Unusual effects of benzodiazepines and cyclodiene insecticides on an expressed invertebrate GABAAreceptor

We have previously reported [(1991) EMBO J. 10, 3239–3245] the sequence of an invertebrate γ‐aminobutyric acid (GABA) type A (GABAA) receptor polypeptide which forms homo‐oligomeric GABA‐gated, bicuculline‐sensitive, chloride‐ion channels upon heterologous expression. We now demonstrate that the benzodiazepines Ro5‐4864 (4′‐chlorodiazepam) and diazepam, that are active at mammalian peripheral benzodiazepine sites, and not those benzodiazepines specific for central sites, directly activate the homo‐oligomeric receptor and evoke larger maximal responses than those elicited by GABA. In addition, members of the cyclodiene class of insecticides block the channel of the receptor in a manner indistinguishable from that of picrotoxin

Preferential activation of HIF-2? adaptive signalling in neuronal-like cells in response to acute hypoxia

Stroke causes severe neuronal damage as disrupted cerebral blood flow starves neurons of oxygen and glucose. The hypoxia inducible factors (HIF-1? and HIF-2?) orchestrate oxygen homeostasis and regulate specific aspects of hypoxic adaptation. Here we show the importance of HIF-2? dependant signalling in neuronal adaptation to hypoxic insult. PC12 and NT2 cells were differentiated into neuronal-like cells using NGF and retinoic acid, and exposed to acute hypoxia (1% O2). Gene and protein expression was analysed by qPCR and immunoblotting and the neuronal-like phenotype was examined. PC12 and NT2 differentiation promoted neurite extension and expression of neuronal markers, NSE and KCC2. Induction of HIF-1? mRNA or protein was not detected in hypoxic neuronal-like cells, however marked induction of HIF-2? mRNA and protein expression was observed. Induction of HIF-1? target genes was also not detected in response to acute hypoxia, however significant induction of HIF-2? transcriptional targets was clearly evident. Furthermore, hypoxic insult dramatically reduced both neurite number and length, and attenuated expression of neuronal markers, NSE and KCC2. This correlated with an increase in expression of the neural progenitor and stem cell-like markers, CD44 and vimentin, suggesting HIF-2? molecular mechanisms could potentially promote regression of neuronal-like cells to a stem-like state and trigger neuronal recovery following ischaemic insult. Our findings suggest the HIF-2? pathway predominates over HIF-1? signalling in neuronal-like cells following acute hypoxia

Multiple genes for neuropeptides and their receptors: co-evolution and physiology

It is now well established that neuropeptide receptors, which are present throughout the {CNS} and in peripheral tissues, frequently exist in a variety of different forms (called subtypes), each of which is encoded by a distinct gene. With the recent identification of new neuropeptide genes, it has become clear that families of neuropeptides also occur, which raises the possibility that specific peptide ligands activate particular receptor subtypes preferentially. This article reviews some of the recent advances in the neuropeptide field and provides evidence in support of three ideas: (1) that different receptor subtypes for a given ligand can be distinguished physiologically; (2) that neuropeptide genes probably arose before the corresponding receptor genes; and (3) that, despite the current wealth of information on neuropeptides and neuropeptide receptors, several new members are likely to be discovered before the beginning of the next millennium

Direct sequencing of lambda DNA from crude lysates using an improved linear amplification technique

We described an improved method for directly sequencing lambda (?) DNA that has been isolated from either crude cleared lysates or plate lysates. This protocol does not require that the DNA be obtained from bacteriophage particles that have been purified by caesium chloride centrifugation. Nanogram quantities of ? DNA are unidirectionally amplified using a radioactively-labelled oligonucleotide primer, and Thermus aquaticus (Taq) DNA polymerase, in the presence of T4 gene 32 protein (gp 32). The amplification/sequencing reactions are then incubated with terminal deoxynucleotidyl transferase (TdT) and all four deoxynucleotide triphosphates to elongate any prematurely-arrested products. This procedure, which is a modification of a previously-published method, results in a significant improvement in the quality and amount of DNA sequence information that can be obtained from ? templates. Although it was developed to sequence DNA directly from ?EMBL3 recombinants, it can also be used with cosmid DNA, M13 and plasmid DNA, and polymerase chain reaction (PCR) amplification products, yielding excellent ladders in each case. In addition, our method resolves the nucleotide sequences of double-stranded plasmid templates that are difficult to determine by conventional dideoxynucleotide sequencing protocols because of 'stalling', in which bands appear at the same position in all four lanes

Localization of the ?1- and ?2-subunit messenger RNAs in chick retina by in situ hybridization predicts the existence of ?-aminobutyric acid type C receptor subtypes

We have amplified partial complementary {DNAs} for the chicken ?-aminobutyric acid type C (GABAC) receptor ?1 and ?2 subunits using the polymerase chain reaction. These nucleotide sequences have been utilized to design specific oligonucleotide probes for the in situ hybridization localization of the corresponding messenger {RNAs} (mRNAs) in the 1-day-old chick retina. Although both transcripts are found almost exclusively in the inner nuclear layer, their distributions differ markedly. From the locations of the hybridization signals, we deduce that the ?1-subunit mRNA is present mainly in bipolar cells and that the ?2-subunit mRNA is present in both amacrine and horizontal cells. These results suggest that the ?1 and ?2 subunits frequently occur in different receptor complexes and, therefore, that subtypes of the {GABAC} receptor exist

GABAAreceptor subtypes: which, where and why?

Synaptic inhibition in the vertebrate central nervous system is mediated predominantly by subtypes of the GABAAreceptor, which comprise different pentameric combinations of the products of 13 genes. In this article, we review the results of recent experiments that are helping to define binding-site determinants, on GABAAreceptors, for various ligands and some clinically-important modulators. New and sometimes conflicting data, on the polypeptide compositions of native subtypes, will also be discussed. Studies such as those described here should ultimately lead to a molecular understanding of receptor–ligand interactions, and the biological basis for the large number of subtypes that appear to exist in brai

Random-primed cDNA synthesis facilitates the isolation of multiple 5'-cDNA ends by RACE

No abstract available

Genetics of oxidative phosphorylation: Allelism studies of mitochondrial loci in the PHO1?OLI2 region of the genome

In a search for new aerobic-growth deficiency mutations affecting mitochondrial energy-conservation two mit? mutations, namely pho-8 and pho-9, have been isolated.The two mutations are allelic with each other, but not allelic with the previously known pho1 mutations although close linkage is indicated.Allelism studies define three distinct PHO loci clustered in this region which also includes the drugresistance loci OSS1, OLI2 and OLI4. The existence of phenotypically-distinct markers makes the region amenable to fine-structure mapping