Louis A. Levy

Elevation in cytosolic free calcium concentration early in myocardial ischemia in perfused rat heart.

Changes in cytosolic free calcium concentration during myocardial ischemia were measured by 19F NMR in 5FBAPTA-loaded perfused rat hearts. The hearts were perfused with Krebs-Henseleit buffer containing 5 μM of the acetoxymethyl ester of 5FBAPTA, which was hydrolyzed by cytosolic esterases to achieve cytosolic concentrations of 5FBAPTA of 0.12 to 0.65 mM. Cytosolic free calcium concentrations were calculated as the product of the ratio of peak areas for bound and free 5FBAPTA in the NMR spectra and the dissociation constant (708 nM). The basal cytosolic calcium concentration, measured in potassium or magnesium arrested hearts, was 252 nM, and the time-average calcium concentration in beating hearts was 630 nM. Following the onset of total ischemia, there was no immediate substantial change in cytosolic calcium despite a rapid decline in creatine phosphate and ATP and a marked increase in inorganic phosphate as monitored by 31P NMR, but by 10 minutes, there was a substantial increase in free calcium concentration. The ratio of peak areas of bound and free 5FBAPTA returned to the preischemic value during reperfusion, and there was no detectable loss of 5FBAPTA from the heart. Creatine phosphate was also restored to its preischemic level during reperfusion. These results indicate that cytosolic free calcium increases during ischemia and is not immediately associated with lethal injury. This increase in cytosolic calcium may activate degradative enzymes that eventually could compromise myocyte viability.

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.

Differentiation and characterization of isomeric polychlorinated biphenyls by gas-liquid chromatography coupled with electron impact and chemical ionization mass spectrometry.

Abstract This report describes the utilization of gas-liquid chromatography coupled with chemical ionization and electron impact mass spectrometry for the characterization and isomeric differentiation of polychlorinated biphenyls (PCBs) from numerous sources. By a comparison of model compounds with very complex mixtures, one is able to gain significant information about the o,o′-chlorine interaction of PCBs as monitored by mass spectrometry. The abudance of the specific (M − Cl)+ ion assisted greatly in the isomeric differentiation of dichloro-, hexachloro-, and heptachlorobiphenyls. Furthermore, by the indepth characterization by mass spectrometry of the Ullman reaction products formed in the preparation of 14C-labeled PCB′s, one may conclude that a very complex mixture of dichloro- and trichlorobiphenyls is formed from a specific pure isomeric iodomonochlorobenzene during the Ullman reaction. As revealed by the use of gas-liquid chromatography coupled with electron impact mass spectrometry, care must be exercised in choosing the type of organic synthetic reactions for the preparations of polychlorinated biphenyls which will be used for biological testing in order to minimize isomeric contamination.

Development of fluorinated, NMR-active spin traps for studies of free radical chemistry

Abstract Five fluorinated analogs of the spin trap phenyl-t-butyl nitrone (PBN) have been synthesized and evaluated for use as NMR-active traps. The introduction of the fluorine substituent allows selective observation of chemical reactions involving the spin traps. Although the paramagnetic adducts themselves are not directly observable by this approach as a consequence of extensive broadening, the reduced forms (hydroxylamines) can be readily observed. NMR studies of the trapping of the phenyl radical produced from the thermal decomposition of phenylazotriphenyl methane have been carried out. The observation of fluorinated benzaldehydes in these studies reflects the formation and subsequent degradation of oxygen-centered radicals under some conditions. Relative trapping efficiencies for the phenyl radical in the series 2-F, 4-F, 2,6-difluoro, 2-CF 3 , and 4-CF 3 substituted PBN analogs have been determined based on an analysis of the 19 F NMR resonance intensities of the reduced phenyl radical adducts. The relatively large proton hyperfine coupling constants observed for 2,6-difluoro and 2-CF 3 PBN analogs allow direct observation by ESR of adduct formation in solutions containing both PBN and either of these analogs. The introduction of fluorine substituents into the trap has only a small effect on trapping efficiency.

The Synthesis Of 2,3,6,7-Tetrasubstituted Naphthalenes: 2,3,6,7-Tetrachloronaphthalene

Abstract The synthesis of 2,3,6,7-tetrasubstituted naphthalenes and naphthalene derivatives via the cycloaddition reaction of 4,5 dichloro-o-quinodimethane has been accomplished.

Automerizations in benzenoid hydrocarbons. New mechanistic insights from the thermal rearrangement of benz[a]anthracene-5-13C.

Abstract Scrambling of the carbon atoms in benz[a]anthracene between positions 5 and 6 has been shown to occur at high temperatures; a mechanism involving thermal generation of a carbene is proposed.

Synthesis of 1-Azabenzanthracene: Epimerization with the Use of 1,3-Dihydrobenzo(c)thiophene -S,S Dioxide as a Source of ortho-Quinodimethane

Abstract The cycloaddition of o-quinodimethane, from 1,3-dihydrobenzo(c)-S,S dioxide, to 5,6-dihydroquinoline yields a mixture of adducts isomeric at the newly created ring junction. The cycloadducts can be dehydrogenated to the fully aromatic 1-azabenzanthracene.

Synthesis of methyl substituted benzanthracenes and benzanthracene derivatives

Abstract Diels-Alder reaction of 1,3-dimethylisobenzofuran with cinnamic esters allows for the synthesis of a variety of 7,12 dimethylbenzanthracene derivatives. The sterically congested 1,7,12 trimethylbanzanthracene was prepared.

Synthesis and Characterization of Two Improved NMR Indicators for Cytosolic Ca2+: 3FBAPTA and 35FBAPTA

Fluorinated derivatives of the calcium‐selective chelator BAPTA [1,2‐bis(2‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid] are popular NMR probes for cytosolic calcium ion concentration. Two such derivatives were synthesized and evaluated, 3FBAPTA [1,2‐bis(2‐amino‐3‐fluorophenoxy)ethane‐ N,N,N′,N′‐tetraacetic acid] and 35FBAPTA [1,2‐bis(2‐amino‐3,5‐difluorophenoxy)ethane‐ N,N,N′,N′‐tetraacetic acid]. The dissociation constant ratios obtained at 37°C were KD3F/KD5F = 0.60 and KD35F/KD5F = 0.88; similar values were obtained at 25°C. Hence 3FBAPTA binds calcium ions more tightly than 5FBAPTA [1,2‐bis(2‐amino‐5‐fluorophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid, the currently most frequently utilized indicator], while the doubly substituted derivative exhibits intermediate affinity. The dissociation rate constants for the calcium complexes measured at 37°C exhibit a similar trend, with k‐15>k‐135>k‐13. Both of the additional derivatives exhibit slow exchange kinetics, and should be superior fluorine NMR probes owing to the decreased T1 values and, in the case of 35FBAPTA, the presence of two calcium‐sensitive fluorine nuclei which are measured simultaneously.