Gene variants associated with obstructive sleep apnea (OSA) in relation to sudden infant death syndrome (SIDS).

BACKGROUND: Both obstructive sleep apnea (OSA) and (at least a fraction of) sudden infant death syndrome (SIDS) are associated with impaired respiration. For OSA, an association with several gene variants was identified. Therefore, our hypothesis is that these polymorphisms might be of relevance in SIDS as well. METHODS: Twenty-four single nucleotide polymorphisms (SNPs) in 21 candidate genes connected to OSA, were genotyped in a total of 282 SIDS cases and 374 controls. Additionally, subgroups based on factors codetermining the SIDS risk (age, sex, season, and prone position) were established and compared as well. RESULTS: Two of the analyzed SNPs showed nominally significant differences between SIDS and control groups: rs1042714 in ADRB2 (adrenoceptor beta 2) and rs1800541 in EDN1 (endothelin 1). In the subgroup analyses, 10 further SNPs gave significant results. Nevertheless, these associations did not survive adjustment for multiple testing. CONCLUSIONS: Our results suggest that there might be a link between SIDS and OSA and its resulting respiratory and cardiovascular problems, albeit this predisposition might be dependent on the combination with other, hitherto unknown gene variants. These findings may encourage replication studies to get a better understanding of this connection.

Evaluation of variation in the phosphoinositide-3-kinase catalytic subunit alpha oncogene and breast cancer risk.

BACKGROUND: Somatic mutations in phosphoinositide-3-kinase catalytic subunit alpha (PIK3CA) are frequent in breast tumours and have been associated with oestrogen receptor (ER) expression, human epidermal growth factor receptor-2 overexpression, lymph node metastasis and poor survival. The goal of this study was to evaluate the association between inherited variation in this oncogene and risk of breast cancer. METHODS: A single-nucleotide polymorphism from the PIK3CA locus that was associated with breast cancer in a study of Caucasian breast cancer cases and controls from the Mayo Clinic (MCBCS) was genotyped in 5436 cases and 5280 controls from the Cancer Genetic Markers of Susceptibility (CGEMS) study and in 30 949 cases and 29 788 controls from the Breast Cancer Association Consortium (BCAC). RESULTS: Rs1607237 was significantly associated with a decreased risk of breast cancer in MCBCS, CGEMS and all studies of white Europeans combined (odds ratio (OR)=0.97, 95% confidence interval (CI) 0.95-0.99, P=4.6 × 10(-3)), but did not reach significance in the BCAC replication study alone (OR=0.98, 95% CI 0.96-1.01, P=0.139). CONCLUSION: Common germline variation in PIK3CA does not have a strong influence on the risk of breast cancer.

Redox signaling in chloroplasts: cleavage of disulfides by an iron-sulfur cluster.

Light generates reducing equivalents in chloroplasts that are used not only for carbon reduction, but also for the regulation of the activity of chloroplast enzymes by reduction of regulatory disulfides via the ferredoxin:thioredoxin reductase (FTR) system. FTR, the key electron/thiol transducer enzyme in this pathway, is unique in that it can reduce disulfides by an iron-sulfur cluster, a property that is explained by the tight contact of its active-site disulfide and the iron-sulfur center. The thin, flat FTR molecule makes the two-electron reduction possible by forming on one side a mixed disulfide with thioredoxin and by providing on the opposite side access to ferredoxin for delivering electrons.

Evidence of presynaptic location and function of the prion protein.

The prion protein (PrP(C)) is a copper-binding protein of unknown function that plays an important role in the etiology of transmissible spongiform encephalopathies. Using morphological techniques and synaptosomal fractionation methods, we show that PrP(C) is predominantly localized to synaptic membranes. Atomic absorption spectroscopy was used to identify PrP(C)-related changes in the synaptosomal copper concentration in transgenic mouse lines. The synaptic transmission in the presence of H(2)O(2), which is known to be decomposed to highly reactive hydroxyl radicals in the presence of iron or copper and to alter synaptic activity, was studied in these animals. The response of synaptic activity to H(2)O(2) was found to correlate with the amount of PrP(C) expression in the presynaptic neuron in cerebellar slice preparations from wild-type, Prnp(0/0), and PrP gene-reconstituted transgenic mice. Thus, our data gives strong evidence for the predominantly synaptic location of PrP(C), its involvement in the regulation of the presynaptic copper concentration, and synaptic activity in defined conditions.

Improved in vitro light activation and assay systems for two spinach chloroplast enzymes.

An improved system for the in vitro light activation of the chloroplast enzymes fructose-1,6-bisphosphatase and NADP-dependent malate dehydrogenase is described. Through the presence of the monothiol beta-mercaptoethanol in the reaction mixtures the activated forms of the enzymes can be stabilized and their activity determined spectrophotometrically.

Studies on the regulatory properties of chloroplast fructose-1,6-bisphosphatase.

The regulatory properties of chloroplast fructose-1,6-bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrolase, (EC 3.1.3.11) were examined with a homogeneous enzyme preparation isolated from spinach leaves. The activation of the enzyme, that was earlier shown to occur via reduced thioredoxin, was found to be accompanied by a structural change that took place more slowly than the rate of catalysis. The recently found deactivation of the thioredoxin-activated enzyme by physiological oxidants such as oxidized glutathione and dehydroascorbic acid was also slow relative to catalysis. Under the conditions used, the activated enzyme showed a pH optimum of about 8.0, whereas the corresponding value for the non-activated form was pH 8.8. The importance of the thioredoxin-linked mechanism of enzyme regulation that is effected through photoreduced ferredoxin and ferredoxin-thioredoxin reductase is discussed in relation to other light-controlled regulatory agents in chloroplasts.

Cloning and sequencing of a corn (Zea mays) nuclear gene coding for the chloroplast specific catalytic subunit of ferredoxin-thioredoxin reductase.

We determined the complete nucleotide sequence of a cDNA clone encoding the smaller, catalytic subunit of ferredoxin-thioredoxin reductase from corn chloroplasts. The translated protein sequence, representing a subunit of 12.9 kDa shows 80% identity with the protein sequence from spinach and contains seven Cys residues all at conserved positions.

The oxidation-reduction properties of spinach thioredoxins f and m and of ferredoxin:thioredoxin reductase.

Oxidation-reduction midpoint potentials have been determined, using cyclic voltammetry, for the active-site disulfide/dithiol couples of spinach thioredoxins f and m and of spinach ferredoxin:thioredoxin reductase (FTR) and for a component likely to be the [4Fe-4S] cluster of FTR. Values for the midpoint potentials (n = 2) of -210 +/- 10 mV were determined for both thioredoxins f and m. Two redox centers were detected in FTR, with midpoint potential values of -230 +/- 10 mV (n = 2) and +340 +/- 30 mV, respectively. Alkylation of the active-site cysteines of FTR by treatment of the enzyme with N-ethylmaleimide (NEM) eliminates the component with the -230 mV midpoint potential, allowing one to assign this value to the active site disulfide/dithiol couple. Inasmuch as the only other electron-carrying center known to be present in FTR is the [4Fe-4S] cluster, it appears likely that the high-potential component can be attributed to this redox moiety. The midpoint potential value of the high-potential feature shifts slightly, to +380 +/- 20 mV, in the NEM-treated enzyme.

Crystal structures of two functionally different thioredoxins in spinach chloroplasts.

Thioredoxins are small ubiquitous proteins which act as general protein disulfide reductases in living cells. Chloroplasts contain two distinct thioredoxins ( f and m) with different phylogenetic origin. Both act as enzyme regulatory proteins but have different specificities towards target enzymes. Thioredoxin f (Trx f), which shares only low sequence identity with thioredoxin m (Trx m) and with all other known thioredoxins, activates enzymes of the Calvin cycle and other photosynthetic processes. Trx m shows high sequence similarity with bacterial thioredoxins and activates other chloroplast enzymes. The here described structural studies of the two chloroplast thioredoxins were carried out in order to gain insight into the structure/function relationships of these proteins. Crystal structures were determined for oxidized, recombinant thioredoxin f (Trx f-L) and at the N terminus truncated form of it (Trx f-S), as well as for oxidized and reduced thioredoxin m (at 2.1 and 2.3 A resolution, respectively). Whereas thioredoxin f crystallized as a monomer, both truncated thioredoxin f and thioredoxin m crystallized as non-covalent dimers. The structures of thioredoxins f and m exhibit the typical thioredoxin fold consisting of a central twisted five-stranded beta-sheet surrounded by four alpha-helices. Thioredoxin f contains an additional alpha-helix at the N terminus and an exposed third cysteine close to the active site. The overall three-dimensional structures of the two chloroplast thioredoxins are quite similar. However, the two proteins have a significantly different surface topology and charge distribution around the active site. An interesting feature which might significantly contribute to the specificity of thioredoxin f is an inherent flexibility of its active site, which has expressed itself crystallographically in two different crystal forms.

Crystallization and preliminary X-ray diffraction studies of the spinach-chloroplast thioredoxin f.

Thioredoxins are low-molecular-mass proteins that function as hydrogen carriers in DNA synthesis and in the transformation of sulfur metabolites. They also act as regulatory proteins in the light-dependent enzyme activation during photosynthesis. F-type thioredoxin from spinach chloroplasts, a monomeric protein of 113 amino acid residues, has been found to specifically activate fructose-1,6-bisphosphatase and other key enzymes of CO2 assimilation. It has been crystallized in the monoclinic system, space group P2(1) with a = 30.6 A, b = 63.1 A, c = 31.6 A and beta = 110.7 degrees. The crystals are suitable for X-ray diffraction studies.

Inhibition of Escherichia coli porphobilinogen synthase using analogs of postulated intermediates.

BACKGROUND: Porphobilinogen synthase is the second enzyme involved in the biosynthesis of natural tetrapyrrolic compounds, and condenses two molecules of 5-aminolevulinic acid (ALA) through a nonsymmetrical pathway to form porphobilinogen. Each substrate is recognized individually at two different active site positions to be regioselectively introduced into the product. According to pulse-labeling experiments, the substrate forming the propionic acid sidechain of porphobilinogen is recognized first. Two different mechanisms for the first bond-forming step between the two substrates have been proposed. The first involves carbon-carbon bond formation (an aldol-type reaction) and the second carbon-nitrogen bond formation, leading to an iminium ion. RESULTS: With the help of kinetic studies, we determined the Michaelis constants for each substrate recognition site. These results explain the Michaelis-Menten behavior of substrate analog inhibitors - they act as competitive inhibitors. Under standard conditions, however, another set of inhibitors demonstrates uncompetitive, mixed, pure irreversible, slow-binding or even quasi-irreversible inhibition behavior. CONCLUSIONS: Analysis of the different classes of inhibition behavior allowed us to make a correlation between the type of inhibition and a specific site of interaction. Analyzing the inhibition behavior of analogs of postulated intermediates strongly suggests that carbon-nitrogen bond formation occurs first.

Analgesic effect of fluproquazone in postoperative patients.

In 4 double-blind, randomized, stratified, parallel group studies, single oral doses of fluproquazone (75 to 200 mg), a new nonsteroidal anti-inflammatory analgesic, were compared with aspirin (1,000 mg) and placebo in a total of 672 hospitalized patients with moderate or severe pain following episiotomy or other surgical interventions. A dose-dependent effect of fluproquazone which was highly significantly superior to placebo and which resembled the effect of aspirin with respect to onset, degree, and duration was noted in all studies. Fluproquazone, 100 to 150 mg, was found to be approximately equiactive to 1,000 mg of aspirin and better tolerated.

Heterodimer formation between thioredoxin f and fructose 1,6-bisphosphatase from spinach chloroplasts.

Chloroplast fructose 1,6-bisphosphatase (FBPase) is activated by reduction of a regulatory disulfide through thioredoxin f (Trx f). In the course of this reduction a transient mixed disulfide is formed linking covalently Trx f with FBPase, which possesses three Cys on a loop structure, two of them forming the redox-active disulfide bridge. The goal of this study was to identify the Cys involved in the transient mixed disulfide. To stabilize this reaction intermediate, mutant proteins with modified active sites were used. We identified Cys-155 of the FBPase as the one engaged in the formation of the mixed disulfide intermediate with Cys-46 of Trx f.

Comparison of the binding sites of plant ferredoxin for two ferredoxin-dependent enzymes.

Differential chemical modification of acidic residues was used to map the binding site of plant ferredoxin (Fd) for the chloroplast enzyme ferredoxin:thioredoxin reductase (FTR). Binding of FTR to Fd inhibits chemical modification of Fd residues D34, D65, E92, E93, E94 and C-terminal A97. The binding site demarcated by these residues differs from that for ferredoxin:NADP+ reductase (FNR). The FTR site includes C-terminal residues but not helix 24-31, which is part of the FNR site. Both sites enclose the [2Fe-2S] cluster.

N-terminal truncation of the variable subunit stabilizes spinach ferredoxin:thioredoxin reductase.

The variable subunit of spinach ferredoxin:thioredoxin reductase (FTR) has an extended N-terminus compared to FTRs from other sources and this was proposed to contribute to the instability of the protein. We constructed two N-terminal truncation mutants of recombinant FTR by removing 16 or 24 residues from the variable subunit. The mutant proteins are readily expressed and show half-saturation values (S(0.5)) for ferredoxin and thioredoxin f comparable to WT. However, truncation increases significantly their stability. Using the stabilized FTR an exposed Cys on its thioredoxin contact surface could be substituted without altering its properties, whereas the replacement of an active site Cys by Ser completely destabilized the protein.

Characterization of the regulatory function of the 46-kDa isoform of Rubisco activase from Arabidopsis.

Arabidopsis Rubisco activase was recently shown to be regulated by redox changes in the larger (46-kDa) isoform specifically mediated by thioredoxin-f [Zhang and Portis (1999) Proc Natl Acad Sci USA 96: 9438-9443]. Reduction greatly increases the activity of the 46-kDa isoform and the native protein at physiological ATP/ADP ratios. In this study we conducted additional experiments to characterize the regulation of Rubisco activase by thioredoxin-f. The K(m) for both ATP hydrolysis and Rubisco activation by the 46-kDa isoform was lowered by 4 to 5-fold after reduction, but the maximum activity was increased by only 10%. Only 0.35 muM thioredoxin-f was required for a half-maximal activity change after a 10 min preincubation and activation with 1 muM was complete after 10 min. Equal amounts of 46-kDa and 43-kDa isoforms were required for a complete inhibition of the Rubisco activation activity after a reduction-oxidation cycle and assay at an ATP/ADP ratio of 3:1, whereas activity was only inhibited by 50% at a 2:1 ratio (43-/46-kDa) of the isoforms. This requirement is consistent with the fact that Arabidopsis normally contains about a 1:1 ratio of the two isoforms at both the mRNA and protein levels. Redox titrations indicated a midpoint potential of -344 mV for the 46-kDa isoform as compared to -342 mV for spinach fructose 1,6-bisphosphatase at pH 7.9, consistent with previous reports indicating that these proteins are co-regulated by light intensity in a similar manner.

The ferredoxin/thioredoxin system: from discovery to molecular structures and beyond.

Experiments initiated in the early 1960s on fermentative bacteria led to the discovery of ferredoxin-dependent alpha-ketocarboxylation reactions that were later found to be key to a new cycle for the assimilation of carbon dioxide in photosynthetic bacteria (the reductive carboxylic acid or reverse citric cycle). The latter finding set the stage for the discovery of a regulatory system, the ferredoxin/thioredoxin system, functional in photosynthesis in chloroplasts and oxygen-evolving photosynthetic prokaryotes. The chloroplast research led to a description of the extraplastidic NADP/thioredoxin system that is now known to function in heterotrophic plant processes such as seed germination and self-incompatibility. Extensions of the fundamental research have begun to open doors to the broad application of thioredoxin in technology and medicine.