Biomedical subjects

Peter Poulsen

Publications and source records attributed to Peter Poulsen.

8 recordsLinked to original sources

PubMed2006

Competitive intra- and extracellular nutrient sensing by the transporter homologue Ssy1p.

Recent studies of Saccharomyces cerevisiae revealed sensors that detect extracellular amino acids (Ssy1p) or glucose (Snf3p and Rgt2p) and are evolutionarily related to the transporters of these nutrients. An intriguing question is whether the evolutionary transformation of transporters into nontransporting sensors reflects a homeostatic capability of transporter-like sensors that could not be easily attained by other types of sensors. We previously found SSY1 mutants with an increased basal level of signaling and increased apparent affinity to sensed extracellular amino acids. On this basis, we propose and test a general model for transporter- like sensors in which occupation of a single, central ligand binding site increases the activation energy needed for the conformational shift between an outward-facing, signaling conformation and an inward-facing, nonsignaling conformation. As predicted, intracellular leucine accumulation competitively inhibits sensing of extracellular amino acids. Thus, a single sensor allows the cell to respond to changes in nutrient availability through detection of the relative concentrations of intra- and extracellular ligand.

2-Isopropylmalate Synthase↗

PubMed2006

[Rhinoconjunctivitis caused by paper tissues].

Rhinoconjunctivitis can be caused by allergenic as well as non-allergenic factors. When no allergy is involved, it is important to ask the patient about possible daily exposure to factors that may irritate the mucous membranes. The work environment may play a part, but other things may also be provoking factors, including paper tissues, which, besides the fibres of the paper itself, may contain chemical substances that irritate the mucous membranes. The treatment is to replace the paper tissues with well-washed cotton handkerchiefs. The use of a nasal steroid may be necessary for a short time.

Conjunctivitis, Allergic↗

PubMed2006

Deletion of RTS1, encoding a regulatory subunit of protein phosphatase 2A, results in constitutive amino acid signaling via increased Stp1p processing.

In Saccharomyces cerevisiae, extracellular amino acids are sensed at the plasma membrane by the SPS sensor, consisting of the transporter homologue Ssy1p, Ptr3p, and the endoprotease Ssy5p. Amino acid sensing results in proteolytic truncation of the transcription factors Stp1p and Stp2p, followed by their relocation from the cytoplasm to the nucleus, where they activate transcription of amino acid permease genes. We screened a transposon mutant library for constitutively signaling mutants, with the aim of identifying down-regulating components of the SPS-mediated pathway. Three isolated mutants were carrying a transposon in the RTS1 gene, which encodes a regulatory subunit of protein phosphatase 2A. We investigated the basal activity of the AGP1 and BAP2 promoters in rts1delta cells and found increased transcription from these promoters, as well as increased Stp1p processing, even in the absence of amino acids. Based on our findings we propose that the phosphatase complex containing Rts1p keeps the SPS-mediated pathway down-regulated in the absence of extracellular amino acids by dephosphorylating a component of the pathway.

Amino Acid Transport Systems↗

PubMed2006

Mapping of an internal protease cleavage site in the Ssy5p component of the amino acid sensor of Saccharomyces cerevisiae and functional characterization of the resulting pro- and protease domains by gain-of-function genetics.

Ssy5p is a 77-kDa protein believed to be a component of the SPS amino acid sensor complex in the plasma membrane of Saccharomyces cerevisiae. Ssy5p has been suggested to be a chymotrypsin-like serine protease that activates the transcription factor Stp1p upon exposure of the yeast to extracellular amino acid. Here we overexpressed and partially purified Ssy5p to improve our understanding of its structure and function. Antibodies against Ssy5p expressed in Escherichia coli were isolated and used to detect Ssy5p processing in S. cerevisiae cells. Partial purification and N-terminal sequencing of processed Ssy5p revealed in vivo cleavage of Ssy5p between amino acids 381 and 382. We also isolated constitutively signaling SSY5 mutants and quantified target promoter activation and Stp1p processing. One mutant contained an amino acid substitution in the prodomain, whereas three others harbored amino acid substitutions in the protease domain. Dose-response analysis indicated that all four mutants exhibited increased basal levels of Stp1p processing. Interestingly, whereas the three constitutive mutants mapping to the protease domain of Ssy5p exhibited the decreased 50% effective concentration (EC(50)) characteristic of constitutive mutations previously found in Ssy1p, Ptr3p, and Ssy5p, the EC(50) of the mutation that maps to the prodomain of Ssy5p remained essentially unchanged. In a model of Ssy5p derived from its similarities with alpha-lytic protease from Lysobacter enzymogenes, the sites corresponding to the mutations in the protease domain are clustered in a region facing the prodomain, suggesting that this region interacts with the prodomain and participates in the conformational dynamics of sensing.

Amino Acid Sequence↗

PubMed2005

Structure function relationships of transgenic starches with engineered phosphate substitution and starch branching.

Potato tuber starch was genetically engineered in the plant by the simultaneous antisense suppression of the starch branching enzyme (SBE) I and II isoforms. Starch prepared from 12 independent lines and three control lines were characterised with respect to structural and physical properties. The lengths of the amylopectin unit chains, the concentrations of amylose and monoesterified phosphate were significantly increased in the transgenically engineered starches. Size exclusion chromatography with refractive index detection (SEC-RI) indicated a minor decrease in apparent molecular size of the amylose and the less branched amylopectin fractions. Differential scanning calorimetry (DSC) revealed significantly higher peak temperatures for gelatinisation and retrogradation of the genetically engineered starches whereas the enthalpies of gelatinisation were lower. Aqueous gels prepared from the transgenic starches showed increased gel elasticity and viscosity. Principle component analysis (PCA) of the data set discriminated the control lines from the transgenic lines and revealed a high correlation between phosphate concentration and amylopectin unit chain length. The PCA also indicated that the rheological characteristics were primarily influenced by the amylose concentration. The phosphate and the amylopectin unit chain lengths had influenced primarily the pasting and rheological properties of the starch gels.

Amylopectin↗

PubMed2005

Constitutive signal transduction by mutant Ssy5p and Ptr3p components of the SPS amino acid sensor system in Saccharomyces cerevisiae.

Amino acids in the environment of Saccharomyces cerevisiae can transcriptionally activate a third of the amino acid permease genes through a signal that originates from the interaction between the extracellular amino acids and an integral plasma membrane protein, Ssy1p. Two plasma membrane-associated proteins, Ptr3p and Ssy5p, participate in the sensing, which results in cleavage of the transcription factors Stp1p and Stp2p, removing 10 kDa of the N terminus of each of them. This confers the transcription factors with the ability to gain access to the nucleus and activate transcription of amino acid permease genes. To extend our understanding of the role of Ptr3p and Ssy5p in this amino acid sensing process, we have isolated constitutive gain-of-function mutants in these two components by using a genetic screening in which potassium uptake is made dependent on amino acid signaling. Mutants which exhibit inducer-independent processing of Stp1p and activation of the amino acid permease gene AGP1 were obtained. For each component of the SPS complex, constitutive signaling by a mutant allele depended on the presence of wild-type alleles of the other two components. Despite the signaling in the absence of inducer, the processing of Stp1p was more complete in the presence of inducer. Dose response assays showed that the median effective concentration for Stp1p processing in the mutant cells was decreased; i.e., a lower inducer concentration is needed for signaling in the mutant cells. These results suggest that the three sensor components interact intimately in a complex rather than in separate reactions and support the notion that the three components function as a complex.

Amino Acids↗

PubMed2004

Invasive pneumococcal disease in Greenland.

INTRODUCTION: The Inuit of Alaska and Canada have a higher incidence of invasive pneumococcal disease than non-inuit. The purpose of the present study was to evaluate incidence and outcome of these infections in Greenland with special reference to pneumococcal serotypes. STUDY DESIGN: Retrospective study of invasive pneumococcal infections in Greenland in the period 1996-2002. METHODS: Cases were defined as patients with positive cultures of Streptococcus pneumoniae from blood and/or CSF samples received at the microbiological laboratory of Dronning Ingrids Hospital Nuuk. Isolates were sent to Statens Serum Institut in Copenhagen for serotyping. Medical charts were reviewed. RESULTS: Fifty one cases were identified. The incidence among Inuit was 54 and among non-inuit 17 per 100,000 per year. Twenty one patients were in the age group 35-49 years and 20 in the age group 50-64 years. Twenty patients had meningitis (incidence 6 per 100,000/year). Seventeen patients died (33%). The most common serotypes were 1 (6 cases) and 12F (8 cases). The mortality rate was significantly higher among patients with 12F than among others (p < 0.01). No patients with serotype 1 died. CONCLUSION: As in Canada and Alaska the incidence of invasive pneumococcal disease, especially meningitis, is high among the Inuit in Greenland. Young and middle aged adults were most frequently affected. Pneumococcal serotype seems to be an important determinant for the outcome of invasive pneumococcal disease.

Adolescent↗

PubMed2003

The structure and specificity of Escherichia coli maltose acetyltransferase give new insight into the LacA family of acyltransferases.

The crystallographic three-dimensional structure of the Escherichia coli maa gene product, previously identified as a maltose O-acetyltransferase (MAT) [Brand, B., and Boos, W. (1991) J. Biol. Chem. 266, 14113-14118] has been determined to 2.15 A resolution by the single anomalous dispersion method using data from a crystal cocrystallized with trimethyllead acetate. It is shown here that MAT acetylates glucose exclusively at the C6 position and maltose at the C6 position of the nonreducing end glucosyl moiety. Furthermore, MAT shows higher affinity toward artificial substrates containing an alkyl or hydrophobic chain as well as a glucosyl unit. The presence of a long hydrophobic patch near the acceptor site provides the structural explanation for this preference. The three-dimensional structure reveals the expected trimeric left-handed parallel beta-helix structure found in all other known hexapeptide repeat enzymes. In particular, the structure shows similarities both overall and at the putative active site to the recently determined structure of galactoside acetyltransferase (GAT), the lacA gene product [Wang, X.-G., Olsen, L. R., and Roderick, S. L. (2002) Structure 10, 581-588]. The structure, together with the new biochemical data, suggests that GAT and MAT are more closely related than previously thought and might have similar cellular functions. However, while GAT is specific for acetylation of galactosyl units, MAT is specific for glucosyl units and is able to acetylate maltooligosaccharides, an important property for biotechnological applications. Structural differences at the acceptor site reflect the differences in substrate specificity.

Acetyltransferases↗