Poisson distribution to assess actinic keratoses in xeroderma pigmentosum.
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Deoxyribonuclease (Pyrimidine Dimer)↗
Biomedical subjects
L Marzella
Publications and source records attributed to L Marzella.
Toxicity of sea nettle (Chrysaora quinquecirrha) fishing tentacle nematocyst venom in cultured rat hepatocytes.
Sea nettle (Chrysaora quinquecirrha) venom (CQV) is known to be toxic to the cardiac, respiratory, renal and hepatic systems in animal models. However, the mechanism of toxicity of CQV on hepatocytes is unknown. We utilized isolated rat hepatocytes in culture and measured percentage total lactate dehydrogenase (LDH) release after direct exposure to CQV. Toxicity of CQV in this system produced a linear multiple-dose response curve as well as a linear single-dose kinetics curve. Neither extracellular calcium concentration nor intracellular calcium chelation had a statistically significant effect on the toxicity of CQV in our hepatocyte model. From these results it appears that CQV does not form large membrane channels similar to complement, nor does calcium appear to play a major role in the mechanism of toxicity in hepatocytes. The isolated rat hepatocyye culture and measure of LDH release provided a relatively simple and reproducible model for examining toxicity of CQV.
Analysis of Variance↗
Role of ischemia in rats with spinal cord injury induced by decompression sickness.
The microsphere technique was used to determine whether blood flow to the central nervous system and various organs is impaired in rats with spinal cord injury induced by decompression sickness. For this purpose cannulas were placed in the left ventricle of the rats for the injection of microspheres and in the tail artery as the reference site for withdrawal of blood for the calculation of cardiac output (CO) and blood flow (BF) and for measurement of blood pressure (BP) and heart rate (HR). The rats were then subjected to a simulated dive that by electrophysiologic criteria rapidly (within 60 min after diving) induces severe neurologic deficits in the cord. Microspheres were used to determine CO and BF before and at 10, 60, and 180 min after diving. CO, BP, and HR were not affected by diving. BF to various regions of the brain, heart, bone, and fat was also not affected by diving. BF decreased in the lung (40%) and skeletal muscle (50%) and increased in spinal cord (20%) at 10 min after diving. At 60 and 180 min after diving the only alterations seen were increases in hepatic arterial and portal BF. Analysis of the distribution of cardiac output showed that diving induced changes that essentially paralleled the BF changes described above. We conclude that perfusion in the central nervous system is maintained in rats with spinal cord injury induced by decompression sickness. These results indicate that ischemia does not play a role in the pathophysiology of neuronal injury in this model of decompression sickness.
Animals↗
Changes in regional vascular resistance and blood volume after hemorrhage in fed and fasted awake rats.
To determine whether fasting alters the response of blood flow to hemorrhage, blood flow was measured by radiolabeled microspheres before and after a 20 ml.kg-1.3 min-1 hemorrhage in fed and fasted chronically cannulated male Sprague-Dawley rats. Restitution of blood volume, as determined by dilution of hematocrit, was attenuated in fasted rats, although the responses of arterial blood pressure, heart rate, cardiac output, and total peripheral resistance were not significantly different. Fasting only affected resting blood flow in the bronchial artery and fat and had no effect on resting vascular resistance in any organ studied. In both fed and fasted rats, hemorrhage led to a significant fall in blood flow to the stomach, small intestine, cecum, colon, spleen, pancreas, kidney, bronchial artery, thymus, and muscle and a rise in blood flow to the adrenals. However, fasting did not significantly alter the response of flow or vascular resistance to these organs. Fasting did alter the blood flow response to hemorrhage in bone, fat, and the hepatic artery. These results demonstrate that 24 h of fasting does not affect the responses of blood flow and vascular resistance to hemorrhage in most organs, even though restitution of blood volume is attenuated.
Animals↗
Assessment of spinal cord trauma using evoked potentials in a rat model of decompression sickness.
A rapid quantitative technique for assessing spinal cord trauma in a rat model of decompression sickness is described. Evoked potentials are measured from the lower limbs of rats before and after dives with compressed air in a hyperbaric chamber. Under chloral hydrate anesthesia, the sciatic nerve is stimulated at the sciatic notch with needle electrodes and platinum/iridium electrodes are used to record the action potentials from the plantar muscles. Analysis showed that the sensory reflex response was markedly depressed in the rats soon after diving and did not recover for up to 5 days. The motor response was similarly affected although to a lesser degree. The latency of the reflex response also became prolonged after 3 days. The significant and complex pattern of neurological dysfunction shown by this electrophysiologic technique validates the use of the rat model for the study of spinal cord decompression sickness. This technique should aid studies aimed at testing new therapies for this disease.
Action Potentials↗
Role of extravascular gas bubbles in spinal cord injury induced by decompression sickness in the rat.
We have evaluated the contribution of extravascular gas bubbles to spinal cord injury in decompression sickness. For this purpose, a model of decompression sickness was developed by subjecting rats to simulated dives using compressed air. Various diving profiles were tested and the presence of spinal cord injury was demonstrated by electrophysiologic measurements. To evaluate the space occupying lesions induced by gas bubbles in the white matter, the spinal cord was fixed by perfusion with 10% buffered formalin. Tissue blocks from cervical, thoracic, and lumbar spinal cords were embedded in paraffin. Tissue sections were double stained with luxol fast blue and hematoxylin and eosin. The space occupying lesions were quantified with a digitizer tablet. The fractional area of the lesions was 0.009% in controls and 0.026% in rats subjected to diving. We conclude that the volume of extravascular free gas present in the cord of rats with spinal decompression sickness is small and that artifacts of tissue preparation contribute to the volume estimate. As far as can be judged from the results in this animal model, the contribution of extravascular gas bubbles to spinal cord decompression injury is minor.
Animals↗
Regional hemodynamics in 1-day-delayed rodent island flaps.
We have studied the recovery of microvascular function in ischemic epigastric skin flaps by characterizing the regional hemodynamics in the axial and random portions of double- and single-pedicle island skin flaps in the rat. Blood flows were measured with radiolabeled microspheres 1 day after bipedicle flaps were elevated and at 4 hr, 72 hr, and 1 week after ligation of one of the pedicles. These hemodynamic measurements were correlated with assessments of angiogenesis and skin necrosis performed by lectin histochemistry and histopathology. Twenty-four hours after the skin flap elevation, the mean blood flow was 0.26 ml.min-1.g-1. After the ligation of one pedicle, the blood flow in the axial portion of the skin flap was unchanged at 4 hr (0.22 ml.min-1.g-1), declined insignificantly at 72 hr (0.15 ml.min-1.g-1), and remained normal at 1 week. In the random portion of the skin flap very little collateral blood flow was present at 4 hr (0.06 ml.min-1.g-1) and 72 hr (0.09 ml.min-1.g-1). Blood flow in the random skin flap returned to normal by 1 week (0.27 ml.min-1.g-1). Reelevation of the skin flap at 1 week caused an insignificant decline (10%) in total blood flow to the skin flap. Skin necrosis was detected histologically at 72 hr only in the lateral portion of the random skin flap, where blood flow was less than 0.03 ml.min-1.g-1. Increased areas of lectin binding to vascular endothelium were seen in the subdermal layers of the skin flap by 1 week, presumably due to angiogenesis.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Regulation of intracellular protein degradation with special reference to lysosomes: role in cell physiology and pathology.
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Animals↗
Hyperbaric oxygen treatment for experimental cyclophosphamide-induced hemorrhagic cystitis.
Acrolein is a toxic metabolite of cyclophosphamide that causes hemorrhagic cystitis in 2 to 40% of treated patients. Hyperbaric oxygen (HBO) is used to treat poorly healing wounds in conditions such as Fournier's gangrene and radiation-induced cystitis. The present study was designed to evaluate the effects of HBO on acute acrolein-induced hemorrhagic cystitis in a rat model. Rats were divided into 4 groups. Group I served as a control and received only HBO prior to sacrifice. Group II received acrolein only, while groups III and IV received acrolein as well as HBO therapy. Hyperbaric oxygen (100% oxygen, 2.8 atmospheres, 90 minutes) was delivered twice a day for 4 days, with group III receiving a fifth HBO treatment just before acrolein and group IV receiving the fifth HBO treatment just after acrolein. After therapy, the amount of urothelial injury was determined morphometrically. Group II untreated rat bladders had only 33% of the urothelium intact after acrolein injury, whereas groups III and IV rat bladders had 93% (p < 0.01) and 55% (p < 0.01) intact urothelium, respectively, after treatment with HBO. The timing of the HBO treatment appeared to be a critical factor, with less injury occurring if the fifth HBO treatment immediately preceded acrolein. These results suggest that HBO may be useful as prophylaxis and treatment of cyclophosphamide-induced hemorrhagic cystitis.
Acrolein↗
Hyperoxia and prolongation of aminoglycoside-induced postantibiotic effect in Pseudomonas aeruginosa: role of reactive oxygen species.
Hyperoxia prolongs the postantibiotic effect (PAE) of the aminoglycoside tobramycin in Pseudomonas aeruginosa. We tested the hypothesis that the PAE is prolonged because hyperoxia increases free radical flux while tobramycin inhibits the induction of antioxidant defenses. Exposure of P. aeruginosa to hyperoxia (100% O2) for 1 h increased superoxide dismutase, catalase, and glutathione levels. In the presence of tobramycin (1x the MIC), the induction of antioxidant defenses by hyperoxia was nearly abrogated. Neither preexposure of P. aeruginosa to hyperoxia nor supplementation with the antioxidants copper(II) (diisopropylsalicylate)2 (superoxide dismutase-like), catalase, or dimethyl sulfoxide abolished prolongation of the PAE of tobramycin induced by hyperoxia.
Anti-Bacterial Agents↗
Regulation of protein degradation in normal and transformed human bronchial epithelial cells in culture.
Protein degradation rates are decreased in some transformed cells of mesenchymal origin. We have tested the generality of this phenomenon and evaluated the role of the lysosomes in this down-regulation. To this end we have compared the induction of lysosomal protein degradation among normal, transformed (BEAS-2B), and transformed tumorigenic (BZR, Calu-1) human bronchial epithelial cells in culture. Serum and/or nutrient deprivation, cell confluency, and Ca2+ were used to modulate lysosomal protein degradation. Protein degradation and synthesis were determined by the release or incorporation of [14C]valine in the cells. Autophagic degradation of cytoplasm by lysosomes was evaluated by ultrastructural morphometry. Basal protein degradation was lower (27%) in two of the transformed cell lines (BEAS-2B and BZR). Incorporation of [14C]valine label was raised approximately 4-fold in the transformed cells. Nutrient deprivation stimulated protein degradation equally (2-fold) in transformed and normal cells. Postconfluency increased (1.5-fold) basal protein degradation in Calu-1 cells and a marked enhancement (4-fold) of degradation occurred during nutrient deprivation. Culture of normal human bronchial epithelial cells in high Ca2+ caused phenotypic changes and increased (30%) the degradation of protein induced by nutrient deprivation. In Calu-1, high Ca2+ caused only phenotypic changes. The volume density (Vd) of autophagic vacuoles and dense bodies in the transformed cells was lower under basal conditions but increased markedly during nutrient deprivation. A marked accumulation of lysosomes also occurred in transformed cells during postconfluency. We conclude that cell transformation lowers basal protein degradation in some human epithelial cells. Lysosomal proteolysis of transformed cells is not down-regulated and can be markedly enhanced during nutritional deprivation by the autophagic degradation pathway.
Analysis of Variance↗
Oxygen tensions and infections: modulation of microbial growth, activity of antimicrobial agents, and immunologic responses.
Oxygen tensions play an important role in the outcome of infections. Oxygen is cidal or static for microorganisms that lack defenses against oxidants. Hyperoxia and hyperbaric oxygen exert antimicrobial effects by increasing the intracellular flux of reactive oxygen species. In bacteria, such species cause DNA strand breaks, degradation of RNA, inhibition of amino acid biosynthesis, and inactivation of membrane transport proteins. Oxygen tensions also affect the activity of antimicrobial agents. In general, hyperoxia potentiates while anaerobiosis decreases the activity of many antimicrobial drugs. With regard to host defenses, hyperoxia elevates oxygen tensions in infected tissues to levels that facilitate oxygen-dependent killing by leukocytes. Prolonged hyperoxia inhibits DNA synthesis in lymphocytes and impairs chemotactic activity, adherence, phagocytic capacity, and generation of the oxidative burst in polymorphonuclear leukocytes and macrophages.
Animals↗
Hyperbaric oxygenation decreases blood flows in normal and septic rats.
The purpose of the study reported here was to characterize the effects of acute exposure to 100% oxygen at a pressure of 202.6 kPa on hemodynamics and organ blood flow in antibiotic-controlled bacterial sepsis. An abscess containing known numbers and strains of live Escherichia coli and Bacteroides fragilis was established in the peritoneal cavity of rats. Sham-operated rats were used as controls. Cardiac output, fractional blood flow distribution, and blood flow were calculated from data obtained using the radiolabeled microsphere technique. Myocardial and renal blood flows were decreased (20-30%) in both experimental groups during hyperbaric oxygen (HBO) exposure. Renal blood flow remained diminished for at least 20 min after rats were removed from the hyperbaric chamber. Adrenal gland perfusion in abscess-containing rats was reduced both during and after HBO exposure. Skeletal muscle blood flow (quadriceps and gastrocnemius muscles) was reduced by one third in both experimental groups 20 min after acute exposure to HBO. The results of this study indicate that changes in organ perfusion induced by HBO are similar in abscess-containing and normal rats. We conclude that HBO does not alter the regulation of blood flow in antibiotic-controlled sepsis.
Animals↗
Pathophysiology of sea nettle (Chrysaora quinquecirrha), envenomation in a rat model and the effects of hyperbaric oxygen and verapamil treatment.
The effect of lethal sea nettle envenomation on the morphology and blood flow in various rat organs was characterized and the influence of two antidotes (hyperbaric oxygen and verapamil) was compared. Either antidote slightly prolonged survival, but the protective effects were not statistically significant. The venom caused no histologic alterations in brain, heart, or lungs but induced hepatic and renal necrosis. Hepatocytes in mid-zonal regions and renal tubular epithelium were the cell types predominantly affected. Hyperbaric oxygen and verapamil did not decrease the hepatic injury. The venom did not influence central hemodynamics until preterminally and it diminished blood flow to brain, but not to liver or kidney. Hyperbaric oxygenation protected against venom-induced decreases in blood flow to the brain. These results add toxic hepatic and renal necrosis and cerebral ischemia to the pathophysiology of envenomation in this model.
Animals↗
Evidence for an early free radical-mediated reperfusion injury in frostbite.
Frostbite is characterized by acute tissue injury induced by freezing and thawing. Initial complete ischemia is followed by reperfusion and later, tissue necrosis. These vascular events support the hypothesis that free radical-mediated reperfusion injury at thawing might contribute to tissue necrosis after frostbite in a manner similar to that seen after normothermic ischemia. To test this hypothesis, rabbit ears were frozen at -21 degrees C for 30, 60, 90, or 120 s and rewarmed at room temperature (22 degrees C). Rabbits were treated "blindly" with saline alone, highly purified, pharmaceutical grade superoxide dismutase (SOD), allopurinol, or deferoxamine. The area of ear necrosis was determined 3 weeks after frostbite by "blinded" morphometry. The administration of SOD at the time of thawing significantly improved viability in ears frozen for 60 and 90 s, but not in those frozen for 30 or 120 s. Deferoxamine also improved viability in ears frozen for 60 s. Allopurinol did not significantly affect ear survival. Electron micrographs showed the appearance of severe endothelial cell injury beginning during freezing and extending through early reperfusion. Later, neutrophil adhesion, erythrocyte aggregation, and microvascular stasis were seen. These findings suggest that free radical-mediated reperfusion injury has a role in frostbite, and quantitate the proportion of the injury that is due to this mechanism.
Allopurinol↗
Hyperoxia prolongs the aminoglycoside-induced postantibiotic effect in Pseudomonas aeruginosa.
The objective of this study was to determine whether hyperoxia enhances aminoglycoside activity against Pseudomonas aeruginosa. The existence of tobramycin-oxygen synergy was determined by using the in vitro postantibiotic effect (PAE). P. aeruginosa strains were incubated for 1 h in medium containing tobramycin at four times the MIC in the following gas mixtures: normoxia (21% O2), hyperoxia (100% O2, 101.3 kPa), or hyperbaric oxygen (100% O2, 274.5 kPa). Tobramycin was removed after 1 h and bacteria were incubated under normoxic conditions; growth rates were measured for 5 h. Exposure of three P. aeruginosa strains to hyperoxia prolonged the PAE of tobramycin approximately twofold compared with the PAE after exposure to normoxia (P less than 0.05). Exposure of P. aeruginosa ATCC 27853 to tobramycin and hyperbaric oxygen prolonged the time required for bacteria to increase 1 log10 CFU/ml compared with the time after exposure for this increase to occur in tobramycin-treated, normoxic or hyperoxic groups (P less than 0.02). Pulse-chase labeling of bacteria with L-[35S]methionine, immediately after removal of tobramycin, showed that protein synthesis rates were decreased compared with those in controls (P = 0.0001). Moreover, in tobramycin-treated groups, hyperoxia and hyperbaric oxygen induced 2- and 16-fold decreases, respectively, in protein synthesis rates compared with normoxia; these results did not achieve statistical significance. In the absence of tobramycin, hyperoxia increased bacterial growth (134%; P less than 0.01) and protein synthesis (24%; not significant) compared with normoxia. Hyperbaric oxygen, however, delayed the growth recovery of bacteria (P less than 0.05). We conclude that hyperoxia enhances the bacteriostatic effects of tobramycin in a synergistic manner.+
Anti-Bacterial Agents↗
Purification and characterization of human lysosomal membrane glycoproteins.
Two human cell lysosomal membrane glycoproteins of approximately 120 kDa, hLAMP-1 and hLAMP-2, were identified by use of monoclonal antibodies prepared against U937 myelomonocytic leukemia cells or blood mononuclear cells. The two glycoproteins were purified by antibody affinity chromatography and each was found to be a major constituent of human spleen cells, representing approximately 0.05% of the total detergent-extractable protein. Both molecules were highly glycosylated, being synthesized as polypeptides of 40 to 45 kDa and cotranslationally modified by the addition of Asn-linked oligosaccharides. NH2-terminal sequence analysis indicated that each was approximately 50% identical to the corresponding mLAMP-1 or mLAMP-2 of mouse cells. Electron microscopic studies of human blood monocytes, HL-60, and U937 cells demonstrated that the principal location of these glycoproteins was intracellular, in vacuoles and lysosomal structures but not in the peroxidase-positive granules of monocytes. Transport of the proteins between organelles was evidenced by their marked accumulation in the membranes of phagolysosomes. A fraction of each glycoprotein was also detected on the plasma membrane of U937 and HL-60 cells but not on a variety of other tissue culture cells. This cell-surface expression may be differentiation related, since the proteins were not detected in the plasma membrane of normal blood monocytes and their expression on U937 and HL-60 cells was reduced when the cells were treated with differentiating agents. Cell-surface expression of both glycoproteins was markedly increased in blood monocytes but not in U937 cells after exposure to the lysosomotropic reagent methylamine HCl, indicating differences in LAMP-associated membrane flow in these cell types.
Amino Acid Sequence↗
Stimulation of autophagic protein degradation by nutrient deprivation in a differentiated murine teratocarcinoma (F9 12-1a) cell line.
We have evaluated the participation of the lysosomal degradation pathway in the increased protein degradation induced by nutrient deprivation in transformed cells. To this end we used a clone, 12-1a, derived from a murine teratocarcinoma cell line (F9 12-1) induced to differentiate by culture in retinoic acid. Culture of 12-1a cells, prelabeled with L-[U-14C]valine, in nutrient-deprived medium (Hanks' balanced salt solution plus Ca++) stimulated the protein degradation rate from 0.9% hr to 1.4% hr. Morphometric analysis demonstrated that during nutrient deprivation, the volume density of lysosomes increased 3-fold; the numerical density of lysosomes increased 2-fold; the mean area of lysosomal profiles increased 1.7-fold (1.40 microns2 vs 0.81 microns2). The volume density and numerical density of the dense bodies tended to decrease by approximately 60% without any change in the mean volume of the dense bodies. These data indicate that nutrient deprivation increases protein degradation in transformed cells by increasing the sequestration of cytoplasm into the lysosomes. The decrease in the number of dense bodies indicates that these structures (also termed residual bodies) are functional in transformed cells and merge with the lysosomes to provide more degradative enzymes to enhance proteolysis. This study provides direct evidence that serum factors and nutrients play a crucial role in modulation of lysosomal protein degradation in transformed cells.
Animals↗