2+ dependent and mediated by the V1-receptor. Since free fatty acids derived from triacylglycerols are the preferred metabolic substrate for the heart and since plasma AVP levels increase in cardiac stress states such as shock, these findings suggest a metabolic function of AVP in cardiovascular stress states.

Uptake, incorporation and metabolism of (3H)triolein in the isolated perfused rabbit heart.

The purpose of these experiments was to study the uptake and metabolism of exogenous triglyceride in the isolated perfused rabbit heart. When infused into the rabbit heart, [9,10-3H(N)]triolein was retained and incorporated into a lipid fraction that had the chromatographic mobility of authentic triolein. Incorporation of labeled triolein was not likely to be the result of a lipoprotein lipase-mediated lipolysis/resynthesis cycle, since: (i) The distribution of radioactivity following administration of [3H]oleic acid was markedly different from the distribution of radioactivity following the administration of [3H]triolein; (ii) heparin was administered to the rabbits at the time of sacrifice; and (iii) the hearts were perfused with a protein-free buffer for 20 min prior to the labelling period. When isoproterenol was administered to hearts labelled with [3H]triolein, there was an increased output of total radioactivity, composed of labelled free fatty acids, diacylglycerol and monoacylglycerol. In these same hearts, there was an increased output of glycerol in response to isoproterenol. However, following the administration of bradykinin or angiotensin II, neither the radioactivity nor the glycerol content of the perfusate was changed. These data suggest that [3H]triolein is selectively incorporated into the triglyceride pool of the isolated perfused rabbit heart. Furthermore, this [3H]triolein is available to hormonally-activated lipolytic enzymes.

The influence of mono- and divalent cations on the cardiac metabolism of arachidonic acid.

Our previous study indicated that, in the isolated rabbit heart, perfusion with Ca2+ free Krebs Henseleit buffer (KHB) results in increased conversion of exogenous arachidonic acid to PGE2 and 6-keto-PGF1 alpha, probably as the result of increased availability of substrate to cyclooxygenase. Since perfusion with Ca2+ free buffer is known to cause alterations in the cardiac content of various mono- and divalent cations, the present study was performed to determine: a) The relationship between the conversion of exogenous arachidonic acid to prostaglandins and cardiac content of Na+, K+, Ca2+ and Mg2+; and b) Whether enhanced arachidonic acid conversion to prostaglandins during Ca2+ free perfusion is due to reduced incorporation of this fatty acid into tissue lipids. Perfusion of the rabbit heart with Ca2+ free buffer produced a significant reduction in the tissue content of Na+, K+, Ca2+ and Mg2+. However, the production of 6-keto-PGF1 alpha from exogenous arachidonic acid was linearly correlated with tissue Mg2+. These observations, together with our finding that perfusion with Ca2+ free KHB reduced the incorporation of [3H] arachidonic acid into tissue lipids, suggests that Ca2+ free perfusion may, by reducing the activity of arachidonyl CoA synthetase (a Mg2+ dependent enzyme), decrease the acylation of arachidonic acid into lipids, thus increasing the availability of arachidonic acid to cyclooxygenase.

Uptake and hormonally induced hydrolysis of [3H]phosphatidyl choline in the isolated rabbit heart.

Tritiated phosphatidyl choline (1-palmitoyl,2-[3H]palmitoyl or 1-stearoyl,2-[3H]arachidonyl) is taken up unchanged by the isolated perfused rabbit heart. Isoproterenol, vasopressin or A23187 enhanced the output of tritium in these hearts, whereas bradykinin, angiotensin II or acetylcholine had no effect. These data demonstrate that [3H]phosphatidyl choline is incorporated directly into a phospholipid pool that is accessible to some, but not all, hormonally stimulated lipases. In hearts labeled with 1-stearoyl,2-[3H]arachidonyl phosphatidyl choline, the radioactivity released by isoproterenol consisted of free fatty acid and lysophosphatidyl choline, indicating that isoproterenol stimulates both phospholipase(s) A1 and A2. Vasopressin and A23187 increased the release of free fatty acid, but not lysophosphatidyl choline, suggesting that these agents stimulate only phospholipase A2.

Regulation by calcium of arachidonic acid metabolism in the isolated perfused rabbit heart.

Activation of beta-adrenergic receptors with isoproterenol fails to stimulate prostaglandin synthesis in the isolated rabbit heart perfused with Ca2+-free Krebs-Henseleit buffer. This lack of response could be due to reduced isoproterenol-stimulated liberation of arachidonic acid, reduced cyclooxygenase activity during Ca2+-free perfusion, or both. To test these hypotheses, we have examined the effect of isoproterenol and exogenous arachidonic acid on prostaglandin output in hearts perfused with Ca2+-containing and nominally Ca2+-free Krebs-Henseleit buffer. In hearts prelabelled with [3H]arachidonic acid and perfused with nominally Ca2+-free buffer, the release of radioactivity following isoproterenol was about 90% less than when Ca2+ was included in the perfusion buffer. When exogenous arachidonic acid was administered to hearts perfused with Ca2+-depleted buffer, the output of both 6-keto-prostaglandin F1 alpha and prostaglandin E2 was about threefold greater than when Ca2+ was present in the perfusion medium. In the absence of Ca2+, the dose response curve for arachidonic acid-induced 6-keto-prostaglandin F1 alpha synthesis underwent a parallel shift to the left, with no change in maximal synthesis levels, and a tenfold reduction in the ED50 of arachidonic acid. The cyclooxygenase activities of cell-free homogenates prepared from hearts perfused with and without Ca2+ were not different from each other and were both insensitive to added Ca2+. Reduction of the Na+ concentration of the perfusion medium to 35 mM resulted in increases in arachidonic acid-induced 6-keto-prostaglandin F1 alpha less than those obtained during Ca2+-free perfusion, but greater than that observed during perfusion with normal Na+ (139 mM) and Ca2+ (2.5 mM). Arachidonic acid induced 6-keto-prostaglandin F1 alpha output was inversely correlated with tissue Ca2+ but not Na+ content. These data suggest that in the absence of perfusate Ca2+, the availability of exogenous arachidonic acid to cyclooxygenase is increased, possibly by reduced incorporation into tissue phospholipids.

Beta adrenergic receptor-stimulated prostaglandin synthesis in the isolated rabbit heart: relationship to extra- and intracellular calcium.

We have investigated the contribution of extra- and intracellular Ca++ and calmodulin to beta adrenergic receptor-stimulated prostaglandin synthesis in the isolated rabbit heart perfused with Krebs-Henseleit buffer. Administration of isoproterenol (100 ng) increased the output of immunoreactive 6-keto-prostaglandin F1 alpha and prostaglandin E2 as well as heart rate and developed tension; the coronary perfusion pressure was reduced. Isoproterenol-induced output of prostaglandins was positively correlated with the extracellular Ca++ concentration (0-5 mM). Infusion of the Ca++ channel blockers diltiazem (22 microM) or nifedipine (0.27 microM) inhibited isoproterenol-stimulated output of prostaglandins and the positive inotropic but not the positive chronotropic effect of the amine. Administration of the intracellular Ca++ antagonists 8-(diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride (23 microM) or ryanodine (1.6 microM) reduced the outflow of prostaglandins and the positive chronotropic and inotropic effect elicited by isoproterenol. The calmodulin inhibitors trifluoperazine (50 microM) or calmidazolium (1 microM) failed to alter isoproterenol-induced output of prostaglandins; trifluoperazine but not calmidazolium reduced the developed tension and coronary perfusion pressure without altering heart rate. The prostaglandin synthesis elicited by arachidonic acid (3 micrograms) was inhibited by indomethacin but not by alterations in extracellular Ca++, Ca++ channel blockers, intracellular Ca++ antagonists or calmodulin inhibitors. These data suggest that activation of beta adrenergic receptors promotes cardiac prostaglandin synthesis and myocardial contractility by increasing the trans-sarcolemmal flux of Ca++, which releases intracellular Ca++.(ABSTRACT TRUNCATED AT 250 WORDS)

Inactivation of herpes simplex viruses by nonionic surfactants.

Nonionic surface-active agents possessing ether or amide linkages between the hydrophillic and hydrophobic portions of the molecule rapidly inactivated the infectivity of herpes simplex viruses. The activity stemmed from the ability of nonionic surfactants to dissolve lipid-containing membranes. This was confirmed by observing surfactant destruction of mammalian cell plasma membranes and herpes simplex virus envelopes. Proprietary vaginal contraceptive formulations containing nonionic surfactants also inactivated herpes simplex virus infectivity. This observation suggests that nonionic surfactants in appropriate formulation could effectively prevent herpes simplex virus transmission.