Barbara Hamilton

Endotoxin, TNF-alpha, interleukin-6 and parameters of the cellular immune system in patients with intraabdominal sepsis

The correlation of endotoxin (ET), tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), and cellular immune parameters with multiple organ failure and lethal outcome in intraabdominal infections was studied in a group of 18 patients with peritonitis, abscess or pancreatitis. Of these patients, 7 developed respiratory failure and 5 died due to multiple septic organ failure. The peak levels of ET (2.7 +/- 1.3 ng/ml) in the course of the disease were followed by moderate increases of TNF-alpha (mean 147 +/- 41 pg/ml) and IL-6 (170 +/- 61 pg/ml) within 2 days. Analysis of the parameters for the last 12 days prior to death or discharge showed, that the patient group with lethal outcome was characterized by significant lower mean plasma levels of TNF-alpha (less than 75 pg/ml versus greater than 160 pg/ml) and IL-6 (less than 130 pg/ml versus greater than 270 pg/ml), as well as high rates of unstimulated thymidine uptake into peripheral mononuclear blood cells (greater than 44000 cpm/8 x 10(6) PMBC/18 h versus less than 24000 cmp), T-lymphocyte depression (CD3; approximately greater than 40% reduction) with lower T-helper/inducer subset cell numbers (mean CD:CD8 ratio 1.0 +/- 0.55 versus 1.8 +/- 0.2) and lower lectin (PHA) stimulation values (1.9 +/- 1.4 versus 4.1 +/- 1.0). These data demonstrate an anergic immune status with low mediator levels and depressed T-lymphocyte function in patients with poor prognosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Functional analysis of the Zn(2)Cys(6) transcription factors Oaf1p and Pip2p. Different roles in fatty acid induction of beta-oxidation in Saccharomyces cerevisiae.

Fatty acid induction of the peroxisomal β-oxidation machinery in Saccharomyces cerevisiaeinvolves transcriptional control of genes regulated by the oleate response element (ORE). Glucose as the preferred carbon source antagonizes this effect. Induction is dependent on the Zn2Cys6 family members Oaf1p and Pip2p, which bind to this element as a heterodimer. We show here by ectopically expressing both components and LexA fusion derivatives that this transcription factor complex is only active in the presence of oleate. In contrast to Pip2p, Oaf1p is responsive to oleate activation in the absence of the other component of the heterodimer. Therefore, it is the exclusive receptor of the oleate signal. Pip2p is active also under noninducing conditions but is effectively inhibited when complexed with Oaf1p in the absence of inducer. It contributes to the trans-activation properties of the Oaf1p-Pip2p heterodimer and is required for efficient binding of Oaf1p to OREs in vivo. Repression of ORE-dependent transcription by glucose occurs via both Oaf1p and Pip2p. By dissecting functional domains of both proteins, we identified a region required for regulated activity of the C-terminal activation domain. These findings allow us to postulate a model for carbon source-regulated transcription of peroxisomal protein genes.

Sequence analysis of pre-ferredoxin-NADP+-reductase cDNA from Cyanophora paradoxa specifying a precursor for a nucleus-encoded cyanelle polypeptide

A cDNA clone for pre-ferredoxin-NADP+ reductase (FNR) was obtained by screening a Cyanophora paradoxa expression library with antibodies specific for cyanelle FNR. The 1.4 kb transcript was derived from a single-copy gene. The precursor (41 kDa) and mature forms (34 kDa) of FNR were identified by western blotting of in vitro translation products and cyanelle extracts, respectively. The derived amino acid sequence of the mature form was corroborated by data from N-terminal protein sequencing and yielded identity scores from 58% to 62% upon comparison with cyanobacterial FNRs. Sequence conservation seemed to be even more pronounced in comparison with enzymes from higher plants, but using the neighbor joining method the C. paradoxa sequence was clearly positioned between the prokaryotic and eukaryotic sequences. The transit peptide of 65 or 66 amino acids appeared to be totally unrelated to those from spinach, pea and ice plant but showed overall characteristics of stroma-targeting peptides.

Regulation of the yeast SPS19 gene encoding peroxisomal 2,4‐dienoyl‐CoA reductase by the transcription factors Pip2p and Oaf1p: β‐oxidation is dispensable for Saccharomyces cerevisiae sporulation in acetate medium

The yeast SPS19 gene encoding the peroxisomally targeted 2,4‐dienoyl‐CoA reductase shares its promoter region (291 bp) with the sporulation‐specific gene SPS18. SPS19 is induced during sporulation in diploids but to a lesser extent than SPS18; under oleate induction conditions, SPS19, but not SPS18, is transcribed via an oleate response element (ORE) independently of ploidy or sporulation. The SPS19 ORE is the binding target of the Pip2p and Oaf1p transcription factors, and an SPS19–lacZ reporter gene, which is highly expressed in oleate‐induced cells, is not activated in haploids devoid of either protein. We examined the expression of CYC1–lacZ reporter constructs carrying the SPS19 and CTA1 OREs in diploids propagated under sporulation conditions and have shown that OREs are not sufficient for heterologous expression during yeast development. In addition, diploids deleted at either PIP2 or OAF1 demonstrated abundant ascosporogenesis, indicating that these genes are not essential for sporulation. A Δpex6 strain lacking peroxisomal structures and one devoid of fatty acyl‐CoA oxidase (Δpox1), the first step in fungal β‐oxidation, were both proficient for sporulation and, hence, β‐oxidation and the peroxisomal compartment containing it are dispensable for meiotic development.

The peroxisomal transporter gene ANT1 is regulated by a deviant oleate response element (ORE): characterization of the signal for fatty acid induction.

Saccharomyces cerevisiae ANT1/YPR128c encodes the peroxisomal adenine nucleotide transporter that provides ATP for intra-peroxisomal activation of medium-chain fatty acids. A lacZ reporter construct comprising the ANT1 promoter was shown to be comparatively more highly expressed in a wild-type strain grown on oleic acid, a long-chain fatty acid, than in pip2Delta(oaf1)Delta mutant cells that are defective in fatty acid induction. The ANT1 promoter was demonstrated to contain a deviant oleate response element (ORE) that could bind the Pip2p-Oaf1p transcription factor and confer activation on a basal CYC1-lacZ reporter gene. Expression of Ant1p as well as other enzymes whose genes are known to be regulated by a canonical ORE was found to be increased in cells grown on lauric acid, a medium-chain fatty acid. We concluded that the signal for induction does not differentiate between long- and medium-chain fatty acids. This signal was independent of beta-oxidation or the biogenesis of the peroxisomal compartment where this process occurs, since a pox1Delta strain blocked in the first and rate-limiting step of beta-oxidation as well as various pex mutant cells devoid of intact peroxisomes produced sufficient amounts of Pip2p-Oaf1p for binding OREs in vitro and for expressing an ORE-driven reporter gene. The signals durability was shown to be related to the concentration of fatty acids in the medium, since a pex6Delta strain expressed an ORE-driven reporter gene at high levels for a longer period than did isogenic wild-type cells. Generation of the signal was also independent of protein synthesis, as demonstrated by cycloheximide treatment.

Degradation of conjugated linoleic acid isomers in the yeast Saccharomyces cerevisiae.

Propagation of Saccharomyces cerevisiae cells in conjugated linoleic acid (CLA) medium resulted in activation of the transcriptional machinery that responds to fatty acids. Cells utilized efficiently trans-10,cis-12 CLA, but not the corresponding cis-9,trans-11 isomer, probably due to the formation of cis-3,trans-5-dienoyl-CoA intermediates that are recalcitrant to beta-oxidation.

A novel element in the promoter of the Saccharomyces cerevisiae gene SPS19 enhances ORE-dependent up-regulation in oleic acid and is essential for de-repression

Abstract In Saccharomyces cerevisiae cells grown on oleic acid, genes encoding enzymes of β-oxidation are induced by the interaction of a transcription factor composed of Pip2p and Oaf1p with an oleate response element (ORE) in their promoters. The SPS19 gene, which encodes peroxisomal 2,4-dienoyl-CoA reductase, an auxiliary β-oxidation enzyme, has been shown previously to be up-regulated by a canonical ORE. To determine whether additional elements contribute to this transcriptional up-regulation, deletion analysis of the SPS19 promoter was conducted using SPS19-lacZ reporter genes. In a reporter construct containing a deletion adjacent to the ORE, transcriptional activation of SPS19 in oleic acid medium was impaired. Together with an additional segment that overlaps a portion of the canonical ORE, this region forms a continuous element (termed UASSPS19) that is essential for de-repression of SPS19 when glucose levels are low. The potentially bi-partite UASSPS19 element was able to initiate bi-directional transcription from a promoterless CYC1-lacZ reporter construct under de-repression conditions, whereas the canonical ORE was not. In oleic acid-containing medium, UASSPS19 stimulated transcription of the reporter gene 2.4-fold compared to the intact SPS19 ORE, but did so only in the presence of Pip2p and Oaf1p. UASSPS19, which is similar to a transcriptional enhancer in the promoter of the sporulation-specific gene SPS4, was shown specifically to bind several proteins, including Pip2p and Oaf1p. We propose that UASSPS19 and other sequences like it are required to enhance the transcriptional effects mediated by more specific response elements.

Fate and role of peroxisomes during the life cycle of the yeast Saccharomyces cerevisiae: inheritance of peroxisomes during meiosis

Abstract Sporulation in the yeast Saccharomyces cerevisiae is a meiotic developmental process that occurs in MATa/MATα heterozygotes in response to nutrient deprivation. Here, the fate and role of peroxisomes during sporulation and germination has been examined by a combination of immunoelectron microscopy and the use of pex mutants defective in peroxisomal functions. Using a green fluorescent protein probe targeted to peroxisomes we show that peroxisomes are inherited through meiosis and that they do not increase in number either during sporulation or spore germination. In addition, there is no requirement for peroxisome degradation prior to spore packaging. Unlike the situation in filamentous fungi, peroxisomes do not proliferate during the yeast life cycle. Functional peroxisomes are dispensable for efficient meiotic development on acetate medium since homozygous Δpex6 diploids sporulated well and produced mature spores that were resistant to diethyl ether. Like haploids, diploid cells can proliferate their peroxisomes in response to oleate as sole carbon source in liquid medium, but under these conditions they do not sporulate. On solid oleate medium, homozygous pex5,Δpex6, and pex7 cells were unable to sporulate efficiently, whereas the wild type was. The results presented here are discussed in terms of the transmission of organelles to progeny cells.