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Biomedical subjects

L M Simon

Publications and source records attributed to L M Simon.

At least 19 recordsLinked to original sources
PubMed1990

Effects of polyethylene terephthalate on yeast alcohol dehydrogenase.

Yeast alcohol dehydrogenase (alcohol: NAD+ oxidoreductase, EC 1.1.1.1) was adsorbed onto polyethylene terephthalate, a synthetic polymer. The effects of the polymer on the properties of the enzyme were studied. The specific activity of the bound enzyme on protein basis was only 1.2 per cent of the specific activity of the soluble enzyme. The optimum pH for the catalytic activity was strongly shifted toward acidic direction. The apparent temperature optimum of the bound enzyme was identical with that of the soluble form. The apparent Michaelis constants of the bound enzyme were higher for both ethanol and NAD+. The conformational stability of the enzyme against heat treatment and urea was decreased as a consequence of adsorption.

Adsorption↗
PubMed1988

Endotoxin and pulmonary cell injury.

The physiopathologic similarity between adult respiratory distress syndrome (ARDS) secondary to sepsis and endotoxin-induced pulmonary abnormalities has provided extensive descriptive information confirming bacterial endotoxin as a factor initiating the heterogeneous pulmonary changes in ARDS. The present studies have used an established in vitro model for pulmonary cell injury to examine bacterial endotoxin 1, as a direct cytotoxic agent on the two major alveolar cell types, pulmonary endothelium and epithelium; 2, as a stimulant of neutrophil-mediated pulmonary cell injury, and 3, to examine effector mechanisms of cell-mediated damage by studying the potential effectiveness of antioxidants and antiproteolytic agents in the inhibition of this process. Endotoxin direct toxicity and stimulation of neutrophil-mediated pulmonary cell injury was observed in both pulmonary cell populations in systems free of activated serum complement. Endothelial cells were observed to be more susceptible to both the direct effect of endotoxin and to neutrophil-mediated injury when compared with epithelial cell derived monolayers. The addition of an antiprotease (soybean trypsin inhibitor [STI]) was superior to antioxidants (catalase, superoxide dismutase) in reducing the neutrophil-mediated endothelial toxicity (stimulated 51CR per cent release) observed. A 92 per cent degree of protection was observed with the highest dose of STI (5 milligrams per milliliter) used. Proteases released by activated neutrophils on endotoxin stimulation appear to be the predominant toxic species responsible for endothelial injury in this system.

Antioxidants↗
PubMed1988

Determination of glycolytic intermediates in a flow injection system using immobilized enzymes.

Some glycolytic enzymes (lactate dehydrogenase, pyruvate kinase, enolase and phosphoglyceromutase) were immobilized on a polyacrylamide-type bead polymer containing carboxylic functional groups activated by water-soluble carbodiimide. The immobilized enzymes were used for the determination of pyruvic acid, phosphoenolpyruvic acid, 2-phosphoglyceric acid and 3-phosphoglyceric acid in a flow injection system. The immobilized lactate dehydrogenase column was repeatedly employed for the determination of pyruvic acid in clinical samples. The results of the flow injection method accorded well in accuracy, sensitivity and reproducibility with those of soluble enzyme analysis.

Enzyme Stability↗
PubMed1986

Immobilization of lactate dehydrogenase on polyacrylamide beads.

Pig muscle lactate dehydrogenase (L-lactate:NAD oxidoreductase, EC 1.1.1.27) was covalently immobilized on polyacrylamide beads containing carboxylic functional groups activated by water-soluble carbodiimide. The effects of immobilization on the catalytic properties and stability of the lactate dehydrogenase were studied. There was no shift in the pH optimum of the immobilized enzyme compared to that of the soluble one. The apparent optimum temperature of the soluble enzyme was 65 degrees C, while that of the immobilized enzyme was between 50 and 65 degrees C. The apparent Km values of the immobilized enzyme with pyruvate and NADH substrates were higher than those of the soluble enzyme. As a result of immobilization, enhanced stabilities were found against heat treatment, changes in pH, and urea denaturation.

Acrylic Resins↗
PubMed1985

Comparative studies on soluble and immobilized rabbit muscle pyruvate kinase.

Rabbit muscle pyruvate kinase was immobilized by covalent attachment to a polyacrylamide support (Akrilex C) containing carboxylic functional groups. As a result of immobilization, the pH optimum for catalytic activity shifted into a more alkaline direction. The apparent Km value with phosphoenolpyruvate increased, and that with ADP slightly decreased. With respect to the stability against urea and thermal inactivation, the immobilized pyruvate kinase seemed to be the more stable at lower urea concentrations and between 45 and 55 degrees C. At 1.5 and 2.5M urea and at higher temperature, there were no marked differences between the soluble and the immobilized enzyme.

Animals↗
PubMed1985

Lung cell oxidant injury: decrease in oxidant mediated cytotoxicity by N-acetylcysteine.

Lung cell damage mediate by polymorphonuclear leukocyte (PMN) reactive oxygen metabolites has been suggested as a pathophysiologic mechanism in a variety of acute and chronic pulmonary disease states, while oxidant injury may be a non-specific cytotoxic mechanism. Reducing agents therefore represent one therapeutic direction for decreasing lung cell injury in several clinical circumstances. N-Acetylcysteine (NAC) is a known antioxidant which can be distributed in soluble form to multiple intrapulmonary sites. We have therefore examined a possible role for NAC against oxidant injury in a controlled in vitro model for oxygen metabolite cytotoxicity. Our data suggest that extracellular NAC is able to protect lung cells against PMN mediated oxidant injury. Pre-exposure of lung cells to NAC results in decreased susceptibility to oxidant damage by increasing intracellular antioxidant defense systems. An increase in extracellular and/or intracellular resistance to toxic oxygen metabolites by NAC may be one approach to the prevention of in vivo lung oxidant injury.

Acetylcysteine↗
PubMed1984

Studies on the properties of glucose-6-phosphatase from carp liver microsomes (Cyprinus carpio L.).

The effects of temperature and pH on the phosphohydrolase activity of carp hepatic glucose-6-phosphatase (EC 3.1.3.9) have been investigated. The enzyme activity was maximum at about 308 K and in the pH range 5-6.5. The apparent Michaelis constant (KM) and Vmax of the reaction with glucose-6-phosphate were found to be 14.8 mM and 2.27 nmol/min/mg protein. The enzyme activity was partly inhibited by EDTA, while in the presence of sufficient PCMB virtually total inhibition was observed.

Animals↗
PubMed1983

Studies on the effect of paraquat on glycogen mobilization in liver of common carp (Cyprinus carpio L.).

1. A herbicide, paraquat (1,1'dimethyl-4,4'-bipyridilium-dichloride) was administered to carp in 0.5-10.0 ppm concentrations, respectively, and blood sugar level, glucose-6-phosphatase and glycogen phosphorylase activities of liver were determined. 2. Paraquat treatment caused an increase of blood sugar level and enhanced phosphorylase and glucose-6-phosphatase activities. 3. Paraquat can induce alterations in endoplasmic reticulum that might contribute to the changes in glucose-6-phosphatase activity, resulting in an increase of blood glucose level and/or all the effects can be attributed to a high level of circulating epinephrine produced by paraquat toxicosis.

Animals↗
PubMed1983

Decreased bactericidal function and impaired respiratory burst in lung macrophages after sustained in vitro hyperoxia.

Lung macrophages (LM) play a crucial role in pulmonary bacterial defense. High inspired oxygen concentrations are used in a variety of diseases and "oxygen toxicity" could impair antibacterial function. We therefore examined the effect of sustained in vitro hyperoxia on LM bactericidal function, and on generation of two bactericidal oxygen metabolites. The LM were cultivated under aerobic (PO2 approximately 140 mmHg) or hyperoxic (PO2 approximately 630 mmHg) conditions for 48 h, and then incubated with Staphylococcus aureus labeled with 3H thymidine for 30 min. Incubated monolayers were processed for measurement of total bacterial uptake and for number of viable intracellular bacteria. Superoxide anion (O2-) and hydrogen peroxide (H2O2) generation was determined in similarly cultivated cells stimulated with opsonized zymosan. The results indicate that the bacterial killing capacity of oxygen-cultivated LM is significantly decreased (p less than 0.001). In addition, a significant (p less than 0.001) decrease in generation of O2- and H2O2 was noted after exposure to high oxygen tensions. The data suggest that decreased bactericidal function after sustained hyperoxia may be due to an impairment of a specific bactericidal mechanism, i.e., an impaired "respiratory burst."

Animals↗
PubMed1982

Lung cell oxidant injury. Enhancement of polymorphonuclear leukocyte-mediated cytotoxicity in lung cells exposed to sustained in vitro hyperoxia.

The oxidant damage of lung tissue during in vivo hyperoxic exposure appears to be amplified by neutrophils that release toxic amounts of oxygen metabolites. In our studies cloned lung epithelial cells (L2 cells), lung fibroblasts, and pulmonary artery endothelial cells were cultured under either ambient (Po(2) approximately 140 torr) or hyperoxic (Po(2) approximately 630 torr) conditions for 48 h (24 h for endothelial cells). After cultivation, phorbol myristate acetate- or opsonized zymosan-stimulated neutrophils were added to the cultivated monolayers for 4 h, and lung cell damage was quantitated using (51)Cr release as an index. The data show that stimulated neutrophils are able to injure the three lung cell lines tested, with endothelial cells being highly susceptible to this injury and L2 cells being slightly more susceptible than lung fibroblasts. The studies also demonstrate that all three lung cell lines exposed to sustained hyperoxia are more susceptible to neutrophil-mediated cytotoxicity than their time-matched air controls. Hydrogen peroxide was the main toxic oxygen metabolite because catalase (2,500 U/ml) completely protected the target cells. Equivalent quantities of hydrogen peroxide generated by glucose oxidase instead of by neutrophils gave a similar degree of target cell injury. Superoxide dismutase at high concentrations (250 mug/ml) provided some protection. Other systems that detoxify oxygen metabolites were without protective effect. These findings indicate that the increase in susceptibility of lung cells to neutrophil-mediated oxidant damage is a toxic effect of hyperoxia on lung cells. This specific manifestation of oxygen damage provides insight into the integration between primary mechanisms (oxygen exposure) and secondary mechanisms (release of oxygen metabolites by neutrophils) with respect to the cellular basis for pulmonary oxygen toxicity.

Animals↗
PubMed1981

Differences in oxygen-dependent regulation of enzymes between tumor and normal cell systems in culture.

Metabolic studies in tumor cells have indicated that bioenergetic regulatory mechanisms geared to acute changes in oxygen availability are abnormal. In the present studies we have examined bioenergetic adaptations to chronic oxygen depletion in culture maintained tumor cells in comparison to normal cell lines. Activities of two key glycolytic enzymes (pyruvate kinase (PyKI) and phosphofructokinase (PFK)) were measured in two tumor cell lines (fibrosarcoma (FS) and Hela) and two normal cell lines (rat lung fibroblasts (RLF) and WI-38) maintained in culture for up to 96 hours under aerobic (PO2 approximately 140) and hypoxic PO2 approximately 15) conditions. Exposure to low O2 tensions for 96 hours resulted in significant increases in PyKi and PFK in both RLF and WI-38, ut did not alter activities of these enzymes in either FS or HeLa cell systems. Activities of two enzymes involved in O2 metabolism (cytochrome oxidase (CyOx) and superoxide dismutase (SOD) were also measured in the two tumor cell lines and in RLF. chronic hypoxia significantly decreased the activities of CyOx and SOD in RLF cell systems but did not alter the activities of these enzymes in the tumor cells. In these studies, the tumor-derived cell lines do not demonstrate specific enzymatic responses to sustained oxygen depletion in vitro noted in normal cell systems, suggesting significant abnormalities in regulatory mechanisms geared to chronic changes in molecular O2.

Cell Line↗
PubMed1979

Effects of sustained oxygen depletion on tissue pyruvate kinase activities in the freshwater turtle, Pseudemys scripta elegans.

1. The effect of sustained (48 hr) oxygen depletion on tissue (brain, heart, skeletal muscle) pyruvate kinase (PyKi) activities was examined in the pond turtle (Pseudemys scripta elegans). 2. PyKi activities in skeletal muscle are significantly increased (from 322 +/- 84 to 450 +/- 95) following 48 hr of tissue hypoxia. PyKi activities in brain may already be elevated under ambient conditions but do not change following prolonged submersion (334 +/- 75 vs 325 +/- 77). Cardiac muscle PyKi is actually decreased (from 135 +/- 35 to 94 +/- 18) under anaerobic conditions. 3. The data suggest that prolonged O2 depletion may increase PyKi biosynthesis in turtle skeletal muscle (subserving enhanced glycolysis), but also demonstrate that factors other than oxygen availability are involved in the regulation of tissue PyKi activities in intact vertebrates.

Animals↗
PubMed1979

Bioenergetic pattern of turtle brain and resistance to profound loss of mitochondrial ATP generation.

The adaptations in the freshwater turtle that permit survival despite prolonged loss of mitochondrial ATP generation were investigated by comparing the bioenergetics of turtle brain slices with rat brain slices. Aerobic turtle brain shows no significant difference in basal levels of total ATP generation compared to rat brain; levels in turtle brain and rat brain were 18.4 +/- 2.8 (SD) and 19.4 +/- 2.2 mumol (100 mg of tissue)-1 hr-1, respectively. However, in turtle brain, a significantly greater fraction of ATP is derived from glycolysis both under aerobic and anaerobic conditions [aerobic turtle (24%) and rat (13%), P less than 0.02; anaerobic, turtle (28%) and rat (18%), P less than 0.05]. The increased glycolytic capacity is related to high levels of rate-limiting glycolytic enzymes, such as pyruvate kinase (EC 2.7.1.40). Turtle brain operates close to glycolytic capacity even under aerobic conditions, and no Pasteur effect can be demonstrated. Quantitatively, anaerobic glycolysis accounts for a maximum of 28% of basal aerobic ATP generation, suggesting that prolonged diving is also accompanied by a reduction in brain energy requirements. The adaptation subserving short-term (natural) diving is an increase in brain glycolytic capacity. The adaptation subserving prolonged diving (days to weeks) may be a reduction in the energy requirements of brain (and other cells).

Animals↗
PubMed1979

Effects of high oxygen exposure on bioenergetics in isolated type II pneumocytes.

O2-mediated alterations in cell energy metabolism may play a role in structural and functional abnormalities described in type II pneumocytes (T-II-P) following in vivo hyperoxia. Bioenergetic alterations produced by hyperoxia (95% O2) were therefore examined in a culture-maintained cell line derived from T-II-P. Exposure of cell monolayers to 95% O2 for 96 h results in a significant decrease in O2 consumption (from 0.52 +/- 0.07 to 0.30 +/- 0.08, P less than 0.01), suggesting impaired mitochondrial energy provision. In addition, there are increased rates of aerobic lactate production (from 2.89 +/- 0.52 to 3.84 +/-0.80, P less than 0.05) with loss of Pasteur effect, indicating a shift to glycolytic metabolism at relatively high PO2's. These metabolic changes are not accompanied by altered activities of critical mitochondrial (cytochrome oxidase) or glycolytic (pyruvate kinase, phosphofructokinase) enzymes. Altered cell bioenergetics following hyperoxia may this represent an important secondary mechanism leading to functional abnormalities in T-II-P.

Aerobiosis↗
PubMed1978

Bioenergetic pattern of isolated type II pneumocytes in air and during hypoxia.

The bioenergetic pattern of a cell clone derived from rat lung with ultrastructural and biochemical characteristics like those of type II pneumocytes (T-II-P), has been studied in a tissue culture system. During air cultivation, these cells have a high rate of aerobic and anaerobic glycolysis associated with high activities of two rate-limiting enzymes in glycolysis (pyruvate kinase [PyKi] and phosphofructokinase [PFK]). This is present despite the rates of oxygen consumption and activities of cytochrome oxidase (CyOx) similar to other lung cells. Presumably the high rate of aerobic glycolysis explains the substantial lactate production previously described in lung slices and in the intact perfused lung. Hypoxic cultivation results in a decrease in CyOx. Acute re-exposure to air does not restore the oxygen consumption to normal, presumably as a result of decreased mitochondrial O(2) utilization associated with decreased CyOx activity. As a result, hypoxically cultivated T-II-P cells have a decreased capacity for mitochondrial ATP generation in air as compared to air-cultivated cells. During hypoxia, aerobic and anaerobic glycolysis are further increased as well as the activities of PyKi and PFK. The high rate of glycolysis and high activities of PyKi and PFK in cultivated T-II-P appear to reflect intrinsic genetic regulation. The decreased CyOx activity and increased PyKi and PFK activities in hypoxic T-II-P appear to reflect alterations in enzyme biosynthesis/biodegradation regulated by O(2) availability.

Animals↗