Effects of GABA on acutely isolated neurons from the gustatory zone of the rat nucleus of the solitary tract

Responses of acutely isolated neurons from the rostral nucleus of the solitary tract (rNST) to GABA receptor agonists and antagonists were investigated using whole-cell recording in current clamp mode. The isolated neurons retain their morphology and can be divided into multipolar, elongate and ovoid cell types. Most rNST neurons (97%), including all three cell types, respond to GABA with membrane hyperpolarization and a reduction in input resistance. The GABA(A) receptor agonist muscimol reduces neuronal input resistance in a concentration-dependent manner, whereas the GABA(B) receptor agonist baclofen had no effect on any of the neurons tested. The GABA and muscimol reversal potentials were both found to be -75 mV Both the GABA competitive antagonist picrotoxin and the GABA(A) receptor antagonist bicuculline block the effect of GABA in a concentration-dependent manner. These results suggest that GABA activates all neurons in the rNST and that inhibitory synaptic activity is important in brainstem processing of gustatory and somatosensory information.      

Replacement of receptor cells in the hamster vomeronasal epithelium after nerve transection

Chemoreceptor cells in the vomeronasal and olfactory epithelium are replaced following experimentally induced degeneration. This study analyzes quantitatively the time course and degree of vomeronasal receptor cell replacement. Unilateral transection of the vomeronasal nerves in adult hamster was used to induce a retrograde degeneration of receptor cells in the vomeronasal organ. Histological measurement of both number of receptor cells and epithelial thickness were made for recovery times from 0 to 60 days. After nerve transection, there was a gradual degeneration of receptor cells, the number decreasing to 50% of control by day 2 and 16% by day 6. During days 7-15 maximum receptor cell replacement was observed. Cell number increased rapidly and reached a peak on day 15. At recovery times of 40-60 days, cell number returned to the control level. Epithelial thickness, however, decreased to 60-70% during the degeneration period (days 4-6) and did not return to control levels. After 40-60 days epithelial thickness remained at 70% of control. These results demonstrate that vomeronasal receptor cells are replaced following degeneration, but epithelial thickness does not return to control levels. These findings suggest that the number of replacement cells is not limited by the reduced thickness of the epithelium, and that recovery mechanisms may function to restore an optimum number of receptor cells.      

Folding and Hydrodynamics of a DNA i-Motif from the c-MYC Promoter Determined by Fluorescent Cytidine Analogs

The four-stranded i-motif (iM) conformation of cytosine-rich DNA has importance to a wide variety of biochemical systems that range from their use in nanomaterials to potential roles in oncogene regulation. The iM structure is formed at slightly acidic pH, where hemiprotonation of cytosine results in a stable C-C⁺ basepair. Here, we performed fundamental studies to examine iM formation from a C-rich strand from the promoter of the human c-MYC gene. We used a number of biophysical techniques to characterize both the hydrodynamic properties and folding kinetics of a folded iM. Our hydrodynamic studies using fluorescence anisotropy decay and analytical ultracentrifugation show that the iM structure has a compact size in solution and displays the rigidity of a double strand. By studying the rates of circular dichroism spectral changes and quenching of fluorescent cytidine analogs, we also established a mechanism for the folding of a random coil oligo into the iM. In the course of determining this folding pathway, we established that the fluorescent dC analogs tC° and PdC can be used to monitor individual residues of an iM structure and to determine the pK_a of an iM. We established that the C-C⁺ hydrogen bonding of certain bases initiates the folding of the iM structure. We also showed that substitutions in the loop regions of iMs give a distinctly different kinetic signature during folding compared with bases that are intercalated. Our data reveal that the iM passes through a distinct intermediate form between the unfolded and folded forms. Taken together, our results lay the foundation for using fluorescent dC analogs to follow structural changes during iM formation. Our technique may also be useful for examining folding and structural changes in more complex iMs.     

Interactions of anilinoacridines with nucleic acids: effects of substituent modifications on DNA-binding properties

Spectroscopic methods are used to probe the interactions of several anilinoacridine analogues with calf thymus DNA over a wide range of temperatures and sodium chloride concentrations. The structurally similar compounds m-AMSA, AMSA (both active as antitumor agents), and o-AMSA (inactive as an antitumor agent) have been widely studied in their abilities to bind DNA in an intercalative manner. Recent studies from this laboratory reveal distinct differences in the thermodynamic binding mechanisms between m-AMSA and o-AMSA (Wadkins & Graves, 1989), with the m-AMSA-DNA interaction being an enthalpy-driven process while the binding of o-AMSA to DNA is characterized by more positive entropy values. To further examine the physical chemical properties associated with these compounds and their correlation with antitumor activities, an in-depth investigation into the thermodynamic parameters of these compounds and structurally related anilinoacridine analogues was performed. These studies demonstrate that substituent type and position on the aniline ring of the anilinoacridines greatly influences both the affinities of these drugs in binding to DNA and dictates whether the DNA binding is an enthalpy- or entropy-driven process. The differences in thermodynamic mechanisms of binding between the two isomers along with molecular modeling studies reveal the electronic and/or steric factors resulting from the positioning of the methoxy substituent group on the anilino ring directs the DNA-binding properties through orientation of the methanesulfonamido group at the 1' position of the aniline ring. The orientation of this substituent group may result in favorable contacts through hydrogen bonding with neighboring base pairs and ultimately influence the biological effectiveness as an antitumor agent.