Down to Earth--everyday uses for European Space Technology.

Every year billions of dollars are spent on research and development activities in virtually every technological sector. Sadly, although many such activities have a wide potential of application, the results of this research are still a long way from commercialisation. However, companies, research organisations and academic institutes are now waking up to the fact that it has become increasingly necessary to exchange, transfer and license the technologies (including software) and knowledge they have developed in order to access new markets and revenue streams. On the other hand, it also becomes ever apparent that companies need to acquire new technologies, particularly the leading-edge technologies being developed within the space industry, in order to exploit their ideas and create new products.

Yeast Cdc42 GTPase and Ste20 PAK-like kinase regulate Sho1-dependent activation of the Hog1 MAPK pathway.

The adaptive response to hyperosmotic stress in yeast, termed the high osmolarity glycerol (HOG) response, is mediated by two independent upstream pathways that converge on the Pbs2 MAP kinase kinase (MAPKK), leading to the activation of the Hog1 MAP kinase. One branch is dependent on the Sho1 transmembrane protein, whose primary role was found to be the binding and translocation of the Pbs2 MAPKK to the plasma membrane, and specifically to sites of polarized growth. The yeast PAK homolog Ste20 is essential for the Sho1-dependent activation of the Hog1 MAP kinase in response to severe osmotic stress. This function of Ste20 in the HOG pathway requires binding of the small GTPase Cdc42. Overexpression of Cdc42 partially complements the osmosensitivity of ste20Delta mutants, perhaps by activating another PAK-like kinase, while a dominant-negative Cdc42 mutant inhibited signaling through the SHO1 branch of the HOG pathway. Since activated Cdc42 translocates Ste20 to sites of polarized growth, the upstream and downstream elements of the HOG pathway are brought together through the membrane targeting function of Sho1 and Cdc42.

The Skn7 response regulator of Saccharomyces cerevisiae interacts with Hsf1 in vivo and is required for the induction of heat shock genes by oxidative stress.

The Skn7 response regulator has previously been shown to play a role in the induction of stress-responsive genes in yeast, e.g., in the induction of the thioredoxin gene in response to hydrogen peroxide. The yeast Heat Shock Factor, Hsf1, is central to the induction of another set of stress-inducible genes, namely the heat shock genes. These two regulatory trans-activators, Hsf1 and Skn7, share certain structural homologies, particularly in their DNA-binding domains and the presence of adjacent regions of coiled-coil structure, which are known to mediate protein-protein interactions. Here, we provide evidence that Hsf1 and Skn7 interact in vitro and in vivo and we show that Skn7 can bind to the same regulatory sequences as Hsf1, namely heat shock elements. Furthermore, we demonstrate that a strain deleted for the SKN7 gene and containing a temperature-sensitive mutation in Hsf1 is hypersensitive to oxidative stress. Our data suggest that Skn7 and Hsf1 cooperate to achieve maximal induction of heat shock genes in response specifically to oxidative stress. We further show that, like Hsf1, Skn7 can interact with itself and is localized to the nucleus under normal growth conditions as well as during oxidative stress.

Thoracoscopic sympathectomy: a one-port technique.

BACKGROUND: The present paper describes the development of a one-port technique for thoracoscopic sympathectomy. METHODS: A 7-mm thorascope with a working channel for diathermy was used. CONCLUSION: A highly cosmetic, simple, safe, day-case procedure is achievable.

The yeast histidine protein kinase, Sln1p, mediates phosphotransfer to two response regulators, Ssk1p and Skn7p.

The Saccharomyces cerevisiae Sln1 protein is a 'two-component' regulator involved in osmotolerance. Two-component regulators are a family of signal-transduction molecules with histidine kinase activity common in prokaryotes and recently identified in eukaryotes. Phosphorylation of Sln1p inhibits the HOG1 MAP kinase osmosensing pathway via a phosphorelay mechanism including Ypd1p and the response regulator, Ssk1p. SLN1 also activates an MCM1-dependent reporter gene, P-lacZ, but this function is independent of Ssk1p. We present genetic and biochemical evidence that Skn7p is the response regulator for this alternative Sln1p signaling pathway. Thus, the yeast Sln1 phosphorelay is actually more complex than appreciated previously; the Sln1 kinase and Ypd1 phosphorelay intermediate regulate the activity of two distinct response regulators, Ssk1p and Skn7p. The established role of Skn7p in oxidative stress is independent of the conserved receiver domain aspartate, D427. In contrast, we show that Sln1p activation of Skn7p requires phosphorylation of D427. The expression of TRX2, previously shown to exhibit Skn7p-dependent oxidative-stress activation, is also regulated by the SLN1 phosphorelay functions of Skn7p. The identification of genes responsive to both classes of Skn7p function suggests a central role for Skn7p and the SLN1-SKN7 pathway in integrating and coordinating cellular response to various types of environmental stress.

Effects of oxygen concentration on the expression of cytochrome c and cytochrome c oxidase genes in yeast.

Oxygen is an important environmental regulator for the transcription of several genes in Saccharomyces cerevisiae, but it is not yet clear how this yeast or other eukaryotes actually sense oxygen. To begin to address this we have examined the effects of oxygen concentration on the expression of several nuclear genes (CYC1, CYC7, COX4, COX5a, COX5b, COX6, COX7, COX8, and COX9) for proteins of the terminal portion of the respiratory chain. COX5b and CYC7 are hypoxic genes; the rest are aerobic genes. We have found that the level of expression of these genes is determined by oxygen concentration per se and not merely the presence or absence of oxygen and that each of these genes has a low oxygen threshold (0. 5-1 microM O2) for expression. For some aerobic genes (COX4, COX5a, COX7, COX8, and COX9) there is a gradual decline in expression between 200 microM O2 (air) and their oxygen threshold. Below this threshold expression drops precipitously. For others (COX5a and CYC1) the level of expression is nearly constant between 200 microM O2 and their threshold and then drops off. The hypoxic genes COX5b and CYC7 are not expressed until the oxygen concentration is below 0.5 microM O2. These studies have also revealed that COX5a and CYC1, the genes for the aerobic isoforms of cytochrome c oxidase subunit V and cytochrome c, and COX5b and CYC7, the genes for the hypoxic isoforms of cytochrome c oxidase subunit V and cytochrome c, are coexpressed at a variety of oxygen concentrations and switch on or off at extremely low oxygen concentrations. By shifting cells from one oxygen concentration to another we have found that aerobic genes are induced faster than hypoxic genes and that transcripts from both types of gene are turned over quickly. These findings have important implications for cytochrome c oxidase function and biogenesis and for models of oxygen sensing in yeast.

The Skn7 response regulator controls gene expression in the oxidative stress response of the budding yeast Saccharomyces cerevisiae.

Deletion of the bacterial two-component response regulator homologue Skn7 results in sensitivity of yeast to oxidizing agents indicating that Skn7 is involved in the response to this type of stress. Here we demonstrate that following oxidative stress, Skn7 regulates the induction of two genes: TRX2, encoding thioredoxin, and a gene encoding thioredoxin reductase. TRX2 is already known to be induced by oxidative stress dependent on the Yap1 protein, an AP1-like transcription factor responsible for the induction of gene expression in response to various stresses. The thioredoxin reductase gene has not previously been shown to be activated by oxidative stress and, significantly, we find that it too is regulated by Yap1. The control of at least TRX2 by Skn7 is a direct mechanism as Skn7 binds to the TRX2 gene promoter in vitro. This shows Skn7 to be a transcription factor, at present the only such eukaryotic two-component signalling protein. Our data further suggest that Skn7 and Yap1 co-operate on the TRX2 promoter, to induce transcription in response to oxidative stress.

Function and expression of flavohemoglobin in Saccharomyces cerevisiae. Evidence for a role in the oxidative stress response.

We have studied the function and expression of the flavohemoglobin (YHb) in the yeast Saccharomyces cerevisiae. This protein is a member of a family of flavohemoproteins, which contain both heme and flavin binding domains and which are capable of transferring electrons from NADPH to heme iron. Normally, actively respiring yeast cells have very low levels of the flavohemoglobin. However, its intracellular levels are greatly increased in cells in which the mitochondrial electron transport chain has been compromised by either mutation or inhibitors of respiration. The expression of the flavohemoglobin gene, YHB1, of S. cerevisiae is sensitive to oxygen. Expression is optimal in hyperoxic conditions or in air and is reduced under hypoxic and anaerobic conditions. The expression of YHB1 in aerobic cells is enhanced in the presence of antimycin A, in thiol oxidants, or in strains that lack superoxide dismutase. All three conditions lead to the accumulation of reactive oxygen species and promote oxidative stress. To study the function of flavohemoglobin in vivo, we created a null mutation in the chromosomal copy of YHB1. The deletion of the flavohemoglobin gene in these cells does not affect growth in either rhoo or rho+ genetic backgrounds. In addition, a rho+ strain carrying a yhb1(-) deletion has normal levels of both cyanide-sensitive and cyanide-insensitive respiration, indicating that the flavohemoglobin does not function as a terminal oxidase and is not required for the function or expression of the alternative oxidase system in S. cerevisiae. Cells that carry a yhb1(-)deletion are sensitive to conditions that promote oxidative stress. This finding is consistent with the observation that conditions that promote oxidative stress also enhance expression of YHB1. Together, these findings suggest that YHb plays a role in the oxidative stress response in yeast.

Cloning and characterisation of the cytochrome c gene of Aspergillus nidulans.

The cytochrome c gene (cycA) of the filamentous fungus Aspergillus nidulans has been isolated and sequenced. The gene is present in a single copy per haploid genome and encodes a polypeptide of 112 amino acid residues. The nucleotide sequence of the A. nidulans cycA gene shows 87% identity to the DNA sequence of the Neurospora crassa cytochrome c gene, and approximately 72% identity to the sequence of the Saccharomyces cerevisiae iso-1-cytochrome c gene (CYC1). The S. cerevisiae CYC1 gene was used as a heterologous probe to isolate the homologous gene in A. nidulans. The A. nidulans cytochrome c sequence contains two small introns. One of these is highly conserved in terms of position, but the other has not been reported in any of the cytochrome c genes so far sequenced. Expression of the cycA gene is not affected by glucose repression, but has been shown to be induced approximately tenfold in the presence of oxygen and three- to fourfold under heat-shock conditions.

Isolation and nucleotide sequence of the 5-aminolevulinate synthase gene from Aspergillus nidulans.

The structural gene for 5-aminolevulinate (ALA) synthase has been cloned and sequenced from the filamentous fungus Aspergillus nidulans using an oligonucleotide probe based on a highly conserved-amino-acid sequence found in ALA synthase genes of a wide range of species. The cloned gene, hemA, has a 5' untranslated mRNA of 92 nucleotides (nt) and one intron (64 nt). The deduced protein sequence (648 amino acids) shows 64% identity to the yeast ALA synthase in the C-terminal region of 453 amino acids. The N-terminal region is typical of ALA synthase proteins in that the specific amino-acid sequence is not conserved but consists of a "leader" region rich in basic amino acids, believed to be involved in mitochondrial targeting, followed by a stretch of largely hydrophobic residues which may allow interaction with the inner mitochondrial membrane. Under the conditions used the transcription of hemA was unaffected by dextrose repression, heat shock, or oxygen levels.