A review of the in vitro and in vivo neurochemical characterization of the NMDA/PCP/Glycine/Ion channel receptor macrocomplex

Conclusions Current neurochemical studies of the NMDA receptor macromolecular complex are yielding new insights into the interactions of the subunits of this complex and the associated potential clinical benefits of selective modulation of these subnits. Such studies offer the great potential for a new generation of pharmacotherapies for a wide range of CNS disorders, including stroke, a condition for which there is currently no effective pharmacological treatment. However, it is essential to understand that the first generation products in this area may not be optimal pharmacotherapies, such that haracterization of possible receptor subtypes and understanding the molecular biology of the component proteins of the receptor complex will be crucial in the design of the optimal pharmacological modulators of the NMDA receptor complex.Special issue dedicated to Dr. Erminio Costa     

Neutral endopeptidase from nuchal ligament of fetal calves

The nuchal ligament of unborn calves contains a neutral endopeptidase that is biochemically and immunologically similar to the neutral endopeptidase (NEP), or enkephalinase, from human kidney. Enzymatic activity was inhibited more than 90% by phosphoramidon (1 microM). The specific activity in membrane fractions, as determined by hydrolysis of the dansylated substrate, DAPGN, was similar in tissue from fetuses of gestational ages ranging from 100 to 280 days. NEP activity in adult ligament tissue, however, was less than 10% of that in fetal tissue. Fibroblasts dissociated from ligament tissue by collagenase displayed less NEP activity than did preparations of intact ligament, and activity was even lower in cultured cells. By contrast, fibroblasts cultured from fetal calf lungs had NEP activity comparable to that in the ligament tissue. When ligament fibroblasts were cultured on subcellular matrices derived from fetal lung fibroblasts the NEP activity increased relative to those cultured on plastic alone. These studies confirm the presence of neutral endopeptidase (NEP) in the nuchal ligament of the fetal calf. The consistent activity through a range of gestational ages and the influence of the subcellular matrix suggest that this enzyme might be involved in growth of the ligament during fetal life.     

Identification of an amber nonsense mutation in the rosy516 gene by germline transformation of an amber suppressor tRNA gene.

Seven xanthine dehydrogenase and cross-reacting material negative Drosophila melanogaster rosy stocks were screened for amber and ochre nonsense mutations. Amber and ochre nonsense suppressors were created by site-directed mutagenesis starting from a wild-type tRNA(Tyr) gene. The suppressor tRNA genes were subcloned into a pUChsneo transformation vector providing heat-shock controlled neomycin resistance. The seven rosy stocks were germline transformed with amber and ochre tDNA(Tyr), and the G1 generation was screened for Geneticin resistance. Surviving rosy516 flies transformed with the amber suppressor showed an eye colour intermediate between the original ry516 stock and the wild-type, suggesting that ry516 is an amber nonsense mutant. This was confirmed by sequencing the relevant part of the ry516 gene; the analysis revealed a C-to-T transition in a CAG glutamine codon at nucleotide 1522 of the wild-type rosy gene.     

In vitro stimulation and inhibition of steroid hormone release from postovulatory follicles of the tilapia,Oreochromis mossambicus

Summary Postovulatory follicles of the tilapia, Oreochromis mossambicus, were incubated with graded doses of salmon gonadotropin to identify the steroid hormones released by this tissue. In addition, the effects of either cytochalasin B or colchicine on steroid hormone release were studied. After the incubation, the tissue was examined by electron microscopy. Postovulatory follicles released testosterone and estradiol-17B in a dose-dependent manner with gonadotropin. There was no detectable release of progesterone or 17a-OH-progesterone. When stimulated with high doses of gonadotropin, the steroidogenic cells showed an increase in smooth endoplasmic reticulum, Golgi complexes, and lipid droplets. Also, microfilaments became arranged in orderly bundles and were found close to the numerous secretory vesicles and lipid droplets. Upon incubation with gonadotropin and either colchicine or cytochalasin B, the cells still appeared steroidogenic, but the filaments were not organized nor associated with vesicles or lipid droplets. Release of steroid hormone decreased significantly. Also in these tissues, vesicles were no longer numerous in the apical region of the granulosa cells, but were located primarily near smooth endoplasmic reticulum and Golgi complexes. This suggests that disruption of the cytoskeleton results in reduced steroid hormone synthesis or release.     

Synthesis and assembly of the cytochrome b-f complex in higher plants

The cytochrome b-f complex is composed of four polypeptide subunits, three of which, cytochrome f, cytochrome b-563 and subunit IV, are encoded in chloroplast DNA and synthesised within the chloroplast, and the fourth, the Rieske FeS protein, is encoded in nuclear DNA and synthesised in the cytoplasm. The assembly of the cytochrome b-f complex therefore requires the interaction of subunits encoded by different genomes. A key role for the nuclear-encoded Rieske FeS protein in the assembly of the complex is suggested by a study of cytochrome b-f complex mutants. The assembly of individual subunits of the complex may be regulated by the availability of prosthetic groups. The genes for the chloroplast-encoded subunits and cDNA clones for the Rieske FeS protein have been isolated and characterised. Cytochrome f and the Rieske FeS protein are synthesised initially with N-terminal presequences required for their correct assembly within the chloroplast. The deduced amino acid sequences of the four subunits have been used to suggest models for the arrangement of the polypeptides in the thylakoid membrane.     

Genes for tRNAAsp,tRNAPro, tRNATyr and two tRNAsSer in wheat mitochondrial DNA

We have begun a systematic search for potential tRNA genes in wheat mtDNA, and present here the sequences of regions of the wheat mitochondrial genome that encode genes for tRNA^Asp (anticodon GUC), tRNA^Pro (UGG), tRNA^Tyr (GUA), and two tRNAs^Ser (UGA and GCU). These genes are all solitary, not immediately adjacent to other tRNA or known protein coding genes. Each of the encoded tRNAs can assume a secondary structure that conforms to the standard cloverleaf model, and that displays none of the structural aberrations peculiar to some of the corresponding mitochondrial tRNAs from other eukaryotes. The wheat mitochondrial tRNA sequences are, on average, substantially more similar to their eubacterial and chloroplast counterparts than to their homologues in fungal and animal mitochondria. However, an analysis of regions 150 nucleotides upstream and 100 nucleotides downstream of the tRNA coding regions has revealed no obvious conserved sequences that resemble the promoter and terminator motifs that regulate the expression of eubacterial and some chloroplast tRNA genes. When restriction digests of wheat mtDNA are probed with ³²P-labelled wheat mitochondrial tRNAs, <20 hybridizing bands are detected, whether enzymes with 4 bp or 6 bp recognition sites are used. This suggests that the wheat mitochondrial genome, despite its large size, may carry a relatively small number of tRNA genes.     

Peptide injections into the amygdala of conscious rats: effects on blood pressure, heart rate and plasma catecholamines

The central nucleus of the amygdala (Ce) mediates cardiovascular and autonomic changes associated with defense or fear responses. At least 16 different neuropeptides have been identified within nerve terminals within the Ce. The role that these peptides play in the Ce regulation of cardiovascular and autonomic function has been assessed. Neuropeptides were microinjected into the region of the Ce and mean arterial pressure (MAP), heart rate (HR) and plasma catecholamine concentrations were measured. Five of the 16 peptides caused changes of MAP and HR. Thyrotropin releasing factor (TRF) and calcitonin gene-related peptide (CGRP) induced increases of MAP and HR. Angiotensin-II (A-II) and somatostatin-28 (SS-28) injection produced increases of MAP and decreases of HR. Bombesin (Bom) injections into the Ce induced an increase of MAP but did not alter HR. Corticotropin releasing factor (CRF), TRF and CGRP were the only peptides found to increase plasma catecholamine concentrations. These results support the conclusion that the Ce contains several peptides that could be involved in the regulation of cardiovascular and autonomic nervous system function. A role of the amygdala in mediating the observed effects of CRF, TRF, CGRP, A-II, SS-28, and Bom is suggested by these studies.