Structure and function of GABA(C) receptors: a comparison of native versus recombinant receptors.

In less than a decade our knowledge of the GABA(C) receptor, a new type of Cl(-)-permeable ionotropic GABA receptor, has greatly increased based on studies of both native and recombinant receptors. Careful comparison of properties of native and recombinant receptors has provided compelling evidence that GABA receptor rho-subunits are the major molecular components of GABA(C) receptors. Three distinct rho-subunits from various species have been cloned and the pattern of their expression in the retina, as well as in various brain regions, has been established. The pharmacological profile of GABA(C) receptors has been refined and more specific drugs have been developed. Molecular determinants that underlie functional properties of the receptors have been assigned to specific amino acid residues in rho-subunits. This information has helped determine the subunit composition of native receptors, as well as the molecular basis underlying subtle variations among GABA(C) receptors in different species. Finally, GABA(C) receptors play a unique functional role in retinal signal processing via three mechanisms: (1) slow activation; (2) segregation from other inhibitory receptors; and (3) contribution to multi-neuronal pathways.

NMDA receptors: from genes to channels.

N-methyl-D-aspartate receptors belong to the family of ionotropic glutamate receptors. NMDA receptors were named after the specific glutamate-like synthetic agonist N-methyl-D-aspartate. In the past decade, an increasing number of functional sites have been discovered and used to refine the operational definition of NMDA receptors. The goal to characterize the molecular substrate underlying the heretofore strictly operationally defined NMDA receptors has come into reach following the cloning of a number of cDNAs coding for NMDA receptor subunits. However, in their review, Nikolaus Sucher and colleagues show that caution should be exercised in comparing the pharmacological properties of recombinant NMDA receptors to those of native neurones. Future work on NMDA receptors will be challenged to reconcile disparate effects obtained with recombinant versus native receptors.

Expression of N-methyl-D-aspartate receptor subunit mRNAs in the rat pheochromocytoma cell line PC12.

The expression of the N-methyl-D-aspartate (NMDA) receptor subunit mRNAs NMDAR1 and NMDAR2A-D was characterized in undifferentiated and nerve growth factor (NGF)-differentiated PC12 cells using Northern blotting, RNase protection assays (RPA) and polymerase chain reaction (PCR). PC12 cells expressed predominately the splice variant NMDAR1-4a and smaller amounts of NMDAR1-1a, NMDAR1-2a and NMDAR1-3a. No splice isoforms containing exon 5 were detected. The NMDAR2C subunit was detected in PC12 cells by Northern blotting and trace amounts of NMDAR2A, B and D were detected by PCR. PC12 cells may be a useful model system for the study of the transcriptional and post-transcriptional regulation of expression of the NMDA receptor subunit genes, including the alternative splicing of NMDAR1 pre-mRNAs.

Developmental and regional expression pattern of a novel NMDA receptor-like subunit (NMDAR-L) in the rodent brain.

A novel NMDA receptor-like (NMDAR-L) cDNA was isolated that contained an open reading frame coding for a predicted polypeptide of 1115 amino acids that shares approximately 27% identity with NMDA receptor subunits. In situ hybridization experiments indicated that NMDAR-L mRNA was expressed in the developing rodent CNS. On postnatal day 1 (P1), NMDAR-L mRNA expression was pronounced in the entorhinal cortex, the subiculum and the thalamus, in layer V of the developing neocortex, in the superior and inferior colliculi, and various regions of the hindbrain, excluding the cerebellum. On P5, NMDAR-L mRNA was expressed in layer V of the neocortex, in the entorhinal cortex, in the subiculum, and in the thalamus. On P14, NMDAR-L mRNA was expressed in layers II-VI of the neocortex, in the entorhinal and piriform cortex, in the subiculum and CA1 field, and in the nucleus of the lateral olfactory tract. In the adult brain, NMDAR-L mRNA was detected predominately in the nucleus of the lateral olfactory tract. Injection of NMDAR-L cRNA into Xenopus oocytes did not lead to the expression of homomeric glutamate-activated channels. However, coinjection of the triple combination of NMDAR-L with NMDAR1 and NMDAR2B cRNAs led to a striking decrease in the current magnitude compared to currents obtained after coexpression of the double combination of NMDAR1 with NMDAR2B. While the function of NMDAR-L remains to be established, its developmental and regional expression pattern suggests that NMDAR-L may influence axonal outgrowth and synaptogenesis during brain development.

Expression of endogenous NMDAR1 transcripts without receptor protein suggests post-transcriptional control in PC12 cells.

Expression of RNA for the NMDAR1 subunit of the N-methyl-D-aspartate receptor was detected by Northern hybridization in both nerve growth factor-differentiated and undifferentiated rat pheochromocytoma (PC12) cells. The NMDA receptor type 1 (NMDAR1) message in PC12 cells was similar in size to that expressed in hippocampal neurons. PC12 cell cDNAs that were amplified by polymerase chain reaction with primers flanking the coding region of NMDAR1 corresponded to the NMDAR1 splice variant NMDA receptor type 1 isoform C (NMDAR1C). Using calcium imaging or patch-clamp recording, no functional NMDA-gated ion channels were found in PC12 cells. A monoclonal antibody against NMDAR1 was developed in order to investigate whether or not NMDAR1 protein was present in PC12 cells. Only trace amounts of NMDAR1 protein were found in native PC12 cells. However, expression of NMDAR1 protein was detected in PC12 cells that were transfected with an expression vector containing an NMDAR1C clone under control of a cytomegalovirus promoter. These findings suggest that the expression of NMDAR1 protein in PC12 cells may be controlled by post-transcriptional mechanisms. The PC12 cell line may serve as a model system for the study of the transcriptional, post-transcriptional, and translational regulation of NMDAR1. Furthermore, the presence of NMDAR1 RNA in a particular cell type may not necessarily indicate expression of NMDAR1 protein.

The accuracy of frozen section in the diagnosis of ovarian neoplasms.

In a retrospective study to determine the accuracy of frozen section diagnoses in ovarian neoplasms, the results of consecutive frozen section diagnoses of 311 ovarian neoplasms from two institutions, New York University Medical Center and State University of New York Medical Center at Brooklyn, from 1980 through 1989 were compared with the final diagnosis results following extensive sampling on permanent sections. The final diagnosis was assumed to be correct for purposes of this study. Ovarian neoplasms were correctly diagnosed on frozen section as either benign or malignant in 292 patients (accuracy of 93.8%). Frozen section diagnoses were incorrect in 11 patients (3.5%). Frozen section diagnosis was deferred in 8 instances (2.6%). The positive predictive value was 100%. The negative predictive value was 95.3%, specificity 100%, and sensitivity 86%. There were no false positives. Of the 11 false negative frozen section diagnoses, 9 (82%) were due to limited sampling for frozen section. We therefore suggest that careful examination with sampling of any suspicious lesions be carried out at the time of surgery for patients with benign frozen section diagnosis, since this may avoid a second staging laparotomy, if the final diagnosis is malignant.

Contractile proteins in Drosophila development.

In summary, we have used a multidisciplinary approach to the analysis of actomyosin-based motility during Drosophila embryogenesis. We have documented the movements of early embryogenesis with modern, video methods. We have characterized the cytoplasmic myosin polypeptide, made specific polyclonal antisera to the molecule, studied its distribution during early embryogenesis, cloned and partially characterized the gene that encodes it, and have recently completed the nucleotide sequence of a nearly full length cDNA that encodes the entire protein-coding region. We have initiated studies on myosin function in living embryos both by direct microinjection of antibodies and through classical genetics. To better understand how myosin function is regulated, we have begun analysis of its light chains. Finally, to investigate the molecular mechanism by which its function is integrated into a labile cytoskeleton, whose architecture is constantly changing, we have also investigated Drosophila spectrins. Together, these studies are designed to shed light on the dynamics of biologic form at the cellular level, with current focus on such complex processes as cytokinesis and morphogenesis.