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Biomedical subjects

L A Levy

Publications and source records attributed to L A Levy.

At least 37 records · Page 2Linked to original sources
PubMed1989

Diethylstilbestrol metabolites and analogs. Stereochemical probes for the estrogen receptor binding site.

Indenestrol A (IA) and indenestrol B (IB) are analogs and metabolites of diethylstilbestrol (DES). These compounds have high binding affinity with the estrogen receptor (ER) but possess weak uterotropic activity. Due to their chemical structures, IA and IB exist as mixtures of enantiomers. We investigated whether the poor biological activity of these compounds was due to differential activity of the enantiomers. We also utilized these compounds as probes to determine the extent of stereochemical sensitivity in the ER ligand binding site. The IA and IB enantiomers were separated to greater than 98% purity using a chiral high pressure liquid chromatography column. Their enantiomeric nature was confirmed by mass spectrometry and NMR. The purified IA enantiomer peak 1 was derivatized with 4-bromobenzoyl chloride. The resulting di(4-bronobenzoate) IA was analyzed by x-ray crystallography and the absolute enantiomeric conformation assigned is C(3)-R. The IA enantiomers designated IA-R and A-S were assayed by competitive binding to cytosolic ER. The competitive binding index was estradiol, 100; DES, 286; IA-Rac (racemic mixture of IA), 143; IA-R, 3; and IA-S, 285; the index showed that ER demonstrates a stereochemical chiral preference. The IB enantiomers did not show a binding preference: IB, 145; IB-1, 100; and IB-2, 143. The differences in the IA enantiomer binding were shown to be due to competitive interactions by Lineweaver-Burk analysis of saturation binding of estradiol to ER in the presence of 1-, 5-, and 10-fold molar excess of competitor. Differences in binding affinity of the enantiomers could be partially explained by differences in the association rate constant (k+1) determined by association rate inhibition studies in which IA-S was 15 times more active than IA-R. Nuclear estrogen receptor levels were measured 1 h after in vivo treatment with doses of 5-20 micrograms/kg. The IA-Rac produced only 60% of the levels is compared with DES. Nuclear ER levels were checked every 30 min up to 2 h with no apparent difference, indicating that the low early levels were not due to a delayed estrogen receptor retention. When the enantiomers were tested individually only a dose of 10 micrograms/kg IA-S translocated ER to a level comparable to DES, while IA-R showed low levels at several doses. These results suggest that the poor biological activity of IA may be related to the differential ER interaction of its enantiomers.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1989

Monitoring cytosolic free magnesium in cultured chicken heart cells by use of the fluorescent indicator Furaptra.

Cytosolic free magnesium concentration [Mg2+]i and its regulation were studied in cultured embryonic chicken heart cells by use of the fluorescent indicator 2-[2-(5-carboxy)oxazole]-5-hydroxy-6-aminobenzofuran-N,N,O-triacet ic acid (Furaptra). The intracellular location of Furaptra was confirmed by its complete release from cells upon addition of saponin. The basal [Mg2+]i, which averaged 0.48 +/- 0.03 mM (n = 31), increased 3-fold on perfusion with sodium-free solution. This increase could not simply be attributed to intracellular sodium-extracellular magnesium exchange because a similar increase in [Mg2+]i occurred with magnesium-free, sodium-free perfusion. Furthermore, the increase in [Mg2+]i was largely attenuated when calcium was removed from the sodium-free perfusate. Thus, a substantial part of the increase in [Mg2+]i that occurs upon sodium-free perfusion is dependent on an increase in cytosolic free calcium (intracellular sodium-extracellular calcium exchange). The data suggest that [Mg2+]i is altered by calcium, most likely due to a competition for intracellular binding sites.

Animals↗
PubMed1989

A fluorescent indicator for measuring cytosolic free magnesium.

The previously developed chelator O-aminophenol-N,N,O-triacetic acid (APTRA) (L. A. Levy, E. Murphy, B. Raju, and R. E. London. Biochemistry 27: 4041-4048, 1988) has been modified to yield a fluorescent analogue which can be utilized as an intracellular probe for ionized Mg2+. The fluorescent analogue, FURAPTRA, with a magnesium dissociation constant of 1.5 mM, is structurally analogous to the calcium chelator fura-2 and exhibits a similar excitation shift on magnesium complexation. Hence, data on the intracellular Mg2+ concentration can be obtained using an analogous ratio method. The acetoxymethyl form of the chelator is readily loaded into cells and has been used to determine a cytosolic free Mg2+ concentration of 0.59 mM for isolated rat hepatocytes. As a consequence of the relatively high levels of cytosolic Mg2+, the problem of ion buffering is much less severe than for the analogous calcium indicators.

Animals↗
PubMed1989

Cytosolic free magnesium concentration in cultured chick heart cells.

Cytosolic free magnesium (Mgi) was measured in embryonic chick heart cells loaded with one of two newly developed 19F nuclear magnetic reasonance (NMR)-sensitive magnesium chelators, 4-methyl,5-fluoro-2-aminophenol-N,N,O-triacetate (MF-APTRA) and 5-fluoro-2-aminophenol-N,N,O-triacetate (5F-APTRA). The cells, embedded in strands of collagen, were superfused at a rate that allowed for solution changes in 2 min. In this preparation 19F- and 31P-NMR spectra were stable for at least 3.5 h. Because Na-coupled Mg countertransport may be a possible mechanism of Mg transport, in some experiments extracellular Na was reduced to 1 mM (choline substituted). This manipulation caused a 2.5-fold increase in Mgi from the basal level of 0.56 mM. A significant proportion of this increase in Mgi could be secondary to an increase in Cai that occurs with low extracellular Na (Nao) perfusion (Nai-Cao exchange). Perfusing cells with nominally Ca-free, 1 mM Na salt solution substantially attenuated the increase in Mgi that occurred with Ca present (1.25 mM) in the low Na (1 mM) solution. Furthermore, perfusion with 1 mM Na, Mg-free salt solution caused a 1.5-fold increase in Mgi, which cannot be attributable to Nai-Mgo exchange. Therefore attempts to describe the regulation of Mgi in heart cells must differentiate between the effects of Nai-Mgo exchange and competition for binding sites that are secondary to stimulation of ion gradient-coupled mechanisms.

Aminophenols↗
PubMed1989

Smoking and peripheral vascular disease. Epidemiology and podiatric perspective.

Virtually no attention has been given in public education efforts to the potentially devastating effects of smoking on the lower extremities. An analysis of the epidemiologic studies that have been done by the Office of the Surgeon General and other clinical investigators has been presented, leading to the conclusion that cigarette smoking is the single most powerful risk factor for peripheral vascular disease. In the care of patients with peripheral vascular disease, the podiatric physician has an opportunity to be a participant in efforts to solve one of the nation's major public health problems.

Arterial Occlusive Diseases↗
PubMed1988

Measurement of cytosolic free magnesium ion concentration by 19F NMR.

Fluorinated derivatives of the chelator o-aminophenol-N,N,O-triacetic acid (APTRA) have been developed, synthesized, and analyzed for use as 19F NMR indicators of free cytosolic magnesium concentration. Magnesium dissociation constants for the 4-fluoro, 5-fluoro, and 4-methyl-5-fluoro species were determined to be 3.1, 0.9, and 0.6 mM, respectively, on the basis of UV absorption measurements at 37 degrees C in 115 mM KCl and 20 mM NaCl, pH 7.1, buffered with 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-tris-(hydroxymethyl)aminomethane. The corresponding pK values, which reflect protonation of the nitrogen atom, were determined by 19F NMR to be 4.15, 5.45, and 5.55, respectively, so that the chelators are insensitive to pH variations near the normal physiological range. The dissociation constants of these chelators for calcium ions are lower than those for magnesium but roughly 2-3 orders of magnitude above typical basal cytosolic free calcium levels, so that calcium ions will not interfere with the determinations of magnesium levels. 19F NMR studies carried out at 339.7 MHz indicate that magnesium ions are in slow exchange with the 5-fluoro and 4-methyl-5-fluoro APTRA derivatives and in fast exchange with the 4-fluoro APTRA derivative. In contrast, calcium ions were found to be in intermediate to fast exchange with all chelators. The apparent anomaly of higher thermodynamic stability of the APTRA complexes for calcium relative to magnesium but lower kinetic stability (higher k-1 values) for the calcium complexes reflects the very different association rates for the two ions. Thus, the magnesium association rates are 3 orders of magnitude slower than those for calcium ions.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium↗
PubMed1987

Estrogen receptor stereochemistry: receptor binding and hormonal responses.

Estrogen stimulation of the uterus elicits a spectrum of biochemical responses which are customarily linked together. DES and certain structural analogs, indenestrol A (IA), indenestrol B (IB), indanestrol (I), and pseudo DES (PD), were used as probes to segregate various genomic responses previously considered interrelated, most notably the events of specific protein synthesis, DNA synthesis, and mitosis. These compounds have poor uterotrophic activity; except for I, they interact specifically with mouse uterine estrogen receptors (ER) with high affinity. All translocate stoichiometrically similar amounts of ER complex to the nucleus. IA and IB possess a single chiral carbon atom and exist as a mixture of enantiomers (ENT). We investigated whether the poor biological activity of IA could be explained by differential activity of the enantiomers. The IA ENT were separated to greater than 98% purity using a chirally active HPLC column. Competitive binding assays to cytosolic ER demonstrated a stereochemical chiral preference. This preference was also evident from nuclear ER translocation experiments. IB was as active as DES to induce mouse uterine glucose 6-phosphate dehydrogenase (G-6-PD), while the other compounds had weak activity. Induction of cytosolic progesterone receptor (PR) was stimulated by all the DES compounds. Ornithine decarboxylase (ODC) was stimulated 600% by DES and 180% by IB; the other compounds had no significant activity. Uterine DNA synthesis was increased by DES and IB. Thymidine autoradiography indicated nuclear labeling was occurring primarily in luminal epithelium. Treatment with PD increased uterine cell height but not cell numbers, suggesting the two responses are not necessarily interdependent as previously thought and may require two separate receptor interactions. Such a probe should be useful in studying the individual events involved in estrogen-induced uterine growth. These data also indicate that induction of ER, PR and G-6-PD are not coupled. Therefore, stimulation of a certain uterine response may depend on the structure of the particular ligand receptor complex formed, and its interaction may be regulated by specificity at the genomic acceptor site.

Animals↗
PubMed1987

Synthesis and characterization of 19F NMR chelators for measurement of cytosolic free Ca.

Fluorine 19 nuclear magnetic resonance (NMR) studies of intracellular fluorinated calcium chelators provide a useful strategy for the determination of cytosolic free calcium levels in cells and perfused organs. However, the fluorinated chelator with the highest affinity for calcium ions which has been described to date. 1,2-bis-(2-amino-5-fluorophenoxy)ethane-N,N,N',N'-tetraacetic acid (5FBAPTA), exhibits a dissociation constant (Kd) value 5- to 10-fold greater than the intracellular calcium concentration levels in most cell types, thus limiting the ability of fluorine NMR to report these concentrations reliably. We have consequently designed and synthesized several fluorinated calcium chelators with higher affinity for calcium. The best of these, 2-(2-amino-4-methyl-5-fluorophenoxy)-methyl-8 aminoquinidine-N,N,N',N'-tetraacetic acid (quinMF), has a Kd value approximately 10 times lower than that of 5FBAPTA. Several of the newly synthesized indicators have different chemical shifts for the calcium complexed and uncomplexed chelators to allow the simultaneous use of two indicators. In addition to providing information about the level of cytosolic free calcium, chelators containing a quinoline ring exhibit considerable sensitivity to magnesium levels and hence have potential application for the determination of cytosolic-magnesium concentrations. Application of these chelators is illustrated by determination of the cytosolic-free calcium level in erythrocytes. Use of quinMF, the chelator with the lowest Kd value, gives a calcium value of 25-30 nM.

Calcium↗
PubMed1985

Relationship of the gastrointestinal system to podiatric diagnosis and therapeutics.

Relationships to the gastrointestinal system can be found in the diagnosis and treatment of problems affecting the pedal extremity. This is due to the fact that parts of the gastrointestinal tract are derived from the same germ layer as portions of the foot. An awareness of these relationships can be useful in determining the etiology, establishing a diagnosis, and forming a plan for management.

Adult↗
PubMed1985

Foot and ankle ulcers associated with hematologic disorders.

Ulcers of the foot and ankle secondary to blood dyscrasias may become more common as a result of modern medicine's ability to keep patients alive long enough to become victims of chronic disease. Also, many pharmaceutic agents have, among their side effects, the ability to produce hematologic complications. The physician should consider that hematologic disorders may be among the etiologic agents responsible for foot and ankle ulcers.

Anemia, Sickle Cell↗
PubMed1983

Parkinsonism: improvement by electroconvulsive therapy.

Not infrequently, patients with parkinsonism either do not respond to antiparkinsonian medications or cannot tolerate those medications because of side effects. We report a patient who, having responded poorly to every medical regimen for parkinsonism, showed marked improvement in gait and balance after electroconvulsive therapy (ECT) for treatment of concomitant severe depression. ECT has been found to increase the sensitivity of postsynaptic dopaminergic receptors and noradrenergic receptors and to facilitate, for a considerable period of time, the neurotransmission along dopaminergic and noradrenergic pathways in the brain. Thus, ECT may represent another important therapeutic modality for patients with parkinsonism.

Aged↗