A giant outburst two years before the core-collapse of a massive star.

The death of massive stars produces a variety of supernovae, which are linked to the structure of the exploding stars. The detection of several precursor stars of type II supernovae has been reported (see, for example, ref. 3), but we do not yet have direct information on the progenitors of the hydrogen-deficient type Ib and Ic supernovae. Here we report that the peculiar type Ib supernova SN 2006jc is spatially coincident with a bright optical transient that occurred in 2004. Spectroscopic and photometric monitoring of the supernova leads us to suggest that the progenitor was a carbon-oxygen Wolf-Rayet star embedded within a helium-rich circumstellar medium. There are different possible explanations for this pre-explosion transient. It appears similar to the giant outbursts of luminous blue variable stars (LBVs) of 60-100 solar masses, but the progenitor of SN 2006jc was helium- and hydrogen-deficient (unlike LBVs). An LBV-like outburst of a Wolf-Rayet star could be invoked, but this would be the first observational evidence of such a phenomenon. Alternatively, a massive binary system composed of an LBV that erupted in 2004, and a Wolf-Rayet star exploding as SN 2006jc, could explain the observations.

Thorough design and pre-trial quality assurance (QA) decrease dosimetric impact of delineation and dose planning variability in the STRICTLUNG and STARLUNG trials for stereotactic body radiotherapy (SBRT) of central and ultra-central lung tumours.

INTRODUCTION: SBRT of central lung tumours implies significant risk of toxicity. We are initiating two phase II trials prescribing 56 Gy/eight fractions to PTV, allowing for dose escalation of GTV. We prioritize organs at risk (OAR) constraints over target coverage, making the treatment plans very sensitive to OAR delineation variations. The aim of this study is to quantify the dosimetric impact of contouring variations and to provide a thorough description of pre-trial quality assurance to be used in upcoming trials to provide consistent clinical care. MATERIALS AND METHODS: Delineation: Seven physicians delineated OAR in three rounds, with evaluations in-between. For each patient case, seven treatment plans, repeatedly using each of the OAR structure sets from the seven physicians, were made and compared to evaluate the dosimetric effect of delineation variability. Treatment planning: Treatment plans for seven cases were made at six departments in two rounds, with discussion in-between. RESULTS: OAR delineation variation between centres resulted in high variabilities in OAR dose for simulated plans and led to potential overdosage of the lobar bronchus (constraint: D0.03cc < 45 Gy), with maximum doses ranging between 58 Gy (first round), and 50 Gy (third round). For mediastinal tissue, the constraint (D0.03cc < 45 Gy) was violated for the majority of the delineations in all three rounds, with maximum doses of 84 Gy (first round), and 72 Gy (third round).For the treatment planning study, the range of the standard deviation for GTV mean dose was 12.8-18.5 Gy (first round) and 2.8-3.5 Gy (second round). CONCLUSIONS: Even small variations in OAR delineation led to high OAR overdosage. The study demonstrates the importance of having extensive QA procedures in place before initiating clinical trials on dose escalation in SBRT.

Roles of GTP and GDP in the regulation of the thyroid adenylate cyclase system.

Effects of guanine nucleotides on the adenylate cyclase activity of thyroid plasma membranes were investigated by monitoring metabolism of the radiolabeled nucleotides by thin-layer chromatography (TLC). When ATP was used as substrate with a nucleotide-regenerating system, TSH stimulated the adenylate cyclase activity in the absence of exogenous guanine nucleotide. Addition of GTP or GDP equally enhanced the TSH stimulation. Effects of GTP and GDP were indistinguishable in regard to their inhibitory effects on NaF-stimulated activities. The results from TLC suggested that GDP could be converted to GTP by a nucleotide-regenerating system. Even in the absence of a nucleotide-regeneration system, addition of GDP to the adenylate cyclase assay mixture resulted in the parallel decrease in ATP levels and formation of GTP indicating that thyroid plasma membrane preparations possessed a transphosphorylating activity. When an ATP analog, App[NH]p, was used as substrate without a nucleotide-regenerating system, no conversion of GDP to GTP was observed. Under such conditions, TSH did not stimulate the adenylate cyclase activity unless exogenous GTP or Gpp[NH]p was added. GDP no longer supported TSH stimulation and caused a slight decrease in the activity. GDP was less inhibitory than Gpp(NH)p to the NaF-stimulated adenylate cyclase activity. These results suggest: (1) TSH stimulation of thyroid adenylate cyclase is absolutely dependent on the regulatory nucleotides. (2) In contrast to GTP, GDP cannot support the coupling of the receptor-TSH complex to the catalytic component of adenylate cyclase. (3) The nucleotide regulatory site is more inhibitory to the stimulation of the enzyme by NaF when occupied by Gpp[NH]p than GDP.

Characteristics of the guanine nucleotide regulatory component of adenylate cyclase in human erythrocyte membranes.

This study probes the structure and mutual interactions of the components of adenylate cyclase. We use a complementation assay which involves the addition of an adenylate cyclase-related guanine nucleotide-binding protein component to a membrane lacking this component to measure guanine nucleotide-stimulated-adenylate cyclase. Instead of using detergent extracts we were able to achieve full complementation by mixing intact membrane preparations in the presence of the nucleotide component. Of particular interest was the human erythrocyte membrane which contains very low amounts of catalytic activity and no measurable beta-adrenergic receptor but has normal amounts of the nucleotide component. This component appears to be the same, by several criteria, as components found in pigeon and turkey erythrocytes and in rat liver plasma membrane. The component confers Gpp(NH)p, fluoride, and GTP stimulation of adenylate cyclase along a single reconstitution curve. It is labeled with NAD by cholera toxin, and has an apparent molecular weight of 39 000 upon sodium dodecyl sulfate gel electrophoresis. The presence of the nucleotide unit in the virtual absence of the active catalytic unit allowed us to determine those properties intrinsic to each unit and those conferred by the association of the units. The nucleotide component binds guanine nucleotides weakly in the human erythrocyte membrane, yet produces persistent activation of adenylate cyclase and tight binding (of Gpp(NH)p) upon combination with the catalytic unit. Treatment of the human erythrocyte membrane with N-ethylmaleimide causes a simultaneous diminution in both Gpp(NH)p and fluoride stimulation in reconstituted activities, suggesting that both activities are conferred by the same component.

Effects of forskolin on adenylate cyclase, cyclic AMP, protein kinase and intermediary metabolism of the thyroid gland.

Forskolin (40 microM) stimulated adenylate cyclase activities of bovine thyroid plasma membranes without the addition of guanine nucleotides. GDP had little effect on the forskolin-stimulated adenylate cyclase activity while Gpp[NH]p (0.1-1.0 microM) decreased it. In the presence of TSH (10 mU/0.11), Gpp[NH]p no longer caused inhibition. Forskolin did not affect phosphodiesterase activities of thyroid homogenates. Forskolin (10 microM) rapidly increased cAMP levels in bovine thyroid slices both in the absence and presence of a phosphodiesterase inhibitor. The effect of TSH (50 mU/ml) on cAMP levels was additive or greater than additive to that of forskolin. An initial 2-h incubation of slices with forskolin did not decrease their subsequent cAMP responses to either forskolin and/or TSH while similar treatment of slices with TSH induced desensitization of the cAMP response to TSH, but not to forskolin. Forskolin (10 microM) as well as TSH (50 mU/ml) activated cAMP-dependent protein kinase of slices in the absence of a phosphodiesterase inhibitor. Although forskolin activated the adenylate cyclase cAMP system, it did not stimulate iodide organification or glucose oxidation, effects which have been attributed to cAMP. In fact, forskolin inhibited these parameters and 32P incorporation into phospholipids as well as their stimulation by TSH. These results indicate that an increase in cAMP levels and cAMP-dependent protein kinase activity in thyroid slices may not necessarily reproduce the effects of TSH on the thyroid.

Effects of phospholipase A2 and filipin on the activation of adenylate cyclase.

Rat liver plasma membranes were incubated with phospholipase A2 (purified from snake venom) or with filipin, a polyene antibiotic, followed by analysis of the binding of glucagon to receptors, effects of GTP on the glucagon-receptor complex, and the activity and responses of adenylate cyclase to glucagon + GTP, GTP, Gpp(NH)p, and F-. Phospholipase A2 treatment resulted in concomitant lossess of glucagon binding and of activation of cyclase by glucagon + GTP. Greater than 85% of maximal hydrolysis of membrane phospholipids was required before significant effects of phospholipase A2 on receptor binding and activity response to glucagon were observed. The stimulatory effects of Gpp(NH)p or F- remained essentially unaffected even at maximal hydrolysis of phospholipids, whereas the stimulatory effect of GTP was reduced. Detailed analysis of receptor binding indicates that phospholipase A2 treatment affected the affinity but not the number of glucagon receptors. The receptors remain sensitive to the effects of GTP on hormone binding. Filipin also caused marked reduction in activation by glucagon + GTP. However, in contrast to phospholipase A2 treatment, the binding of glucagon to receptors was unaffected. The effect of GTP on the binding process was also not affected. The most sensitive parameter of activity altered by filipin was stimulation by GTP or Gpp(NH)p; basal and fluoride-stimulated activities were least affected. It is concluded from these findings that phospholipase A2 and filipin, as was previously shown with phospholipase C, are valuable tools for differentially affecting the components involved in hormone, guanyl nucleotide, and fluoride action on hepatic adenylate cyclase.

Biochemical properties of cloned lipases from the Pseudomonas family.

Three Pseudomonas lipases, representing three subfamilies, were analysed for pH optima, destabilization by EGTA and surfactants, phospholipase and cholesterolesterase side activities. All the Pseudomonas lipases tested showed alkaline pH optima. The Pseudomonas cepacia and the P. pseudoalcaligenes lipases were totally inhibited by EGTA at pH 9, and the latter was also fully inhibited at pH 7. The lipase from P. mendocina was not inhibited by EGTA at any of the pH values tested. These findings indicate that a calcium binding site exists in some of the Pseudomonas lipases. The P. pseudoalcaligenes, P. cepacia and P. mendocina lipases were inhibited by the anionic surfactant SDS at concentrations between 0.01-0.5 mg/ml. The P. pseudoalcaligenes and P. cepacia lipases were not inhibited by the nonionic surfactant Brij35 in concentration up to 1 mg/ml, whereas the lipase from P. mendocina was inhibited at 0.1 mg/ml. The P. pseudoalcaligenes and P. cepacia lipases were found to possess high cholesterol esterase activity. P. pseudoalcaligenes lipase was further found to have high phospholipase activity. Ten Pseudomonas lipase sequences were compared by automatic sequence alignment. On the basis of sequence identity we have classified Pseudomonas lipases into five subfamilies.

Multiple responses to administration of liposome-encapsulated hemoglobin (LEH): Effects on hematopoiesis and serum IL-6 levels.

Liposome-encapsulated hemoglobin (LEH) has been tested in animals as an oxygen-carrying red cell substitute and has been shown to be beneficial in the treatment of hemorrhagic shock. The effects of LEH on immune responses have not been studied thoroughly in any well-controlled model. Using a murine model, we evaluated nephrotoxicity and hepatotoxicity as well as immune function parameters following LEH administration. Following intravenous administration of LEH, 1) a serum spike of interleukin-6 (IL-6) occurred in mice at 4-8 hours, with no elevation of IL-1, tumor necrosis factor (TNF), or interferon-gamma (IFN-gamma); 2) the serum liver function enzymes SGOT (AST, aspartate aminotransferase) and SGPT (ALT, alanine aminotransferase) were elevated at 48 hours; 3) only a slight increase in serum antibody to bovine hemoglobin was observed; and 4) increased hematopoietic activity was observed in the spleen and bone marrow. The finding that only IL-6 but not the associated TNF, IL-1, or IFN-gamma is secreted in vivo following LEH administration is novel and may have significance in defining the mechanisms underlying specific adverse responses observed with LEH administration in animals.

A new wound healing agent--sphingosylphosphorylcholine.

Spingosylphosphorylcholine (lysosphingomyelin or SPC) is an effective and broad spectrum cell growth promoting agent and a candidate for evaluation on wound healing. The effect of SPC on full-thickness excision and incision wounds in genetically healing-impaired diabetic (db/db) mice was evaluated by measurement of wound area, skin strength, and tissue histology. The effect on cell proliferation was measured in vivo by incorporation of bromo-deoxyuridine and in vitro by [3H] thymidine incorporation. SPC increased the rate of wound closure, with a statistically significant improvement in measured wound areas (p < 0.02, compared with vehicle controls). The optimum concentration was 2-3 microM. SPC, alone and in combination with insulin, stimulated DNA synthesis in cells known to participate in wound healing, including microvascular endothelial cells. In vivo, SPC stimulated proliferation of keratinocytes, fibroblasts, endothelial cells, and cells around sebaceous glands and hair follicles at day 2-4 postwound, resulting in a complete re- epithelialization and profound granulation tissue formation in excisional and incisional wound sites of db.db and db/+ mice. Quantitative assessment of wound tissue section morphology indicated that SPC induced up to a 3-fold increase in the numbers of mitotic cells, resulted in smaller cross-sectional scar area, and led to more normalized tissue in the wound sites. SPC had no deleterious effect on wound skin strength. In conclusion, the acceleration of dermal wound healing animal models suggests that SPC could be an interesting candidate for clinical application.

Transfection with aFGF cDNA improves wound healing.

Somatic gene therapy is a potentially useful strategy for the delivery of growth factors or cytokines to enhance wound healing. Experimental excisional and incisional wounds in impaired-healing diabetic mice (db/db) were treated with aFGF and with a plasmid coding for aFGF. A eukaryotic expression plasmid composed of the Hst signal peptide sequence in-frame with the human aFGF sequence was used. Transfection of tissues was accomplished either by direct plasmid uptake or by uptake facilitated with cationic liposomes. The results show that the closure of excisional wounds was significantly accelerated (p < 0.05) by topical application of human recombinant aFGF or by transfection with the aFGF plasmid but not by vehicle or control plasmid not containing the aFGF sequence. In incisional wounds, aFGF or transfection with the plasmid significantly increased the wound-breaking strength compared to their corresponding controls (p < 0.05). Quantitative histology of the plasmid-treated incisional wound sections revealed improved wound quality. The transcription of mRNA from human aFGF cDNA in the incisional wound tissue extracts was confirmed by RT-PCR, and the expressed aFGF was detected by immune dot blot and immunohistochemistry assays. The transfection was a transient process with a peak at 9 d in db/+ (littermates of the diabetic mice) incisional wounds, at 36 d in db/db incisional wounds, and at 27 d in db/db excisional wounds. Cells transfected with human aFGF occupied up to 6.4% of the transectional area in the wound sites. Thus, aFGF gene delivery resulted in both gene expression and a functional improvement in healing.

Effect of thyrotropin-induced desensitization of bovine thyroid adenylate cyclase on the nucleotide regulatory protein.

The stimulation of adenylate cyclase by TSH was decreased 50-60% in crude membranes prepared from homogenates of bovine thyroid slices that had previously been incubated for 2 h with the hormone. The diminished response was not associated with any significant change in the binding capacity or affinity for 125I-labeled TSH. The apparent affinities of the desensitized adenylate cyclase for TSH or GTP were not different from those of the enzyme prepared from thyroid slices that had been incubated without TSH. Decreased adenylate cyclase responses to NaF, cholera toxin, or guanyl-5'-yl-imidodiphosphate were also observed in the desensitized membrane, whereas the enzyme responses to prostaglandin E1, GTP, or forskolin were not decreased. However, desensitization caused no decrease in the cholera toxin-catalyzed ADP ribosylation of the 40,000 mol wt polypeptide guanine nucleotide-binding component of the adenylate cyclase. The desensitized membranes showed basal adenylate cyclase activity similar to that of the control membranes using adenyl-5'-yl-imidodiphosphate as substrate in the absence of a nucleotide-regenerating system. These results suggest that the in vitro TSH-induced desensitization of thyroid adenylate cyclase reflects an alteration in the activation processes of the nucleotide regulatory protein.

Role of cellular Ca++ in phosphorylation of 21 K and 19 K polypeptides in cultured thyroid cells: effects of phorbol ester, trifluoperazine, and 8-diethylamino-octyl-3,4,5-trimethoxybenzoate hydrochloride.

Cultured dog thyroid cells contain 21 and 19 kilodalton (K) phosphoproteins which by several criteria have been identified as light chains of myosin (MLC). TSH causes a reduction in the phosphorylation state of the 21 K-19 K proteins, at least in part through activating adenylate cyclase and increasing cAMP levels. We now report that 12-O-tetradecanoyl-phorbol-13-acetate (TPA) also decreases the 21 K-19 K protein phosphorylation state, but in contrast to that due to TSH, the TPA-induced decrease is not associated with elevated cAMP levels. The effect of TPA was not additive to that of TSH. Because Ca++ is a major factor regulating MLC kinase and TPA-stimulated protein kinase C in other systems, the role of Ca++ in the phosphorylation of the 21 and 19 K polypeptides in dog thyroid was examined. In intact cells, both (8-diethylamino)-octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8) (1 X 10(-4) M) and trifluoperazine (TFP) (4 X 10(-5) M) increase basal 21 K-19 K protein phosphorylation and inhibit the decrease in phosphorylation caused by TSH and TPA without affecting cAMP levels. Ionophore A23187 (5 X 10(-6) M) counteracts TMB-8- and TFP-stimulated phosphorylation as well as TMB-8 and TFP inhibition of TSH- and TPA-reduced 21 K-19 K phosphorylation. Incubation of 32PO4-labeled dog thyroid cells in the absence of extracellular Ca++ or with verapamil does not significantly affect basally phosphorylated 21 K-19 K proteins or the decreased 21 K-19 K phosphorylation state caused by TSH. These results strongly suggest that the phosphorylation state of the 21 and 19 K proteins is affected more significantly by intracellular Ca++ pools than by extracellular Ca++, and implicate a kinase(s) other than Ca++-calmodulin-dependent MLC kinase in the phosphorylation of MLC in the dog thyroid.

Dephosphorylation of 19K and 21K polypeptides in response to thyroid-stimulating hormone in cultured thyroid cells.

Cultured dog thyroid cells incubated with [32P] phosphate contain at least two phosphoproteins of 19 and 21 kDalton (K), as determined by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Myosin light chain appears to be a component of the 19K and 21K phosphoproteins by the following criteria: 1) coextraction with myosin heavy chain from Triton-insoluble cytoskeletons with KCl-ATP, 2) coisolation with myosin heavy chain by immunoprecipitation, and 3) purification of undenatured myosin with pyrophosphate-agarose gel electrophoresis. The phosphorylation state of these proteins is decreased by incubation of cells with TSH. In the basal state, the 19K and 21K proteins from Triton-insoluble cytoskeleton fractions contain 0.86 +/- 0.07 (+/- SE) mol phosphate/mol protein, which is reduced to 0.34 +/- 0.03 in TSH-treated cells. TSH-induced dephosphorylation occurs in 1 min with 2.5 mU/ml TSH and reaches a maximum at 15 min. This TSH effect appears to be mediated by cAMP, since it is mimicked by (Bu)2cAMP, forskolin, cholera toxin, and prostaglandin E1 and is potentiated by isobutylmethylxanthine. Carbamylcholine, ionophore A23187, and norepinephrine, which inhibit TSH stimulation of cAMP, have no effect on basal phosphorylation of the 19K and 21K proteins, but do inhibit the effect of TSH.

Morphological and biochemical responses of cultured thyroid cells to thyrotropin.

In the thyroid gland, TSH stimulates cAMP formation, exocytosis of precursor (noniodinated) thyroglobulin, endocytosis of thyroglobulin, and proteolytic processing of the thyroglobulin to form thyroid hormones. In this report we describe TSH effects on cAMP levels, microtubules, microfilaments, myosin fibrils, and the morphology of cultured thyroid follicle cells. The cells were normally cultured in the presence of 10 mU/ml TSH, and fresh TSH produced no stimulation when assayed for cAMP production in a 15-min assay. When such cells were cultured for up to 72 h in the absence of TSH and then assayed for cAMP production, the basal levels were much reduced, but fresh TSH stimulated cAMP levels half-maximally at 1 mU/ml and up to 50-fold at 20 mU/ml. Microtubules, myosin fibers, and microfilaments were demonstrated by indirect immunofluorescent staining. Fluorescent staining of fibers was observed in cells fixed before lysis and in cells lysed before fixation. In control cells grown without hormone, microtubules originated near the nucleus and extended to the cell periphery. Myosin-containing fibrils traversed the cell or radiated from foci. Microfilaments spanned the cell in a stress fiber pattern. After incubation with 20 mU/ml TSH and 4 mM isobutylmethylxanthine (IBMX) for 10-20 min, the microtubules in up to half of the cells appeared altered and more granular, and the cell periphery was scalloped. After 15-30 min with TSH and IBMX, normal myosin fibers were replaced with a fine lattice-work, peripheral staining disappeared, and the proportion of nonfibrous myosin increased. Stress fibers demonstrated with antibody to actin also disappeared, and the peripheral structures observed in normal cells became fragmented. Incubation with forskolin or TSH and IBMX for 2-3 h resulted in arborization of 30-60% of the cells that contained bundles of microtubules, myosin fibers, or microfilaments into dendrite-like processes and increased staining near the nucleus. At 5 h, more than 80% of the cells were arborized. These morphological changes were less pronounced with IBMX alone and minimal with TSH alone. The time course of cAMP levels observed basally or after TSH, forskolin, or TSH and IBMX was consistent with the relative effects of these agents on arborization. These studies are consistent with effects of cAMP on microtubules, myosin-containing fibrils, and microfilaments and may provide a basis for the morphological response to TSH.

Phospholipid-sensitive Ca2+-dependent protein kinase from bovine thyroid: characteristics and subcellular distribution of the enzyme and its substrates.

Ca2+-phosphatidylserine-dependent protein kinase activity was demonstrated in whole thyroid homogenates, cytosol, particulate, and membrane fractions. Although Ca2+-phospholipid-dependent protein kinase was difficult to detect in purified thyroid plasma membranes, an EGTA extract of such membranes had this activity. While phosphatidylinositol did not stimulate the enzyme, it enhanced the response to phosphatidylserine in the presence of 10 mmol/L free Ca2+. The enzyme was active at concentrations of free Ca2+ as low as 1 mumol/L but was inhibited at Ca2+ in excess of 500 mumol/L. Bovine thyroid contained endogenous substrates of 38,000 and 33,000 daltons for thyroid or purified spleen Ca2+-phospholipid-dependent protein kinase. The 38,000 dalton polypeptide was present in all the subcellular fractions while the 33,000 dalton substrate was present only in the whole homogenate and cytosolic fraction. The 38,000 dalton polypeptide, like the Ca2+-phospholipid-dependent protein kinase, was released from thyroid plasma membranes by EGTA. Phosphorylation of this substrate was rapid, highly sensitive to Ca2+, and inhibited by chlorpromazine (100 mumol/L) and trifluoperazine chlorpromazine (100 mumol/L) and trifluoperazine (100 mumol/L). Several substrates of a phospholipid-independent, Ca2+-dependent protein kinase with molecular weights of 51,000, 76,000, and 96,000 were also observed. This Ca2+-phospholipid-dependent protein phosphorylation system may be important in the membrane-associated functions of the thyroid.

Functional and immunohistochemical evaluation of porcine neonatal islet-like cell clusters.

Porcine neonatal islet-like cell clusters (NICCs) may be an attractive source of insulin-producing tissue for xenotransplantation in type I diabetic patients. We examined the functional and immunohistochemical outcome of the islet grafts in vitro during long-term culture and in vivo after transplantation to athymic nude mice. On average we obtained 29,000 NICCs from each pancreas. In a perifusion system, NICCs responded poorly to a glucose challenge alone, but 10 mmol/L arginine elicited a fourfold increase in insulin secretion and 16.7 mmol/L glucose + 10 mmol/L arginine caused a sevenfold increase in insulin section indicating some sensitivity towards glucose. Hormone content as well as the number of hormone-containing cells increased for the first 14 days of culture. When NICCs were stained for hormones, proliferation (Ki67), and duct cells (CK7), some insulin- and glucagon-positive cells co-stained for proliferation. However no co-staining was observed between insulin- and glucagon-positive cells or between hormone-and CK-positive cells. Following transplantation of 2000 NICCs under the renal capsule of diabetic nude mice, BG levels were normalized within an average of 13 weeks. Oral and IP glucose tolerance tests revealed a normal or even faster clearance of a glucose load compared with normal controls. Immunohistochemical examination of the grafts revealed primarily insulin-positive cells. In summary, in vitro, NICCs responded to a challenge including glucose and arginine. There was a potential for expansion of the beta-cell mass of NICCs in vitro as well as in vivo where NICCs eventually may normalize blood glucose of diabetic mice.

Electric cautery lowers the contamination threshold for infection of laparotomies.

BACKGROUND: Interplay between wound resistance factors and bacterial innoculum determines the risk of surgical infection. Since cautery causes more damage than the scalpel, our hypothesis is that lower numbers of bacteria are required to infect wounds made by electric cautery than to infect wounds made with a scalpel. METHODS: Abdominal fascia was incised in 375 rats by cold knife, cutting current, or coagulation current. Wounds were innoculated with increasing numbers of bacteria and histologically scored at 7 days for necrosis, inflammation, and abscess. RESULTS: Coagulation current causes more inflammation, necrosis, and abscesses than the scalpel at all bacterial levels. Electric cutting current is intermediate, causing more damage than the scalpel only after contamination reached 10(5). Above this threshold most wounds were infected in all groups. CONCLUSIONS: Electric coagulation current should be used only when the need for meticulous hemostasis outweighs the considerably increased risk of infection. Electric cutting current is less destructive but also less hemostatic; indications for its use are difficult to identify.

Effects of phorbol esters on endothelial cell microfilaments: laser scanning confocal microscopy and quantitative morphometry of dose dependent changes.

Phorbol esters are known to alter microfilaments but it is not clear if the changes correspond to modulation of the phosphoinositide turnover/protein kinase C system. The novel technique of laser scanning confocal epifluorescence was used to study fiber orientation in phorbol ester treated cells. We treated endothelial cells with control agents and agents known to stimulate protein kinase C: 4 alpha-phorbol, phorbol 12-myristate 13-acetate (PMA), phorbol dibutyrate (PDB), or lipopolysaccharide. After incubation with the test agents, the endothelial cell microfilaments were stained with rhodamine pholloidin and viewed by conventional epifluorescence and by laser scanning confocal epifluorescence microscopy. The images obtained by the confocal microscopy corresponded to a thin optical section through the cells, 300 nm or more in thickness. The microfilaments extended predominantly in the plane of focus. After exposure of the cells to phorbol esters, the stress fibers became more nearly parallel in arrangement or were shortened, but remained in the plane of focus. The modification of microfilaments in response to phorbol esters was quantitated by a single blind analysis. In order to compare the morphological changes with a biochemical action of the phorbol esters, we measured phosphoinositide turnover. The dose-dependence of morphological changes was compared and contrasted to the dose-dependent effect of phorbol esters on bradykinin-stimulated phosphoinositide turnover. PMA had about the same EC50 (1-5 nM) for both biochemical and morphological processes. PDB was less potent in inducing the disruption of microfilament structure than in inhibiting phosphoinositide turnover. Lipopolysaccharide was ineffective in inducing a morphological change under these conditions. A simple activation of protein kinase C is insufficient to explain the dose-dependent effects of phorbol esters. Thus a morphometric analysis can help distinguish the potency of cytoskeleton modulators.