Uptake, Metabolism, and Release of N-[3H]-Acetylaspartylglutamate by the Avian Retina

N-Acetylaspartylglutamate (NAAG) is a nervous system-specific dipeptide that is released from retinal neurons on depolarization. In the present study, extracellular metabolism, uptake, and release of [3H]NAAG were examined in the chick retina. After in vitro incubation with NAAG radiolabeled in the glutamate moiety, [3H]glutamate and [3H]NAAG increased in retinal cells through time- and temperature-dependent processes, which were reduced in the absence of extracellular sodium. Coincubation of cells with [3H]NAAG and aspartylglutamate or phosphate resulted in the decreased extracellular appearance of [3H]glutamate, produced by hydrolysis of radiolabeled NAAG, and a consequent increased availability of [3H]NAAG for transport into the retinal cells. When this tissue was incubated with radiolabeled NAAG, glutamate, glutamine, or aspartate under similar conditions, only [3H]NAAG served as a significant source for the appearance of intracellular [3H]NAAG. These data support the conclusion that [3H]NAAG can be transported into retinal cells, whereas [3H]glutamate transport is the predominant process after release of this amino acid from NAAG by extracellular peptidase activities. After uptake, [3H]NAAG entered a cellular pool, from which the peptide was secreted under depolarizing conditions and in a calcium-dependent manner.     

Virtual intermediates in photosynthetic electron transfer.

We explore the possibility of virtual transfer in the primary charge separation of photosynthetic bacteria within the context of several types of experimental data. We show that the peak that might be expected in the virtual rate as electric fields vary the intermediate state energy is severely broadened by coupling to high-frequency modes. The Stark absorption kinetics data are thus consistent with virtual transfer in the primary charge separation. High-frequency coupling also makes the temperature dependence weak over a wide range of parameters. We demonstrate that Stark fluorescence anisotropy data, usually taken as evidence of virtual transfer, can in fact be consistent with two-step transfer. We suggest a two-pulse excitation experiment to quantify the contributions from two-step and virtual transfer. We show that virtual absorption into a charge transfer state can make a substantial contribution to the Stark absorption spectrum in a way that is not related to any derivative of the absorption spectrum.     

Unique biphasic band shape of the visible circular dichroism of bacteriorhodopsin in purple membrane: Excitons,multiple transitions or protein heterogeneity?

Over a decade and a half ago, when the first visible membrane suspension circular dichroic (CD) spectrum of the purple membrane (PM) was presented, two mechanisms were proposed to account for the observed biphasic shaped CD band: (a) excitonic interactions among the retinals of the sole protein bacteriorhodopsin (bR) in the crystalline structure of the PM, and (b) combination of CD bands with opposite rotational strengths due to a retinal-apoprotein heterogeneity of the bR molecules or due to two possible close-lying long-wavelength transitions of the retinal of the bR with opposite rotational strengths. Since that time, an impressive body of experimental and theoretical evidence has been accumulated, mostly consistent with an exciton model but many at serious odds with any heterogeneity or multiple transition model. Recently, a number of articles have appeared reporting analyses of new experimental observations which are proposed to cast serious doubts on the viability of the exciton model, and therefore, may revive the heterogeneity or multiple transition model as an explanation for the unique shape of the CD band of the PM. The intent of this article is to demonstrate that if all observations found in literature baring on this question are considered in toto and in a consistent manner, they can be interpreted without exception by excitons, and furthermore, that there is no plausible evidence available to warrant the revival of the heterogeneity or multiple transition model as an explanation for the unique shape of the biphasic CD band of the PM.     

Alpha-1-adrenergic modulation of K and Cl transport in bovine retinal pigment epithelium.

Intracellular microelectrode techniques were used to characterize the electrical responses of the bovine retinal pigment epithelium (RPE)-choroid to epinephrine (EP) and several other catecholamines that are putative paracrine signals between the neural retina and the RPE. Nanomolar amounts of EP or norepinephrine (NEP), added to the apical bath, caused a series of conductance and voltage changes, first at the basolateral or choroid-facing membrane and then at the apical or retina-facing membrane. The relative potency of several adrenergic agonists and antagonists indicates that EP modulation of RPE transport begins with the activation of apical alpha-1-adrenergic receptors. The membrane-permeable calcium (Ca2+) buffer, amyl-BAPTA (1,2-bis(o-aminophenoxy)-ethane-N,N,N',N' tetraacetic acid) inhibited the EP-induced voltage and conductance changes by approximately 50-80%, implicating [Ca2+]i as a second messenger. This conclusion is supported by experiments using the Ca2+ ionophore A23187, which mimics the effects of EP. The basolateral membrane voltage response to EP was blocked by lowering cell Cl, by the presence of DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid) in the basal bath, and by current clamping VB to the Cl equilibrium potential. In the latter experiments the EP-induced conductance changes were unaltered, indicating that EP increases basolateral membrane Cl conductance independent of voltage. The EP-induced change in basolateral Cl conductance was followed by a secondary decrease in apical membrane K conductance (approximately 50%) as measured by delta [K]o-induced diffusion potentials. Decreasing apical K from 5 to 2 mM in the presence of EP mimicked the effect of light on RPE apical and basolateral membrane voltage. These results indicate that EP may be an important paracrine signal that provides exquisite control of RPE physiology.     

Amide isosteres of lexitropsins: synthesis, DNA binding characteristics and sequence selectivity of thioformyldistamycin.

The synthesis and properties of an amide isostere of the antibiotic distamycin, thioformyldistamycin 3 is described. Compound 3 exists predominantly in the E conformation of the thioamide group in freshly prepared DMSO solution but is converted into the Z form, predicted by molecular mechanics to be more stable, on standing for 24 h. The coalescence temperature in DMSO is 110 degrees C by 1H-NMR. The thioformyl moiety of 3 is resistant to both peptidase action and acid treatment. Complementary strand MPE footprinting on a EcoRI/Hind III restriction fragment of pBR322 DNA demonstrated that either E or Z forms of 3 give a single set of footprints very similar to that of the parent antibiotic with strongest protection at TAAG and TATTAT with moderately strong protection at ATTT and AAAA. The strength of binding of 3 and distamycin from delta Tm measurements to either poly.d(AT) or calf thymus DNA is comparable. Molecular modeling predicted a preferred conformation for 3 wherein the C = S bond has a torsional angle of 110 degrees with the pyrrole ring. The energy difference between this conformation and the E form is less than 1 kcal/mole. In contrast the E-form has an energy 17.3 kcal/mole greater than the Z and a value of 26.3 kcal/mole was calculated for the energy barrier between the two isomers.     

Glutathione-related inhibition of prostaglandin metabolism

Placental homogenates contain a heat-stable, dialyzable fraction which specifically inhibits two placental enzymes, both of which possess 15-hydroxyprostaglandin dehydrogenase and 9-ketoprostaglandin reductase activities. The inhibition of the two enzymes is the same. The inhibitor has been resolved into two components by gel filtration on a column of Sephadex LH-20. The component which eluted first has been identified as oxidized glutathione (GSSG), the other as a glutathione-containing material (GSX). Inhibition of the 15-hydroxyprostaglandin dehydrogenase activity is competitive with respect to the prostaglandin substrate (K_i^GSSG = 26 μM, K_i^GSX = 1.4 μM). Inhibition of the 9-ketoprostaglandin reductase activity is also competitive with respect to the prostaglandin substrate (K_i^GSSG = 68 μM). The most effective inhibitor of the 15-hydroxyprostaglandin dehydrogenase is the prostaglandin A₁-glutathione adduct (K_i = 0.27 μM). This compound is not a substrate for oxidation of the 15-hydroxyl group but it is the best substrate found to date for reduction of the 9-keto function.     

Versatile Method for Evaluating a Cell Culture by Various Morphological Techniques

Cellulose acetate is a versatile material for evaluating cells grown under identical conditions by various morphological techniques. This inexpensive material is transparent, easily cut to size and shape, nontoxic to cell cultures, and resistant to most chemicals used in histochemistry and in scanning and transmission electron microscopy. Samples may be obtained during and after the culture process. Cellulose acetate slides can be mounted directly over glass slides for direct observation and are easily peeled off plastic blocks for electron microscopy, leaving the cells behind. Relative disadvantages include its autofluorescence and a tendency to soften in strong acids or pure solutions of organic solvents such as xylene and propylene oxide.