Maintenance of recombinant type A gamma-aminobutyric acid receptor function: role of protein tyrosine phosphorylation and calcineurin

In the present study, rundown of gamma-aminobutyric acid (GABA)-activated Cl- channels was studied in recombinant GABAA receptors stably expressed in human embryonic kidney cells (HEK 293), with conventional whole-cell and amphotericin B-perforated patch recording. When [ATP]i was lowered to 1 mM and resting [Ca++]i was buffered to a relatively high level, the response of alpha 3 beta 2 gamma 2 GABAA receptors to relatively low [GABA] (up to 50 microM) did not show rundown in the whole-cell configuration. However, high [GABA] (greater than 200 microM) induced significant rundown, which was observed by decreases in both the maximum GABA-induced current and GABA EC50. Rundown was prevented completely with a solution containing 4 mM Mg(++)-ATP and low resting [Ca++]i, or during perforated patch recording. The magnitude of rundown was comparable in alpha 1 beta 2 gamma 2 and beta 2 gamma 2 receptors. Neither stimulation nor inhibition of protein kinase A or protein kinase C had a significant effect on rundown. However, sodium metavanadate, an inhibitor of protein tyrosine phosphatase, significantly reduced rundown. In addition, inhibition of protein tyrosine kinase activity by either genistein or lavendustin A induced rundown of the GABA response. Inhibition of the Ca++/calmodulin-dependent phosphatase calcineurin with fenvalerate also prevented rundown of the response to GABA. Our results demonstrate that rundown of GABAA receptor function is concentration-dependent, due to depletion of ATP and/or unbuffered [Ca++]i, and does not depend on the presence or subtype of the alpha subunit. We propose that protein phosphorylation at a tyrosine kinase-dependent site, and a distinct unidentified site, which is dephosphorylated by calcineurin, maintains the function of GABAA receptors.     

Breast disease: dynamic spiral MR imaging

The cochleo- and tonotopic organization of the second auditory area (AII) was investigated in cats anaesthetized with pentobarbital using a combination of macro- and microelectrode recording technique. The results obtained following electrical stimulation of the neural fibres innervating different regions of the organ of Corti indicate the existence of two complete representations of the cochlea in area AII: one in the dorsocaudal portion, the other in its ventrorostral portion. These two cortical representations of the cochlea differ in size and spatial orientation. The dorsocaudal projection area extends over a distance of 2.6-3.2 mm from the basal to the apical focus and is arc-shaped. The spatial orientation of cochlea representation within the dorsocaudal region of AII is similar to that described in AI, in that stimulation of the cochlea base results in maximal responses in the more rostral portion of AII and stimulation of the apex evokes cortical responses more caudally. The ventrorostral region within AII is smaller (1.4-2.5 mm length), and has the opposite cochleotopic orientation (base and apex stimulation represented caudally and rostrally, respectively). In both AII zones, there was a proportionally greater cortical representation of basilar membrane than of middle and apical portions. Although two distinct zones with the overall cochleotopic pattern described above were noted in all cats, their precise size and location considerably varied in different animals. Using microelectrode recordings, a cortical tonotopic organization can be observed that was consistent with and expanded on the earlier cochleotopic data. Within the dorsocaudal region of AII, neurons with higher best frequency responses were located in more rostral regions, while those with lower best frequencies were located caudally. An orderly progression of best frequency responses was noted as serial recordings carried out along the full extent of the representation. Neurons within the ventrorostral region of AII also displayed an orderly progression of best frequencies, but in the opposite direction, with higher best frequencies noted more caudally and lower best frequencies more rostrally.     

Quantitative imaging of TATA-binding protein in living yeast cells

We describe the quantitative monitoring of TATA-binding protein (TBP) localization and expression in living Saccharomyces cerevisiae cells. We replaced the endogenous TBP with a green fluorescent protein (GFP) x TBP fusion, which was imaged quantitatively by laser scanning confocal microscopy (LSCM). When GFP x TBP expression was altered by using various promoters, the levels measured by LSCM correlated well with the levels determined by immunoblot of whole cell extract protein. These results show that GFP x TBP imaging not only offers a method of measurement equivalent to a more conventional technique but also provides real-time quantitation in living cells and subcellular localization information. Time-lapse confocal imaging of GFP x TBP in mitotic yeast cells revealed that it remains localized to the nucleus and displays an asymmetric distribution (1:0.7) between mother and daughter cells. Based on this and data from a mutant which underexpresses GFP x TBP, we suggest that intracellular levels of TBP are near rate-limiting for growth and viability.     

Analytical performance of the Tandem-R free PSA immunoassay measuring free prostate-specific antigen

The analytical performance of the Tandem-R free PSA assay available from Hybritech Inc. was evaluated. Comparison of recoveries of purified free (unbound) prostate-specific antigen (PSA) diluted in female serum in the Tandem-R free PSA assay and the Tandem-R (total) PSA assay demonstrated a link in calibration between the assays and an accurate determination of percent free PSA. The cross-reactivity of the assay to purified PSA-alpha 1-antichymotrypsin was determined to be < 1%. The minimum-detectable concentration was < 0.05 microgram/L. The within-run and between-day CVs were < or = 5% for samples with > 0.3 microgram/L free PSA. Dilution and recovery showed no significant deviations from linearity across the assay range. The assay was insensitive to interference from blood components. The Tandem-R free PSA kit was shown to be an accurate, precise, and reliable assay for the measurement of free PSA.     

A method for functional magnetic resonance imaging of olfaction

A method for generating olfactory stimuli for humans within a functional magnetic resonance imaging (fMRI) experimental design is described. The system incorporates a nasal-mask in which the change from odorant to no-odorant conditions occurs in less than 500 ms and is not accompanied by visual, auditory, tactile, or thermal cues. The mask provides an ordorant-free environment following prolonged ordorant presence. Specific imaging parameters that are conducive to the study of the human olfactory system are described. In a pilot study performed using these methods, the specific patterns of activation observed converged with published experimental and clinical findings.     

Antigenic structure of soluble herpes simplex virus (HSV) glycoprotein D correlates with inhibition of HSV infection

Soluble forms of herpes simplex virus (HSV) glycoprotein D (gD) block viral penetration. Likewise, most HSV strains are sensitive to gD-mediated interference by cells expressing gD. The mechanism of both forms of gD-mediated inhibition is thought to be at the receptor level. We analyzed the ability of different forms of soluble, truncated gD (gDt) to inhibit infection by different strains of HSV-1 and HSV-2. Strains that were resistant to gD-mediated interference were also resistant to inhibition by gDt, thereby suggesting a link between these two phenomena. Virion gD was the major viral determinant for resistance to inhibition by gDt. An insertion-deletion mutant, gD-1(delta 290-299t), had an enhanced inhibitory activity against most strains tested. The structure and function of gDt proteins derived from the inhibition-resistant viruses rid1 and ANG were analyzed. gD-1(ridlt) and gD-1(ANGt) had a potent inhibitory effect on plaque formation by wild-type strains of HSV but, surprisingly, little or no effect on their parental strains. As measured by quantitative enzyme-linked immunosorbent assay with a diverse panel of monoclonal antibodies, the antigenic structures of gD-1(rid1t) and gD-1(ANGt) were divergent from that of the wild type yet were similar to each other and to that of gD-1 (delta 290-299t). Thus, three different forms of gD have common antigenic changes that correlate with enhanced inhibitory activity against HSV. We conclude that inhibition of HSV infectivity by soluble gD is influenced by the antigenic conformation of the blocking gDt as well as the form of gD in the target virus.