Octopamine stabilizes conduction reliability of an unmyelinated axon during hypoxic stress.

Mechanisms that could mitigate the effects of hypoxia on neuronal signaling are incompletely understood. We show that axonal performance of a locust visual interneuron varied depending on oxygen availability. To induce hypoxia, tracheae supplying the thoracic nervous system were surgically lesioned and action potentials in the axon of the descending contralateral movement detector (DCMD) neuron passing through this region were monitored extracellularly. The conduction velocity and fidelity of action potentials decreased throughout a 45-min experiment in hypoxic preparations, whereas conduction reliability remained constant when the tracheae were left intact. The reduction in conduction velocity was exacerbated for action potentials firing at high instantaneous frequencies. Bath application of octopamine mitigated the loss of conduction velocity and fidelity. Action potential conduction was more vulnerable in portions of the axon passing through the mesothoracic ganglion than in the connectives between ganglia, indicating that hypoxic modulation of the extracellular environment of the neuropil has an important role to play. In intact locusts, octopamine and its antagonist, epinastine, had effects on the entry to, and recovery from, anoxic coma consistent with octopamine increasing overall neural performance during hypoxia. These effects could have functional relevance for the animal during periods of environmental or activity-induced hypoxia.

SELENBP1 expression in the prefrontal cortex of subjects with schizophrenia.

Selenium binding protein 1 (SELENBP1) messenger RNA (mRNA) has previously been shown to be upregulated in the brain and blood from subjects with schizophrenia. We aimed to validate these findings in a new cohort using real-time PCR in Brodmann's Area (BA) 9, and to determine the disease specificity of increased SELENBP1 expression by measuring SELENBP1 mRNA in subjects with major depressive disorder and bipolar disorder. We then extended the study to include other cortical regions such as BA8 and BA44. SELENBP1 mRNA was higher in BA9 (P = 0.001), BA8 (P = 0.003) and BA44 (P = 0.0007) from subjects with schizophrenia. Conversely, in affective disorders, there was no significant difference in SELENBP1 mRNA in BA9 (P = 0.67), suggesting that the upregulation may be diagnosis specific. Measurement of SELENBP1 protein levels showed that changes in mRNA did not translate to changes in protein. In addition, chronic treatment of rats with antipsychotics did not significantly affect the expression of Selenbp1 in the cortex (P = 0.24). Our data show that elevated SELENBP1 transcript expression is widespread throughout the prefrontal cortex in schizophrenia, and confirm that this change is a consistent feature of schizophrenia and not a simple drug effect.

Placental CLIC3 is increased in fetal growth restriction and pre-eclampsia affected human pregnancies.

Chloride intracellular channel (CLIC) proteins constitute a subgroup of the glutathione-S-transferase (GSTs) superfamily. In humans, the CLIC family of proteins consists of six members, designated CLIC 1-6, which have a conserved C-terminal 240 residue module and one major transmembrane domain. CLIC proteins regulate fundamental cellular processes including regulation of chloride ion concentration, stabilization of cell membrane potential, trans-epithelial transport, regulation of cell volume and stimulation of apoptotic processes in response to cellular stress. Previously, we described the expression profile of a member of the CLIC family of proteins, CLIC3, in human placentae and fetal membranes. In the current study, we determined CLIC3 expression in placentae from pregnancies complicated with either fetal growth restriction (FGR, n=19), pre-eclampsia (PE, n=16) or both FGR and PE combined (n=12) compared to gestation-matched controls (n=13) using real-time PCR and a CLIC3 specific immunoassay. Significantly increased CLIC3 mRNA and protein were detected in placental extracts from pregnancies with FGR, PE and PE with FGR compared to controls. Our results suggest that increased expression of CLIC3 may play a role in abnormal placental function associated with the human pregnancy disorders FGR and PE.

Treating schizophrenia: novel targets for the cholinergic system.

Cognitive deficits in patients with schizophrenia are the biggest obstacle to achieving an independent and productive lifestyle, with these deficits being refractory to current drug treatments. Significantly, both nicotinic and muscarinic receptors (cholinoceptors) have been shown to have an important role in cognition and are therefore viewed as potential therapeutic targets for drugs designed to lessen cognitive deficits. Importantly, the demonstration that acetylcholinesterase inhibitors, which result in higher synaptic levels of acetylcholine, can reduce the cognitive deficits of schizophrenia suggested that under-stimulation of cholinoceptors could be associated with the cognitive deficits associated with this disorder. This has lead to a focus on the development of receptor agonists, partial agonists and allosteric agonists that can be used to stimulate cholinergic pathways and thus reduce the cognitive deficits of schizophrenia. In addition, muscarinic receptors have now been associated with the modulation of dopamine and may constitute an alternative target for the treatment of psychoses. Given these exciting new therapeutic initiatives, this review will outline current evidence that involves the cholinoceptors in the pathophysiology of schizophrenia and how these data can inform on approaches to more targeted treatments for the disorder.

Expression and cellular localisation of chloride intracellular channel 3 in human placenta and fetal membranes.

Chloride channels regulate the movement of a major cellular anion and are involved in fundamental processes that are critical for cell viability. Regulation of intracellular chloride is achieved by multiple classes of channel proteins. One class of putative channels are the chloride intracellular channel (CLIC) family. Evidence suggests that several CLICs are expressed in human placenta, although their roles in this tissue are not certain. Northern blot analysis has shown that CLIC3 is highly expressed in placenta relative to other human tissues; however, its cellular distribution is not known. This study used microarray expression profiling to clarify which CLICs are expressed in human placenta and RT-PCR, Western blot and immunohistochemistry to determine the expression pattern of CLIC3 in human placenta and fetal membranes. Placentas and fetal membranes were obtained from term pregnancies after delivery and placental tissue was obtained from first trimester following either chorionic villous sampling or elective pregnancy termination. Trophoblast cells were isolated from first trimester and term placentas and placental endothelial cells were isolated from term placentas. Microarray expression profiling identified high expression of mRNA for CLICs 1, 3 and 4 in the isolated first trimester and term trophoblast cells. High mRNA expression in the isolated endothelial cells was also found for CLICs 1 and 4, but not CLIC3. Low expression was found for CLIC5 in all three types of isolated cells. RT-PCR confirmed that CLIC3 mRNA was expressed in trophoblast cells at both gestational ages, but was not present in endothelial cells. CLIC3 mRNA was also identified in whole placental extracts at both gestational ages and in term amnion and choriodecidua. Immunohistochemistry using a chicken anti-human CLIC3 antibody localised strong CLIC3-specific staining to the syncytiotrophoblast and villous cytotrophoblast cells in both first trimester and term placentas, and weaker staining in extravillous trophoblast cells in first trimester. In fetal membranes at term strong CLIC3-specific staining was localised to chorionic trophoblast cells, with weaker staining in amniotic epithelial and decidual cells. It was previously shown that chloride uptake was increased into cells that had been transfected with CLIC3. CLIC3 may facilitate chloride ion movement and the regulation of cellular processes associated with the movement of chloride in the placental and fetal membrane cells in which it is expressed.

Protein-protein interactions in the complex between the enhancer binding protein NIFA and the sensor NIFL from Azotobacter vinelandii.

The enhancer binding protein NIFA and the sensor protein NIFL from Azotobacter vinelandii comprise an atypical two-component regulatory system in which signal transduction occurs via complex formation between the two proteins rather than by the phosphotransfer mechanism, which is characteristic of orthodox systems. The inhibitory activity of NIFL towards NIFA is stimulated by ADP binding to the C-terminal domain of NIFL, which bears significant homology to the histidine protein kinase transmitter domains. Adenosine nucleotides, particularly MgADP, also stimulate complex formation between NIFL and NIFA in vitro, allowing isolation of the complex by cochromatography. Using limited proteolysis of the purified proteins, we show here that changes in protease sensitivity of the Q linker regions of both NIFA and NIFL occurred when the complex was formed in the presence of MgADP. The N-terminal domain of NIFA adjacent to the Q linker was also protected by NIFL. Experiments with truncated versions of NIFA demonstrate that the central domain of NIFA is sufficient to cause protection of the Q linker of NIFL, although in this case, stable protein complexes are not detectable by cochromatography.

Isolation and properties of the complex between the enhancer binding protein NIFA and the sensor NIFL.

In Azotobacter vinelandii, activation of nif gene expression by the transcriptional regulatory enhancer binding protein NIFA is controlled by the sensor protein NIFL in response to changes in levels of oxygen and fixed nitrogen in vivo. NIFL is a novel redox-sensing flavoprotein which is also responsive to adenosine nucleotides in vitro. Inhibition of NIFA activity by NIFL requires stoichiometric amounts of the two proteins, implying that the mechanism of inhibition is by direct protein-protein interaction rather than by catalytic modification of the NIFA protein. The formation of the inhibitory complex between NIFL and NIFA may be regulated by the intracellular ATP/ADP ratio. We show that adenosine nucleotides promote complex formation between purified NIFA and NIFL in vitro, allowing isolation of the NIFL-NIFA complex. The complex can also be isolated from cell extracts containing coexpressed NIFL and NIFA in the presence of MgADP. Removal of the nucleotide causes dissociation of the complex. Experiments with truncated proteins demonstrate that the amino-terminal domain of NIFA and the C-terminal region of NIFL potentiate the ADP-dependent stimulation of NIFL-NIFA complex formation.

A simple PCR-based method for scoring the ph1b deletion in wheat.

The amplified fragment length polymorphism (AFLP) technique was used to isolate DNA sequences present in the euploid wheat Chinese Spring but not in the Chinese Spring ph1b mutant (which has a deletion of the Ph1 gene, a suppressor of homoeologous chromosome pairing). The polymorphic DNA fragments identified by AFLP were then cloned, sequenced, and used to design two primer pairs. These primers were used in a PCR-based assay to specifically amplify products from the Chinese Spring euploid but not from the ph1b mutant. This PCR assay can be carried out from extracted genomic DNA or directly from alkaline-treated wheat leaves, and the reaction products can be scored on a plus-minus basis, making the screening amenable to automation. The reliability of the assay was tested using a F1-derived doubled-haploid population of 55 lines which segregate for the ph1b deletion. This PCR-screening technique is less time and labour consuming, and more accurate and reliable, than cytologically based conventional methods.

Conservation of fine-scale DNA marker order in the genomes of rice and the Triticeae.

DNA markers distribute over large chromosomal regions exhibit conservation of order (collinearity) in different cereal species, but it is not known whether this is maintained on a finer scale, i.e. < or = 2 cM. To address this, sets of two or more genetically linked DNA markers were localised to yeast artificial chromosomes containing rice DNA inserts. Linkage analysis of these DNA markers in barley revealed complete correspondence with their genetic order in rice, the distance between linked sequences on rice chromosomes being < 1.6 cM or < or = 1 + 10(6) bp (1 Mb). Thus, DNA markers separated in this range are collinear in rice, barley and, by inference, other members of the Triticeae. These results are discussed with respect to the use of rice as a key system for the isolation of cereal genes.

A family of related sequences associated with (TTTAGGG)n repeats are located in the interstitial regions of wheat chromosomes.

A family of related sequences associated with (TTTAGGG)n repeats has been cloned from the wheat cultivar Chinese Spring. These sequences reveal a high level of polymorphism between wheat varieties when used as restriction fragment length polymorphism (RFLP) probes. Although this family of sequences contains motifs homologous to the repeats in the telomeres of wheat, they are located at interstitial sites on wheat chromosomes.

Key features of cereal genome organization as revealed by the use of cytosine methylation-sensitive restriction endonucleases.

Unlike mammalian genomes, cereal (Gramineae) genomes exhibit little suppression of CpG dinucleotides. In cereal genomes, however, most of the numerous potential recognition sites for CpG methylation-sensitive restriction enzymes are methylated. Analysis of cereal genomic libraries and of regions flanking genes indicates that unmethylated NotI sites are useful landmarks for regions containing genes/single-copy sequences. Studies of a rye chromosome arm indicate that its pericentromeric region has a reduced density of unmethylated NotI (and MluI) sites and therefore of genes. Unmethylated MluI and NruI sites are distributed nonrandomly in the genomes of wheat, barley, and rice. Analysis of the genomic blocks defined by these sites in wheat and barley indicates that they are most likely to have arisen by amplification. These observations form the basis of a proposed model for the organization and evolution of the wheat, barley, and rice genomes.

Targeting deletion (homoeologous chromosome pairing locus) or addition line single copy sequences from cereal genomes.

We describe here a protocol for obtaining clones containing sequences present in low copy-number from genomic DNA where moderately and highly repeated sequences predominate. Specific chromosomal regions can be targeted by using deletion or addition line material. We have used this protocol to identify a sequence which has been deleted in both the tetraploid and hexaploid wheat mutants for the homoeologous chromosome pairing locus.

HpaII library indicates 'methylation-free islands' in wheat and barley.

A library of wheat genomic DNA HpaII tiny fragments (HTF), sized below 500 bp, has been constructed. Of the clones in the library 80% belong to the single/low-copy category, while 12% of the clones are nuclear repetitive sequences and 8% originate from the chloroplast and mitochondrial DNA. This result shows a substantial enrichment in the single/low-copy sequences of the wheat genome, which contains at least 80% repetitive sequences. Twenty-nine random single/lowcopy clones were analysed further for wheat chromosome location, cross-hybridisation to barley DNA and their association with rare-cutting, C-methylation-sensitive restriction sites. The results show that the HTF clones are associated more frequently than expected with NotI, MluI, NruI and PstI sites in wheat and barley genomic DNA. The 12% repetitive fraction of the clones contain both moderately and highly repetitive sequences, but no tandemly repeated sequences. The level of enrichment for single/low-copy sequences indicates that libraries of this type are a valuable source of probes for RFLP mapping. In addition, the close association of the HTF clones with rare-cutting restriction enzyme sites ensures that HTF clones will have a useful role in the construction of long-range physical maps in wheat.