Candidaemia in Sweden: a nationwide prospective observational survey.

A prospective observational nationwide investigation was performed from September 2005 to August 2006 to study the epidemiology of candidaemia in Sweden. From 385 patients, 403 isolates were recovered, yielding an incidence of 4.2 cases per 100 000 inhabitants. Candida albicans was the most common species (61%), followed by Candida glabrata (20%) and Candida parapsilosis (9%). The rates of resistance to fluconazole were ≤ 1% in C. albicans and 6-29% in non-albicans species other than C. glabrata and Candida krusei. Resistance to voriconazole was rare, except for C. glabrata and C. krusei. Only three isolates had reduced susceptibility to amphotericin B, and one had reduced susceptibility to caspofungin.

Tumor suppressor Fbxw7 regulates TGFß signaling by targeting TGIF1 for degradation.

Transforming growth factor-ß (TGFß) signaling regulates multiple cellular processes, including extracellular matrix production, cell growth, apoptosis and differentiation. Dysfunction of TGFß signaling has been implicated in various human disorders ranging from vascular diseases to cancer. TGFß signaling is negatively regulated by the transcriptional repressor TGFß-induced factor 1 (TGIF1). The tumor suppressor Fbxw7 is the substrate-recognition factor of a ubiquitin ligase that targets multiple proteins for degradation, including c-Myc, cyclin E, c-Jun and Notch. Here, we describe that TGIF1 is targeted for degradation by Fbxw7 in a phosphorylation-dependent manner. Inactivation of Fbxw7 results in the accumulation of phosphorylated TGIF1 molecules and repression of TGFß-dependent transcription. Cancer cell lines with inactivating mutations in Fbxw7 show enhanced levels of TGIF1 and attenuated TGFß-dependent signaling. Importantly, inactivation of Fbxw7 attenuates TGFß-dependent regulation of cell growth and migration. Taken together, our results suggest that Fbxw7 is a novel regulator of TGFß signaling.

Histone acetyltransferases interact with and acetylate p70 ribosomal S6 kinases in vitro and in vivo.

The 70kDa ribosomal protein S6 kinases (S6K1 and S6K2) play important roles in the regulation of protein synthesis, cell growth and survival. S6Ks are activated in response to mitogen stimulation and nutrient sufficiency by the phosphorylation of conserved serine and threonine residues. Here we show for the first time, that in addition to phosphorylation, S6Ks are also targeted by lysine acetylation. Following mitogen stimulation, S6Ks interact with the p300 and p300/CBP-associated factor (PCAF) acetyltransferases. S6Ks can be acetylated by p300 and PCAF in vitro and S6K acetylation is detected in cells expressing p300. Furthermore, it appears that the acetylation sites targeted by p300 lie within the divergent C-terminal regulatory domains of both S6K1 and S6K2. Acetylation of S6K1 and 2 is increased upon the inhibition of class I/II histone deacetylases (HDACs) by trichostatin-A, while the enhancement of S6K1 acetylation by nicotinamide suggests the additional involvement of sirtuin deacetylases in S6K deacetylation. Both expression of p300 and HDAC inhibition cause increases in S6K protein levels, and we have shown that S6K2 is stabilized in cells treated with HDAC inhibitors. The finding that S6Ks are targeted by histone acetyltransferases uncovers a novel mode of crosstalk between mitogenic signalling pathways and the transcriptional machinery and reveals additional complexity in the regulation of S6K function.

Scaling of the low-temperature dephasing rate in Kondo systems.

We present phase coherence time measurements in quasi-one-dimensional Ag wires doped with Fe Kondo impurities of different concentrations n_{s}. Because of the relatively high Kondo temperature T_{K} approximately 4.3 K of this system, we are able to explore a temperature range from above T_{K} down to below 0.01T_{K}. We show that the magnetic contribution to the dephasing rate gamma_{m} per impurity is described by a single, universal curve when plotted as a function of T/T_{K}. For T>0.1T_{K}, the dephasing rate is remarkably well described by recent numerical results for spin S=1/2 impurities. At lower temperature, we observe deviations from this theory. Based on a comparison with theoretical calculations for S>1/2, we discuss possible explanations for the observed deviations.

CTP:phosphocholine cytidylyltransferase, a new sterol- and SREBP-responsive gene.

The CTP:phosphocholine cytidylyltransferase (CT) gene encodes the rate-controlling enzyme in the phosphatidylcholine biosynthesis pathway. CTalpha mRNA levels, like farnesyl diphosphate synthase and the LDL receptor, are repressed when human or rodent cells are incubated with exogenous sterols and induced when cells are incubated in lipid-depleted medium. A putative sterol response element (SRE) was identified 156 bp upstream of the transcription start site of the CTalpha gene. Electrophoretic mobility shift assays demonstrate that recombinant SREBP-1a binds to the wild-type SRE identified in the CTalpha promoter but not to oligonucleotides containing two mutations in the SRE. In other studies, a luciferase reporter construct under the control of the murine CTalpha proximal promoter was transiently transfected into cells. The activity of the reporter was repressed after addition of sterols to the medium and induced when the cells were incubated in lipid-depleted medium. The activity of the CTalpha-luciferase reporter was also induced when cells were cotransfected with plasmids encoding either SREBP-1a or SREBP-2. In contrast, no induction was observed under the same conditions when the CTalpha promoter-reporter gene contained two mutations in the SRE. In addition, the induction of the wild-type CTalpha promoter-reporter gene that occurs in cells incubated in lipid-depleted medium is attenuated when dominant-negative SREBP is cotransfected into the cells. These studies demonstrate that transcription of the CTalpha gene is inhibited by sterols and activated by mature forms of SREBP. We conclude that SREBP-regulated genes are involved not only in the synthesis of cholesterol, fatty acids, triglycerides, and NADPH, but also, as shown here, in the synthesis of phospholipids.

Association of beta2-adrenoceptor Gln27Glu variant with body weight but not hypertension.

Beta2-adrenoceptor (beta2-ADR)-mediated vasodilatation decreases vascular reactivity and blood pressure (BP) and chromosome 5 where its gene (ADRB2R) resides and shows linkage to hypertension (HT). A Gln27Glu ADRB2R variant confers resistance to agonist-induced desensitization and enhanced vasodilator response to isoprenaline. Therefore, we carried out a case-control study in a cohort of HT and normotensive (NT) Anglo-Celtic Australian white subjects whose parents had a similar BP status as the subjects. Glu27 frequency was 0.41 in 108 HT and 0.42 in 141 NT (chi2 = 0.05, P = .82). Within the HT group, the Glu27 allele was more prevalent in 61 subjects who were overweight (body mass index [BMI] > or = 25 kg/m2) compared with 41 who were lean (BMI <25 kg/m2); ie, 0.49 v 0.31, respectively (chi2 = 6.4, P = .012). Furthermore, Glu27 tracked with elevation in BMI in these subjects: 24 +/- 4 kg/m2, 27 +/- 5 kg/m2, and 28 +/- 5 kg/m2 for Gln/Gln, Gln/Glu, and Glu/Glu, respectively (P = .0058 by one-way ANOVA). Thus, the Gln27Glu beta2-ADR variant is excluded in HT, but might influence body weight.

The transcriptional co-activator P/CAF potentiates TGF-beta/Smad signaling.

Smads perform pivotal functions in the intracellular signaling of transforming growth factor-beta (TGF-beta). TGF-beta-mediated activation of TGF-beta type I receptor stimulates the phosphorylation of Smad2 and Smad3 and subsequent heteromeric complex formation with Smad4. The heteromeric Smad complexes translocate into the nucleus where they, in co-operation with co-activators and co-repressors, regulate transcriptional responses. Here we investigated the possible co-activator function of P/CAF in TGF-beta/Smad signaling. P/CAF was found to interact directly with Smad3 in vitro. Moreover, Smad2 and Smad3 interacted with P/CAF upon TGF-beta type I receptor activation in cultured mammalian cells. The interaction involves the MH2 domain of Smad3 and the N-terminal region of P/CAF. P/CAF potentiated the transcriptional activity of heterologous Gal4-Smad2 and Gal4-Smad3 fusion proteins. In addition, P/CAF potentiated the TGF-beta/Smad3-induced transcriptional responses, which could be further enhanced by co-activators p300 and Smad4. P/CAF may, therefore, activate Smad-mediated transcriptional responses independently or in co-operation with p300/CBP. Our results indicate a direct physical and functional interplay between two negative regulators of cell proliferation, Smad3 and P/CAF.

YY1 is a negative regulator of transcription of three sterol regulatory element-binding protein-responsive genes.

Ying Yang 1 (YY1) is shown to bind to the proximal promoters of the genes encoding 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase, farnesyl diphosphate (FPP) synthase, and the low density lipoprotein (LDL) receptor. To investigate the potential effect of YY1 on the expression of SREBP-responsive genes, HepG2 cells were transiently transfected with luciferase reporter constructs under the control of promoters derived from either HMG-CoA synthase, FPP synthase, or the LDL receptor genes. The luciferase activity of each construct increased when HepG2 cells were incubated in lipid-depleted media or when the cells were cotransfected with a plasmid encoding mature sterol regulatory element-binding protein (SREBP)-1a. In each case, the increase in luciferase activity was attenuated by coexpression of wild-type YY1 but not by coexpression of mutant YY1 proteins that are known to be defective in either DNA binding or in modulating transcription of other known YY1-responsive genes. In contrast, incubation of cells in lipid-depleted media resulted in induction of an HMG-CoA reductase promoter-luciferase construct by a process that was unaffected by coexpression of wild-type YY1. Electromobility shift assays were used to demonstrate that the proximal promoters of the HMG-CoA synthase, FPP synthase, and the LDL receptor contain YY1 binding sites and that YY1 displaced nuclear factor Y from the promoter of the HMG-CoA synthase gene. We conclude that YY1 inhibits the transcription of specific SREBP-dependent genes and that, in the case of the HMG-CoA synthase gene, this involves displacement of nuclear factor Y from the promoter. We hypothesize that YY1 plays a regulatory role in the transcriptional regulation of specific SREBP-responsive genes.

Sterols and isoprenoids: signaling molecules derived from the cholesterol biosynthetic pathway.

Compounds derived from the isoprenoid/cholesterol biosynthetic pathway have recently been shown to have novel biological activities. These compounds include certain sterols, oxysterols, farnesol, and geranylgeraniol, as well as the diphosphate derivatives of isopentenyl, geranyl, farnesyl, geranylgeranyl, and presqualene. They regulate transcriptional and post-transcriptional events that in turn affect lipid synthesis, meiosis, apoptosis, developmental patterning, protein cleavage, and protein degradation.

Solid phase organic synthesis of piperazinone containing enkephalin mimetics: a readily derivatized, traceless scaffold.

The solid phase synthesis of a series of piperazinone-derived Leu-enkephalin analogues is presented. The initial step in the synthesis involved the N-alkylation of Wang resin bound N-(4-tert-butyloxy-phenethyl)-glycine with D or L Boc-serine-beta-lactone (the Vederas lactone). The resulting carboxylic acid was then coupled to a variety of monosubstituted benzylamine derivatives using benzotriazol-1-yloxy-tris(dimethyl-amino)phosphonium hexafluorophosphate (the BOP reagent) to yield a series of resin bound tertiary amides. Treatment with 5% H2O in TFA resulted in the facile cleavage, deprotection, and cyclization of this linear precursor to yield a series of piperazinones (compounds 1-8).

Analysis of cytochrome P450 metabolites of arachidonic and linoleic acids by liquid chromatography-mass spectrometry with ion trap MS.

We have used reversed phase-high performance liquid chromatography-mass spectrometry (RP-HPLC-MS) with an ion trap mass spectrometer to study the metabolism of arachidonic and linoleic acids by human recombinant cytochrome P450 (CYP) enzymes. We first recorded the MS2 spectra of the carboxylate anions of epoxides, diols, omega-side chain, and bisallylic hydroxy fatty acids of arachidonic, octadeuterated arachidonic, and linoleic acids. The metabolites formed by CYP2C9 and CYP2C19 were then studied. CYP2C9 converted arachidonic and linoleic acids to epoxides/diols and monohydroxy fatty acids. Some hydroxyeicosatetraenoic acids (HETEs) were studied in detail to investigate the oxygenation mechanism. Incubation of CYP2C9 under oxygen-18 gas showed that all HETEs had incorporated oxygen-18 to the same degree. Chiral HPLC showed that CYP2C9 formed 15R-HETE (72% of the R enantiomer), 13S-HETE (90%), and 11R-HETE (57%). RP-HPLC-MS analysis revealed that CYP2C19 oxygenated arachidonic acid to 19-HETE, 14,15-epoxyeicosatrienoic acid (EET), and 8,9-EET as main metabolites. The method was sufficiently sensitive to identify arachidonic acid metabolites formed by some other isozymes. RP-HPLC-MS with MS2 seems to be useful for rapid identification of fatty acid metabolites in complex mixtures formed by cytochrome P450.

Signaling molecules derived from the cholesterol biosynthetic pathway: mechanisms of action and possible roles in human disease.

The association of high plasma cholesterol levels with the development of atherosclerosis is well known. The metabolic pathways that are regulated by cholesterol and the mechanisms involved are less well understood. Recent studies have identified not only cholesterol, but also oxysterols and isoprenoids, derived from the cholesterol biosynthetic pathway, as new signaling molecules. The transcriptional and post-transcriptional regulation of specific genes and metabolic pathways by these newly discovered signaling molecules may be important in the development of human disease and forms the topic of this review.

Human geranylgeranyl diphosphate synthase: isolation of the cDNA, chromosomal mapping and tissue expression.

We report the nucleotide sequence of human geranylgeranyl diphosphate (GGPP) synthase cDNA isolated from a fetal heart library. The 2.5 kb cDNA encodes a protein of 34 kDa. The protein contains six domains that have been identified previously in many other prenyltransferases. Recombinant, purified histidine-tagged protein exhibited the enzymatic properties associated with GGPP synthase, namely the synthesis of GGPP from farnesyl diphosphate and isopentenyl diphosphate. Transient transfection of mammalian cells with a plasmid encoding the putative GGPP synthase resulted in a 55-fold increase in GGPP synthase activity. Taken together, these results establish that the cDNA encodes the mammalian GGPP synthase protein. The mRNA for GGPP synthase was expressed ubiquitously. Of the 16 human tissues examined, the highest expression of the mRNA was in testis. The mRNA levels in cultured HeLa cells were unaffected by alterations in cellular sterol levels and contrasted with the significant regulation of isopentenyl diphosphate synthase mRNA under these same conditions. Fluorescent in situ hybridization was used to map the single gene encoding human GGPP synthase to chromosome 1q43.

CBP is required for sterol-regulated and sterol regulatory element-binding protein-regulated transcription.

Cells were transfected with luciferase reporter genes, under the control of promoters derived from either the farnesyl diphosphate (FPP) synthase, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase, HMG-CoA reductase, or low density lipoprotein receptor genes. The increase in luciferase activity that occurred when cells were either incubated in sterol-depleted medium or cotransfected with a cDNA encoding sterol regulatory element-binding protein (SREBP)-1a was prevented by coexpression of wild-type E1A or a Gal4-CBP (1-451) fusion protein. The inhibitory effect of E1A was overcome by coexpression of CBP. The increase in reporter gene activity noted above was not affected when the cells were cotransfected with cDNAs that encoded either a mutant E1A that is unable to interact with the transcriptional activator CBP or Gal4-CBP fusion proteins encoding separate fragments of CBP, which span the remainder of the CBP molecule. A preformed SREBP-1a:[32P]DNA complex bound specifically to membrane-immobilized GST-CBP fusion proteins that contained amino-terminal portions of CBP. In order to investigate the role of CBP in the regulation of endogenous genes, we isolated stable transformants that express Gal4-CBP(1-451) in response to added doxycycline. Induction of endogenous FPP synthase and HMG-CoA synthase mRNAs, in response to cellular cholesterol depletion, was prevented when cells expressed Gal4-CBP(1-451). We conclude that when cells are incubated in the absence of sterols, the transcriptional activation of the HMG-CoA synthase, HMG-CoA reductase, FPP synthase, and low density lipoprotein receptor genes is dependent on a specific interaction between SREBP, which is bound to the promoter DNA, and the amino-terminal domain (amino acids 1-451) of CBP.

Synergistic activation of transcription by nuclear factor Y and sterol regulatory element binding protein.

The current studies define the role of three distinct cis-elements in the proximal promoter of the rat farnesyl diphosphate (FPP) synthase gene. The three cis-elements, a sterol regulatory element 3 (SRE-3) flanked by an ATTGG motif (inverted CCAAT box), and a CCAAT box, form a sterol regulatory unit that is necessary and sufficient for sterol-regulated expression of FPP synthase promoter-reporter genes. FPP synthase promoter-reporter genes, that contain promoters with either wild-type nucleotide sequences or mutations in one or more of the three cis-elements, were transiently transfected into CV-1 cells. The activity of the wild-type promoter-reporter gene increased when the cells were incubated in sterol-depleted media or when the cells were co-transfected with a plasmid encoding the mature form of SRE binding protein (SREBP-1a). The results with the mutant promoter-reporter genes demonstrated that all three cis-elements were necessary for normal expression/regulation of the reporter gene by either sterols or by co-expressed SREBP-1a. Gel mobility shift assays demonstrated that the synergistic binding of SREBP-1a to SRE-3 was dependent on the binding of recombinant nuclear factor Y (NF-Y) to the DNA, consistent with the in vivo regulation studies.

Identification of glycerol-3-phosphate acyltransferase as an adipocyte determination and differentiation factor 1- and sterol regulatory element-binding protein-responsive gene.

We demonstrate that the mRNA levels of glycerol-3-phosphate acyltransferase (GPAT), a mitochondrial enzyme catalyzing the initial step in glycerolipid synthesis, are induced during the differentiation of 3T3-L1 preadipocytes to adipocytes and following ectopic expression of rat adipocyte determination and differentiation factor 1 (ADD1), a protein with high homology to the human sterol regulatory element-binding protein-1 (SREBP-1). The increase in GPAT mRNA levels that occurs during differentiation is partially prevented by ectopic expression of a dominant negative form of ADD1. Nucleotide sequences corresponding to the proximal promoter of the murine mitochondrial GPAT gene (Jerkins, A. A., Liu, W. R., Lee, S., and Sul, H. S. (1995) J. Biol. Chem. 270, 1416-1421) bound SREBP-1a and NF-Y in electromobility shift assays. In addition, GPAT promoter-luciferase reporter genes were stimulated by co-expression of SREBP-1a. This increase was attenuated when either a dominant negative form of NF-Y was co-transfected into the cells or when the GPAT promoter contained mutations in the putative binding sites for SREBP-1a or NF-Y. These studies demonstrate that the regulated expression of the mitochondrial GPAT gene requires both NF-Y and ADD1/SREBPs. Thus, SREBPs/ADD1 regulate not only genes involved in cholesterol homeostasis and fatty acid synthesis but also a key enzyme in glycerolipid synthesis.

Administration of G-CSF to healthy subjects: the effects on eosinophil counts and mobilization of eosinophil granule proteins.

Any influence of G-CSF on eosinophils is mostly negative, although reports which have studied this relationship are few with varied results. The aim of this study was to investigate the influence of G-CSF administration to healthy subjects on eosinophils in peripheral blood. Blood eosinophil counts, serum levels of eosinophil cationic protein (ECP), eosinophil peroxidase (EPO) and eosinophil protein X (EPX), as well as cell morphology were studied. 14 healthy volunteers received 7.5 microg (n = 8) or 10 microg/kg body weight (n = 6) G-CSF daily for six consecutive days. ECP and EPX were assessed by specific RIAs and EPO by a specific FEIA. Cell morphology was examined by electron microscopy. During G-CSF administration, eosinophil counts increased from 0.22 +/- 0.04 x 10(9)/l to 0.61 +/- 0.098 x 10(9)/l (P = 0.001), serum ECP from 12.39 +/- 2.45 microg/l to 61.82 +/- 7.38 microg/l (P = 0.0014), serum EPX from 28.05 +/- 4.54 microg/l to 87.86 +/- 9.84 microg/l (P = 0.002) and serum EPO from 8.89 +/- 2.2 microg/l to 19.98 +/- 5.1 microg/l (P = 0.003). All variables returned gradually to initial values after discontinuation of G-CSF. Distinct changes in the morphology of secondary granules were observed 24 h after G-CSF administration. The granules became irregular and their matrix less electron dense. We conclude that administration of G-CSF to healthy humans increases the number of circulating eosinophils and affects the mobilization of eosinophil granule proteins.

Multiple primary malignant tumors in a national cancer registry--reliability of reporting.

During the years 1958-1988, 808,522 individuals were registered in the Swedish national population-based cancer register with a total of 933,900 primary malignant tumors. Roughly 11% of the tumors reported to the Swedish Cancer Registry in 1988 were found in persons earlier registered for another primary malignancy. One hundred of the individuals registered with multiple primary malignant tumors were randomly selected for a study of the reliability of reporting of multiple malignancies. Medical records and when necessary histopathological slides and other relevant diagnostic material for each malignancy were collected and the diagnoses reevaluated. Three persons had to be excluded; thus 97 cases with 209 reported malignancies were analysed. Of these, 94% of the first, 98% of the second and 79% of the third malignancy were accepted. Twelve of the reported tumors were not accepted as malignant, five were benign or cancer in situ, five were incorrectly reported as new primaries and two were a second incorrect registration of a previously registered malignancy. All 97 persons had at least one malignant tumor, in 90% of the persons all reported diagnoses were accepted and 93% had multiple primary malignancies. The results of this quality control study indicate that suitable data are available in the Swedish Cancer Registry for investigations related to the occurrence of multiple primary malignancies in a large unselected population. The Swedish Cancer Registry, to which all newly diagnosed malignancies by law have to be reported, comprise today close to one million individuals with registered malignancies.