Baclofen destabilises breathing during sleep in healthy humans: A randomised, controlled, double-blind crossover trial.

Aims: Periodic breathing is frequent in patients with severe heart failure. Apart from being an indicator of severity, periodic breathing has its own deleterious consequences (sleep-related oxygen desaturations, sleep fragmentation), which justifies attempts to correct it irrespective of the underlying disease. Animal models and human data suggest that baclofen can reconfigure respiratory central pattern generators.

A multi-gene phylogeny for Stachybotrys evidences lack of trichodiene synthase (tri5) gene for isolates of one of three intrageneric lineages.

Members of the mitosporic fungal form-genus Stachybotrys, including common indoor contaminants Stachybotrys chartarum, Stachybotrys echinata and Stachybotrys chlorohalonata, are capable of producing potent, protein synthesis-inhibiting, trichothecene mycotoxins. A combined multi-gene approach was used to investigate relationships among species of Stachybotrys against which the presence/absence of the trichothecene biosynthetic pathway gene, trichodiene synthase (tri5), was evaluated. Phylogenetic analyses partitioned species of Stachybotrys into three strongly supported lineages, two of which contained common indoor taxa.

Whole genome amplification using single-primer PCR.

Comprehensive genomic molecular analyses require relatively large DNA amounts that are often not available from forensic, clinical and other crucial biological samples. Numerous methods to amplify the whole genome have been proposed for cancer, forensic and taxonomic research. Unfortunately, when using truly random primers for the initial priming step, all of these procedures suffer from high background problems for sub-nanogram quantities of input DNA.

Real time PCR profiling of 330 human micro-RNAs.

The small size of miRNAs makes profiling of all the 462 known human miRNAs difficult using single cell samples. Recently, we showed that judicious sequence partitioning between RT primers and second strand synthesis primers permitted multiplexed RT-PCR amplification of miRNA in very small samples to allow individual real time PCR quantification. Here, we show that zip coding the primers and TaqMan probes with sequences specific to each miRNA greatly improves reaction specificity, which permits the profiling of all miRNAs in a single multiplexed RT-PCR reaction.

Multiplexing RT-PCR for the detection of multiple miRNA species in small samples.

MicroRNAs are short (approximately 22 nucleotides), non-coding RNAs that play critical roles in gene regulation and may be used as rapid precise diagnostic indicators of early stages of cancer. The small size of these RNAs makes detection of multiple microRNA species in very small samples problematic. Here we investigate the parameters associated with multiplexing RT-PCR to obtain relative abundance profiles of multiple microRNAs in small sample sizes down to the amount of RNA found in a single cell.

Genotypic variation in Penicillium chysogenum from indoor environments.

We examined 198 isolates of P. chysogenum recovered from 109 houses in Wallaceburg, Ontario, and 25 culture collection isolates including seven ex-type strains. Multilocus genotypes were determined by heteroduplex mobility assay of regions spanning introns in acetyl co-enzyme A synthase, beta-tubulin, thioredoxin reductase and the internal transcribed spacer regions of the nuclear ribosomal subrepeat.

Ancient tripartite coevolution in the attine ant-microbe symbiosis.

The symbiosis between fungus-growing ants and the fungi they cultivate for food has been shaped by 50 million years of coevolution. Phylogenetic analyses indicate that this long coevolutionary history includes a third symbiont lineage: specialized microfungal parasites of the ants' fungus gardens. At ancient levels, the phylogenies of the three symbionts are perfectly congruent, revealing that the ant-microbe symbiosis is the product of tripartite coevolution between the farming ants, their cultivars, and the garden parasites.

Fur regulates the expression of iron-stress genes in the cyanobacterium Synechococcus sp. strain PCC 7942.

A homologue of the 'ferric uptake regulation' gene (fur) was isolated from the cyanobacterium Synechococcus sp. strain PCC 7942 by an Escherichia coli-based 'in vivo repression assay'. The assay uses a reporter-gene construct containing the promoter region of the iron-regulated cyanobacterial gene isiA, fused to the coding region for chloramphenicol acetyltransferase.

Structure of the trigonal form of recombinant oxidized flavodoxin from Anabaena 7120 at 1.40 A resolution.

The oxidized recombinant flavodoxin from the cyanobacterium Anabaena 7120 has been crystallized in a trigonal form. The recombinant protein has an identical primary structure to that purified directly from Anabaena, which functions as a substitute for ferredoxin in an iron-deficient environment for electron transfer from photosystem I to ferredoxin-NADP(+) reductase. X-ray data to 1.

The nucleotide sequence of the Tn5271 3-chlorobenzoate 3,4-dioxygenase genes (cbaAB) unites the class IA oxygenases in a single lineage.

The nucleotide sequence of the 3-chlorobenzoate 3,4-dioxygenase genes, designated cbaAB, from the transposon Tn5271 was determined. The function of the two sequenced open reading frames was evaluated by mutagenesis and expression in vivo to show that the cbaA and cbaB genes code for dioxygenase and reductase proteins, respectively. Comparison of the deduced amino acid sequences of the cbaAB genes with sequences for other oxygenases revealed a clearly defined lineage among the class IA oxygenases that shows several unique features.

Cloning, sequencing and transcriptional studies of the genes for cytochrome c-553 and plastocyanin from Anabaena sp. PCC 7120.

In some cyanobacteria and eukaryotic algae, cytochrome c-553 (c-552) and plastocyanin function as alternative electron carriers between the cytochrome b6-f complex and Photosystem I. In these organisms plastocyanin is the electron carrier under copper-replete conditions, and cytochrome c-553 is the electron carrier during copper deprivation. In this paper we report the cloning, sequencing and transcriptional analysis of the genes for cytochrome c-553 and plastocyanin from Anabaena sp.

Photosystem II genes isiA, psbDI and psbC in Anabaena sp. PCC 7120: cloning, sequencing and the transcriptional regulation in iron-stressed and iron-repleted cells.

Under conditions of iron deprivation cyanobacteria produce flavodoxin to replace ferredoxin as the terminal electron acceptor of photosynthesis. In unicellular cyanobacteria, the gene for flavodoxin is the second open reading frame in a dicistronic operon whose transcription is tightly regulated by iron. The first gene, isiA, produces a protein that is very similar to CP43, a chlorophyll-binding, antenna protein of the photosystem II reaction center.

Effect of PCR conditions on the formation of heteroduplex and single-stranded DNA products in the amplification of bacterial ribosomal DNA spacer regions.

PCR amplifications of 16S/23S rDNA spacer regions were carried out from conserved 16S and 23S sequences for genomic DNA samples from strains representing 16 bacterial species (12 genera). Multiple products were produced containing conserved homologous sequences at the 3' and 5' ends, separated by highly variable internal spacer sequences. These products cross-hybridized forming heteroduplex DNA structures containing double-stranded ends surrounding an internal single-stranded loop.

Variation in the ribosomal internal transcribed spacers (ITS1 and ITS2) among eight taxa of the Mimulus guttatus species complex.

The internal transcribed spacer region (ITS1 and ITS2) of the 18S-25S nuclear ribosomal DNA sequence and the intervening 5.8S region were sequenced from three individuals in each of eight taxa of the Mimulus guttatus species complex. Three discrete variants, or "types," of ITS sequences were found, among which 30%-40% of sites differed, compared with 1%-2% within types.

High evolutionary divergence of the 5.8S ribosomal DNA in Mimulus glaucescens (Scrophulariaceae).

Ribosomal DNA sequences for the ITS 1, 5.8S, ITS 2 and adjoining regions of the 18S and 25S were obtained from Mimulus glaucescens (Scrophulariaceae) via cloned PCR products. The spacer sequences were completely unrelated to other plant taxa, although spacer lengths were approximately the same.

Rapid identification of bacteria on the basis of polymerase chain reaction-amplified ribosomal DNA spacer polymorphisms.

To facilitate genus and species level identification of a broad range of bacteria without the requirement of presumptive identification, we have developed a unified set of primers and polymerase chain reaction conditions to amplify spacer regions between the 16S and 23S genes in the prokaryotic rRNA genetic loci. Spacer regions within these loci show a significant level of length and sequence polymorphism across both genus and species lines. A generic pair of priming sequences was selected for the amplification of these polymorphisms from highly conserved sequences in the 16S and 23S genes occurring adjacent to these polymorphic regions.

An iron stress operon involved in photosynthetic electron transport in the marine cyanobacterium Synechococcus sp. PCC 7002.

The iron-stress-induced genes isiA and isiB have been cloned and sequenced from the marine unicellular cyanobacterium Synechococcus sp. PCC 7002. These genes code for a photosystem II chlorophyll-binding protein and flavodoxin respectively.

Chlorobenzoate catabolic transposon Tn5271 is a composite class I element with flanking class II insertion sequences.

The structure of a transposon specifying the biodegradation of chlorobenzoate contaminants is described. Tn5271 is a 17-kilobase (kb) transposon that resides in the plasmid or chromosome of Alcaligenes sp. strain BR60 and allows this organism to grow on 3- and 4-chlorobenzoate.

In vitro production of large single-stranded templates for DNA sequencing.

A method has been developed for the preparation of large single-stranded DNA sequencing templates from primary cloning plasmids or cosmids. The method utilizes the separate action of T7 Gene 6 exonuclease and exonuclease III to generate large quantities of single-stranded template for each strand of a large-cloned fragment. Since the procedure is intended for use on primary clones, it avoids the time-consuming subcloning steps associated with most sequencing programs.

Hydrogen-1, carbon-13, and nitrogen-15 NMR spectroscopy of Anabaena 7120 flavodoxin: assignment of beta-sheet and flavin binding site resonances and analysis of protein-flavin interactions.

Sequence-specific 1H and 13C NMR assignments have been made for residues that form the five-stranded parallel beta-sheet and the flavin mononucleotide (FMN) binding site of oxidized Anabaena 7120 flavodoxin. Interstrand nuclear Overhauser enhancements (NOEs) indicate that the beta-sheet arrangement is similar to that observed in the crystal structure of the 70% homologous long-chain flavodoxin from Anacystis nidulans [Smith et al. (1983) J.

Cytochrome c-553 is not required for photosynthetic activity in the cyanobacterium Synechococcus.

In cyanobacteria, the water-soluble cytochrome c-553 functions as a mobile carrier of electrons between the membrane-bound cytochrome b6-f complex and P-700 reaction centers of Photosystem I. The structural gene for cytochrome c-553 (designated cytA) of the cyanobacterium Synechococcus sp. PCC 7942 was cloned, and the deduced amino acid sequence was shown to be similar to known cyanobacterial cytochrome c-553 proteins.

Cloning and characterization of an Anacystis nidulans R2 superoxide dismutase gene.

The E. coli iron superoxide dismutase gene (sodB) was utilized as a heterologous probe to isolate a superoxide dismutase (sod) gene from Anacystis nidulans R2. Nucleotide sequence analysis revealed a 603 bp open reading frame with deduced amino acid sequence similar to other sod genes and to cyanobacterial superoxide dismutase amino-terminal sequences.

A class-I intron in a cyanelle tRNA gene from Cyanophora paradoxa: phylogenetic relationship between cyanelles and plant chloroplasts.

Cyanelles are photosynthetic organelles which are considered as intermediates between cyanobacteria and chloroplasts, and which have been found in unicellular eukaryotes such as Cyanophora paradoxa. The nucleotide sequence of a 667-bp region of the cyanelle genome from Cyanophora paradoxa containing genes coding for tRNA(UUCGlu) and tRNA(UAALeu) has been determined. The gene coding for tRNA(UAALeu) is split by a 232-bp intron which has a secondary structure typical for class-I structured introns and which is closely related to the intron located in the corresponding gene from liverwort and higher plant chloroplasts.

Characterization of a cyanobacterial iron stress-induced gene similar to psbC.

Recently we have reported that the flavodoxin gene from the cyanobacterium Anacystis nidulans R2 is transcribed as part of an iron stress-induced operon containing multiple mRNA species (D. E. Laudenbach, M.