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Biomedical subjects

L Levine

Publications and source records attributed to L Levine.

At least 73 records · Page 4Linked to original sources

Hypohydration does not impair skeletal muscle glycogen resynthesis after exercise.

The purpose of this investigation was to examine the effects of moderate hypohydration (HY) on skeletal muscle glycogen resynthesis after exhaustive exercise. On two occasions, eight males completed 2 h of intermittent cycle ergometer exercise (4 bouts of 17 min at 60% and 3 min at 80% of maximal O2 consumption/10 min rest) to reduce muscle glycogen concentrations (control values 711 +/- 41 mumol/g dry wt). During one trial, cycle exercise was followed by several hours of light upper body exercise in the heat without fluid replacement to induce HY (-5% body wt); in the second trial, sufficient water was ingested during the upper body exercise and heat exposure to maintain euhydration (EU). In both trials, 400 g of carbohydrate were ingested at the completion of exercise and followed by 15 h of rest while the desired hydration level was maintained. Muscle biopsy samples were obtained from the vastus lateralis immediately after intermittent cycle exercise (T1) and after 15 h of rest (T2). During the HY trial, the muscle water content was lower (P less than 0.05) at T1 and T2 (288 +/- 9 and 265 +/- 5 ml/100 g dry wt, respectively; NS) than during EU (313 +/- 8 and 301 +/- 4 ml/100 g dry wt, respectively; NS). Muscle glycogen concentration was not significantly different during EU and HY at T1 (200 +/- 35 vs. 251 +/- 50 mumol/g dry wt) or T2 (452 +/- 34 vs. 491 +/- 35 mumol/g dry wt). These data indicate that, despite reduced water content during the first 15 h after heavy exercise, skeletal muscle glycogen resynthesis is not impaired.

Adolescent↗

Fluid replacement during sustained activity in the heat: nutrient solution vs. water.

This study examined the thermoregulatory and hydrational status of men during sustained activity in a hot-dry (37 degrees C, 20% rh) environment while they consumed only a nutrient solution (nutrient), or consumed only colored, flavored water (control). Eleven heat acclimated young men attempted 24-h sustained activity experiments. These experiments consisted of alternating 45-min bouts of treadmill walking (410 W, approximately 30% VO2max) and rest (including sedentary activity). Data were analyzed through 13 h (after 13 h subjects began to discontinue testing). No significant differences between trials were observed for metabolic rate, fluid intake, skin or rectal temperature, sweating rate, plasma volume (as indicated by hemoglobin concentration) or plasma glucose concentrations. By the 8th h plasma osmolality was higher and by the 11th h plasma free fatty acids were lower during the nutrient trial compared to the control. In separate experiments with nine different men, the gastric emptying rates of the nutrient solution and water were compared during exercise (55% VO2max) in the heat (35 degrees C, 20% rh). The gastric emptying rates of the nutrient solution and water were similar (approximately 20 ml.min-1). These data indicate that during 13 h of sustained activity in a hot environment, the nutrient solution and water provided similar thermoregulatory and hydrational benefits.

Adult↗

Effects of the protein kinase inhibitors, staurosporine and K-252a, on PGI2 production by rat liver cells (the C-9 cell line).

Staurosporine and K-252a, known inhibitors of several protein kinases, stimulated PGI2 production (measured as 6-keto-PGF1 alpha) in rat liver cells (the C-9 cell line). Preincubation of the rat liver cells with staurosporine or K-252a enhanced the PGI2 production stimulated by 12-O-tetradecanoylphorbol-13-acetate (TPA), platelet activating factor (PAF) and the Ca2(+)-ionophore A-23187, but not the PGI2 synthesis stimulated by exogenous arachidonic acid. These results suggest that phosphorylation of some proteins or certain amino acids on a protein can regulate arachidonic acid metabolism probably in the pathway leading to deesterification of phospholipids.

6-Ketoprostaglandin F1 alpha↗

Leukotriene and prostaglandin production in rat brain synaptosomes treated with phospholipase A2 neurotoxins and enzymes.

beta-Bungarotoxin (beta-BuTX) and notexin cause an irreversible blockade of neurotransmitter release through specific and potent effects at the presynaptic nerve terminal, however, the mechanism of action is uncertain. We examined the effects of beta-BuTX and notexin on LT and PG production in rat cerebrocortical synaptosomes in order to determine if eicosanoid production might mediate or regulate the pharmacological actions of these phospholipase A2 (PLA2) neurotoxins. The effects of the PLA2 enzymes isolated from Naja naja atra and Naja nigricollis snake venoms (which are not presynaptic selective) on LT and PG production were compared with the effects of beta-BuTX and notexin. N. n. atra PLA2, beta-BuTX, and notexin (all 50 nM) produced a time dependent rise in free fatty acids as measured in synaptic plasma membranes isolated from treated synaptosomes. Both the PLA2 neurotoxins and enzymes stimulated LTC4, LTB4, and PGE2 production, as measured by radioimmunoassay. In all cases, the PLA2 enzymes were more potent than the PLA2 neurotoxins. This observation correlates with their relative enzymatic potencies, as measured by free fatty acid generation. EDTA and BSA antagonized PLA2 induced LTB4 production and BSA also antagonized PLA2 induced PGE2 production. These results suggest that stimulation of eicosanoid production does not mediate the potent and specific presynaptic actions of beta-BuTX and notexin.

Animals↗

Antibodies and radioimmunoassays for phosphoserine, phosphothreonine and phosphotyrosine. Serologic specificities and levels of the phosphoamino acids in cytoplasmic fractions of rat tissues.

For antibody production, the O-phosphorylated derivative of tyrosine, threonine, or serine was covalently linked to succinylated bovine albumin via the carbodiimide reaction. Each conjugate was then complexed with methylated bovine albumin for immunization of rabbits. To determine binding, the corresponding O-phosphorylated [3H]amino acids were chemically synthesized. In addition, these 3H-phosphorylated derivatives were acylated (with succinic or acetic anhydride) to obtain ligands whose structures resemble those present in the immunogen. The acylated ligands bound to their respective antibodies more effectively: in some cases binding was about three orders of magnitude greater than their non-acylated counterparts. Radioimmunoassays were therefore developed using the N-succinyl-[3H]phosphoamino acids. When the unlabeled N-succinyl-phosphorylated amino acids were used as inhibitors in the homologous immune systems, 50% displacement of the labeled ligand was found with 0.06, 0.27 or 0.8 pmol of the tyrosine, threonine, or serine derivative, respectively. The antibodies were highly specific for the homologous hapten; the requirement for the phosphate group on the acylated amino acid was essentially absolute. Antibody content (expressed as mg/ml serum) and apparent binding constants for the N-succinyl derivatives in individual bleedings of immune sera were 1.9 and 1 X 10(10) M-1 for phosphotyrosine, 0.825 and 6 X 10(8) M-1 for phosphothreonine, and 0.150 and 2 X 10(8) M-1 for phosphoserine. The radioimmunoassays were used to quantitate the phosphoamino acids in cytoplasmic fractions of rat tissue extracts. The production of antibodies to phosphorylated O-tyrosine has been reported previously, but to our knowledge, this represents the first report of antibodies specific for O-phosphorylated serine and threonine residues.

Animals↗

Lithium carbonate for aggressive behavior or affective instability in ten brain-injured patients.

Lithium carbonate (LiCO3) was used to treat 10 brain-injured patients with severe, unremitting, aggressive, combative, or self destructive behavior or severe affective instability. Five patients had a dramatic response that resulted in significant improvement in their participation in a rehabilitative program. One other patient had a moderate response. A seventh patient improved dramatically, but regressed after 7 wk. Three other patients had neurotoxic side effects that precluded continued use of the medication. Two of them were simultaneously taking neuroleptic agents. These case reports provide further evidence that LiCO3 can be a useful medication in the treatment of aggressive behavior and affective instability after brain injury, but that it has significant potential for neurotoxicity in this population, particularly when used in conjunction with neuroleptic agents.

Adult↗

Nuclear power plants and natural populations of Mexican Drosophila.

With the worldwide proliferation of nuclear power plants has come the need to study the biological effects of the operation of the reactors on surrounding populations. We have begun a long-term study of the sibling species Drosophila melanogaster and D. simulans in the area of Laguna Verde in the state of Veracruz in Mexico. Laguna Verde, on the Gulf of Mexico about 75 km north of the city of Veracruz, is the location of the country's first nuclear power plant. This plant has not yet gone "on-line." The species have been collected from two sites, one of which is south of the reactor and is in the path of the prevailing north to south wind flow. The other collecting site is west of the plant. The species are being studied for the following: species frequency, desiccation resistance, vagility, proportion of larvae pupating, pupation height, and egg to adult survival after irradiation. To date we have noted both spatial and seasonal differences in a number of these characteristics. The information being gathered will serve as base-line data for monitoring the future operation of the nuclear power plant.

Animals↗

A flavonoid inhibitor of 5-lipoxygenase inhibits leukotriene production following ischemia in gerbil brain.

Leukotrienes C4 and D4 are arachidonic acid metabolites that constrict blood vessels and enhance vascular permeability; their biosynthesis is initiated by the reaction of arachidonic acid with 5-lipoxygenase enzyme. After bilateral carotid artery occlusion for 15 minutes and reperfusion of the gerbil brain for 15 minutes, we determined the brain tissue concentrations of leukotrienes C4 and D4 by radioimmunoassay; they had increased from a baseline concentration of less than 1 to a mean +/- SEM concentration of 12.8 +/- 3.9 pmol/g brain. We also studied the effect of a flavonoid 5-lipoxygenase inhibitor on leukotriene production in the reperfused gerbil brain. A water-soluble flavonoid (5-hexyloxy-3',4'-dihydroxy-6,7-dimethoxyflavone 4'-disodium phosphate) was administered intravenously at a dose of 200 mg/kg body wt; 15 minutes later, both carotid arteries were occluded. The enhanced production of leukotrienes C4 and D4 in the reperfused brain was reduced by approximately 80% (from a mean +/- SEM of 12.8 +/- 3.9 to 2.2 +/- 1.3 pmol/g brain) in the presence of the 5-lipoxygenase inhibitor. The flavonoid did not affect the production of prostaglandin D2, the concentration of which also increased in the reperfused ischemic brain.

Animals↗

Two squamous cell carcinomas not associated with humoral hypercalcemia produce a potent bone resorption-stimulating factor which is interleukin-1 alpha.

Conditioned medium (CM) from two squamous cell carcinoma cell lines, SCC-9 and SCC-13, stimulated bone resorption in neonatal mouse calvariae in organ culture. Enhanced bone resorption induced by CM was associated with an increased production of prostaglandin-E2 (PGE2) by the calvariae. Complete inhibition of stimulated PGE2 synthesis by indomethacin only partially inhibited bone resorption-stimulating activity (BRSA) in the CM. Neither SCC-9 nor SCC-13 CM stimulated cAMP production in rat osteosarcoma cells (ROS 17/2.8). The BRSA in CM was completely inhibited by an antibody to interleukin-1 alpha (IL-1 alpha). Fractionation of SCC-9 CM by gel filtration and HPLC ion exchange chromatography revealed a single peak of BRSA and PGE2 synthesis-stimulating activity at 17-20K (termed SCMII). In mouse calvariae, SCMII increased medium Ca2+ and PGE2 in a dose-dependent manner at concentrations from 20 ng protein/ml to a maximum of 500 ng protein/ml. Preincubation of SCMII with antibody to IL-1 alpha completely inhibited SCMII-induced bone resorption. SCMII also enhanced thymocyte proliferation with activity that was equivalent to 353 U/ml IL-1. Antibodies to IL-1 beta and tumor necrosis factor had no effect on SCMII-induced bone resorption. Using specific enzyme-linked immunosorbent assays for IL-1 alpha and IL-1 beta, IL-1 alpha was measured in high concentrations in both crude and partially purified fractions of SCC-9 and SCC-13 CM. In contrast, IL-1 beta was either undetectable or present in amounts below those that stimulate bone resorption. In addition, SCMII did not enhance cAMP production in bone cells. We conclude that the BRSA produced by the two squamous cell carcinoma cell lines SCC-9 and SCC-13 is IL-1 alpha.

Adenylyl Cyclases↗

Prostaglandins E2 and E2 alpha are elevated in saliva of cystic fibrosis patients.

Several products of arachidonic acid metabolism were assayed in whole saliva of patients with cystic fibrosis, a disease affecting primarily the exocrine glands. The levels of prostaglandins (PG) E2 and F2 alpha were elevated fourfold in patients with cystic fibrosis, compared with a control group (mean +/- SEM for PGE2 1.13 +/- 0.22 vs 0.37 +/- 0.08 ng/ml saliva, p less than 0.005; and for PGE2 alpha 0.84 +/- 0.27 vs 0.17 +/- 0.03 ng/ml saliva, p less than 0.025). The levels of PGI2, hydroxyeicosatetraenoic acids, and leukotrienes showed no significant differences between the two groups. These changes were not present in individuals heterozygous for the cystic fibrosis gene. Study of appropriate control groups suggested that the changes that we observed in patients with cystic fibrosis could not be attributed to their lung disease or to the medications these patients were receiving. Our findings indicate that metabolism of arachidonic acid is abnormal in cystic fibrosis.

Adolescent↗

Selective inhibition by minoxidil of prostacyclin production by cells in culture.

The effect of minoxidil on arachidonic acid metabolism by cells in culture was studied. In bovine aorta endothelial cells, treatment with minoxidil in the presence of various stimulators of arachidonic acid metabolism was accompanied by a dose-dependent inhibition of prostacyclin production (measured as 6-keto-prostaglandin F1 alpha). Synthesis of the other cyclooxygenase products (prostaglandins E2, F2 alpha and thromboxane) was not inhibited. When the bovine aorta endothelial cells were stimulated by the Ca2+ ionophore A-23187, the inhibition was seen as early as 2 min. Minoxidil also inhibited prostacyclin production by a second cell line of bovine aorta endothelial cells (the established CPAE cell line), bovine aorta smooth muscle cells, porcine aorta endothelial cells, and rat liver cells (the C-9 cell line)--the latter, less effectively. Again, formation of all the other cyclooxygenase products studied was not inhibited. Minoxidil did not affect significantly prostaglandin E2 and F2 alpha production by newborn rat keratinocytes (the NBR cell line)--a cell that does not produce PGI2. The clinical, biochemical, and pharmacologic implications are discussed.

6-Ketoprostaglandin F1 alpha↗

A radioimmunoassay for palytoxin.

Palytoxin, labelled with 125I-Bolton-Hunter reagent on its terminal amino group, bound specifically to rabbit anti-palytoxin. The extent of binding increased progressively with repeated immunizations. After absorption of the rabbit IgGs with a goat anti-rabbit IgG, binding was reduced greater than 95%. For 50% inhibition of binding in the 125I-palytoxin-antipalytoxin reaction 0.27 pmoles of unlabelled palytoxin was required. Maitotoxin, teleocidin, okadaic acid, debromoaplysiatoxin and 12-O-tetradecanoylphorbol-13-acetate, when tested at 10-100-fold higher concentrations than palytoxin did not affect binding. Palytoxin's serologic activity was stable after 60 min exposure to 100 degrees C and after 60 min exposure to 0.1 N HCl at 50 degrees C, but its capacity to stimulate the arachidonic acid metabolism of rat liver cells was reduced after the 60 min exposure to 0.1 N HCl treatments at 35 degrees C or 0.01 N HCl at 50 degrees C. The average binding constant (K0) as determined by separation of antibody-bound palytoxin from free palytoxin by the double antibody technique was 4.9 x 10(9) M-1 at 0 degrees C. This apparent average association constant increased with increasing temperature suggesting that palytoxin's epitope, most likely hydrophilic, is bound to H2O and the H2O is displaced before binding to the antibody's paratope.

Acrylamides↗

Production of antibodies and development of a radioimmunoassay for okadaic acid.

An okadaic acid immunogen, prepared by conjugation of okadaic acid to bovine albumin with carbodiimide, was used to immunize two rabbits. The rabbits responded by producing antibodies that neutralized okadaic acid's stimulation of arachidonic acid metabolism and this neutralization increased during the course of immunization. The immune sera bound 3H-okadaic acid and this binding also increased with repeated immunization. After absorption of the rabbit IgG with a goat anti-rabbit IgG, binding was reduced greater than 99%. The binding of okadaic acid to the antibodies in one antiserum was inhibited by as little as 0.2 pmoles of unlabelled okadaic acid. The apparent association constant for binding with this antiserum was 4.17 x 10(9) M-1 (35 degrees C). Maitotoxin, teleocidin, 12-O-tetradecanoylphorbol-13-acetate, aplysiatoxin, palytoxin and brevetoxin B when tested at 29, 228, 168, 169, 3.7 and 112 pmole levels, respectively, did not inhibit binding. The serologic and biological activities of okadaic acid after incubation for 60 min in 0.01 N HCl at 35 degrees C or at 100 degrees C at pH 7.2 were unaffected.

6-Ketoprostaglandin F1 alpha↗

A radioimmunoassay for the teleocidins using 26 (2'-aminoethylthio)-tetrahydroteleocidin A-2 as hapten.

A teleocidin A-2 derivative, 26 (2'-aminoethylthio)-tetrahydroteleocidin A-2, induced ornithine decarboxylase in mouse skin and inhibited the specific binding of [3H]12-O-tetradecanoyl-phorbol-13-acetate to a mouse skin particulate fraction with a potency that was weaker than that of teleocidin A-2 and stronger than that of (-)-indolactam-V. To obtain antibodies, 26 (2'-aminoethylthio)-tetrahydroteleocidin A-2 was covalently linked to bovine albumin with a carbodiimide and the resulting conjugate used for immunization of rabbits. Antibodies directed toward teleocidin were produced as measured by neutralization of teleocidin's capacity to stimulate arachidonic acid metabolism in rat liver cells (the C-9 cell line). An 125I-labeled ligand was prepared by reaction of the same derivative with radiolabeled Bolton-Hunter reagent. The antibodies bound this radiolabeled hapten, and the binding increased progressively with repeated immunizations. After absorption of the rabbit IgG with a goat anti-rabbit IgG, binding was reduced greater than 95%. The binding of the 125I-labeled ligand to the antibodies of one rabbit had an apparent average association constant of 2.84 x 10(9) M-1 at 0 degrees C. The serologic specificity of the antiserum was characterized by measuring the inhibition of binding by several teleocidins of varying structure as well as by other tumor promoters and toxins. The rank order of inhibitory activity expressed as concentration required for 50% inhibition (IC50) was for 26 (2'-aminoethylthio)-tetrahydroteleocidin A-2'(0.56 pmol) greater than teleocidin A-1 (6.5 pmol) greater than or equal to teleocidin A-2 (7.3 pmol) greater than (-)-indolactam-V (3.7 nmol) greater than teleocidin B-4 (13 nmol). Maitotoxin, aplysiatoxin, palytoxin, mezerein, okadaic acid and 12-O-tetradecanoylphorbol-13-acetate did not inhibit at the levels tested.

Animals↗

Blood components contribute to rise in gerbil brain levels of leukotriene-like immunoreactivity after ischemia and reperfusion.

The mean +/- SEM concentrations of immunoreactive leukotriene C4 and D4 (iLTD4) and prostaglandin D2 (iPGD2) increased from 3.0 +/- 1.2 and 0.71 +/- 0.33 to 16.3 +/- 4.7 and 3.0 +/- 1.14 ng/g forebrain, respectively (p less than 0.05, iLTD4; p less than 0.01, iPGD2), in the forebrains of 12 gerbils after 15 minutes of bilateral common carotid artery occlusion and 15 minutes of reperfusion. Removal of blood from ischemic brain of 11 gerbils by intracardiac perfusion with ice-cold saline for 10 minutes decreased iLTD4 concentrations significantly to 7.0 +/- 0.9 (p less than 0.05) but did not change iPGD2 concentrations. Severe granulocytopenia (4.98 +/- 1.79 to 0.05 +/- 0.03 x 10(3)/mm3, p less than 0.01) in seven gerbils following intraperitoneal injection of 50 mg/kg busulfan was associated with decreased iLTD4 accumulation in the brain to 3.46 +/- 1.36 ng/g forebrain (p less than 0.01). Taken together, our results suggest that blood components (most likely leukocytes) are a source of leukotriene-like immunoreactivity in the ischemic and reperfused brain.

Agranulocytosis↗

Evidence for multiple bone resorption-stimulating factors produced by normal human keratinocytes in culture.

Conditioned medium from cultured normal human foreskin keratinocytes enhanced the release of calcium from neonatal mouse calvaria in organ culture. Unfractionated keratinocyte-conditioned medium (KCM) stimulated bone resorption in a dose-dependent manner, but it did not increase the concentration of prostaglandin E2 (PGE2) in the bone culture medium until a maximal dose of KCM for resorption was used. Furthermore, inhibitors of PGE2 synthesis, indomethacin, ibuprofen, and piroxicam, did not inhibit KCM-induced calcium release. High concentrations of KCM increased cAMP production by calvaria in the presence of isobutylmethylxanthine, but the increase was small compared with that produced by a dose of bovine PTH that caused a similar level of bone resorption. The bone resorption-stimulating activity of KCM was not lost after incubation at 56 C for 60 min, but it was lost after heating at 100 C for 10 min. Fractionation of KCM by gel filtration chromatography revealed two distinct peaks of bone resorption-stimulating activity. One peak, KCMI, caused a significant increase in bone resorption at 2 micrograms protein/ml. KCMI did not increase medium PGE2, and inhibition of PGE2 synthesis in bone had no effect on KCMI-induced bone resorption. KCMI failed to increase cAMP production by human osteosarcoma SaOS-2 cells. Another peak, KCMII, caused a dose-dependent increase in bone resorption, and a significant increase in medium calcium was noted at a 20-fold lower concentration (0.1 microgram protein/ml) than with KCMI. In contrast to KCMI, the increase in bone resorption stimulated by KCMII was accompanied by a parallel increase in the production of PGE2, and inhibition of PGE2 synthesis completely inhibited the bone resorption-stimulating activity of KCMII. KCMII also caused an increase in cAMP production by SaOS-2 cells. We conclude that KCM contains at least two distinct bone resorption-stimulating factors, one of which acts via a PG-mediated mechanism and the other by a PG-independent pathway.

Animals↗

Use of minoxidil to demonstrate that prostacyclin is not the mediator of bone resorption stimulated by growth factors in mouse calvariae.

Growth factors, such as human transforming growth factor-alpha (hTGF alpha) and epidermal growth factor, as well as human tumor necrosis factor (hTNF) stimulate the resorption of bone in neonatal mouse calvariae in organ culture via a prostaglandin (PG)-mediated pathway. In response to such factors mouse calvariae produce substantial quantities of prostaglandin E2 (PGE2) and prostacyclin (PGI2). We have selectively inhibited the production of PGI2, but not PGE2, using the drug minoxidil and have measured the effects on stimulated bone resportion and arachidonic acid metabolism. The increased production of 6-keto-PGF1 alpha (6k-PGF1 alpha), the hydrolytic product of PGI2, stimulated by recombinant hTGF alpha and hTNF as well as murine epidermal growth factor was inhibited by minoxidil. There was no inhibition by minoxidil of PGE2 production. Despite essentially complete inhibition of stimulated 6k-PGF1 alpha formation, there was no inhibition of bone resorption. The possibility was investigated that growth factors and TNF enhanced enzymatic conversion of PGI2 to 6k-PGE1, which stimulates bone resorption in mouse calvariae with a potency about one fourth that of PGE2. Enzymatic conversion of PGI2 to 6k-PGE1 is inhibited by rutin. Rutin did not inhibit bone resorption stimulated by hTGF alpha or hTNF. We conclude, on the basis of these new results and previously published data, that the cyclooxygenase product that acts as the mediator of bone resorption enhanced by growth factors and TNF in mouse calvariae is probably PGE2.

6-Ketoprostaglandin F1 alpha↗